Class 4-6 Lyases, Isomerases, Ligases: EC 4-6 (Springer Handbook of Enzymes, Supplement Volume S7)

Springer Handbook of Enzymes Supplement Volume S7 Dietmar Schomburg and Ida Schomburg (Eds.) Springer Handbook of En...

Author: Antje Chang | Dietmar Schomburg | Ida Schomburg

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Springer Handbook of Enzymes Supplement Volume S7

Dietmar Schomburg and Ida Schomburg (Eds.)

Springer Handbook of Enzymes Supplement Volume S7 Class 4–6 Lyases, Isomerases, Ligases EC 4–6 coedited by Antje Chang

Second Edition

13

Professor Dietmar Schomburg e-mail: [email protected] Dr. Ida Schomburg e-mail: [email protected]

Technical University Braunschweig Bioinformatics & Systems Biology Langer Kamp 19b 38106 Braunschweig Germany

Dr. Antje Chang e-mail: [email protected]

Library of Congress Control Number: 2009927508

ISBN 978-3-540-85706-8

2nd Edition Springer Berlin Heidelberg New York

The first edition was published as the “Enzyme Handbook, edited by D. and I. Schomburg”.

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springer.com # Springer-Verlag Berlin Heidelberg 2009 Printed in Germany The use of general descriptive names, registered names, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and free for general use. The publisher cannot assume any legal responsibility for given data, especially as far as directions for the use and the handling of chemicals and biological material are concerned. This information can be obtained from the instructions on safe laboratory practice and from the manufacturers of chemicals and laboratory equipment. Cover design: Erich Kirchner, Heidelberg Typesetting: medionet Publishing Services Ltd., Berlin Printed on acid-free paper

2/3141m-5 4 3 2 1 0

Preface

Today, as the full information about the genome is becoming available for a rapidly increasing number of organisms and transcriptome and proteome analyses are beginning to provide us with a much wider image of protein regulation and function, it is obvious that there are limitations to our ability to access functional data for the gene products – the proteins and, in particular, for enzymes. Those data are inherently very difficult to collect, interpret and standardize as they are widely distributed among journals from different fields and are often subject to experimental conditions. Nevertheless a systematic collection is essential for our interpretation of genome information and more so for applications of this knowledge in the fields of medicine, agriculture, etc. Progress on enzyme immobilisation, enzyme production, enzyme inhibition, coenzyme regeneration and enzyme engineering has opened up fascinating new fields for the potential application of enzymes in a wide range of different areas. The development of the enzyme data information system BRENDAwas started in 1987 at the German National Research Centre for Biotechnology in Braunschweig (GBF), continued at the University of Cologne from 1996 to 2007, and then returned to Braunschweig, to the Technical University, Institute of Bioinformatics & Systems Biology. The present book “Springer Handbook of Enzymes” represents the printed version of this data bank. The information system has been developed into a full metabolic database. The enzymes in this Handbook are arranged according to the Enzyme Commission list of enzymes. Some 5,000 “different” enzymes are covered. Frequently enzymes with very different properties are included under the same EC-number. Although we intend to give a representative overview on the characteristics and variability of each enzyme, the Handbook is not a compendium. The reader will have to go to the primary literature for more detailed information. Naturally it is not possible to cover all the numerous literature references for each enzyme (for some enzymes up to 40,000) if the data representation is to be concise as is intended. It should be mentioned here that the data have been extracted from the literature and critically evaluated by qualified scientists. On the other hand, the original authors’ nomenclature for enzyme forms and subunits is retained. In order to keep the tables concise, redundant information is avoided as far as possible (e.g. if Km values are measured in the presence of an obvious cosubstrate, only the name of the cosubstrate is given in parentheses as a commentary without reference to its specific role). The authors are grateful to the following biologists and chemists for invaluable help in the compilation of data: Cornelia Munaretto and Dr. Antje Chang. Braunschweig Spring 2009

Dietmar Schomburg, Ida Schomburg

VII

List of Abbreviations

A Ac ADP Ala All Alt AMP Ara Arg Asn Asp ATP Bicine C cal CDP CDTA CMP CoA CTP Cys d dDFP DNA DPN DTNB DTT EC E. coli EDTA EGTA ER Et EXAFS FAD FMN Fru Fuc G Gal

adenine acetyl adenosine 5’-diphosphate alanine allose altrose adenosine 5’-monophosphate arabinose arginine asparagine aspartic acid adenosine 5’-triphosphate N,N’-bis(2-hydroxyethyl)glycine cytosine calorie cytidine 5’-diphosphate trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid cytidine 5’-monophosphate coenzyme A cytidine 5’-triphosphate cysteine deoxy(and l-) prefixes indicating configuration diisopropyl fluorophosphate deoxyribonucleic acid diphosphopyridinium nucleotide (now NAD+ ) 5,5’-dithiobis(2-nitrobenzoate) dithiothreitol (i.e. Cleland’s reagent) number of enzyme in Enzyme Commission’s system Escherichia coli ethylene diaminetetraacetate ethylene glycol bis(-aminoethyl ether) tetraacetate endoplasmic reticulum ethyl extended X-ray absorption fine structure flavin-adenine dinucleotide flavin mononucleotide (riboflavin 5’-monophosphate) fructose fucose guanine galactose

IX

List of Abbreviations

GDP Glc GlcN GlcNAc Gln Glu Gly GMP GSH GSSG GTP Gul h H4 HEPES His HPLC Hyl Hyp IAA IC 50 Ig Ile Ido IDP IMP ITP Km lLeu Lys Lyx M mM mMan MES Met min MOPS Mur MW NAD+ NADH NADP+ NADPH NAD(P)H

X

guanosine 5’-diphosphate glucose glucosamine N-acetylglucosamine glutamine glutamic acid glycine guanosine 5’-monophosphate glutathione oxidized glutathione guanosine 5’-triphosphate gulose hour tetrahydro 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid histidine high performance liquid chromatography hydroxylysine hydroxyproline iodoacetamide 50% inhibitory concentration immunoglobulin isoleucine idose inosine 5’-diphosphate inosine 5’-monophosphate inosine 5’-triphosphate Michaelis constant (and d-) prefixes indicating configuration leucine lysine lyxose mol/l millimol/l metamannose 2-(N-morpholino)ethane sulfonate methionine minute 3-(N-morpholino)propane sulfonate muramic acid molecular weight nicotinamide-adenine dinucleotide reduced NAD NAD phosphate reduced NADP indicates either NADH or NADPH

List of Abbreviations

NBS NDP NEM Neu NMN NMP NTP oOrn pPBS PCMB PEP pH Ph Phe PHMB PIXE PMSF p-NPP Pro Q10 Rha Rib RNA mRNA rRNA tRNA Sar SDS-PAGE Ser T tH Tal TDP TEA Thr TLCK Tm TMP TosTPN Tris Trp TTP Tyr U

N-bromosuccinimide nucleoside 5’-diphosphate N-ethylmaleimide neuraminic acid nicotinamide mononucleotide nucleoside 5’-monophosphate nucleoside 5’-triphosphate orthoornithine paraphosphate-buffered saline p-chloromercuribenzoate phosphoenolpyruvate -log10[H+ ] phenyl phenylalanine p-hydroxymercuribenzoate proton-induced X-ray emission phenylmethane-sulfonylfluoride p-nitrophenyl phosphate proline factor for the change in reaction rate for a 10 C temperature increase rhamnose ribose ribonucleic acid messenger RNA ribosomal RNA transfer RNA N-methylglycine (sarcosine) sodium dodecyl sulfate polyacrylamide gel electrophoresis serine thymine time for half-completion of reaction talose thymidine 5’-diphosphate triethanolamine threonine Na-p-tosyl-l-lysine chloromethyl ketone melting temperature thymidine 5’-monophosphate tosyl- (p-toluenesulfonyl-) triphosphopyridinium nucleotide (now NADP+ ) tris(hydroxymethyl)-aminomethane tryptophan thymidine 5’-triphosphate tyrosine uridine

XI

List of Abbreviations

U/mg UDP UMP UTP Val Xaa XAS Xyl

XII

mmol/(mg*min) uridine 5’-diphosphate uridine 5’-monophosphate uridine 5’-triphosphate valine symbol for an amino acid of unknown constitution in peptide formula X-ray absorption spectroscopy xylose

Index of Recommended Enzyme Names

EC-No.

Recommended Name

4.2.3.18 6.4.1.6 4.2.1.112 4.1.1.78 6.3.1.10 6.3.5.10 6.3.2.27 4.2.1.110 4.99.1.5 6.3.2.26 6.3.2.28 4.1.3.38 6.3.5.8 4.3.1.21 4.2.3.24 4.2.1.111 4.2.2.15 4.2.3.9 4.1.99.7 5.3.3.15 6.3.5.6 6.3.1.12 6.1.1.23 4.1.99.11 4.2.1.106 4.2.3.13 5.3.99.8 5.4.99.18 6.3.4.18 6.3.3.4 4.2.3.8 4.2.2.19 4.2.2.20 4.2.2.21 4.1.3.40 4.2.3.5 6.6.1.2 5.5.1.12 4.2.1.104 6.3.2.30 6.3.2.29 4.2.1.100 4.2.1.102 4.2.1.103

abietadiene synthase . . . . . . . . . . . . . . . . . . . . . . acetone carboxylase . . . . . . . . . . . . . . . . . . . . . . acetylene hydratase . . . . . . . . . . . . . . . . . . . . . . . acetylenedicarboxylate decarboxylase . . . . . . . . . . . . . . . adenosylcobinamide-phosphate synthase . . . . . . . . . . . . . adenosylcobyric acid synthase (glutamine-hydrolysing) . . . . . . . aerobactin synthase . . . . . . . . . . . . . . . . . . . . . . aldos-2-ulose dehydratase . . . . . . . . . . . . . . . . . . . . aliphatic aldoxime dehydratase. . . . . . . . . . . . . . . . . . N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase. . . . L-amino-acid a-ligase. . . . . . . . . . . . . . . . . . . . . . aminodeoxychorismate lyase. . . . . . . . . . . . . . . . . . . aminodeoxychorismate synthase (transferred to EC 2.6.1.85) . . . . . aminodeoxygluconate ammonia-lyase (deleted, identical to EC 4.3.1.9) amorpha-4,11-diene synthase . . . . . . . . . . . . . . . . . . 1,5-anhydro-D-fructose dehydratase. . . . . . . . . . . . . . . . anhydrosialidase . . . . . . . . . . . . . . . . . . . . . . . . aristolochene synthase . . . . . . . . . . . . . . . . . . . . . aristolochene synthase (transferred to EC 4.2.3.9) . . . . . . . . . . ascopyrone tautomerase. . . . . . . . . . . . . . . . . . . . . asparaginyl-tRNA synthase (glutamine-hydrolysing) . . . . . . . . D-aspartate ligase . . . . . . . . . . . . . . . . . . . . . . . aspartate-tRNAAsn ligase . . . . . . . . . . . . . . . . . . . . benzylsuccinate synthase . . . . . . . . . . . . . . . . . . . . bile-acid 7a-dehydratase . . . . . . . . . . . . . . . . . . . . (+)-d-cadinene synthase . . . . . . . . . . . . . . . . . . . . capsanthin/capsorubin synthase . . . . . . . . . . . . . . . . . 5-(carboxyamino)imidazole ribonucleotide mutase . . . . . . . . . 5-(carboxyamino)imidazole ribonucleotide synthase . . . . . . . . (carboxyethyl)arginine b-lactam-synthase . . . . . . . . . . . . . casbene synthase . . . . . . . . . . . . . . . . . . . . . . . . chondroitin B lyase . . . . . . . . . . . . . . . . . . . . . . . chondroitin-sulfate-ABC endolyase . . . . . . . . . . . . . . . . chondroitin-sulfate-ABC exolyase . . . . . . . . . . . . . . . . chorismate lyase . . . . . . . . . . . . . . . . . . . . . . . . chorismate synthase . . . . . . . . . . . . . . . . . . . . . . cobaltochelatase . . . . . . . . . . . . . . . . . . . . . . . . copalyl diphosphate synthase . . . . . . . . . . . . . . . . . . cyanase . . . . . . . . . . . . . . . . . . . . . . . . . . . . cyanophycin synthase (L-arginine-adding) . . . . . . . . . . . . . cyanophycin synthase (L-aspartate-adding) . . . . . . . . . . . . cyclohexa-1,5-dienecarbonyl-CoA hydratase . . . . . . . . . . . . cyclohexa-1,5-dienecarbonyl-CoA hydratase (transferred to EC 4.2.1.100) cyclohexyl-isocyanide hydratase . . . . . . . . . . . . . . . . .

Page 276 657 118 1 592 651 606 111 465 600 609 49 644 376 307 115 131 219 62 512 628 597 562 66 100 250 514 548 625 622 215 152 159 162 57 202 675 551 91 616 610 80 86 87

XIII

Index of Recommended Enzyme Names

4.4.1.25 4.3.3.4 4.3.3.3 4.1.1.85 4.2.3.4 4.1.3.37 4.1.1.86 4.1.99.12 4.3.1.22 4.1.1.84 4.2.1.108 4.2.3.10 5.5.1.13 4.2.3.19 4.3.1.20 4.2.3.2 4.6.1.15 4.2.3.22 4.2.3.23 4.2.2.14 6.3.1.11 6.1.1.24 6.3.5.7 4.6.1.14 6.3.5.9 4.1.3.39 5.4.4.1 5.4.4.3 4.2.1.105 4.4.1.22 4.1.1.83 4.1.1.80 4.2.99.19 4.4.1.23 4.99.1.6 4.2.2.17 4.2.2.18 5.4.4.2 5.1.1.17 4.2.3.27 4.4.1.20 4.2.2.16 4.2.3.16 4.2.3.20 4.1.99.10 4.2.3.25 4.2.3.26 6.1.1.25 6.6.1.1 5.1.3.21 4.6.1.12 4.2.3.3 4.2.1.109

XIV

L-cysteate sulfo-lyase . . . . . . . . . . . . . . . . . . . . . deacetylipecoside synthase . . . . . . . . . . . . . . . . . . . deacetylisoipecoside synthase. . . . . . . . . . . . . . . . . . 3-dehydro-L-gulonate-6-phosphate decarboxylase . . . . . . . . . 3-dehydroquinate synthase . . . . . . . . . . . . . . . . . . . 1-deoxy-D-xylulose 5-phosphate synthase (transferred to EC 2.2.1.7) diaminobutyrate decarboxylase . . . . . . . . . . . . . . . . . 3,4-dihydroxy-2-butanone-4-phosphate synthase . . . . . . . . . 3,4-dihydroxyphenylalanine reductive deaminase . . . . . . . . . D-dopachrome decarboxylase . . . . . . . . . . . . . . . . . . ectoine synthase . . . . . . . . . . . . . . . . . . . . . . . (-)-endo-fenchol synthase . . . . . . . . . . . . . . . . . . . ent-copalyl diphosphate synthase . . . . . . . . . . . . . . . . ent-kaurene synthase . . . . . . . . . . . . . . . . . . . . . erythro-3-hydroxyaspartate ammonia-lyase. . . . . . . . . . . . ethanolamine-phosphate phospho-lyase . . . . . . . . . . . . . FAD-AMP lyase (cyclizing) . . . . . . . . . . . . . . . . . . . germacradienol synthase. . . . . . . . . . . . . . . . . . . . germacrene-A synthase . . . . . . . . . . . . . . . . . . . . glucuronan lyase . . . . . . . . . . . . . . . . . . . . . . . glutamate-putrescine ligase. . . . . . . . . . . . . . . . . . . glutamate-tRNAGln ligase. . . . . . . . . . . . . . . . . . . . glutaminyl-tRNA synthase (glutamine-hydrolysing) . . . . . . . . glycosylphosphatidylinositol diacylglycerol-lyase . . . . . . . . . hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing) 4-hydroxy-2-oxovalerate aldolase . . . . . . . . . . . . . . . . (hydroxyamino)benzene mutase . . . . . . . . . . . . . . . . 3-(hydroxyamino)phenol mutase . . . . . . . . . . . . . . . . 2-hydroxyisoflavanone dehydratase . . . . . . . . . . . . . . . S-(hydroxymethyl)glutathione synthase . . . . . . . . . . . . . 4-hydroxyphenylacetate decarboxylase . . . . . . . . . . . . . . 4-hydroxyphenylpyruvate decarboxylase . . . . . . . . . . . . . 2-hydroxypropyl-CoM lyase (transferred to EC 4.4.1.23). . . . . . . 2-hydroxypropyl-CoM lyase . . . . . . . . . . . . . . . . . . indoleacetaldoxime dehydratase. . . . . . . . . . . . . . . . . inulin fructotransferase (DFA-I-forming). . . . . . . . . . . . . inulin fructotransferase (DFA-III-forming) . . . . . . . . . . . . isochorismate synthase . . . . . . . . . . . . . . . . . . . . isopenicillin-N epimerase . . . . . . . . . . . . . . . . . . . isoprene synthase . . . . . . . . . . . . . . . . . . . . . . . leukotriene-C4 synthase . . . . . . . . . . . . . . . . . . . . levan fructotransferase (DFA-IV-forming) . . . . . . . . . . . . (4S)-limonene synthase . . . . . . . . . . . . . . . . . . . . (R)-limonene synthase. . . . . . . . . . . . . . . . . . . . . (-)-(4S)-limonene synthase (transferred to EC 4.2.3.16) . . . . . . . S-linalool synthase . . . . . . . . . . . . . . . . . . . . . . R-linalool synthase . . . . . . . . . . . . . . . . . . . . . . lysine-tRNAPyl ligase . . . . . . . . . . . . . . . . . . . . . magnesium chelatase . . . . . . . . . . . . . . . . . . . . . maltose epimerase . . . . . . . . . . . . . . . . . . . . . . 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase . . . . . . methylglyoxal synthase . . . . . . . . . . . . . . . . . . . . methylthioribulose 1-phosphate dehydratase . . . . . . . . . . .

413 381 379 22 194 48 31 70 377 18 104 227 557 281 373 182 451 295 301 127 595 572 638 441 645 53 523 533 97 405 15 6 329 407 473 141 145 526 481 320 388 134 267 288 65 311 317 578 665 495 415 185 109

Index of Recommended Enzyme Names

4.2.3.15 4.1.99.9 5.3.99.9 6.4.1.7 4.2.2.22 4.2.3.7 4.99.1.7 4.6.1.13 5.4.2.10 4.1.1.82 4.4.1.19 4.1.99.8 4.2.3.14 5.3.3.13 4.4.1.18 6.1.1.26 4.2.3.12 4.4.1.21 5.1.3.22 4.2.3.11 4.3.1.18 4.3.1.17 5.1.1.18 4.99.1.3 4.99.1.4 5.4.99.17 4.2.1.113 4.4.1.24 4.1.1.79 4.2.3.17 4.3.1.16 4.1.2.42 4.3.1.19 4.2.3.1 4.1.1.81 5.3.3.14 4.2.1.101 6.2.1.34 4.2.1.107 5.1.3.23 4.1.2.41 4.2.3.21

myrcene synthase . . . . . . . . . . . . . . . . . . . myrcene synthase (transferred to EC 4.2.3.15) . . . . . . . neoxanthin synthase . . . . . . . . . . . . . . . . . . 2-oxoglutarate carboxylase . . . . . . . . . . . . . . . pectate trisaccharide-lyase. . . . . . . . . . . . . . . . pentalenene synthase . . . . . . . . . . . . . . . . . . phenylacetaldoxime dehydratase . . . . . . . . . . . . . phosphatidylinositol diacylglycerol-lyase . . . . . . . . . phosphoglucosamine mutase. . . . . . . . . . . . . . . phosphonopyruvate decarboxylase . . . . . . . . . . . . phosphosulfolactate synthase . . . . . . . . . . . . . . pinene synthase (transferred to EC 4.2.3.14) . . . . . . . . pinene synthase . . . . . . . . . . . . . . . . . . . . polyenoic fatty acid isomerase . . . . . . . . . . . . . . prenylcysteine lyase (transferred to EC 1.8.3.5) . . . . . . . pyrrolysine-tRNAPyl ligase . . . . . . . . . . . . . . . 6-pyruvoyltetrahydropterin synthase . . . . . . . . . . . S-ribosylhomocysteine lyase . . . . . . . . . . . . . . . L-ribulose-5-phosphate 3-epimerase . . . . . . . . . . . sabinene-hydrate synthase . . . . . . . . . . . . . . . . D-serine ammonia-lyase . . . . . . . . . . . . . . . . . L-serine ammonia-lyase . . . . . . . . . . . . . . . . . serine racemase . . . . . . . . . . . . . . . . . . . . sirohydrochlorin cobaltochelatase . . . . . . . . . . . . sirohydrochlorin ferrochelatase . . . . . . . . . . . . . squalene-hopene cyclase . . . . . . . . . . . . . . . . o-succinylbenzoate synthase . . . . . . . . . . . . . . . sulfolactate sulfo-lyase . . . . . . . . . . . . . . . . . sulfopyruvate decarboxylase . . . . . . . . . . . . . . . taxadiene synthase . . . . . . . . . . . . . . . . . . . threo-3-hydroxyaspartate ammonia-lyase . . . . . . . . . D-threonine aldolase . . . . . . . . . . . . . . . . . . threonine ammonia-lyase . . . . . . . . . . . . . . . . threonine synthase . . . . . . . . . . . . . . . . . . . threonine-phosphate decarboxylase . . . . . . . . . . . . trans-2-decenoyl-[acyl-carrier protein] isomerase . . . . . trans-feruloyl-CoA hydratase . . . . . . . . . . . . . . trans-feruloyl-CoA synthase . . . . . . . . . . . . . . . 3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA hydratase . . UDP-2,3-diacetamido-2,3-dideoxyglucuronic acid 2-epimerase vanillin synthase . . . . . . . . . . . . . . . . . . . . vetispiradiene synthase . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

264 64 517 662 169 211 476 421 519 12 385 63 256 502 384 583 235 400 497 231 348 332 486 455 460 536 123 411 4 272 330 42 356 173 9 508 82 590 102 499 39 292

XV

Description of Data Fields

All information except the nomenclature of the enzymes (which is based on the recommendations of the Nomenclature Committee of IUBMB (International Union of Biochemistry and Molecular Biology) and IUPAC (International Union of Pure and Applied Chemistry) is extracted from original literature (or reviews for very well characterized enzymes). The quality and reliability of the data depends on the method of determination, and for older literature on the techniques available at that time. This is especially true for the fields Molecular Weight and Subunits. The general structure of the fields is: Information – Organism – Commentary – Literature The information can be found in the form of numerical values (temperature, pH, Km etc.) or as text (cofactors, inhibitors etc.). Sometimes data are classified as Additional Information. Here you may find data that cannot be recalculated to the units required for a field or also general information being valid for all values. For example, for Inhibitors, Additional Information may contain a list of compounds that are not inhibitory. The detailed structure and contents of each field is described below. If one of these fields is missing for a particular enzyme, this means that for this field, no data are available.

1 Nomenclature EC number The number is as given by the IUBMB, classes of enzymes and subclasses defined according to the reaction catalyzed. Systematic name This is the name as given by the IUBMB/IUPAC Nomenclature Committee Recommended name This is the name as given by the IUBMB/IUPAC Nomenclature Committee Synonyms Synonyms which are found in other databases or in the literature, abbreviations, names of commercially available products. If identical names are frequently used for different enzymes, these will be mentioned here, cross references are given. If another EC number has been included in this entry, it is mentioned here.

XVII

Description of Data Fields

CAS registry number The majority of enzymes have a single chemical abstract (CAS) number. Some have no number at all, some have two or more numbers. Sometimes two enzymes share a common number. When this occurs, it is mentioned in the commentary.

2 Source Organism For listing organisms their systematic name is preferred. If these are not mentioned in the literature, the names from the respective literature are used. For example if an enzyme from yeast is described without being specified further, yeast will be the entry. This field defines the code numbers for the organisms in which the enzyme with the respective EC number is found. These code numbers (form <_>) are displayed together with each entry in all fields of BRENDA where organism-specific information is given.

3 Reaction and Specificity Catalyzed reaction The reaction as defined by the IUBMB. The commentary gives information on the mechanism, the stereochemistry, or on thermodynamic data of the reaction. Reaction type According to the enzyme class a type can be attributed. These can be oxidation, reduction, elimination, addition, or a name (e.g. Knorr reaction) Natural substrates and products These are substrates and products which are metabolized in vivo. A natural substrate is only given if it is mentioned in the literature. The commentary gives information on the pathways for which this enzyme is important. If the enzyme is induced by a specific compound or growth conditions, this will be included in the commentary. In Additional information you will find comments on the metabolic role, sometimes only assumptions can be found in the references or the natural substrates are unknown. In the listings, each natural substrate (indicated by a bold S) is followed by its respective product (indicated by a bold P). Products are given with organisms and references included only if the respective authors were able to demonstrate the formation of the specific product. If only the disappearance of the substrate was observed, the product is included without organisms of references. In cases with unclear product formation only a ? as a dummy is given. Substrates and products All natural or synthetic substrates are listed (not in stoichiometric quantities). The commentary gives information on the reversibility of the reaction,

XVIII

Description of Data Fields

on isomers accepted as substrates and it compares the efficiency of substrates. If a specific substrate is accepted by only one of several isozymes, this will be stated here. The field Additional Information summarizes compounds that are not accepted as substrates or general comments which are valid for all substrates. In the listings, each substrate (indicated by a bold S) is followed by its respective product (indicated by a bold P). Products are given with organisms and references included if the respective authors demonstrated the formation of the specific product. If only the disappearance of the substrate was observed, the product will be included without organisms or references. In cases with unclear product formation only a ? as a dummy is given. Inhibitors Compounds found to be inhibitory are listed. The commentary may explain experimental conditions, the concentration yielding a specific degree of inhibition or the inhibition constant. If a substance is activating at a specific concentration but inhibiting at a higher or lower value, the commentary will explain this. Cofactors, prosthetic groups This field contains cofactors which participate in the reaction but are not bound to the enzyme, and prosthetic groups being tightly bound. The commentary explains the function or, if known, the stereochemistry, or whether the cofactor can be replaced by a similar compound with higher or lower efficiency. Activating Compounds This field lists compounds with a positive effect on the activity. The enzyme may be inactive in the absence of certain compounds or may require activating molecules like sulfhydryl compounds, chelating agents, or lipids. If a substance is activating at a specific concentration but inhibiting at a higher or lower value, the commentary will explain this. Metals, ions This field lists all metals or ions that have activating effects. The commentary explains the role each of the cited metal has, being either bound e.g. as Fe-S centers or being required in solution. If an ion plays a dual role, activating at a certain concentration but inhibiting at a higher or lower concentration, this will be given in the commentary. Turnover number (min- 1) The kcat is given in the unit min-1 . The commentary lists the names of the substrates, sometimes with information on the reaction conditions or the type of reaction if the enzyme is capable of catalyzing different reactions with a single substrate. For cases where it is impossible to give the turnover number in the defined unit (e.g., substrates without a defined molecular weight, or an undefined amount of protein) this is summarized in Additional Information.

XIX

Description of Data Fields

Specific activity (U/mg) The unit is micromol/minute/milligram of protein. The commentary may contain information on specific assay conditions or if another than the natural substrate was used in the assay. Entries in Additional Information are included if the units of the activity are missing in the literature or are not calculable to the obligatory unit. Information on literature with a detailed description of the assay method may also be found. Km-Value (mM) The unit is mM. Each value is connected to a substrate name. The commentary gives, if available, information on specific reaction condition, isozymes or presence of activators. The references for values which cannot be expressed in mM (e.g. for macromolecular, not precisely defined substrates) are given in Additional Information. In this field we also cite literature with detailed kinetic analyses. Ki-Value (mM) The unit of the inhibition constant is mM. Each value is connected to an inhibitor name. The commentary gives, if available, the type of inhibition (e.g. competitive, non-competitive) and the reaction conditions (pH-value and the temperature). Values which cannot be expressed in the requested unit and references for detailed inhibition studies are summerized under Additional information. pH-Optimum The value is given to one decimal place. The commentary may contain information on specific assay conditions, such as temperature, presence of activators or if this optimum is valid for only one of several isozymes. If the enzyme has a second optimum, this will be mentioned here. pH-Range Mostly given as a range e.g. 4.0–7.0 with an added commentary explaining the activity in this range. Sometimes, not a range but a single value indicating the upper or lower limit of enzyme activity is given. In this case, the commentary is obligatory. Temperature optimum ( C) Sometimes, if no temperature optimum is found in the literature, the temperature of the assay is given instead. This is always mentioned in the commentary. Temperature range ( C) This is the range over which the enzyme is active. The commentary may give the percentage of activity at the outer limits. Also commentaries on specific assay conditions, additives etc.

XX

Description of Data Fields

4 Enzyme Structure Molecular weight This field gives the molecular weight of the holoenzyme. For monomeric enzymes it is identical to the value given for subunits. As the accuracy depends on the method of determination this is given in the commentary if provided in the literature. Some enzymes are only active as multienzyme complexes for which the names and/or EC numbers of all participating enzymes are given in the commentary. Subunits The tertiary structure of the active species is described. The enzyme can be active as a monomer a dimer, trimer and so on. The stoichiometry of subunit composition is given. Some enzymes can be active in more than one state of complexation with differing effectivities. The analytical method is included. Posttranslational modifications The main entries in this field may be proteolytic modification, or side-chain modification, or no modification. The commentary will give details of the modifications e.g.: – proteolytic modification <1> (<1>, propeptide Name) [1]; – side-chain modification <2> (<2>, N-glycosylated, 12% mannose) [2]; – no modification [3]

5 Isolation / Preparation / Mutation / Application Source / tissue For multicellular organisms, the tissue used for isolation of the enzyme or the tissue in which the enzyme is present is given. Cell-lines may also be a source of enzymes. Localization The subcellular localization is described. Typical entries are: cytoplasm, nucleus, extracellular, membrane. Purification The field consists of an organism and a reference. Only references with a detailed description of the purification procedure are cited. Renaturation Commentary on denaturant or renaturation procedure. Crystallization The literature is cited which describes the procedure of crystallization, or the X-ray structure.

XXI

Description of Data Fields

Cloning Lists of organisms and references, sometimes a commentary about expression or gene structure. Engineering The properties of modified proteins are described. Application Actual or possible applications in the fields of pharmacology, medicine, synthesis, analysis, agriculture, nutrition are described.

6 Stability pH-Stability This field can either give a range in which the enzyme is stable or a single value. In the latter case the commentary is obligatory and explains the conditions and stability at this value. Temperature stability This field can either give a range in which the enzyme is stable or a single value. In the latter case the commentary is obligatory and explains the conditions and stability at this value. Oxidation stability Stability in the presence of oxidizing agents, e.g. O2, H2 O2, especially important for enzymes which are only active under anaerobic conditions. Organic solvent stability The stability in the presence of organic solvents is described. General stability information This field summarizes general information on stability, e.g., increased stability of immobilized enzymes, stabilization by SH-reagents, detergents, glycerol or albumins etc. Storage stability Storage conditions and reported stability or loss of activity during storage.

References Authors, Title, Journal, Volume, Pages, Year.

XXII

Acetylenedicarboxylate decarboxylase

4.1.1.78

1 Nomenclature EC number 4.1.1.78 Systematic name acetylenedicarboxylate carboxy-lyase (pyruvate-forming) Recommended name acetylenedicarboxylate decarboxylase Synonyms EC 4.2.1.72 acetylenedicarboxylate hydrase hydratase, acetylenedicarboxylate CAS registry number 72561-10-5

2 Source Organism <1> <2> <3> <4> <5> <6>

Pseudomonas sp. (no sequence specified) [1] no activity in Escherichia coli [1] no activity in Pseudomonas fluorescens [1] no activity in Clostridium cylindrosporum [1] no activity in Rattus norvegicus [1] no activity in Clostridium kluyveri [1]

3 Reaction and Specificity Catalyzed reaction acetylenedicarboxylate + H2 O = pyruvate + CO2 Reaction type addition of H2 O decarboxylation Natural substrates and products S acetylene dicarboxylate <1> (Reversibility: ir) [1] P pyruvate + CO2

1

Acetylenedicarboxylate decarboxylase

Substrates and products S acetylene dicarboxylate <1> (Reversibility: ir) [1] P pyruvate + CO2 Inhibitors Al(NO3 )3 <1> [1] BaCl2 <1> [1] CN- <1> [1] CoCl2 <1> [1] EDTA <1> [1] Fe(NO3 )3 <1> [1] HgCl2 <1> [1] NiCl2 <1> [1] ZnSO4 <1> [1] p-chloromercuribenzoate <1> [1] Cofactors/prosthetic groups Additional information <1> (<1> no requirement [1]) [1] Specific activity (U/mg) 463 <1> [1] Km-Value (mM) 0.07 <1> (acetylene dicarboxylate, <1> 23 C, pH 7.3 [1]) [1] pH-Optimum 7.3-7.8 <1> [1] pH-Range 6.5-8.5 <1> [1] Temperature optimum ( C) 23 <1> (<1> enzyme assay at [1]) [1] Temperature range ( C) 20 <1> [1]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <1> [1] Purification <1> [1]

6 Stability pH-Stability 7.5 <1> (<1> highest stability at 3 C for 17 h [1]) [1]

2

4.1.1.78

4.1.1.78

Acetylenedicarboxylate decarboxylase

General stability information <1>, loss of stabililty by freezing and thawing [1] Storage stability <1>, -20 C, 0.005 mM 2-mercaptoethanol, 10 days, less than 10% loss of activity [1]

References [1] Yamada, E.W; Jakoby, W.B.: Enzymatic utilization of acetylenic compounds. I. An enzyme converting acetylenedicarboxylic acid to pyruvate. J. Biol. Chem., 233, 706-711 (1958)

3

Sulfopyruvate decarboxylase

4.1.1.79

1 Nomenclature EC number 4.1.1.79 Systematic name sulfopyruvate carboxy-lyase (2-sulfoacetaldehyde-forming) Recommended name sulfopyruvate decarboxylase Synonyms decarboxylase, sulfopyruvate sulfopyruvate decarboxylase CAS registry number 303155-97-7

2 Source Organism <1> Methanococcus jannaschii (UNIPROT accession number: P58415) [1]

3 Reaction and Specificity Catalyzed reaction 3-sulfopyruvate = 2-sulfoacetaldehyde + CO2 Reaction type decarboxylation Natural substrates and products S 3-sulfopyruvate <1> (<1> fourth step in biosynthesis of coenzyme M [1]) (Reversibility: ?) [1] P 2-sulfoacetaldehyde + CO2 <1> [1] Substrates and products S 3-sulfopyruvate <1> (<1> fourth step in biosynthesis of coenzyme M [1]) (Reversibility: ?) [1] P 2-sulfoacetaldehyde + CO2 <1> [1] Cofactors/prosthetic groups thiamine diphosphate <1> (<1> required [1]) [1]

4

4.1.1.79

Sulfopyruvate decarboxylase

Km-Value (mM) 0.64 <1> (sulfopyruvate) [1]

4 Enzyme Structure Molecular weight 210000 <1> (<1> gel filtration [1]) [1] Subunits dodecamer <1> (<1> a6 b6 , 6 * 17000 + 6 * 23000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Cloning <1> (expression in Escherichia coli) [1]

6 Stability Oxidation stability <1>, inactivation by oxygen at 80 C and reactivation by reduction with dithionite [1]

References [1] Graupner, M.; Xu, H.; White, R.H.: Identification of the gene encoding sulfopyruvate decarboxylase, an enzyme involved in biosynthesis of coenzyme M. J. Bacteriol., 182, 4862-4867 (2000)

5

4-Hydroxyphenylpyruvate decarboxylase

4.1.1.80

1 Nomenclature EC number 4.1.1.80 Systematic name 4-hydroxyphenylpyruvate carboxy-lyase (4-hydroxyphenylacetaldehydeforming) Recommended name 4-hydroxyphenylpyruvate decarboxylase Synonyms decarboxylase, 4-hydroxyphenylpyruvate p-hydroxyphenylpyruvate decarboxylase CAS registry number 109300-96-1

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10>

Eschscholzia californica (no sequence specified) [1] Berberis regeliana (no sequence specified) [1] Thalictrum minus (no sequence specified) [1] Berberis stolonifera (no sequence specified) [1] Berberis julianae (no sequence specified) [1] Thalictrum dipterocarpum (no sequence specified) [1] Tinospora caffra (no sequence specified) [1] Tinospora cordifolia (no sequence specified) [1] Berberis canadensis (no sequence specified) [1] Berberis aristata (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction 4-hydroxyphenylpyruvate = 4-hydroxyphenylacetaldehyde + CO2 Reaction type decarboxylation

6

4.1.1.80

4-Hydroxyphenylpyruvate decarboxylase

Natural substrates and products S 4-hydroxyphenylpyruvate <4, 9> (<4,9> involved in tyrosine metabolism, precursor of tyrosine, early steps of isoquinoline alkaloid biosynthesis [1]) (Reversibility: ?) [1] P 4-hydroxyphenylacetaldehyde + CO2 <4, 9> (<4,9> phenylacetaldehydes furnish the benzylic moiety of the isoquinoline alkaloids in their biosynthesis, formation of the 1-benzylisoquinoline structure requires the condensation of p-hydroxyphenylacetaldehyde with dopamine to form norcoclaurine [1]) [1] Substrates and products S 4-hydroxyphenylpyruvate <4, 9> (<4,9> involved in tyrosine metabolism, precursor of tyrosine, early steps of isoquinoline alkaloid biosynthesis [1]) (Reversibility: ?) [1] P 4-hydroxyphenylacetaldehyde + CO2 <4, 9> (<4,9> phenylacetaldehydes furnish the benzylic moiety of the isoquinoline alkaloids in their biosynthesis, formation of the 1-benzylisoquinoline structure requires the condensation of p-hydroxyphenylacetaldehyde with dopamine to form norcoclaurine [1]) [1] Specific activity (U/mg) 0.001271 <4> [1] Additional information <1, 2, 3, 5, 6, 7, 8, 10> (<1,2,3,5,6,7,8,10> values given as pkat/litre of suspension culture [1]) [1] Km-Value (mM) 0.67 <4> (4-hydroxyphenylpyruvate) [1] pH-Optimum 6.5 <4> [1] Temperature optimum ( C) 30 <4> (<4> assay at [1]) [1]

4 Enzyme Structure Molecular weight 30000 <4> (<4> HPLC gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue callus <9> [1] cell suspension culture <1, 2, 3, 4, 5, 6, 7, 8, 10> [1] Purification <4> (29fold partial purification) [1]

7

4-Hydroxyphenylpyruvate decarboxylase

4.1.1.80

References [1] Rueffer, M.; Zenk, M.H.: Distant precursors of benzylisoquinoline alkaloids and their enzymatic formation. Z. Naturforsch. C, 42, 319-332 (1987)

8

Threonine-phosphate decarboxylase

4.1.1.81

1 Nomenclature EC number 4.1.1.81 Systematic name l-threonine O-3-phosphate carboxy-lyase [(R)-1-aminopropan-2-yl-phosphate-forming] Recommended name threonine-phosphate decarboxylase Synonyms CobD <2, 4> [1, 2, 3] CobD gene product <2> [1] l-threonine-O-3-phosphate decarboxylase CAS registry number 205069-45-0

2 Source Organism <1> <2> <3> <4>

Salmonella typhimurium (no sequence specified) [2] Salmonella enterica (no sequence specified) [1, 3] no activity in eukaryota [1] Salmonella typhimurium (UNIPROT accession number: P97084) (<4> gene ispS [2]) [2]

3 Reaction and Specificity Catalyzed reaction l-threonine O-3-phosphate = (R)-1-aminopropan-2-yl phosphate + CO2 Natural substrates and products S l-threonine O-3-phosphate <1, 2> (Reversibility: ?) [1, 2] P (R)-1-aminopropan-2-yl phosphate + CO2 S l-threonine O-3-phosphate <2, 4> (<4> adenosylcobalamin biosynthesis, end product of the corrin ring biosynthetic pathway is 5-deoxyadenosylcobinamide phosphate, not 5-deoxyadenosylcobinamide [2]; <2> biosynthetic pathway of cobalamin, essential cofactor for many living organisms, only synthesized de novo by bacteria and archaea [1]; <2> de novo synthesis of enzymatic cofactors, cobalamin biosynthetic pathway [3]) (Reversibility: ?) [1, 2, 3]

9

Threonine-phosphate decarboxylase

4.1.1.81

P (R)-1-amino-2-propan-2-yl phosphate + CO2 <2, 4> (<2> precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin [1,3]) [1, 2, 3] Substrates and products S l-threonine O-3-phosphate <1, 2> (Reversibility: ?) [1, 2] P (R)-1-aminopropan-2-yl phosphate + CO2 S l-threonine O-3-phosphate <2, 4> (<4> enzyme shows stereospecificity for the l-isomer, unable to decarboxylate the d-isomer [2]; <4> adenosylcobalamin biosynthesis, end product of the corrin ring biosynthetic pathway is 5-deoxyadenosylcobinamide phosphate, not 5-deoxyadenosylcobinamide [2]; <2> biosynthetic pathway of cobalamin, essential cofactor for many living organisms, only synthesized de novo by bacteria and archaea [1]; <2> de novo synthesis of enzymatic cofactors, cobalamin biosynthetic pathway [3]) (Reversibility: ?) [1, 2, 3] P (R)-1-amino-2-propan-2-yl phosphate + CO2 <2, 4> (<2> precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin [1,3]) [1, 2, 3] S Additional information <4> (<4> CobD does not have lactaldehyde aminotransferase activity [2]) (Reversibility: ?) [2] P ? <4> [2]

4 Enzyme Structure Molecular weight 40800 <2, 4> (<4> predicted from nucleotide sequence [2]) [1, 2] Subunits dimer <2> [1, 3]

5 Isolation/Preparation/Mutation/Application Purification <2> [1, 3] Crystallization <2> (crystals belong to space group I222 with unit cell dimensions a : 66.6 A, b : 103.2 A, and c : 117.1 A, apo-CobD crystals belong to space group I222 with unit cell dimensions a : 76.0 A, b : 103.3 A, and c : 109.3 A) [3] <2> (crystals grown from hanging drops, CobD crystallizes in orthorhombic space group I222, unit cell dimensions a = 67.96 A, b = 101.55 A, and c = 117.23 A) [1] Cloning <2> (overproduced in Escherichia coli) [1] <4> (cloned, sequenced and overexpressed) [2]

10

4.1.1.81

Threonine-phosphate decarboxylase

References [1] Cheong, C.G.; Bauer, C.B.; Brushaber, K.R.; Escalante-Semerena, J.C.; Rayment, I.: Three-dimensional structure of the l-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica. Biochemistry, 41, 4798-4808 (2002) [2] Brushaber, K.R.; O’Toole, G.A.; Escalante-Semerena, J.C.: CobD, a novel enzyme with l-threonine-O-3-phosphate decarboxylase activity, is responsible for the synthesis of (R)-1-amino-2-propanol O-2-phosphate, a proposed new intermediate in cobalamin biosynthesis in Salmonella typhimurium LT2. J. Biol. Chem., 273, 2684-2691 (1998) [3] Cheong, C.G.; Escalante-Semerena, J.C.; Rayment, I.: Structural studies of the l-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica: The apo, substrate, and product-aldimine complexes. Biochemistry, 41, 9079-9089 (2002)

11

Phosphonopyruvate decarboxylase

4.1.1.82

1 Nomenclature EC number 4.1.1.82 Systematic name 3-phosphonopyruvate carboxy-lyase (2-phosphonoacetaldehyde-forming) Recommended name phosphonopyruvate decarboxylase Synonyms PnPy decarboxylase <1, 4> [1, 3] Ppd <3> [2] Ppyr decarboxylase <2> [4] CAS registry number 151662-34-9

2 Source Organism <1> Streptomyces hygroscopicus (no sequence specified) [3] <2> Bacteroides fragilis (UNIPROT accession number: Q9F768) [4] <3> Streptomyces viridochromogenes (UNIPROT accession number: O86938) [2] <4> Streptomyces hygroscopicus (UNIPROT accession number: Q54271) [1]

3 Reaction and Specificity Catalyzed reaction 3-phosphonopyruvate = 2-phosphonoacetaldehyde + CO2 Natural substrates and products S 3-phosphonopyruvate <1, 2, 3, 4> (<4> key enzyme in the biosynthesis of C-P bond compounds, overview [1]) (Reversibility: ir) [1, 2, 3, 4] P 2-phosphonoacetaldehyde + CO2 Substrates and products S 3-phosphonopyruvate <1, 2, 3, 4> (<4> key enzyme in the biosynthesis of C-P bond compounds, overview [1]) (Reversibility: ir) [1, 2, 3, 4] P 2-phosphonoacetaldehyde + CO2

12

4.1.1.82

Phosphonopyruvate decarboxylase

S pyruvate <2> (<2> 0.5% of 3-phosphonopyruvate kcat [4]) (Reversibility: ?) [4] P acetaldehyde + CO2 S sulfopyruvate <2> (<2> 0.5% of 3-phosphonopyruvate kcat [4]) (Reversibility: ?) [4] P sulfoacetaldehyde + CO2 Inhibitors sulfopyruvate <2> (<2> competitive inhibition [4]) [4] Cofactors/prosthetic groups thiamine diphosphate <1, 2, 3> (<3> primary sequence of Ppd contains signature typical for enzymes requiring thiamine diphosphate [2]) [2,3,4] Metals, ions Ca2+ <2> (<2> required for activity [4]) [4] Mg2+ <1, 2> (<1,2> required for activity [3,4]) [3, 4] Mn2+ <2> (<2> required for activity [4]) [4] Turnover number (min–1) 10.2 <2> (3-phosphonopyruvate, <2> 25 C, pH 7.3, 5 mM MgCl2 , 1 mM MnCl2 , 0.2 mM thiamine diphosphate [4]) [4] Km-Value (mM) 0.0032 <2> (3-phosphonopyruvate, <2> 25 C, pH 7.3, 5 mM MgCl2 , 1 mM MnCl2 , 0.2 mM thiamine diphosphate [4]) [4] 0.013 <2> (Mn2+ , <2> 25 C, pH 7.3 [4]) [4] 0.013 <2> (thiamine diphosphate, <2> 25 C, pH 7.3 [4]) [4] 0.078 <2> (Ca2+ , <2> 25 C, pH 7.3 [4]) [4] 0.082 <2> (Mg2+ , <2> 25 C, pH 7.3 [4]) [4] Ki-Value (mM) 0.2 <2> (sulfopyruvate, <2> 25 C, pH 7.0 [4]) [4]

4 Enzyme Structure Molecular weight 120000 <2> (<2> gel filtration [4]) [4] 135000 <1> (<1> gel filtration [3]) [3] Subunits ? <3> (<3> x * 41000, deduced from nucleotide sequence [2]) [2] tetramer <1> (<1> 4 * 36000, SDS-PAGE [3]) [3] trimer <2> (<2> 3 * 40000, SDS-PAGE [4]; <2> 3 * 41199, MALDI- TOF mass spectroscopy [4]) [4]

13

Phosphonopyruvate decarboxylase

4.1.1.82

5 Isolation/Preparation/Mutation/Application Purification <1> (ammonium sulfate, DEAE-cellulose, TSK-gel Phenyl-5PW, gel filtration, Mono Q) [3] <2> (recombinant Ppyr decarboxylase, ammonium sulfate, DEAE-Sepahrose, hydroxylapatite) [4] Cloning <2> (expression in Escherichia coli) [4] <3> [2] <4> (gene bcpC, located in the bialaphos biosynthetic gene cluster, DNA and amino acid sequence determination and analysis, complementation of an enzyme-deficient mutant strain of Streptomyces wedmorensis NP-7, enzyme expression in Streptomyces lividans) [1] Engineering D258A <2> (<2> 9% of wild-type kcat for 3-phosphonopyruvate [4]) [4] D260A <2> (<2> 0.1% of wild-type kcat for 3-phosphonopyruvate [4]) [4] E213A <2> (<2> 5% of wild-type kcat for 3-phosphonopyruvate [4]) [4]

6 Stability Storage stability <1>, 4 C, 50 mM Tris-HCl, pH 7.5, 3 weeks, no loss of activity [3]

References [1] Nakashita, H.; Kozuka, K.; Hidaka, T.; Hara, O.; Seto, H.: Identification and expression of the gene encoding phosphonopyruvate decarboxylase of Streptomyces hygroscopicus. Biochim. Biophys. Acta, 1490, 159-162 (2000) [2] Schwartz, D.; Recktenwald, J.; Pelzer, S.; Wohlleben, W.: Isolation and characterization of the PEP-phosphomutase and the phosphonopyruvate decarboxylase genes from the phosphinothricin tripeptide producer Streptomyces viridochromogenes Tu494. FEMS Microbiol. Lett., 163, 149-157 (1998) [3] Nakashita, H.; Watanabe, K.; Hara, O.; Hidaka, T.; Seto, H.: Studies on the biosynthesis of bialaphos. Biochemical mechanism of C-P bond formation: discovery of phosphonopyruvate decarboxylase which catalyzes the formation of phosphonoacetaldehyde from phosphonopyruvate. J. Antibiot., 50, 212-219 (1997) [4] Zhang, G.; Dai, J.; Lu, Z.; Dunaway-Mariano, D.: The phosphonopyruvate decarboxylase from Bacteroides fragilis. J. Biol. Chem., 278, 41302-41308 (2003)

14

4-Hydroxyphenylacetate decarboxylase

4.1.1.83

1 Nomenclature EC number 4.1.1.83 Systematic name 4-hydroxyphenylacetate carboxy-lyase (4-methylphenol-forming) Recommended name 4-hydroxyphenylacetate decarboxylase Synonyms 4-Hpd <1> [2] p-Hpd <1> [2] p-hydroxyphenylacetate decarboxylase <1, 2, 3, 4> [2, 3] pHPA decarboxylase <1> [2] CAS registry number 340137-18-0

2 Source Organism <1> <2> <3> <4>

Clostridium difficile Clostridium difficile Clostridium difficile Clostridium difficile

(no sequence specified) [1, 2] (UNIPROT accession number: Q84F16) [3] (UNIPROT accession number: Q84F15) [3] (UNIPROT accession number: Q84F14) [3]

3 Reaction and Specificity Catalyzed reaction (4-hydroxyphenyl)acetate + H+ = 4-methylphenol + CO2 Reaction type decarboxylation Substrates and products S (3,4-dihydroxyphenyl)acetate + H+ <1> (Reversibility: ?) [2] P 4-methylcatechol + CO2 S (4-hydroxyphenyl)acetate + H+ <1, 2, 3, 4> (Reversibility: ?) [1, 2, 3] P 4-methylphenol + CO2

15

4-Hydroxyphenylacetate decarboxylase

4.1.1.83

Inhibitors 4-hydroxyphenylacetamide <1> (<1> competitive [2]) [2] 4-hydroxymandelate <1> [2] NaCl <1> (<1> 800 mM, 50% inactivation [2]) [2] O2 <1> (<1> readily and irreversibly inhibited [2]) [2] Cofactors/prosthetic groups Additional information <1> (<1> indications for an as yet unidentified low molecular weight cofactor that is required for catalytic activity [2]) [2] Specific activity (U/mg) 0.315 <1> [2] Km-Value (mM) 0.5 <1> ((3,4-dihydroxyphenyl)acetate) [2] 2.8 <1> ((4-hydroxyphenyl)acetate) [2] Ki-Value (mM) 0.4 <1> ((3,4-dihydroxyphenyl)acetate) [2] 0.48 <1> (4-hydroxymandelate) [2] 0.7 <1> (4-Hydroxyphenylacetamide) [2]

4 Enzyme Structure Molecular weight 200000 <1> (<1> gel filtration [2]) [2] 460000 <2, 3, 4> (<2,3,4> hetero-octameric catalytically competent complex, gel filtration [3]) [3] Subunits dimer <1, 2, 3, 4> (<1> a,a’, the smaller unit is a C-terminally truncated form, 1 * 110000 + 1 * 105000, SDS-PAGE [2]; <2,3,4> b2 , decarboxylase purified from Clostridium difficile is an almost inactive homo-dimeric protein [3]) [2, 3] octamer <2, 3, 4> (<2,3,4> b4 g4 , small subunit HpdC has a molecular weight of 9598 Da as determined by MALDI TOF MS, the recombinant enzyme is a hetero-octameric catalytically competent complex [3]) [3] Posttranslational modification phosphoprotein <2, 3, 4> (<2,3,4> phosphorylation of the small subunit is responsible for th change in oligomeric state from inactive homo-dimeric protein of Clostridium difficile to the recombinant hetero-octameric catalytically competent complex [3]) [3]

16

4.1.1.83

4-Hydroxyphenylacetate decarboxylase

5 Isolation/Preparation/Mutation/Application Purification <1> [2] <2> [3] <3> [3] <4> [3] Cloning <2> (cloning of three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme, expressed in Escherichia coli) [3] <3> (cloning of three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme, expressed in Escherichia coli) [3] <4> (cloning of three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme, expressed in Escherichia coli) [3]

6 Stability pH-Stability 6-9 <1> (<1> 0 C in presence of 1 mM sodium sulfide and 5 mM ammonium sulfate [2]) [2] Temperature stability 30 <1> (<1> half-life: 15 min, in presence of 1 mM sodium sulfide and 5 mM ammonium sulfate [2]) [2] Oxidation stability <1>, readily and irreversibly inhibited by O2 [2] Storage stability <1>, 0 C, 100 mM Tris/HCl, pH 7.5, 5 mM ammonium sulfate, 1 mM magnesium chloride, more than 90% of the activity is recovered after 5 days [2]

References [1] D’Ari, L.; Barker, H.A.: p-Cresol formation by cell-free extracts of Clostridium difficile. Arch. Microbiol., 143, 311-312 (1985) [2] Selmer, T.; Andrei, P.I.: p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of pcresol. Eur. J. Biochem., 268, 1363-1372 (2001) [3] Andrei, P.I.; Pierik, A.J.; Zauner, S.; Andrei-Selmer, L.C.; Selmer, T.: Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase. A small subunit, HpdC, is essential for catalytic activity. Eur. J. Biochem., 271, 2225-2230 (2004)

17

D-Dopachrome

decarboxylase

4.1.1.84

1 Nomenclature EC number 4.1.1.84 Systematic name d-dopachrome carboxy-lyase (5,6-dihydroxyindole-forming) Recommended name d-dopachrome decarboxylase Synonyms d-dopachrome tautomerase <4> [2] d-tautomerase <4> (<4> distinct from EC 5.3.3.12, dopachrome isomerase, and from EC 5.3.2.1, phenylpyruvate tautomerase or MIF [4]) [4] phenylpyruvate tautomerase <2> (<2> identical with phenylpyruvate tautomerase, EC 5.3.2.1 [9]) [9] CAS registry number 184111-06-6

2 Source Organism <1> <2> <3> <4> <5> <6>

Homo sapiens (no sequence specified) [6] Rattus norvegicus (no sequence specified) [9] Bos taurus (no sequence specified) [6] Homo sapiens (UNIPROT accession number: P30046) [2, 4, 7, 8] Mus musculus (UNIPROT accession number: O35215) [5] Rattus norvegicus (UNIPROT accession number: P80254) [1,3]

3 Reaction and Specificity Catalyzed reaction d-dopachrome = 5,6-dihydroxyindole + CO2 d-dopachrome = 5,6-dihydroxyindole-2-carboxylic acid Reaction type intramolecular oxidoreduction Substrates and products S 4-hydroxyphenylpyruvate <2> (<2> enol form [9]) (Reversibility: ?) [9] P 4-hydroxyphenylpyruvate <2> (<2> keto form [9]) [9]

18

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D-Dopachrome

decarboxylase

d-dopachrome <1, 2, 3, 4, 6> (Reversibility: ?) [1, 2, 3, 6, 7, 9] 5,6-dihydroxyindole + CO2 <1, 2, 3, 4, 6> [1, 2, 3, 6, 7, 9] phenylpyruvate <2> (<2> enol form [9]) (Reversibility: ?) [9] phenylpyruvate <2> (<2> keto form [9]) [9] indole-3-pyruvate <2> (<2> enol form [9]) (Reversibility: ?) [9] indole-3-pyruvate <2> (<2> keto form [9]) [9] Additional information <6> (<6> no substrates are: l-dopachrome, dopaminochrome, l-a-methyldopachrome, d-a-methyldopachrome [1]) (Reversibility: ?) [1] P ? <6> [1]

S P S P S P S

Inhibitors 4-hydroxyphenylpyruvate <2> (<2> competitive [9]) [9] Additional information <1> (<1> no inhibition by serum or plasma [6]) [6] Specific activity (U/mg) 0.5 <6> (<6> pH 6.0, 25 C [1]) [1] 100 <4> (<4> 25 C, pH 6.5, wild type enzyme [2]) [2] Km-Value (mM) 0.42 <1> (d-dopachrome, <1> 25 C, pH 6.5, wild type enzyme [2]) [2] 0.44 <1> (d-dopachrome, <1> 25 C, pH 6.5, P1A mutant [2]) [2] 1.5 <6> (d-dopachrome, <6> pH 6.0, 25 C [1]) [1] pH-Optimum 6-9 <6> (<6> broad [1]) [1]

4 Enzyme Structure Molecular weight 37000 <6> (<6> gel filtration [1,3]) [1, 3] Subunits ? <4> (<4> x * 13000, SDS-PAGE [2]) [2] dimer <4> (<4> and trimer, crystallization data [8]) [8] trimer <4, 6> (<6> 3 * 12000, SDS-PAGE [1]; <6> 3 * 12500, SDS-PAGE [3]; <4> and dimer, crystallization data [8]; <4> homotrimer, crystallization data [4]) [1, 3, 4, 8]

5 Isolation/Preparation/Mutation/Application Localization cytosol <2> [9] soluble <1, 6> [1, 6] Purification <2> [1]

19

D-Dopachrome

decarboxylase

4.1.1.84

Crystallization <4> [4] <4> (dimeric and trimeric enzyme and its selenomethyl derivative) [8] Cloning <1> (cDNA of 566 base pairs, expressed in Escherichia coli) [2] Engineering P1A <4> (<4> enzyme activity extremely decreased [2]) [2] Application medicine <4> (<4> strong correlation and covariation of enzyme activity with macrophage migration inhibitory factor, MIF, upon UVB-induction [7]) [7]

6 Stability Temperature stability 45 <6> (<6> 5 min, loss of activity [1]) [1] Storage stability <6>, -20 C, pH 5.5-9.0, several weeks, stable [1] <6>, 25 C, overnight, stable [1]

References [1] Odh, G.; Hindemith, A.; Rosengren, A-M.; Rosengren, E.; Rorsman, H.: Isolation of a new tautomerase monitored by the conversion of d-dopachrome to 5,6-dihydroxyindole. Biochem. Biophys. Res. Commun., 197, 619-624 (1993) [2] Nishihira, J.; Fujinaga, M.; Kuriyama, T.; Suzuki, M.; Sugimoto, H.; Nakagawa, A.; Tanaka, I.; Sakai, M.: Molecular cloning of human d-dopachrome tautomerase cDNA: N-terminal proline is essential for enzyme activation. Biochem. Biophys. Res. Commun., 243, 538-544 (1998) [3] Yoshida, H.; Nishihira, J.; Suzuki, M.; Hikichi, K.: NMR characterization of physicochemical properties of rat d-dopachrome tautomerase. Biochem. Mol. Biol. Int., 42, 891-899 (1997) [4] Sugimoto, H.; Taniguchi, M.; Nakagawa, A.; Tanaka, I.; Suzuki, M.; Nishihira, J.: Crystal structure of human d-dopachrome tautomerase, a homologue of macrophage migration inhibitory factor, at 1.54 A resolution. Biochemistry, 38, 3268-3279 (1999) [5] Kuriyama T, Fujinaga M, Koda T, Nishihira J.: Cloning of the mouse gene for d-dopachrome tautomerase.. Biochim. Biophys. Acta, 1388, 506-512 (1998) [6] Bjoerk, P.; Aman, P.; Hindemith, A.; Odh, G.; Jacobsson, L.; Rosengren, E.; Rorsman, H.: A new enzyme activity in human blood cells and isolation of the responsible protein (d-dopachrome tautomerase) from erythrocytes. Eur. J. Haematol., 57, 254-256 (1996)

20

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D-Dopachrome

decarboxylase

[7] Sonesson, B.; Rosengren, E.; Hansson, A.S.; Hansson, C.: UVB-induced inflammation gives increased d-dopachrome tautomerase activity in blister fluid which correlates with macrophage migration inhibitory factor. Exp. Dermatol., 12, 278-282 (2003) [8] Sugimoto, H.; Taniguchi, M.; Nakagawa, A.; Tanaka, I.; Suzuki, M.; Nishihira, J.: Crystallization and preliminary x-ray analysis of human d-dopachrome tautomerase. J. Struct. Biol., 120, 105-108 (1997) [9] Rosengren, E.; Thelin, S.; Aman, P.; Hansson, C.; Jacobsson, L.; Rorsman, H.: The protein catalysing the conversion of d-dopachrome to 5,6-dihydroxyindole is a phenylpyruvate tautomerase (EC 5.3.2.1). Melanoma Res., 7, 517518 (1997)

21

3-Dehydro-L-gulonate-6-phosphate decarboxylase

4.1.1.85

1 Nomenclature EC number 4.1.1.85 Systematic name 3-dehydro-l-gulonate-6-phosphate carboxy-lyase (l-xylulose-5-phosphateforming) Recommended name 3-dehydro-l-gulonate-6-phosphate decarboxylase Synonyms 3-keto-l-gulonate 6-phosphate decarboxylase <1, 2, 3> [1, 2, 3, 4, 5, 6, 7] KGPDC <1, 2, 3> [1, 2, 3, 4, 5, 6] SgaH <2> [7] SgbH <2> [7] UlaD <2> [7] Additional information <1, 2, 3> (<1,2,3> the enzyme belongs to the orotidine 5-monophosphate decarboxylase OMPDC suprafamily [3,4,5,6]; <1> the enzyme belongs to the orotidine 5-monophosphate decarboxylase OMPDC suprafamily, overview [1,2]) [1, 2, 3, 4, 5, 6] CAS registry number 406722-60-9

2 Source Organism <1> Escherichia coli (no sequence specified) [1, 2, 4] <2> Escherichia coli K-12 (no sequence specified) [3, 5, 6, 7] <3> Methylomonas aminofaciens (no sequence specified) [5]

3 Reaction and Specificity Catalyzed reaction 3-dehydro-l-gulonate 6-phosphate + H+ = l-xylulose 5-phosphate + CO2 (<1> active site structure, substrate binding structure, formation of a cis-1,2enediolate anion intermediate, intermediate structure and reaction mechanism involving residues Lys64, Asp67, and His136, overview [2]; <2> active site structure, substrate binding structure, structure-function relationship, overview [6]; <2> catalytic mechanism, formation and Mg2+ -stabilization of an cis-1,2-enediolate anion intermediate, important residues for the stereospecific

22

4.1.1.85

3-Dehydro-L-gulonate-6-phosphate decarboxylase

exchange of the pro-1S hydrogen are Lys64, Asp67, Glu112, Arg139, and Lys64, the latter stabilizes the intermediate via hydrogen bonds to O1 and O2 of the intermediate involving conserved active site residue Asp67, His136 takes part in hydrogen exchange with solvent of the 1-hydroxymethylene group of the product, overview [3]; <1> formation and stabilization of an 1,2-enediolate anion intermediate, protonation of the intermediate involving residues Glu112, His136, and Arg139, active site structure-function relationship, overview [4]; <1> formation and stabilization of an enediolate anion intermediate is part of reaction mechanism, mechanism overview, the active site is located at the dimer interface, structure-function relationship, overview [1]) Natural substrates and products S 3-dehydro-l-gulonate 6-phosphate + H+ <1, 2, 3> (<2> SgaH/UlaD and SgbH [7]; <2> step in the catabolic pathway of l-ascorbate utilization [3,5]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7] P l-xylulose 5-phosphate + CO2 S Additional information <2> (<2> enzyme catalyzes one step in the l-ascorbate utilization pathway, l-ascorbate is converted to l-xylulose, overview [7]) (Reversibility: ?) [7] P ? Substrates and products S 3-dehydro-l-gulonate 6-phosphate + H+ <1, 2, 3> (<2> stereochemistry [3,5]; <1> substrate binding structure [2]; <2> SgaH/UlaD and SgbH [7]; <2> step in the catabolic pathway of l-ascorbate utilization [3,5]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7] P l-xylulose 5-phosphate + CO2 S Additional information <2, 3> (<2> enzyme catalyzes one step in the lascorbate utilization pathway, l-ascorbate is converted to l-xylulose, overview [7]; <2> enzymes SgaH/UlaD and SgbH are not active with 3-dehydro-l-gulonate [7]; <3> the d-arabino-hex-3-ulose 6-phosphate synthase of Methylomonas aminofaciens also performs the 3-keto-l-gulonate 6phosphate decarboxylase reaction with lower activity than for the preferred reaction catalyzing Mg2+ -dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate, proton exchange reaction rates, overview [5]; <2> the enzyme also performs the Methylomonas aminofaciens d-arabino-hex-3-ulose 6-phosphate synthase reaction with low activity catalyzing Mg2+ -dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate, while the Methylomonas aminofaciens enzyme also performs the 3-keto-l-gulonate 6-phosphate decarboxylase reaction, overview [6]; <2> the enzyme also performs the Methylomonas aminofaciens d-arabino-hex-3-ulose 6-phosphate synthase reaction with low activity catalyzing Mg2+ -dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate, while the Methylomonas aminofaciens enzyme also performs the 3-keto-l-gulonate 6-phosphate decarboxylase reaction, proton exchange reaction rates, overview [5]) (Reversibility: ?) [5, 6, 7] P ?

23

3-Dehydro-L-gulonate-6-phosphate decarboxylase

4.1.1.85

Inhibitors l-gulonate 6-phosphate <1> (<1> binding structure involving Glu3 3 and Asp62 and Mg2+ [1]) [1] l-xylitol 5-phosphate <1> (<1> binding structure [2]) [2] Metals, ions Mg2+ <1, 2> (<1,2> dependent on [2,3,4,5]; <1> dependent on, binding structure involving Glu33 and Asp62, overview [1]) [1, 2, 3, 4, 5, 7] Turnover number (min–1) 0.19 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant D67A/H136A [3]) [3] 0.2 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112Q/H136A/R139V [3]) [3] 0.21 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112A/H136A/R139V [3]) [3] 0.23 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112Q/H136A [3]) [3] 0.26 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A/E112A/H136A/R139V [3]) [3] 0.3 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112A [3]) [3] 0.45 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112A/H136A [3]) [3] 0.75 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A/H136A [3]) [3] 1.1 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant D67N [3]) [3] 1.2 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A/R139V [3]) [3] 1.3 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant D67N/H136A [3]) [3] 1.4 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant H136Q [3]) [3] 2.4 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112D/R139V/T169A [5]; <2> pH 7.5, 25 C, recombinant mutants D67A and H136A [3]) [3, 5] 2.9 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A/E112Q/H136A [3]) [3] 4 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112Q [3]) [3] 4.2 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64R/H136A [3]) [3] 4.3 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant H136N [3]) [3] 8.3 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A [3]) [3]

24

4.1.1.85

3-Dehydro-L-gulonate-6-phosphate decarboxylase

8.9 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant R139V [3]) [3] 9.2 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant H136A/R139V [3]) [3] 15 <3> (3-dehydro-l-gulonate 6-phosphate, <3> pH 7.5, 25 C, recombinant enzyme [5]) [5] 17 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64R [3]) [3] 51 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant wild-type enzyme [3,5]; <2> SgaH, pH 7.5, 25 C [7]) [3, 5, 7] 64 <2> (3-dehydro-l-gulonate 6-phosphate, <2> SgbH, pH 7.5, 25 C [7]) [7] Specific activity (U/mg) Additional information <2> [7] Km-Value (mM) 0.15 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112Q/H136A [3]) [3] 0.22 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112Q/H136A/R139V [3]) [3] 0.27 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant D67A/H136A [3]) [3] 0.3 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutants K64R and D67N/H136A [3]) [3] 0.31 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112Q [3]) [3] 0.34 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112A [3]) [3] 0.36 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant R139V [3]) [3] 0.37 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant H136N [3]) [3] 0.41 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant D67N [3]) [3] 0.44 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64R/H136A [3]) [3] 0.5 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant D67A [3]) [3] 0.51 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A [3]) [3] 0.52 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant H136Q [3]) [3] 0.55 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A/E112Q/H136A [3]) [3] 0.58 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A/H136A [3]) [3] 0.65 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112A/H136A [3]) [3]

25

3-Dehydro-L-gulonate-6-phosphate decarboxylase

4.1.1.85

0.67 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant wild-type enzyme [3,5]) [3, 5] 0.7 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant H136A [3]) [3] 0.77 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112A/H136A/R139V [3]) [3] 0.9 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant K64A/R139V [3]) [3] 0.91 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutant E112D/R139V/T169A [5]) [5] 1.4 <2> (3-dehydro-l-gulonate 6-phosphate, <2> pH 7.5, 25 C, recombinant mutants H136A/R139V and K64A/E112A/H136A/R139V [3]) [3] 5.4 <3> (3-dehydro-l-gulonate 6-phosphate, <3> pH 7.5, 25 C, recombinant enzyme [5]) [5] Additional information <2> (<2> analysis of kinetics and activity of the mutant enzymes, overview [3]) [3] pH-Optimum 7.5 <2, 3> (<2,3> assay at [3,5,7]) [3, 5, 7] Temperature optimum ( C) 25 <2, 3> (<2,3> assay at [3,5,7]) [3, 5, 7]

4 Enzyme Structure Subunits dimer <1, 2> (<2> (b/a)8 -barrel enzyme [3,5]; <1> (b/a)8 -barrel enzyme, the active site is located at the dimer interface [1]) [1, 3, 5]

5 Isolation/Preparation/Mutation/Application Purification <1> (recombinant His-tagged UlaD from strain BL21(DE3), the His-tag is removed by thrombin) [1] <2> (recombinant N-terminally His10-tagged SgbH from strain BL21(DE3) to homogeneity, the His-tag is removed by thrombin) [7] Crystallization <1> (purified recombinant enzyme complexed with l-gulonate 6-phosphate, l-threonohydroxamate 4-phosphate, and l-xylitol 5-phosphate, analogues of the substrate, enediolate intermediate, and product, as well as with the product l-xylulose 5-phosphate, 15 mg/ml protein in 50 mM HEPES, pH 7.5, 5 mM MgCl2 , 100 mM NaCl, micro-batch method, 0.01 ml protein solution mixed with equal volume of crystallization solution containing 16% monomethyl PEG 5000, 100 mM Bis-Tris propane, pH 7.0, and 5 mM MgCl2 , with

26

4.1.1.85

3-Dehydro-L-gulonate-6-phosphate decarboxylase

25 mM ligand, X-ray diffraction structure determination and analysis at 1.2, 1.8, 1.7, and 1.8 A resolution, respectively) [2] <1> (purified recombinant mutant enzymes K64A, H136A, E112Q, and E112Q/H136A, in complex with reaction intermediate analogue l-threonohydroxamate 4-phosphate, 15 mg/ml protein in 50 mM HEPES, pH 7.5, 5 mM MgCl2 , 100 mM NaCl, 0.01 ml protein solution mixed with equal volume of crystallization solution containing 16% monomethyl PEG 5000, 100 mM BisTris propane, pH 7.0, 5 mM MgCl2 , and 25 mM l-threonohydroxamate 4phosphate, X-ray diffraction structure determination and analysis at 1.7, 1.9, 1.8, and 1.9 A resolution, respectively) [4] <1> (purified recombinant wild-type and selenomethionine-labeled enzymes, free or complexed with inhibitor l-gulonate 6-phosphate, 15 mg/ml protein in 50 mM HEPES, pH 7.5, 5 mM MgCl2 , 100 mM NaCl, room temperature, micro-batch method, 0.01 ml protein solution mixed with equal volume of crystallization solution containing 18% monomethyl PEG 5000, 50 mM NaH2 PO4, 50 mM K2 HPO4, and 50 mM Bis-Tris propane, pH 7.0, with or without 20 mM inhibitor l-gulonate 6-phosphate, crystals appear a few days after microseeding, growing for 1 week, X-ray diffraction structure determination and analysis at 2.0 A resolution) [1] <2> (purified recombinant wild-type and mutant enzymes E112D/R139V, E112D/T169A, and E112D/R139V/T169A, 15 mg/ml protein in 50 mM HEPES, pH 7.5, 5 mM MgCl2 , 100 mM NaCl, micro-batch method, 0.01 ml protein solution mixed with equal volume of crystallization solution containing 16% monomethyl PEG 5000, 100 mM Bis-Tris propane, pH 7.0, 5 mM MgCl2 , and 25 mM l-xylulose 5-phosphate or d-ribulose 5-phosphate, cryoprotection of crystals in 15% monomethylPEG 5000, 100 mM PIPES, pH 7.0, 15% ethylene glycol, 200 mM NaCl, and 50 mM l-threonohydroxamate 4-phosphate or 50 mM d-ribulose 5-phosphate, X-ray diffraction structure determination and analysis at 1.6-1.8 A resolution, molecular replacement) [6] Cloning <1> (expression of mutant enzymes in strain BLR(DE3)recA-strain) [4] <1> (gene ulaD, expression as His-tagged protein in strain BL21(DE3), and as selenomethionine-labeled enzyme) [1] <2> (expression of N-terminally His10-tagged wild-type and mutant enzymes in enzyme-deficient strain BLR(DE3)) [3, 5, 6] <2> (gene sgbH, expression in strain BL21(DE3) as N-terminally His10tagged protein) [7] <3> (expression of N-terminally His10-tagged wild-type and mutant enzymes in enzyme-deficient strain BLR(DE3)) [5] Engineering D67A <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] D67A/H136A <2> (<2> site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme [3]) [3] D67N <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3]

27

3-Dehydro-L-gulonate-6-phosphate decarboxylase

4.1.1.85

D67N/H136A <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] E112A <2> (<2> site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme [3]) [3] E112A/H136A <2> (<2> site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme [3]) [3] E112A/H136A/R139V <2> (<2> site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme [3]) [3] E112D/R139V <2> (<2> site-directed mutagenesis, mutations alter the Escherichia coli residues to those of Methylomonas aminofaciens d-arabinohex-3-ulose 6-phosphate synthase, a homologous enzyme, also altering the enzyme activity of the 3-keto-l-gulonate 6-phosphate decarboxylase performing th Mg2+ -dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate with highly increased activity compared to the wildtype enzyme [6]) [6] E112D/R139V/T169A <2> (<2> site-directed mutagenesis, mutations alter the Escherichia coli residues to those of Methylomonas aminofaciens d-arabino-hex-3-ulose 6-phosphate synthase, a homologous enzyme, also altering the enzyme activity of the 3-keto-l-gulonate 6-phosphate decarboxylase performing th Mg2+ -dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate with highly increased activity compared to the wildtype enzyme [5,6]) [5, 6] E112D/R139V/T169A/R192A <2> (<2> site-directed mutagenesis, mutations alter the Escherichia coli residues to those of Methylomonas aminofaciens darabino-hex-3-ulose 6-phosphate synthase, a homologous enzyme, also altering the enzyme activity of the 3-keto-l-gulonate 6-phosphate decarboxylase performing th Mg2+ -dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate with highly increased activity compared to the wild-type enzyme [5]) [5] E112D/T169A <2> (<2> site-directed mutagenesis, mutations alter the Escherichia coli residues to those of Methylomonas aminofaciens d-arabinohex-3-ulose 6-phosphate synthase, a homologous enzyme, also altering the enzyme activity of the 3-keto-l-gulonate 6-phosphate decarboxylase performing th Mg2+ -dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate with highly increased activity compared to the wildtype enzyme [6]) [6] E112Q <1, 2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]; <1> site-directed mutagenesis, crystal structure determination and analysis, the mutant enzyme shows altered reaction intermediate binding at the active site, reaction mechanism, and stereochemistry [4]) [3, 4] E112Q/H136A <1, 2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]; <1> site-directed mutagenesis, crystal structure determination and analysis, the mutant enzyme shows altered reaction intermediate binding at the active site, reaction mechanism, and stereochemistry [4]) [3, 4]

28

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3-Dehydro-L-gulonate-6-phosphate decarboxylase

E112Q/H136A/R139V <2> (<2> site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme [3]) [3] E33K <2> (<2> site-directed mutagenesis, inactive mutant [3]) [3] H136A <1, 2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]; <1> site-directed mutagenesis, crystal structure determination and analysis, the mutant enzyme shows altered reaction intermediate binding at the active site, reaction mechanism, and stereochemistry [4]) [3, 4] H136A/R139V <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] H136N <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] H136Q <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] K64A <1, 2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]; <1> site-directed mutagenesis, crystal structure determination and analysis, the mutant enzyme shows altered reaction intermediate binding at the active site, reaction mechanism, and stereochemistry [4]) [3, 4] K64A/E112A/H136A/R139V <2> (<2> site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme [3]) [3] K64A/E112Q/H136A <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] K64A/H136A <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] K64A/R139V <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] K64R <2> (<2> site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme [3]) [3] K64R/H136A <2> (<2> site-directed mutagenesis, reduced activity compared to the wild-type enzyme [3]) [3] R139V <2> (<2> site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme [3]) [3]

References [1] Wise, E.; Yew, W.S.; Babbitt, P.C.; Gerlt, J.A.; Rayment, I.: Homologous (b/ a)8 -barrel enzymes that catalyze unrelated reactions: orotidine 5’-monophosphate decarboxylase and 3-keto-l-gulonate 6-phosphate decarboxylase. Biochemistry, 41, 3861-3869 (2002) [2] Wise, E.L.; Yew, W.S.; Gerlt, J.A.; Rayment, I.: Structural evidence for a 1,2enediolate intermediate in the reaction catalyzed by 3-keto-l-gulonate 6phosphate decarboxylase, a member of the orotidine 5’-monophosphate decarboxylase suprafamily. Biochemistry, 42, 12133-12142 (2003) [3] Yew, W.S.; Wise, E.L.; Rayment, I.; Gerlt, J.A.: Evolution of enzymatic activities in the orotidine 5’-monophosphate decarboxylase suprafamily: mechan-

29

3-Dehydro-L-gulonate-6-phosphate decarboxylase

4.1.1.85

istic evidence for a proton relay system in the active site of 3-keto-l-gulonate 6-phosphate decarboxylase. Biochemistry, 43, 6427-6437 (2004) [4] Wise, E.L.; Yew, W.S.; Gerlt, J.A.; Rayment, I.: Evolution of enzymatic activities in the orotidine 5’-monophosphate decarboxylase suprafamily: crystallographic evidence for a proton relay system in the active site of 3-keto-lgulonate 6-phosphate decarboxylase. Biochemistry, 43, 6438-6446 (2004) [5] Yew, W.S.; Akana, J.; Wise, E.L.; Rayment, I.; Gerlt, J.A.: Evolution of enzymatic activities in the orotidine 5’-monophosphate decarboxylase suprafamily: enhancing the promiscuous d-arabino-hex-3-ulose 6-phosphate synthase reaction catalyzed by 3-keto-l-gulonate 6-phosphate decarboxylase. Biochemistry, 44, 1807-1815 (2005) [6] Wise, E.L.; Yew, W.S.; Akana, J.; Gerlt, J.A.; Rayment, I.: Evolution of enzymatic activities in the orotidine 5’-monophosphate decarboxylase suprafamily: structural basis for catalytic promiscuity in wild-type and designed mutants of 3-keto-l-gulonate 6-phosphate decarboxylase. Biochemistry, 44, 1816-1823 (2005) [7] Yew, W.S.; Gerlt, J.A.: Utilization of l-ascorbate by Escherichia coli K-12: assignments of functions to products of the yjf-sga and yia-sgb operons. J. Bacteriol., 184, 302-306 (2002)

30

Diaminobutyrate decarboxylase

4.1.1.86

1 Nomenclature EC number 4.1.1.86 Systematic name l-2,4-diaminobutanoate carboxy-lyase (propane-1,3-diamine-forming) Recommended name diaminobutyrate decarboxylase Synonyms DABA AT <18> [1] DABA DC <1, 2, 3, 4, 5, 6, 8, 11, 12, 13, 16, 17, 19> [2, 3, 4, 5, 7, 11] DABA decarboxylase <1, 7, 9, 10, 14, 15, 19> [6, 8, 10, 11] DABA-DC <4> [9] l-2,4-diaminobutyrate decarboxylase <1, 2, 3, 4, 5, 6, 8, 11, 12, 13, 16, 17, 19> [2, 3, 4, 5, 6, 7, 11] l-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase <18> [1] l-2,4-diaminobutyric acid decarboxylase <4, 7, 9, 10, 14, 15> [8, 9, 10] Additional information <18> (<18> the enzyme belongs to the subgroup II of the aminotransferases [1]) [1] CAS registry number 110277-62-8

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11> <12> <13>

Acinetobacter calcoaceticus (no sequence specified) [3, 6] Serratia marcescens (no sequence specified) [4] Klebsiella pneumoniae (no sequence specified) [4] Enterobacter aerogenes (no sequence specified) [4, 9] Enterobacter cloacae (no sequence specified) [4] Citrobacter freundii (no sequence specified) [4] Vibrio costicola (no sequence specified) [8] Enterobacter agglomerans (no sequence specified) [4] Vibrio cholerae (no sequence specified) [8] Vibrio alginolyticus (no sequence specified) [8, 10] Klebsiella oxytoca (no sequence specified) [4] Serratia liquefaciens (no sequence specified) [4] Acinetobacter baumannii (no sequence specified) [3, 7]

31

Diaminobutyrate decarboxylase

<14> <15> <16> <17> <18> <19>

4.1.1.86

Vibrio proteolyticus (no sequence specified) [8] Vibrio anguillarum (no sequence specified) [8] Acinetobacter baumannii (UNIPROT accession number: Q43908) [2] Enterobacter aerogenes (UNIPROT accession number: Q9S0P8) [5] Acinetobacter baumannii (UNIPROT accession number: P56744) [1] Acinetobacter calcoaceticus (UNIPROT accession number: A41817) [11]

3 Reaction and Specificity Catalyzed reaction l-2,4-diaminobutanoate = propane-1,3-diamine + CO2 Natural substrates and products S l-2,4-diaminobutanoate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19> (<10> propane-1,3-diamine is a precursor for norspermidine, the enzyme is involved in biosynthesis of norspermidine [10]; <4> the enzyme is involved in polyamine biosynthesis [9]) (Reversibility: ?) [1, 3, 4, 5, 6, 7, 8, 9, 10, 11] P propane-1,3-diamine + CO2 Substrates and products S 4-aminobutanoate <18> (Reversibility: ?) [1] P propylamine + CO2 S l-2,4-diaminobutanoate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19> (<19> specific for [11]; <10> propane-1,3-diamine is a precursor for norspermidine, the enzyme is involved in biosynthesis of norspermidine [10]; <4> the enzyme is involved in polyamine biosynthesis [9]; <10> stereospecific reaction, no activity with the d-isomer [8]) (Reversibility: ?) [1, 3, 4, 5, 6, 7, 8, 9, 10, 11] P propane-1,3-diamine + CO2 (<10> product identification [8]; <1> GC-MS product identification [6]) S l-2,4-diaminobutanoate + 2-oxoglutarate <18> (<18> the enzyme is highly specific for 2-oxoglutarate [1]) (Reversibility: r) [1] P l-aspartic b-semialdehyde + l-glutamic acid S l-lysine <18> (Reversibility: ?) [1] P 1,5-diaminopentane + CO2 S l-ornithine <18> (<18> low activity [1]) (Reversibility: ?) [1] P 1,4-diaminobutane + CO2 S Additional information <10, 18, 19> (<10> no activity with 2,3-diaminopropionate, ornithine, lysine, or arginine [10]; <19> no activity with 4-Nacetyl-l-2,4-diaminobutanoate, l-2,3-diaminopropionate, l-ornithine, and l-lysine [11]; <18> substrate specificity of the purified recombinant enzyme, l-2,3-diaminopropionic acid is a poor substrate [1]) (Reversibility: ?) [1, 10, 11] P ?

32

4.1.1.86

Diaminobutyrate decarboxylase

Inhibitors Ag+ <10> (<10> over 90% inhibition at 1 mM [10]) [10] Ca2+ <10> (<10> 10% inhibition at 1 mM [10]) [10] carboxymethoxylamine <1, 4> (<4> PLP-dependent inhibitor, 42% inibition at 0.5 mM, 65% inhibition at 1 mM [9]; <1> PLP-dependent inhibitor, 42% inibition at 0.5 mM, 78% inhibition at 1 mM [6]) [6, 9] Co2+ <2, 4, 10, 19> (<10> highly inhibitory at 20 mM [8]) [4, 8, 11] Cu2+ <10> (<10> highly inhibitory at 20 mM [8]) [8] Fe2+ <10> (<10> over 90% inhibition at 1 mM [10]; <10> highly inhibitory at 20 mM [8]) [8, 10] Hg2+ <10> (<10> over 90% inhibition at 1 mM [10]) [10] Mn2+ <2, 4, 10, 19> (<10> highly inhibitory at 20 mM [8]) [4, 8, 11] Ni2+ <2, 4, 19> [4, 11] Pb2+ <10> (<10> highly inhibitory at 20 mM [8]) [8] Sn2+ <10> (<10> highly inhibitory at 20 mM [8]) [8] Zn2+ <2, 4, 10, 19> (<10> over 90% inhibition at 1 mM [10]; <10> highly inhibitory at 20 mM [8]) [4, 8, 10, 11] propane-1,3-diamine <10> (<10> product inhibition, about 50% at 5 mM [10]) [10] Additional information <10> (<10> no inhibition by N-ethylmaleimide at up to 20 mM [8]; <10> the enzyme is not affected by GTP, ATP, DTT, Na+ or K+ [10]) [8, 10] Cofactors/prosthetic groups pyridoxal 5’-phosphate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19> (<4> absolutely required for activity, highly activating in pesence of 20 mM Mg2+ [9]; <17> binds to Lys307 [5]; <1> maximal activity at 0.1 mM PLP [6]; <19> maximal activity at 0.1 mM PLP, depletion leads to irreversible loss of 70% activity [11]; <10> required, depletion leads to over 25% irreversible loss of activity [10]) [1,3,4,5,6,7,8,9,10,11] Activating compounds DTT <4> (<4> activates 8fold at 5-7 mM [9]) [9] Additional information <1, 2, 4, 10> (<1,10> l-2,4-diaminobutanoate induces enzyme expression [6,10]; <2,4> no activation by ATP or GTP [4]) [4, 6, 10] Metals, ions Ca2+ <2, 4, 19> (<2,4> about 65% of the activity with Mg2+ [4]; <4> activates 14fold at 20 mM [9]; <19> activates, preferred divalent cation [11]) [4, 9, 11] Cu2+ <4> (<4> activates 7fold at 20 mM [9]) [9] Mg2+ <1, 2, 4, 7, 9, 10, 14, 15, 19> (<19> activates [11]; <10> 30% activation at 20 mM [8]; <2> required, activates 17fold at 10 mM [4]; <4> required, activates 20fold at 20 mM, enhances activation by PLP [9]; <4> required, activates 7fold at 10 mM [4]; <10> stimulates 30% at 20 mM [10]) [4, 6, 8, 9, 10, 11] Mn2+ <4> (<4> activates 5fold at 20 mM [9]) [9]

33

Diaminobutyrate decarboxylase

4.1.1.86

Sr2+ <2, 4, 19> (<19> activates [11]; <2, 4> about 65% of the activity with Mg2+ [4]) [4, 11] Zn2+ <4> (<4> activates 3fold at 20 mM [9]) [9] Additional information <10, 19> (<10> enzyme is not affected by Ca2+ [8]; <19> no effect by Na+ and K+ [11]) [8, 11] Specific activity (U/mg) 0.00036 <15> (<15> cell extract [8]) [8] 0.00071 <9> (<9> cell extract [8]) [8] 0.0011 <14> (<14> cell extract [8]) [8] 0.0014 <7> (<7> cell extract [8]) [8] 0.0022 <6> (<6> enzyme in cell extract [4]) [4] 0.0039 <8> (<8> enzyme in cell extract [4]) [4] 0.00392 <12> (<12> enzyme in cell extract [4]) [4] 0.0042 <11> (<11> enzyme in cell extract [4]) [4] 0.005 <4> (<4> enzyme in cell extract [4]) [4] 0.0051 <2> (<2> enzyme in cell extract [4]) [4] 0.0052 <5> (<5> enzyme in cell extract [4]) [4] 0.0058 <1> [3] 0.0071 <3> (<3> enzyme in cell extract [4]) [4] 0.0089 <13> [3] 0.012 <13> (<13> recombinant enzyme in Escherichia coli [7]) [7] 0.76 <1> (<1> partially purified enzyme [6]) [6] 1.2 <4> (<4> partially purified enzyme [9]) [9] 3.9 <2> (<2> purified enzyme [4]) [4] 4.21 <10> (<10> purified enzyme [10]) [10] 11.8 <4> (<4> purified enzyme [4]) [4] 17.2 <19> (<19> purified enzyme [11]) [11] 29.5 <10> (<10> partially purified enzyme [8]) [8] Additional information <18> (<18> DABA AT activity compared to other Acinetobacter strains [1]) [1] Km-Value (mM) 0.0056 <10> (pyridoxal 5’-phosphate, <10> pH 8.3, 37 C [10]) [10] 0.081 <10> (l-2,4-diaminobutanoate, <10> pH 8.3, 37 C [10]) [10] 0.13 <10> (l-2,4-diaminobutanoate, <10> pH 8.3, 37 C [8]) [8] 0.3 <10> (dl-2,4-diaminobutanoate, <10> pH 8.3, 37 C [8]) [8] 0.3 <4> (Mg2+ , <4> pH 8.0, 37 C [4]) [4] 0.32 <2, 4> (l-2,4-diaminobutanoate, <2> pH 8.0, 37 C [4]; <4> pH 8.3, 37 C [9]) [4, 9] 0.91 <1> (l-2,4-diaminobutanoate, <1> pH 8.5, 37 C [6]) [6] 1.07 <4> (l-2,4-diaminobutanoate, <4> pH 8.0, 37 C [4]) [4] 1.2 <2> (Mg2+ , <2> pH 8.0, 37 C [4]) [4] 1.51 <19> (l-2,4-diaminobutanoate, <19> pH 8.5, 37 C [11]) [11] pH-Optimum 7.5-8.8 <10> (<10> broad optimum [10]) [10] 7.8-8 <4> [9]

34

4.1.1.86

Diaminobutyrate decarboxylase

8-8.2 <2, 4> [4] 8-8.5 <10> [8] 8.3 <7, 9, 14, 15> (<7,9,14,15> assay at [8]) [8] 8.5 <1, 13, 18> (<1,13,18> assay at [1,3,6]) [1, 3, 6] 8.5-8.8 <19> [11] pH-Range 5.8-10 <10> (<10> about half maximal activity at pH 6.5 and pH 9.3 [10]) [10] 6-9 <4> (<4> 43% of maximal activity at pH 7.3, 60% at pH 9.0 [9]) [9] 7.2-8.2 <2, 4> (<2,4> narrow pH range, the enzyme is almost inactive at pH 7.0 [4]) [4] 7.8-9.3 <19> (<19> half maximal activity at pH 7.8 and pH 9.3 [11]) [11] Temperature optimum ( C) 37 <1, 4, 7, 9, 13, 14, 15, 18> (<1,4,7,9,13,14,15,18> assay at [1,3,6,7,8,9]) [1, 3, 6, 7, 8, 9] 37-40 <2, 4> [4] 45 <10, 19> [8, 11] Additional information <13> (<13> maximal growth temperature of the organism is 44 C [3]) [3]

4 Enzyme Structure Molecular weight 100000 <2, 4> (<2,4> gel filtration [4]) [4] 108000 <19> (<19> gel filtration [11]) [11] 450000 <10> (<10> gel filtration [10]) [10] Subunits ? <1, 13, 16, 17, 18> (<13,16> x * 53000, recombinant enzyme, SDS-PAGE [2,7]; <18> x * 47423, DNA sequence calculation [1]; <1,13> x * 53000, approximately, SDS-PAGE [3]; <17> x * 53659, DNA sequence calculation [5]) [1, 2, 3, 5, 7] dimer <2, 4, 19> (<19> 2 * 53000, SDS-PAGE [11]; <2,4> 2 * 51000, SDSPAGE [4]) [4, 11] tetramer <10> (<10> 4 * 109000, SDS-PAGE [10]) [10]

5 Isolation/Preparation/Mutation/Application Purification <1> (native enzyme 168fold by ammonium sulfate fractionation, ion exchange chromatography, and gel filtration) [6] <2> (native enzyme 886fold to homogeneity by two steps of ion exchange chromatography, gel filtration, hydroxyapatite chromatography, and another two steps of ion exchange chromatography) [4]

35

Diaminobutyrate decarboxylase

4.1.1.86

<4> (native enzyme 1750fold to homogeneity by two steps of ion exchange chromatography, gel filtration, hydroxyapatite chromatography, and another two steps of ion exchange chromatography) [4] <4> (native enzyme 360fold) [9] <10> (3660fold to homogeneity by ammonium sulfate fractionation, gel filtration, and two steps of ion exchange chromatography) [10] <10> (native enzyme 22.7fold by ammonium sulfate fractionation and anion exchange chromatography) [8] <13> [3] <16> (recombinant enzyme from Escherichia coli strain HB101) [2] <18> (recombinant enzyme from Escherichia coli strain HB101 to homogeneity by ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydroxylapatite chromatography) [1] <19> (native enzyme 2150fold to homogeneity by ammonium sulfate fractionation, two steps of ion exchange chromatography, and two steps of gel filtration) [11] Cloning <13> (DNA sequence determination and analysis, promoter determination, restriction mapping, functional expression in Escherichia coli strain XL1Blue) [7] <16> (DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain HB101) [2] <17> (gene ddc, DNA and amino acid sequence determination and analysis, restriction mapping, expression in Escherichia coli strain HB101) [5] <18> (gene dat, DNA and amino acid sequence determination and analysis, overexpression in Escherichia coli strain HB101) [1] Engineering K198R <17> (<17> site-directed mutagenesis, mutant shows 96% of the wildtype activity [5]) [5] K307R <17> (<17> site-directed mutagenesis, mutant is catalytically inactive and shows slightly reduced molecular weight compared to the wild-type enzyme due to lacking pyridoxal 5’-phosphate [5]) [5]

6 Stability pH-Stability 6.5-7.5 <2, 4> (<2,4> stable for 12 h at 4 C in 20 mM potassium phosphate [4]) [4] Temperature stability 37 <10> (<10> completely stable for up to 90 min [8]) [8] 40 <2, 4, 19> (<19> purified enzyme, stable for more than 15 min at pH 8.5 [11]; <2,4> stable up to, purified enzyme [4]) [4, 11] 45 <10> (<10> loss of 18% and 55% after 30 min and 2 h, respectively [8]) [8] 50 <2, 4> (<2,4> above, rapid inactivation [4]) [4]

36

4.1.1.86

Diaminobutyrate decarboxylase

General stability information <10>, bovine serum albumin stabilizes at below 0.1 mg/ml [8] <10>, freezing and thawing of the purified enzyme causes almost complete loss of activity [10] <10>, pyridoxal 5’-phosphate is required for stability during storage [10] <2, 4>, freezing and thawing of purified enzyme causes severe loss of activity [4] Storage stability <2>, 1 C, purified enzyme, 20 mM potassium phosphate, pH 7.5, 0.04 mM pyridoxal 5’-phosphate, and 0.02% NaN3 , loss of 65% activity within 1 week [4] <4>, 1 C, purified enzyme, 20 mM potassium phosphate, pH 7.5, 0.04 mM pyridoxal 5’-phosphate, and 0.02% NaN3 , 2 weeks without loss of activity [4] <10>, 4 C, purified enzyme, pH 7.5, in presence of pyridoxal 5’-phosphate and NaN3 , 3 weeks, stable [10]

References [1] Ikai, H.; Yamamoto, S.: Identification and analysis of a gene encoding l-2,4diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3diaminopropane production pathway in Acinetobacter baumannii. J. Bacteriol., 179, 5118-5125 (1997) [2] Ikai, H.; Yamamoto, S.: Sequence analysis of the gene encoding a novel l2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases. Arch. Microbiol., 166, 128-131 (1996) [3] Yamamoto, S.; Ikai, H.; Uesugi, T.; Horie, A.; Hirai, Y.: Occurrence and antigenic heterogeneity of l-2,4-diaminobutyrate decarboxylase in Acinetobacter species. Biol. Pharm. Bull., 18, 454-456 (1995) [4] Yamamoto, S.; Mutoh, N.; Ikai, H.; Nagasaka, M.: Occurrence of a novel l2,4-diaminobutyrate decarboxylase activity in some species of Enterobacteriaceae, and purification and characterization of the enzymes of Enterobacter aerogenes and Serratia marcescens. Biol. Pharm. Bull., 19, 1298-1303 (1996) [5] Yamamoto, S.; Mutoh, N.; Tsuzuki, D.; Ikai, H.; Nakao, H.; Shinoda, S.; Narimatsu, S.; Miyoshi, S.I.: Cloning and characterization of the ddc homolog encoding l-2,4-diaminobutyrate decarboxylase in Enterobacter aerogenes. Biol. Pharm. Bull., 23, 649-653 (2000) [6] Yamamoto, S.; Tsuzaki, Y.; Kishi, R.; Nakao, H.: Occurrence of l-2,4-diaminobutyrate decarboxylase activity in Acinetobacter. Chem. Pharm. Bull., 39, 2451-2453 (1991) [7] Ikai, H.; Yamamoto, S.: Cloning and expression in Escherichia coli of the gene encoding a novel l-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii. FEMS Microbiol. Lett., 124, 225-228 (1994)

37

Diaminobutyrate decarboxylase

4.1.1.86

[8] Yamamoto, S.; Suemoto, Y.; Seito, Y.; Nakao, H.; Shinoda, S.: The presence of l-2,4-diaminobutyric acid decarboxylase activity in Vibrio species: a new biosynthetic pathway for 1,3-diaminopropane. FEMS Microbiol. Lett., 35, 289-293 (1986) [9] Nakao, H.; Takeuchi, K.; Shinoda, S.; Yamamoto, S.: l-2,4,-Diaminobutyric acid decarboxylase activity responsible for the formation of 1,3-diaminopropane in Enterobacter aerogenes. FEMS Microbiol. Lett., 70, 61-66 (1990) [10] Nakao, H.; Ishii, M.; Shinoda, S.; Yamamoto, S.: Purification and some properties of a novel l-2,4-diaminobutyric acid decarboxylase from Vibrio alginolyticus. J. Gen. Microbiol., 135, 345-351 (1989) [11] Yamamoto, S.; Tsuzaki, Y.; Tougou, K.; Shinoda, S.: Purification and characterization of l-2,4-diaminobutyrate decarboxylase from Acinetobacter calcoaceticus. J. Gen. Microbiol., 138, 1461-1465 (1992)

38

Vanillin synthase

4.1.2.41

1 Nomenclature EC number 4.1.2.41 Systematic name 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propionyl-CoA:vanillin lyase (acetyl-CoA-forming) Recommended name vanillin synthase CAS registry number 197462-63-8

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8>

Bacillus subtilis (no sequence specified) [1] Escherichia coli (no sequence specified) [1] Pseudomonas fluorescens (no sequence specified) [1, 3] Corynebacterium glutamicum (no sequence specified) [1] Pseudomonas acidovorans (no sequence specified) [1, 3] Pseudomonas cepacia (no sequence specified) [1] Streptomyces viridosporus (no sequence specified) [2] Streptomyces setonii (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propionyl-CoA = vanillin + acetyl-CoA Reaction type reversal of an aldol condensation Natural substrates and products S 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propionyl-CoA <1, 2, 3, 4, 5, 6, 7, 8> (Reversibility: ?) [1, 2, 3] P vanillin + acetyl-CoA

39

Vanillin synthase

4.1.2.41

Substrates and products S 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propionyl-CoA <1, 2, 3, 4, 5, 6, 7, 8> (Reversibility: ?) [1, 2, 3] P vanillin + acetyl-CoA S 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propionyl-CoA + H2 O <3, 7> (Reversibility: ?) [1, 2, 3] P 4-hydroxy-3-methoxybenzaldehyde + acetyl-CoA S 4-hydroxy-3-methoxy-phenyl-b-hydroxypropionyl-CoA + H2 O <3> (Reversibility: ?) [3] P vanillin + acetylCoA + feruloylCoA <3> [3] S feruloyl-CoA + H2 O <3, 5> (Reversibility: ?) [3] P vanillin + acetyl-CoA pH-Optimum 6 <7> [2] pH-Range 5.4-8 <7> [2]

4 Enzyme Structure Molecular weight 31000 <3> (<3> gel filtration [3]) [3]

5 Isolation/Preparation/Mutation/Application Cloning <3> (gene encoding an enoyl-SCoA hydratase/lyase isolated, cloned and heterologously expressed in Escherichia coli JM109) [3] Application synthesis <3, 7> (<3> biotechnological production of vanillin [3]; <7> useful for commercial microbial batch fermentation of vanillin, economical production of vanillic acid instead of chemical oxidation of vanillin [2]) [2, 3]

References [1] Narbad, A.; Gasson, M.J.: Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly isolated strain of Pseudomonas fluorescens. Microbiology, 144, 1397-1405 (1998) [2] Pometto, A.L.; Crawford, D.L.: Whole-cell bioconversion of vanillin to vanillic acid by Streptomyces viridosporus. Appl. Environ. Microbiol., 45, 15821585 (1983)

40

4.1.2.41

Vanillin synthase

[3] Gasson, M.J.; Kitamura, Y.; McLauchlan, W.R.; Narbad, A.; Parr, A.J.; Parsons, E.L.; Payne, J.; Rhodes, M.J.; Walton, N.J.: Metabolism of ferulic acid to vanillin. A bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of a hydroxycinnamic acid SCoA thioester. J. Biol. Chem., 273, 4163-4170 (1998)

41

D-Threonine

aldolase

4.1.2.42

1 Nomenclature EC number 4.1.2.42 Systematic name d-threonine acetaldehyde-lyase (glycine-forming) Recommended name d-threonine aldolase Synonyms d-TA <1> [3] d-threonine aldolase <1> [3] DTA <3> [6] low specificity d-TA <1, 2> (<1,2> acts on the d-isomer [2,4]) [2, 4] low specificity d-threonine aldolase <4> [7] CAS registry number 87588-22-5

2 Source Organism <1> <2> <3> <4>

Arthrobacter sp. (no sequence specified) [1, 3, 4, 5] Alcaligenes xylosoxidans (no sequence specified) [2] Xanthomonas oryzae (no sequence specified) [6] Arthrobacter sp. (UNIPROT accession number: O82872) [7]

3 Reaction and Specificity Catalyzed reaction d-allothreonine = glycine + acetaldehyde d-threonine = glycine + acetaldehyde (<1> mechanism [3]) Natural substrates and products S d-threonine <1, 2> (Reversibility: r) [2, 4] P glycine + acetaldehyde <1, 2> [2, 4] Substrates and products S d-allo-threonine <1, 3, 4> (Reversibility: r) [5, 6, 7] P glycine + acetaldehyde

42

4.1.2.42

S P S P S P S P S P S P S P S P S P S P S P

S P

S P S P S P S P S P S

D-Threonine

aldolase

d-b-3,4-dihydroxyphenylserine <1> (Reversibility: ?) [5] ? d-b-3,4-methylenedioxyphenylserine <1> (Reversibility: ?) [5] ? d-b-hydroxy-a-aminovaleric acid <1> (Reversibility: ?) [5] glycine + propionaldehyde d-b-phenylserine <1> (Reversibility: ?) [5] glycine + benzaldehyde d-threonine <1, 2, 3, 4> (<1> stereospecific for d-b-hydroxyamino acids [3]) (Reversibility: r) [2, 3, 4, 5, 6, 7] glycine + acetaldehyde <1, 2> [2, 3, 4] dl-erythro-b-(3,4-methylenedioxyphenylserine) <4> (Reversibility: r) [7] ? dl-erythro-phenylserine <4> (Reversibility: r) [7] glycine + benzaldehyde dl-threo-b-(3,4-dihydroxyphenylserine) <4> (Reversibility: r) [7] ? dl-threo-b-(3,4-methylenedioxyphenylserine) <4> (Reversibility: r) [7] ? dl-threo-phenylserine <4> (Reversibility: r) [7] glycine + benzaldehyde glycine + (4R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde <3> (Reversibility: r) [6] (2R,3R)-2-amino-3-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-3-hydroxypropanoic acid + (2R,3S)-2-amino-3-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]3-hydroxypropanoic acid glycine + (4S)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde <3> (Reversibility: ?) [6] (2R,3R)-2-amino-3-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-3-hydroxypropanoic acid + (2R,3S)-2-amino-3-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-3hydroxypropanoic acid glycine + 2-methylpropanal <3> (Reversibility: r) [6] (2R,3S)-2-amino-3-hydroxy-4-methylpentanoic acid + (2R,3R)-2-amino3-hydroxy-4-methylpentanoic acid glycine + 2-nitrobenzaldehyde <3> (Reversibility: r) [6] (2R,3S)-2-amino-3-hydroxy-3-(2-nitrophenyl)propanoic acid + (2R,3R)2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid glycine + 3-hydroxybenzaldehyde <3> (Reversibility: r) [6] (2R,3S)-2-amino-3-hydroxy-3-(3-hydroxyphenyl)propanoic acid + (2R,3R)2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid glycine + 4-fluoro-3-nitrobenzaldehyde <3> (Reversibility: r) [6] (2R,3S)-2-amino-3-(4-fluoro-3-nitrophenyl)-3-hydroxypropanoic acid + (2R,3R)-2-amino-3-(4-fluoro-3-nitrophenyl)-3-hydroxypropanoic acid glycine + 4-methylbenzaldehyde <3> (Reversibility: r) [6] (2R,3S)-2-amino-3-hydroxy-3-(4-methylphenyl)propanoic acid + (2R,3R)2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid glycine + 4-nitrobenzaldehyde <3> (Reversibility: r) [6]

43

D-Threonine

aldolase

4.1.2.42

P (2R,3S)-2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid + (2R,3R)2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid S glycine + acetaldehyde <1, 3, 4> (Reversibility: r) [5, 6, 7] P d-threonine + d-allo-threonine S glycine + benzaldehyde <3> (Reversibility: r) [6] P (2R,3S)-2-amino-3-hydroxy-3-phenylpropanoic acid + (2R,3R)-2-amino3-hydroxy-3-phenylpropanoic acid S glycine + butyraldehyde <3> (Reversibility: r) [6] P (2R,3S)-2-amino-3-hydroxyhexanoic acid + (2R,3R)-2-amino-3-hydroxyhexanoic acid S glycine + hexanaldehyde <3> (Reversibility: r) [6] P (2R,3R)-2-amino-3-hydroxyoctanoic acid + (2R,3S)-2-amino-3-hydroxyoctanoic acid S glycine + octanaldehyde <3> (Reversibility: r) [6] P (2R,3R)-2-amino-3-hydroxydecanoic acid + (2R,3S)-2-amino-3-hydroxydecanoic acid S glycine + phenylacetaldehyde <3> (Reversibility: r) [6] P (2R,3S)-2-amino-3-hydroxy-4-phenylbutanoic acid + (2R,3R)-2-amino-3hydroxy-4-phenylbutanoic acid S Additional information <3> (<3> no activity with but-2-enal, 4-hydroxybenzaldehyde [6]) (Reversibility: ?) [6] P ? Inhibitors acetonitrile <3> (<3> inactivates [6]) [6] dimethylformamide <3> (<3> inactivates [6]) [6] Additional information <1> (<1> enzyme completely loses activity in absence of either pyridoxal 5’-phosphate or divalent cations [5]) [5] Cofactors/prosthetic groups pyridoxal 5’-phosphate <1, 2, 3, 4> (<1> required [5]; <2> dependent [2]; <1> Lys59 forms a Schiff base with pyridoxal phosphate [3]; <4> binds 1 mol of pyridoxal 5’-phosphate per mol of subunit, Lys59 is the cofactor binding site [7]; <3> required to activate glycine [6]) [2,3,4,5,6,7] Metals, ions Co2+ <1> (<1> requirement for a divalent cation [4]; <1> divalent cation required: Co2+, Ni2+ , Mg2+ or Mn2+ [5]) [4, 5] Mg2+ <1, 3> (<3> required [6]; <1> requirement for a divalent cation [4]; <1> divalent cation required: Co2+, Ni2+ , Mg2+ or Mn2+ [5]) [4, 5, 6] Mn2+ <1, 4> (<1> requirement for a divalent cation, binds 1 mol Mn2+ per mol of subunit [4]; <4> activates, can bind 1 mol of Mn2+ per mol of subunit [7]; <1> divalent cation required: Co2+, Ni2+ , Mg2+ or Mn2+ [5]) [4, 5, 7] Ni2+ <1> (<1> divalent cation required: Co2+ , Ni2+ , Mg2+ or Mn2+ [5]) [5] Specific activity (U/mg) 35.7 <4> [7] 38.6 <2> [2]

44

4.1.2.42

D-Threonine

aldolase

Km-Value (mM) 0.94 <3> (d-threonine) [6] 0.99 <3> (d-allo-threonine) [6] 1.2 <4> (dl-threo-b-(3,4-dihydroxyphenylserine), <4> pH 8.0, 30 C, with Mn2+ [7]) [7] 1.4 <4> (dl-threo-b-(3,4-dihydroxyphenylserine), <4> pH 8.0, 30 C, without Mn2+ [7]) [7] 1.8 <4> (dl-threo-b-(3,4-methylenedioxyphenylserine), <4> pH 8.0, 30 C, with Mn2+ [7]; <4> pH 8.0, 30 C, without Mn2+ [7]) [7] 2 <4> (dl-erythro-b-(3,4-methylenedioxyphenylserine), <4> pH 8.0, 30 C, without Mn2+ [7]) [7] 2.5 <4> (dl-erythro-b-(3,4-methylenedioxyphenylserine), <4> pH 8.0, 30 C, with Mn2+ [7]) [7] 3.81 <1> (d-threonine) [5] 4.3 <4> (d-allo-threonine, <4> pH 8.0, 30 C, without Mn2+ [7]) [7] 4.4 <1, 4> (d-allo-threonine, <4> pH 8.0, 30 C, with Mn2+ [7]) [4, 7] 4.9 <4> (dl-erythro-phenylserine, <4> pH 8.0, 30 C, without Mn2+ [7]) [7] 5.3 <4> (dl-erythro-phenylserine, <4> pH 8.0, 30 C, with Mn2+ [7]) [7] 5.4 <4> (dl-threo-phenylserine, <4> pH 8.0, 30 C, with Mn2+ [7]) [7] 5.9 <4> (dl-threo-phenylserine, <4> pH 8.0, 30 C, without Mn2+ [7]) [7] 8.4 <1, 4> (d-threonine, <4> pH 8.0, 30 C, with Mn2+ [7]) [4, 7] 8.8 <4> (d-threonine, <4> pH 8.0, 30 C, without Mn2+ [7]) [7] 14 <1> (d-allo-threonine) [5] pH-Optimum 7.5 <3> (<3> aldol addition [6]) [6] 8 <3> (<3> cleavage reaction [6]) [6] 8-9 <1> [4] Temperature optimum ( C) 37 <3> [6] 65 <1> [4]

4 Enzyme Structure Molecular weight 51000 <1> [4, 5] Subunits ? <4> (<4> x * 40000, SDS-PAGE [7]) [7] monomer <1> (<1> 1 * 40000 [4]; <1> 1 * 51000 [5]) [4, 5]

45

D-Threonine

aldolase

4.1.2.42

5 Isolation/Preparation/Mutation/Application Purification <1> [1, 5] <2> (homogeneity) [2] <3> (recombinant) [6] <4> (recombinant) [7] Cloning <1> [1] <2> [2] <3> (overexpression in Escherichia coli) [6] <4> (expression in Escherichia coli) [7] Application medicine <1, 2> (<2> efficient biocatalyst for resolution of l-X-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinsons disease [2]; <1> low-specificity threonine aldolase can be used in production of l-threo-3-[4-(methylthio)phenylserine], an intermediate for synthesis of antibiotics florfenicol and thiamphenicol [1]) [1, 2]

6 Stability Temperature stability 50 <1> (<1> 100% activity after 15 min [4]) [4]

References [1] Liu, J.Q.; Odani, M.; Dairi, T.; Itoh, N.; Shimizu, S.; Yamada, H.: A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution. Appl. Microbiol. Biotechnol., 51, 586-591 (1999) [2] Liu, J.Q.; Odani, M.; Yasuoka, T.; Dairi, T.; Itoh, N.; Kataoka, M.; Shimizu, S.; Yamada, H.: Gene cloning and overproduction of low-specificity d-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug. Appl. Microbiol. Biotechnol., 54, 44-51 (2000) [3] Paiardini, A.; Contestabile, R.; D’Aguanno, S.; Pascarella, S.; Bossa, F.: Threonine aldolase and alanine racemase: novel examples of convergent evolution in the superfamily of vitamin B6 -dependent enzymes. Biochim. Biophys. Acta, 1647, 214-219 (2003) [4] Liu, J.Q.; Dairi, T.; Itoh, N.; Kataoka, M.; Shimizu, S.; Yamada, H.: Diversity of microbial threonine aldolases and their application. J. Mol. Catal. B, 10, 107115 (2000)

46

4.1.2.42

D-Threonine

aldolase

[5] Kataoka, M.; Ikemi, M.; Morikawa, T.; Miyoshi, T.; Nishi, K.-I.; Wada, M.; Yamada, H.; Shimizu, S.: Isolation and characterization of d-threonine aldolase, a pyridoxal-5’-phosphate-dependent enzyme from Arthrobacter sp. DK38. Eur. J. Biochem., 248, 385-393 (1997) [6] Kimura, T.; Vassilev, V.P.; Shen, G.-J.; Wong, C.-H.: Enzymic synthesis of bhydroxy-a-amino acids based on recombinant d- and l-threonine aldolases. J. Am. Chem. Soc., 119, 11734-11742 (1997) [7] Liu, J.-Q.; Dairi, T.; Itoh, N.; Kataoka, M.; Shimizu, S.; Yamada, H.: A novel metal-activated pyridoxal enzyme with a unique primary structure, low specificity d-threonine aldolase from Arthrobacter sp. strain DK-38. Molecular cloning and cofactor characterization. J. Biol. Chem., 273, 16678-16685 (1998)

47

1-Deoxy-D-xylulose 5-phosphate synthase

1 Nomenclature EC number 4.1.3.37 (transferred to EC 2.2.1.7) Recommended name 1-deoxy-d-xylulose 5-phosphate synthase

48

4.1.3.37

Aminodeoxychorismate lyase

4.1.3.38

1 Nomenclature EC number 4.1.3.38 Systematic name 4-amino-4-deoxychorismate pyruvate-lyase (4-aminobenzoate-forming) Recommended name aminodeoxychorismate lyase Synonyms 4-amino-4-deoxychorismate lyase ADC lyase <2, 3> [8] ADCL enzyme X CAS registry number 132264-33-6

2 Source Organism <1> <2> <3> <4>

Escherichia coli (no sequence specified) [1, 3, 4, 5, 6, 7] Arabidopsis thaliana (no sequence specified) [8] Lycopersicon esculentum (no sequence specified) [8] Escherichia coli (UNIPROT accession number: P28305) [2]

3 Reaction and Specificity Catalyzed reaction 4-amino-4-deoxychorismate = 4-aminobenzoate + pyruvate Reaction type b-elimination Natural substrates and products S 4-amino-4-deoxychorismate <1, 2, 3> (<2,3> last step of the 4-aminobenzoate branch [8]) (Reversibility: ?) [1, 3, 8] P 4-aminobenzoate + pyruvate <1> [1, 3]

49

Aminodeoxychorismate lyase

4.1.3.38

S d-alanine + pyridoxal 5’-phosphate <1> (<1> l-alanine and other d- and l-amino acids tested are inert as substrates of transamination [6]) (Reversibility: r) [6] P pyridoxamine 5’-phosphate + pyruvate <1> [6] Substrates and products S 4-amino-4-deoxychorismate <1, 2, 3, 4> (<1> re-face specificity [5]; <2,3> last step of the 4-aminobenzoate branch [8]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] P 4-aminobenzoate + pyruvate <1, 4> [1, 2, 3, 4, 5, 6, 7] S d-alanine + pyridoxal 5’-phosphate <1> (<1> l-alanine and other d- and l-amino acids tested are inert as substrates of transamination [6]) (Reversibility: r) [6] P pyridoxamine 5’-phosphate + pyruvate <1> [6] Inhibitors Additional information <2> (<2> no feedback inhibition by physiological concentrations of 4-aminobenzoate, its glucose ester, or folates [8]) [8] Cofactors/prosthetic groups pyridoxal 5’-phosphate <1, 4> (<1> tightly bound [5]) [2,4,5,6,7] Temperature optimum ( C) 30 <1> (<1> assay at [6]) [6] 37 <1> (<1> assay at [1,3]) [1, 3]

4 Enzyme Structure Molecular weight 50000 <1, 4> (<1,4> gel filtration [1,2,3]) [1, 2, 3] Subunits dimer <1, 4> (<1> 2 * 25000, SDS-PAGE [1]; <4> 2 * 29700, most likely dimeric, SDS-PAGE [2]; <1> 2 * 29715 [5]) [1, 2, 5]

5 Isolation/Preparation/Mutation/Application Localization plastid <2, 3> [8] Purification <1> (DEAE-Sephacel, phenyl-Sepharose column, Superose 6 gel filtration column, Mono Q, Superose 12 column, yield 400-800fold) [1] <1> (fractionation with ammonium sulfate pH 7.5, DEAE-Sephacel column, butyl-Sepharose 4B column, Gigapite column) [6] <1> (fractionation with ammonium sulfate, DEAE-Toyopearl column, butylToyopearl column and Mono Q column) [5]

50

4.1.3.38

Aminodeoxychorismate lyase

<1> (reactive yellow 3-agarose column, Mono Q HR 5/5 FPLC column, Superose 12HR 10/30 FPLC column, Mono Q HR 5/20 FPLC column, Aquapore RP300 C8 HPLC column: 4100fold to near homogeneity) [3] Crystallization <1> (small yellow prisms crystals in the unliganded form obtained using the sparse-matrix method along with the hanging-drop vapor-difussion method) [5] Cloning <1> (expression in Escherichia coli JM109) [5, 6] <2> (cDNAs are shown to encode functional enzymes by complementation of an Escherichia coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert 4-amino-4-deoxychorismate to 4-aminobenzoate. The full-length Arabidopsis ADC lyase polypeptide is translocated into isolated pea chloroplasts and, when fused to Green Fluorescent Protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments) [8] <3> (cDNAs are shown to encode functional enzymes by complementation of an Escherichia coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert 4-amino-4-deoxychorismate to 4-aminobenzoate) [8] <4> (expression in Escherichia coli MC1000 cells) [2]

References [1] Ye, Q.Z.; Liu, J.; Walsh, C.T.: p-Aminobenzoate synthesis in Escherichia coli: purification and characterization of PabB as aminodeoxychorismate synthase and enzyme X as aminodeoxychorismate lyase. Proc. Natl. Acad. Sci. USA, 87, 9391-9395 (1990) [2] Green, J.M.; Merkel, W.K.; Nichols, B.P.: Characterization and sequence of Escherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme. J. Bacteriol., 174, 5317-5323 (1992) [3] Green, J.M.; Nichols, B.P.: p-Aminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC. J. Biol. Chem., 266, 12971-12975 (1991) [4] Viswanathan, V.K.; Green, J.M.; Nicholas, B.P.: Kinetic characterization of 4amino 4-deoxychorismate synthase from Escherichia coli. J. Bacteriol., 177, 5918-5923 (1995) [5] Nakai, T.; Mizutani, H.; Miyahara, I.; Hirotsu, K.; Takeda, S.; Jhee, K.H.; Yoshimura, T.; Esaki, N.: Three-dimensional structure of 4-amino-4-deoxychorismate lyase from Escherichia coli. J. Biochem., 128, 29-38 (2000) [6] Jhee, K.H.; Yoshimura, T.; Miles, E.W.; Takeda, S.; Miyahara, I.; Hirotsu, K.; Soda, K.; Kawata, Y.; Esaki, N.: Stereochemistry of the transamination reaction catalyzed by aminodeoxychorismate lyase from Escherichia coli: close relationship between fold type and stereochemistry. J. Biochem., 128, 679686 (2000)

51

Aminodeoxychorismate lyase

4.1.3.38

[7] Nichols, B.P.; Green, J.M.: Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase. J. Bacteriol., 174, 5309-5316 (1992) [8] Basset, G.J.; Ravanel, S.; Quinlivan, E.P.; White, R.; Giovannoni, J.J.; Rebeille, F.; Nichols, B.P.; Shinozaki, K.; Seki, M.; Gregory, J.F., 3rd; Hanson, A.D.: Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase. Plant J., 40, 453-461 (2004)

52

4-Hydroxy-2-oxovalerate aldolase

4.1.3.39

1 Nomenclature EC number 4.1.3.39 Systematic name 4-hydroxy-2-oxovalerate pyruvate-lyase (acetaldehyde-forming) Recommended name 4-hydroxy-2-oxovalerate aldolase Synonyms 4-hydroxy-2-ketovalerate aldolase <1> [1] DmpFG <1> [2, 3] HOA <1, 2> [1, 4] CAS registry number 37325-52-3

2 Source Organism <1> Pseudomonas sp. (no sequence specified) [1, 2, 3] <2> Pseudomonas putida (UNIPROT accession number: P51017) [4]

3 Reaction and Specificity Catalyzed reaction 4-hydroxy-2-oxopentanoate = pyruvate + acetaldehyde (<1> reaction mechanism [2]; <1> active site structure, molecular channeling of substrate and intermediate [3]) Natural substrates and products S 4-hydroxy-2-oxovalerate <1, 2> (<1> last but one step in meta-cleavage pathway for catechol degradation [2,3]; <2> step in a meta pathway [4]) (Reversibility: ?) [1, 2, 3, 4] P pyruvate + acetaldehyde S Additional information <1> (<1> the enzyme acts in an enzyme complex with the aldehyde dehydrogenase, EC 1.2.1.10, performs the final step in meta-cleavage pathway for catechol degradation [2,3]) (Reversibility: ?) [2, 3] P ?

53

4-Hydroxy-2-oxovalerate aldolase

4.1.3.39

Substrates and products S 4-hydroxy-2-oxovalerate <1, 2> (<1> last but one step in meta-cleavage pathway for catechol degradation [2,3]; <2> step in a meta pathway [4]; <2> enzyme acts stereospecifically on the l-enantiomer [4]; <1> the enzyme utilizes the l-(S)-isomer or the racemate [1]) (Reversibility: ?) [1, 2, 3, 4] P pyruvate + acetaldehyde S Additional information <1, 2> (<1> the enzyme acts in an enzyme complex with the aldehyde dehydrogenase, EC 1.2.1.10, performs the final step in meta-cleavage pathway for catechol degradation [2,3]; <1,2> the enzyme acts in an enzyme complex with the aldehyde dehydrogenase, EC 1.2.1.10, which acts on acetaldehyde to form acetyl-CoA [1,4]; <1> the enzyme acts in an enzyme complex with the aldehyde dehydrogenase, EC 1.2.1.10, which acts on acetaldehyde to form acetyl-CoA, molecular channeling of substrate and intermediate [3]; <1> the enzyme acts in an enzyme complex with the aldehyde dehydrogenase, EC 1.2.1.10, which acts on acetaldehyde to form acetyl-CoA, product channeling mechanism [2]) (Reversibility: ?) [1, 2, 3, 4] P ? Inhibitors Zn2+ <1> (<1> strong inhibition [1]) [1] Activating compounds NAD+ <1> (<1> stimulates slightly [1]) [1] NADH <1> (<1> stimulates [1]) [1] Metals, ions Mn2+ <1, 2> (<1> stimulates 6-8fold at 1 mM [1]) [1, 4] Additional information <1> (<1> no effect by Mg2+ and Ca2+ [1]) [1] Specific activity (U/mg) 0.015 <2> (<2> recombinant enzyme in extracts of different recombinant strains, activity depends on the plasmid vector used for expression and the aldehyde dehydrogenase, EC 1.2.1.10, overview [4]) [4] 4.2 <1> (<1> purified enzyme [1]) [1] pH-Optimum 8 <2> (<2> assay at [4]) [4] 8.5-9 <1> [1] pH-Range 6.5-9 <1> [1] Temperature optimum ( C) 25 <1, 2> (<1,2> assay at [1,4]) [1, 4]

54

4.1.3.39

4-Hydroxy-2-oxovalerate aldolase

4 Enzyme Structure Molecular weight 148000 <1> (<1> enzyme in complex with aldehyde dehydrogenase, EC 1.2.1.10, gel filtration [1]) [1] Subunits dimer <1> (<1> 1 * 32500 + 1 * 37500, active unit is an enzyme complex of aldehyde dehydrogenase and 4-hydroxy-2-ketovalerate aldolase, SDS-PAGE and crystal structure [3]) [3] tetramer <1> (<1> 2 * 35000 + 2 * 40000, enzyme complex of aldehyde dehydrogenase and 4-hydroxy-2-ketovalerate aldolase, SDS-PAGE [1]) [1] Additional information <1> (<1> enzyme complex structure analysis [3]; <1> the enzyme forms a heterodimer of 71 kDa with the aldehyde dehydrogenase, EC 1.2.1.10 [2]; <1> the enzyme is encoded in the dmp operon with aldehyde dehydrogenase, EC 1.2.1.10, with which it forms an enzyme complex [1]) [1, 2, 3]

5 Isolation/Preparation/Mutation/Application Purification <1> (co-purification with aldehyde dehydrogenase, EC 1.2.1.10, to homogeneity in a 3-step chromatographic procedure involving NAD+ affinity chromatography) [2] <1> (co-purification with aldehyde dehydrogenase, EC 1.2.1.10, to homogeneity in a 5-step chromatographic procedure) [1] Crystallization <1> (purified enzyme complex of 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating), protein solution contains 9 mg/ml protein in 50 mM Tris-HCl, pH 7.4, 1 mM DTT, and 2 mM NAD+, mixing of equal volumes of 0.001 ml of protein and reservoir solutions, the latter contains 15% PEG 8000, 100 mM ammonium sulfate, and 100 mM PIPES, pH 7.5, 3 days, soaking of crystals in 5 mM samarium acetate and 0.25 mM PCMBS, X-ray diffraction structure determination and analysis at 2.1 A resolution) [2] <1> (purified selenomethionine-labeled enzyme complex of 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating), protein solution contains 9 mg/ml protein in 50 mM Tris-HCl, pH 7.4, 1 mM DTT, and 2 mM NAD+, mixing of equal volumes of 0.001 ml of protein and reservoir solutions, the latter contains 18% PEG 8000, 100 mM ammonium sulfate, and 100 mM PIPES, pH 7.5, 3 days, crystals are used for microseeding, cryoprotection by soaking in mother liquor with 20% v/v 2-methyl-2,4-pentanediol, X-ray diffraction structure determination and analysis at 1.7 A resolution) [3]

55

4-Hydroxy-2-oxovalerate aldolase

4.1.3.39

Cloning <1> (gene dmpG encodes the enzyme in the dmp operon together with aldehyde dehydrogenase, EC 1.2.1.10, with which it forms an enzyme complex) [1] <2> (DNA and amino acid sequence determination and analysis of the gene nahM from the naphthalene catabolic plasmid pWW60-22 of strain NCIMB9816, gene nahM is organized in the nahNLOMK operon of nahOH genes, genetic organization analysis, overview, overexpression in Escherichia coli strain BL21(DE3) using different plasmid vectors, overview) [4]

6 Stability Storage stability <1>, -80 C, purified native enzyme, stable for at least 6 months [1] <1>, 4-6 C, purified native enzyme, stable for at least 6 days [1]

References [1] Powlowski, J.; Sahlman, L.; Shingler, V.: Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600. J. Bacteriol., 175, 377-385 (1993) [2] Manjasetty, B.A.; Croteau, N.; Powlowski, J.; Vrielink, A.: Crystallization and preliminary x-ray analysis of dmpFG-encoded 4-hydroxy-2-ketovalerate aldolase-aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600. Acta Crystallogr. Sect. D, 57, 582-585 (2001) [3] Manjasetty, B.A.; Powlowski, J.; Vrielink, A.: Crystal structure of a bifunctional aldolase-dehydrogenase: sequestering a reactive and volatile intermediate. Proc. Natl. Acad. Sci. USA, 100, 6992-6997 (2003) [4] Platt, A.; Shingler, V.; Taylor, S.C.; Williams, P.A.: The 4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded by the nahM and nahO genes of the naphthalene catabolic plasmid pWW60-22 provide further evidence of conservation of meta-cleavage pathway gene sequences. Microbiology, 141 (Pt 9), 2223-2233 (1995)

56

Chorismate lyase

4.1.3.40

1 Nomenclature EC number 4.1.3.40 Systematic name chorismate pyruvate-lyase (4-hydroxybenzoate-forming) Recommended name chorismate lyase Synonyms Rv2949c enzyme <2> [6] chorismate pyruvate-lyase <1> [5, 7, 8, 9] chorismate-pyruvate lyase <3> [1] CAS registry number 157482-18-3

2 Source Organism <1> Escherichia coli (no sequence specified) [2, 3, 4, 5, 7, 8, 9] <2> Mycobacterium tuberculosis (no sequence specified) [6] <3> Escherichia coli (UNIPROT accession number: P26602) [1]

3 Reaction and Specificity Catalyzed reaction chorismate = 4-hydroxybenzoate + pyruvate Natural substrates and products S chorismate <1, 3> (<3> first step in ubiquinone biosynthesis [1]; <1> ubiquinone biosynthetic pathway [2]) (Reversibility: ?) [1, 2, 9] P 4-hydroxybenzoate + pyruvate Substrates and products S chorismate <1, 2, 3> (<3> first step in ubiquinone biosynthesis [1]; <1> ubiquinone biosynthetic pathway [2]) (Reversibility: ?) [1, 2, 4, 6, 9] P 4-hydroxybenzoate + pyruvate S chorismate + <1> (Reversibility: ?) [2] P 4-hydroxybenzoate + pyruvate

57

Chorismate lyase

4.1.3.40

Inhibitors 3-carboxymethylaminomethyl-4-hydroxybenzoate <1> [2] 4-hydroxybenzaldehyde <1> [2] 4-hydroxybenzoate <1, 2> (<1> product inhibition [2]; <2> strong product inhibition [6]) [2, 6] vanillate <1> [2, 4] Metals, ions Mg2+ <2> (<2> required [6]) [6] Turnover number (min–1) 0.102 <2> (chorismate, <2> pH 7.5, 37 C, recombinant enzyme produced in Mycobacterium smegmatis [6]) [6] 0.215 <2> (chorismate, <2> pH 7.5, 37 C, recombinant enzyme produced in Escherichia coli [6]) [6] 0.82 <3> (chorismate, <3> pH 7.5, 37 C [1]) [1] 1.3 <1> (chorismate, <1> pH 7.5, mutant enzyme C14S/C81S, initial rate analysis [2]) [2] 1.4 <1> (chorismate, <1> pH 7.5, mutant enzyme C14S/C81S, progress curve analysis [2]) [2] 1.5 <1> (chorismate, <1> pH 7.5, wild-type enzyme, initial rate analysis [2]) [2] 1.7 <1> (chorismate, <1> pH 7.5, wild-type enzyme, progress curve analysis [2]) [2] Km-Value (mM) 0.00197 <2> (chorismate, <2> pH 7.5, 37 C, recombinant enzyme produced in Mycobacterium smegmatis [6]) [6] 0.0097 <3> (chorismate, <3> pH 7.5, 37 C [1]) [1] 0.028 <1> (chorismate, <1> pH 7.5, wild-type enzyme, initial rate analysis [2]) [2] 0.029 <1> (chorismate, <1> pH 7.5, wild-type enzyme, progress curve analysis [2]) [2] 0.037 <1> (chorismate, <1> pH 7.5, mutant enzyme C14S/C81S, initial rate analysis [2]) [2] 0.039 <1> (chorismate, <1> pH 7.5, mutant enzyme C14S/C81S, progress curve analysis [2]) [2] 0.0396 <2> (chorismate, <2> pH 7.5, 37 C, recombinant enzyme produced in Escherichia coli [6]) [6] Ki-Value (mM) 0.001 <2> (4-hydroxybenzoate, <2> pH 7.5, 37 C [6]) [6] 0.0021 <1> (4-hydroxybenzoate, <1> pH 7.5, wild-type enzyme [2]) [2] 0.26 <1> (vanillate, <1> pH 7.5, wild-type enzyme [2]) [2] 0.33 <1> (4-hydroxybenzaldehyde, <1> pH 7.5, wild-type enzyme [2]) [2] 0.4 <1> (3-carboxymethylaminomethyl-4-hydroxybenzoate, <1> pH 7.5, wild-type enzyme [2]) [2]

58

4.1.3.40

Chorismate lyase

pH-Optimum 7.5 <2> [6]

4 Enzyme Structure Molecular weight 17000 <3> (<3> gel filtration [1]) [1] Subunits ? <1> (<1> x * 18000, SDS-PAGE [5]; <1> x * 18776, calculation from nucleotide sequence [5]) [5] monomer <3> (<3> 1 * 17000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue Additional information <1> (<1> expressed in hairy root cultures of Lithospermum erythrorhizon [7]) [7] Purification <1> (wild-type and mutant enzyme C14 S/C81S) [2] <2> (recombinant enzyme produced in Escherichia coli and Mycobacterium smegmatis) [6] <3> [1] Crystallization <1> (1.0 A crystal structure of the enzyme-product complex, 2.0 A structure of the G90A mutant enzyme with bound product, 2.4 A structure of the enzyme complexed with the inhibitor vanillate, 1.9 A structure of the G90A mutant enzyme with the inhibitor vanillate) [4] <1> (structure of wild-type enzyme, mutant enzyme C14S and C14S/C81S in enzyme-product complex. Structure is determined by heavy atom methods using the single mutant in its orthorhombic crystal form, and subsequently solved by molecular replacement in both the wild-type and double-mutant forms) [3] Cloning <1> [5] <1> (expressed in Lithospermum erythrorhizon under the control of the strong (ocs)3mas-promoter) [7] <1> (expression in Nicotiana tabacum under control of the constitutive plant promoter. The gene product is targeted into the plastid by fusing it to the sequence for the chloroplast transit peptide of the small subunit of ribulose1,5-bisphosphate carboxylase/oxygenase. Transgenic plants show high chorismate pyruvate-lyase activity and accumulate 4-hydroxybenzoate as b-glucosides, with the glucose attached to either the hydroxy or the carboxyl function of 4-hydroxybenzoate. The total content of 4-hydroxybenzoate glucosides

59

Chorismate lyase

4.1.3.40

is approximately 0.52% of dry weight, which exceeds the content of untransformed plants by at least a factor of 1000) [8] <1> (the ubiC gene is integrated into the chloroplast genome of Nicotiana tabacum under the control of the light-regulated psbA 5’-untranslated region. the limitation for 4-hydroxybenzoate production in nuclear-transformed plants is the activity of chorismate pyruvate-lyase activity. The process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest) [9] <2> (production of recombinant enzyme in Escherichia coli and Mycobacterium smegmatis) [6] <3> [1] Engineering C14S/C81S <1> (<1> mutation causes greatly improved solution behavior and minor effect on enzyme activity [2]) [2] G90A <1> (<1> the KM -value for the substrate chorismate is unaffected, the Kp for the product is altered [4]) [4]

6 Stability Storage stability <2>, -20 C, in presence of 10% glycerol the purified recombinant enzyme is stable for at least 3 weeks [6]

References [1] Nichols, B.P.; Green, J.M.: Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase. J. Bacteriol., 174, 5309-5316 (1992) [2] Holden, M.J.; Mayhew, M.P.; Gallagher, D.T.; Vilker, V.L.: Chorismate lyase: kinetics and engineering for stability. Biochim. Biophys. Acta, 1594, 160-167 (2002) [3] Gallagher, D.T.; Mayhew, M.; Holden, M.J.; Howard, A.; Kim, K.J.; Vilker, V.L.: The crystal structure of chorismate lyase shows a new fold and a tightly retained product. Proteins, 44, 304-311 (2001) [4] Smith, N.; Roitberg, A.E.; Rivera, E.; Howard, A.; Holden, M.J.; Mayhew, M.; Kaistha, S.; Gallagher, D.T.: Structural analysis of ligand binding and catalysis in chorismate lyase. Arch. Biochem. Biophys., 445, 72-80 (2006) [5] Siebert, M.; Bechthold, A.; Melzer, M.; May, U.; Berger, U.; Schroder, G.; Schroder, J.; Severin, K.; Heide, L.: Ubiquinone biosynthesis. Cloning of the genes coding for chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyl transferase from Escherichia coli. FEBS Lett., 307, 347-350 (1992) [6] Stadthagen, G.; Kordulakova, J.; Griffin, R.; Constant, P.; Bottova, I.; Barilone, N.; Gicquel, B.; Daffe, M.; Jackson, M.: p-Hydroxybenzoic acid synthesis in Mycobacterium tuberculosis. J. Biol. Chem., 280, 40699-40706 (2005)

60

4.1.3.40

Chorismate lyase

[7] Kçhle, A.; Sommer, S.; Yazaki, K.; Ferrer, A.; Boronat, A.; Li, S.M.; Heide, L.: High level expression of chorismate pyruvate-lyase (UbiC) and HMG-CoA reductase in hairy root cultures of Lithospermum erythrorhizon. Plant Cell Physiol., 43, 894-902 (2002) [8] Siebert, M.; Sommer, S.; Li, S.M.; Wang, Z.X.; Severin, K.; Heide, L.: Genetic engineering of plant secondary metabolism. Accumulation of 4-hydroxybenzoate glucosides as a result of the expression of the bacterial ubiC gene in tobacco. Plant Physiol., 112, 811-819 (1996) [9] Viitanen, P.V.; Devine, A.L.; Khan, M.S.; Deuel, D.L.; Van Dyk, D.E.; Daniell, H.: Metabolic engineering of the chloroplast genome using the Escherichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis. Plant Physiol., 136, 4048-4060 (2004)

61

Aristolochene synthase

1 Nomenclature EC number 4.1.99.7 (transferred to EC 4.2.3.9) Recommended name aristolochene synthase

62

4.1.99.7

Pinene synthase

4.1.99.8

1 Nomenclature EC number 4.1.99.8 (transferred to EC 4.2.3.14) Recommended name pinene synthase

63

Myrcene synthase

1 Nomenclature EC number 4.1.99.9 (transferred to EC 4.2.3.15) Recommended name myrcene synthase

64

4.1.99.9

(-)-(4S )-Limonene synthase

4.1.99.10

1 Nomenclature EC number 4.1.99.10 (transferred to EC 4.2.3.16) Recommended name (-)-(4S )-limonene synthase

65

Benzylsuccinate synthase

4.1.99.11

1 Nomenclature EC number 4.1.99.11 Systematic name benzylsuccinate fumarate-lyase (toluene-forming) Recommended name benzylsuccinate synthase Synonyms (R)-benzylsuccinate synthase <1, 2> [7] BSS <1> [8] CAS registry number 209264-18-6

2 Source Organism <1> <2> <3> <4> <5>

Thauera aromatica (no sequence specified) [4, 6, 7, 8] Azoarcus sp. (no sequence specified) [1, 3, 5, 7] Methanosaeta sp. (no sequence specified) [2] Methanospirillum sp. (no sequence specified) [2] Desulfotomaculum (no sequence specified) [2]

3 Reaction and Specificity Catalyzed reaction benzylsuccinate = toluene + fumurate (<2> stereospecific radical addition of toluene to fumarate, enzyme carries a stable organic free radical, most probably located on glycine residue 828 [1]) Reaction type addition Natural substrates and products S toluene + fumarate <1, 2, 3, 4, 5> (<1> highly stereospecific, enantiomer purity for (R-)-(+)-benzylsuccinate 99% [4]) (Reversibility: ?) [1, 2, 3, 4, 5, 6] P benzylsuccinate <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6]

66

4.1.99.11

Benzylsuccinate synthase

Substrates and products S 2-fluorotoluene + fumarate <2> (Reversibility: ?) [3] P (2-fluorobenzyl)succinate <2> [3] S 3-fluorotoluene + fumarate <2> (Reversibility: ?) [3] P (3-fluorobenzyl)succinate <2> [3] S 4-fluorotoluene + fumarate <2> (Reversibility: ?) [3] P (4-fluorobenzyl)succinate <2> [3] S [d3 -methyl]toluene + fumarate <1> (<1> deuterium is transferred from toluene to the C-3 pro-(S) position of benzylsuccinate, implying that the addition of toluene to the double bond of fumarate is syn [8]) (Reversibility: ?) [8] P ? S [d3 -methyl]toluene + maleate <1> (<1> addition of toluene occurs in anti fashion. Formation of the C-3 radical of the benzylsuccinate as an intermediate, in which rotation about the C-2-C-3 bond can occur to relieve the sterically unfavorable cis conformation [8]) (Reversibility: ?) [8] P ? S benzaldehyde + fumarate <2> (Reversibility: ?) [3] P benzoylsuccinate <2> [3] S m-cresol + fumarate <1, 2> (Reversibility: ?) [7] P (3-hydroxybenzyl)succinic acid S m-xylene + fumarate <1, 2> (Reversibility: ?) [7] P (3-methylbenzyl)succinic acid S m-xylene-d6 + fumarate <2> (Reversibility: ?) [3] P (3-methylbenzyl)succinate-d6 <2> [3] S o-cresol + fumarate <1, 2> (Reversibility: ?) [7] P (2-hydroxybenzyl)succinic acid S o-xylene + fumarate <1, 2> (Reversibility: ?) [7] P (2-methylbenzyl)succinic acid S o-xylene-d10 + fumarate <2> (Reversibility: ?) [3] P (2-methylbenzyl)succinate-d10 <2> [3] S p-cresol + fumarate <1, 2> (Reversibility: ?) [7] P (4-hydroxybenzyl)succinic acid S p-xylene + fumarate <1, 2> (Reversibility: ?) [7] P (4-methylbenzyl)succinic acid S p-xylene-d10 + fumarate <2> (Reversibility: ?) [3] P (4-methylbenzyl)succinate-d10 <2> [3] S toluene + fumarate <1, 2, 3, 4, 5> (<1> highly stereospecific, enantiomer purity for (R-)-(+)-benzylsuccinate 99% [4]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7] P benzylsuccinate <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6] S toluene + maleate <2> (Reversibility: ?) [3] P benzylsuccinate <2> [3] Cofactors/prosthetic groups flavin <1> (<1> indicated by fluorescence spectroscopy, chemical nature unknown [6]) [6]

67

Benzylsuccinate synthase

4.1.99.11

Specific activity (U/mg) 0.0002 <1> [6] 0.003 <2> [3] 0.016 <3, 4, 5> [2] pH-Optimum 8 <1> [6]

4 Enzyme Structure Molecular weight 220000 <1> (<1> gel filtration [6]) [6] 260000 <2> (<2> gel filtration [3]) [3] Subunits tetramer <1, 2> (<2> a, a, b, g, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE [3]; <1> a, a, b, g, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE [6]) [3, 6]

5 Isolation/Preparation/Mutation/Application Source/tissue culture condition:toluene-grown cell <1> [8] Purification <1> (DEAE-Sepharose, hydroxyapatite, anion exchange chromatography, enzyme severely inactivated during purification) [6] <2> (hydroxyapatite, gel filtration, 95% loss of specific activity after gel filtration) [3] Cloning <1> (expressed in Escherichia coli) [6]

6 Stability Oxidation stability <1>, rapidly inactivated by oxygen with a half-life of 20-30 s upon exposure to air [6]

References [1] Krieger, C.J.; Rosebooms, W.; Albrachts, P.J.; Spormann, A.M.: A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T. J. Biol. Chem., 276, 12924-12927 (2001)

68

4.1.99.11

Benzylsuccinate synthase

[2] Beller, H.R.; Edwards, E.A.: Anaerobic toluene activation by benzylsuccinate synthase in a highly enriched methanogenic culture. Appl. Environ. Microbiol., 66, 5503-5505 (2000) [3] Beller, H.R.; Spormann, A.M.: Substrate range of benzylsuccinate synthase from Azoarcus sp. strain T. FEMS Microbiol. Lett., 178, 147-153 (1999) [4] Leutwein, C.; Heider, J.: Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and b-oxidation of the first intermediate, (R)(+)-benzylsuccinate. Microbiology, 145, 3265-3271 (1999) [5] Beller, H.R.; Spormann, A.M.: Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product. J. Bacteriol., 180, 5454-5457 (1998) [6] Leuthner, B.; Leutwein, C.; Schultz,H.; Hçrth, P.; Haehnel, W.; Schiltz, E.; Schgger, H.; Heider, J.: Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalaysing the first step in anaerobic toluene metabolism. Mol. Microbiol., 28, 615-628 (1998) [7] Verfrth, K.; Pierik, A.J.; Leutwein, C.; Zorn, S.; Heider, J.: Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and m-xylene metabolism. Arch. Microbiol., 181, 155-162 (2004) [8] Qiao, C.; Marsh, E.N.G.: Mechanism of benzylsuccinate synthase: Stereochemistry of toluene addition to fumarate and maleate. J. Am. Chem. Soc., 127, 8608-8609 (2005)

69

(-)-(4S )-Limonene synthase

4.1.99.10

1 Nomenclature EC number 4.1.99.10 (transferred to EC 4.2.3.16) Recommended name (-)-(4S )-limonene synthase

65

Benzylsuccinate synthase

4.1.99.11

1 Nomenclature EC number 4.1.99.11 Systematic name benzylsuccinate fumarate-lyase (toluene-forming) Recommended name benzylsuccinate synthase Synonyms (R)-benzylsuccinate synthase <1, 2> [7] BSS <1> [8] CAS registry number 209264-18-6

2 Source Organism <1> <2> <3> <4> <5>

Thauera aromatica (no sequence specified) [4, 6, 7, 8] Azoarcus sp. (no sequence specified) [1, 3, 5, 7] Methanosaeta sp. (no sequence specified) [2] Methanospirillum sp. (no sequence specified) [2] Desulfotomaculum (no sequence specified) [2]

3 Reaction and Specificity Catalyzed reaction benzylsuccinate = toluene + fumurate (<2> stereospecific radical addition of toluene to fumarate, enzyme carries a stable organic free radical, most probably located on glycine residue 828 [1]) Reaction type addition Natural substrates and products S toluene + fumarate <1, 2, 3, 4, 5> (<1> highly stereospecific, enantiomer purity for (R-)-(+)-benzylsuccinate 99% [4]) (Reversibility: ?) [1, 2, 3, 4, 5, 6] P benzylsuccinate <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6]

66

4.1.99.11

Benzylsuccinate synthase

Substrates and products S 2-fluorotoluene + fumarate <2> (Reversibility: ?) [3] P (2-fluorobenzyl)succinate <2> [3] S 3-fluorotoluene + fumarate <2> (Reversibility: ?) [3] P (3-fluorobenzyl)succinate <2> [3] S 4-fluorotoluene + fumarate <2> (Reversibility: ?) [3] P (4-fluorobenzyl)succinate <2> [3] S [d3 -methyl]toluene + fumarate <1> (<1> deuterium is transferred from toluene to the C-3 pro-(S) position of benzylsuccinate, implying that the addition of toluene to the double bond of fumarate is syn [8]) (Reversibility: ?) [8] P ? S [d3 -methyl]toluene + maleate <1> (<1> addition of toluene occurs in anti fashion. Formation of the C-3 radical of the benzylsuccinate as an intermediate, in which rotation about the C-2-C-3 bond can occur to relieve the sterically unfavorable cis conformation [8]) (Reversibility: ?) [8] P ? S benzaldehyde + fumarate <2> (Reversibility: ?) [3] P benzoylsuccinate <2> [3] S m-cresol + fumarate <1, 2> (Reversibility: ?) [7] P (3-hydroxybenzyl)succinic acid S m-xylene + fumarate <1, 2> (Reversibility: ?) [7] P (3-methylbenzyl)succinic acid S m-xylene-d6 + fumarate <2> (Reversibility: ?) [3] P (3-methylbenzyl)succinate-d6 <2> [3] S o-cresol + fumarate <1, 2> (Reversibility: ?) [7] P (2-hydroxybenzyl)succinic acid S o-xylene + fumarate <1, 2> (Reversibility: ?) [7] P (2-methylbenzyl)succinic acid S o-xylene-d10 + fumarate <2> (Reversibility: ?) [3] P (2-methylbenzyl)succinate-d10 <2> [3] S p-cresol + fumarate <1, 2> (Reversibility: ?) [7] P (4-hydroxybenzyl)succinic acid S p-xylene + fumarate <1, 2> (Reversibility: ?) [7] P (4-methylbenzyl)succinic acid S p-xylene-d10 + fumarate <2> (Reversibility: ?) [3] P (4-methylbenzyl)succinate-d10 <2> [3] S toluene + fumarate <1, 2, 3, 4, 5> (<1> highly stereospecific, enantiomer purity for (R-)-(+)-benzylsuccinate 99% [4]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7] P benzylsuccinate <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6] S toluene + maleate <2> (Reversibility: ?) [3] P benzylsuccinate <2> [3] Cofactors/prosthetic groups flavin <1> (<1> indicated by fluorescence spectroscopy, chemical nature unknown [6]) [6]

67

Benzylsuccinate synthase

4.1.99.11

Specific activity (U/mg) 0.0002 <1> [6] 0.003 <2> [3] 0.016 <3, 4, 5> [2] pH-Optimum 8 <1> [6]

4 Enzyme Structure Molecular weight 220000 <1> (<1> gel filtration [6]) [6] 260000 <2> (<2> gel filtration [3]) [3] Subunits tetramer <1, 2> (<2> a, a, b, g, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE [3]; <1> a, a, b, g, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE [6]) [3, 6]

5 Isolation/Preparation/Mutation/Application Source/tissue culture condition:toluene-grown cell <1> [8] Purification <1> (DEAE-Sepharose, hydroxyapatite, anion exchange chromatography, enzyme severely inactivated during purification) [6] <2> (hydroxyapatite, gel filtration, 95% loss of specific activity after gel filtration) [3] Cloning <1> (expressed in Escherichia coli) [6]

6 Stability Oxidation stability <1>, rapidly inactivated by oxygen with a half-life of 20-30 s upon exposure to air [6]

References [1] Krieger, C.J.; Rosebooms, W.; Albrachts, P.J.; Spormann, A.M.: A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T. J. Biol. Chem., 276, 12924-12927 (2001)

68

4.1.99.11

Benzylsuccinate synthase

[2] Beller, H.R.; Edwards, E.A.: Anaerobic toluene activation by benzylsuccinate synthase in a highly enriched methanogenic culture. Appl. Environ. Microbiol., 66, 5503-5505 (2000) [3] Beller, H.R.; Spormann, A.M.: Substrate range of benzylsuccinate synthase from Azoarcus sp. strain T. FEMS Microbiol. Lett., 178, 147-153 (1999) [4] Leutwein, C.; Heider, J.: Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and b-oxidation of the first intermediate, (R)(+)-benzylsuccinate. Microbiology, 145, 3265-3271 (1999) [5] Beller, H.R.; Spormann, A.M.: Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product. J. Bacteriol., 180, 5454-5457 (1998) [6] Leuthner, B.; Leutwein, C.; Schultz,H.; Hçrth, P.; Haehnel, W.; Schiltz, E.; Schgger, H.; Heider, J.: Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalaysing the first step in anaerobic toluene metabolism. Mol. Microbiol., 28, 615-628 (1998) [7] Verfrth, K.; Pierik, A.J.; Leutwein, C.; Zorn, S.; Heider, J.: Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and m-xylene metabolism. Arch. Microbiol., 181, 155-162 (2004) [8] Qiao, C.; Marsh, E.N.G.: Mechanism of benzylsuccinate synthase: Stereochemistry of toluene addition to fumarate and maleate. J. Am. Chem. Soc., 127, 8608-8609 (2005)

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1 Nomenclature EC number 4.1.99.12 Systematic name d-ribulose 5-phosphate formate-lyase (l-3,4-dihydroxybutan-2-one 4-phosphate-forming) Recommended name 3,4-dihydroxy-2-butanone-4-phosphate synthase Synonyms 3,4-dihydroxy-2-butanone 4-phosphate synthase <2, 5, 7> [2, 5, 10, 12, 13, 16] DBPS <5> [10] DHBP synthase <3, 8> [9, 14] DHBPS <2, 5, 7> [2, 12, 15] l-3,4-dihydroxy-2-butanone 4-phosphate synthase <4> [6, 7] Rib3 <3> [9] RibA <1> [11] dihydroxybutanone phosphate synthase <3, 6> [1, 9] Additional information <1> (<1> cf. EC 3.5.4.25 [11]) [11]

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8>

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Bacillus subtilis (no sequence specified) [11] Escherichia coli (no sequence specified) [3, 5, 13, 15, 16] Saccharomyces cerevisiae (no sequence specified) [9] Candida guilliermondii (no sequence specified) [6, 7] Methanococcus jannaschii (no sequence specified) [2,8,10,15] Magnaporthe grisea (no sequence specified) [1,4] Candida albicans (UNIPROT accession number: AY504626) [12] Escherichia coli K12 (UNIPROT accession number: P0A7J0) [14]

4.1.99.12

3,4-Dihydroxy-2-butanone-4-phosphate synthase

3 Reaction and Specificity Catalyzed reaction d-ribulose 5-phosphate = formate + l-3,4-dihydroxybutan-2-one 4-phosphate (<2> reaction mechanism [13]; <2,4,8> catalytic mechanism [5,7,14]; <5> active site and reaction mechanism, structure-function relationship, structural coordination spheres are important, residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity, Glu185 is essential for catalytic activity [10]; <5> active site architecture [2]; <6> active site structure and catalytic mechanism, modeling of the substrate ribulose 5-phosphate bound in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site, the catalytic reaction involves residues Asp41, Cys66, and Glu174, and the Asp99-His136 dyad [4]; <2> catalytic reaction mechanism, substrate recognition and active site structure, active site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg2+ cofactor within the active site [16]; <5> reaction mechanism involving intramolecular skeletal rearrangement, a cluster of charged amino acid residues comprising Arg25, Glu26 and Glu28, Asp21 and Asp30 is essential for catalytic activity, as well as His164 and Glu185 [8]; <7> reaction mechanism, the enzyme possesses an essential acidic active-site loop [12]; <1> RibA is a bifunctional enzyme possessing 3,4-dihydroxy-2-butanone 4-phosphate synthase activity located in the N-terminal half of the protein and GTP cyclohydrolase II activity, EC 3.5.4.25, of the C-terminal domain, overview [11]) Natural substrates and products S d-ribulose 5-phosphate <1, 2, 3, 4, 5, 6, 7, 8> (<4> a step in the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino6-ribitylamino-2,4(1H,3H)-pyrimidinedione requiring a phosphorylated 4-carbon intermediate, designated as compound X [6]; <4> a step in the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requiring a phosphorylated 4-carbon intermediate, designated as compound X, analysis of the riboflavin biosynthetic pathway, overview [7]; <5> DHBPS supplies the building blocks for the assembly of the xylene ring of the vitamin B2 , riboflavin, all eight C atoms of the xylene moiety are derived from the product of the enzyme [2]; <1> rate-limiting step in riboflavin biosynthesis [11]; <2> step in biosynthesis of vitamin B2 , riboflavin [3]; <5> step in the biosynthesis of riboflavin, vitamin B2 , ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions [10]; <2> the enzyme is important in riboflavin biosynthesis, overview [13]; <8> the enzyme is important in the riboflavin biosynthesis, the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, is formed by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) with 3,4-dihydroxy-2-butanone 4-phosphate, overview [14]; <7> the enzyme is in-

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volved in riboflavin biosynthesis [12]; <2, 5> the enzyme is involved in the pathway of riboflavin biosynthesis [5, 15]; <2> the enzyme is involved in the pathway of riboflavin biosynthesis, overview [16]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] P formate + l-3,4-dihydroxy-2-butanone-4-phosphate S Additional information <1, 3> (<1> RibA is the rate limiting enzyme in an industrial riboflavin producing strain [11]; <3> the enzyme is involved in the riboflavin biosynthetic pathway, but has a second unrelated function in expression of mitochondrial respiration [9]) (Reversibility: ?) [9, 11] P ? Substrates and products S d-ribulose 5-phosphate <1, 2, 3, 4, 5, 6, 7, 8> (<8> substrate preparation, overview [14]; <4> a step in the formation of the riboflavin precursor, 6,7dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione requiring a phosphorylated 4-carbon intermediate, designated as compound X [6]; <4> a step in the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino2,4(1H,3H)-pyrimidinedione requiring a phosphorylated 4-carbon intermediate, designated as compound X, analysis of the riboflavin biosynthetic pathway, overview [7]; <5> DHBPS supplies the building blocks for the assembly of the xylene ring of the vitamin B2 , riboflavin, all eight C atoms of the xylene moiety are derived from the product of the enzyme [2]; <1> rate-limiting step in riboflavin biosynthesis [11]; <2> step in biosynthesis of vitamin B2 , riboflavin [3]; <5> step in the biosynthesis of riboflavin, vitamin B2 , ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions [10]; <2> the enzyme is important in riboflavin biosynthesis, overview [13]; <8> the enzyme is important in the riboflavin biosynthesis, the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, is formed by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) with 3,4-dihydroxy-2-butanone 4-phosphate, overview [14]; <7> the enzyme is involved in riboflavin biosynthesis [12]; <2,5> the enzyme is involved in the pathway of riboflavin biosynthesis [5,15]; <2> the enzyme is involved in the pathway of riboflavin biosynthesis, overview [16]; <5> a dimetal center is involved in substrate binding, structure-function relationship, overview [10]; <4> carbon atoms 1-3 of the enzyme product correspond to carbon atoms 1-3 of the substrate, whereas C-4 of the product stems from C-6 of the substrate. Carbon atom 4 of the substrate is released as formate together with the hydrogen atom attached to it. The skeletal rearrangement which leads to the loss of C-4 and the direct linkage between C-3 and C-6 of the substrate is an intramolecular reaction, the hydrogen atom at C-3 of the enzyme product is introduced from solvent water [7]; <5> substrate binding structure involving a dimetal center, overview [2]; <5> the reaction in-

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volves an intramolecular skeletal rearrangement, NMR study [8]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] P formate + l-3,4-dihydroxy-2-butanone-4-phosphate (<4> product identification by GC-MS [7]; <4> product identification by NMR and CD spectroscopy [6]; <5> the enzyme converts ribulose 5-phosphate into 3,4-dihydroxy-2-butanone 4-phosphate, while its C4 atom is released as formate [2]) S Additional information <1, 3, 6> (<1> RibA is the rate limiting enzyme in an industrial riboflavin producing strain [11]; <3> the enzyme is involved in the riboflavin biosynthetic pathway, but has a second unrelated function in expression of mitochondrial respiration [9]; <1> RibA is a bifunctional enzyme possessing 3,4-dihydroxy-2-butanone 4-phosphate synthase activity located in the N-terminal half of the protein and GTP cyclohydrolase II activity, EC 3.5.4.25, of the C-terminal domain, overview [11]; <6> the position of the metal cofactors and the substrate’s phosphate group are further stabilized by an extensive hydrogen-bond and saltbridge network, overview [4]) (Reversibility: ?) [4, 9, 11] P ? Inhibitors EDTA <1, 4, 8> (<8> complete inhibition [14]; <4> complete inhibition, reversible by Mg2+ [6]) [6, 11, 14] Cofactors/prosthetic groups Additional information <8> (<8> no cofactor required [14]) [14] Metals, ions Ca2+ <5, 7> (<5> binding structure, overview [10]; <5> Ca2+ binding requires the binding of the substrate, binding involves Glu204, Glu97, and Asn207 [2]; <7> not involved in catalysis, stabilize the enzyme [12]) [2, 10, 12] Mg2+ <1, 2, 4, 5, 6, 7, 8> (<2, 8> required [5,14]; <7> essential for activity [12]; <2,4> required for activity [6,13,16]; <5> binding structure, overview [10]; <2> required, cannot be substituted by Ca2+ , KM : 0.99 mM [3]; <6> required, two Mg2+ cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules [4]) [3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 16] Mn2+ <6> (<6> binding structure, overview [4]) [4] sulfate <6> (<6> binding structure at the active site, overview [4]) [4] Zn2+ <5, 6, 7> (<5, 6> binding structure, overview [4, 10]; <5> bound at His164, binding involves His206, Glu132, and Glu128 [2]; <7> not involved in catalysis, stabilize the enzyme [12]) [2, 4, 10, 12] Additional information <5, 6> (<5> substrate binding structure involving a dimetal center, overview [2]; <5> the enzyme depends on divalent metal ions, a dimetal center is involved in substrate binding [10]; <6> the position of the metal cofactors and the substrate’s phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network, overview [4]) [2, 4, 10]

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Specific activity (U/mg) 0.152 <2> (<2> purified recombinant enzyme [5]) [5] 0.174 <5> (<5> purified enzyme [8]) [8] 0.283 <8> (<8> purified recombinant enzyme, assay method development, overview [14]) [14] 0.332 <7> (<7> purified recombinant enzyme [12]) [12] 14 <4> (<4> purified enzyme [6]) [6] Additional information <2> (<2> development of a spectrophotometric/colorimetric assay method, overview [3]) [3] pH-Optimum 7 <4> (<4> assay at [7]) [7] 7.5 <2, 4, 7, 8> (<2,4,7,8> assay at [3,6,12,14]) [3, 6, 12, 14] 7.5-7.7 <5> (<5> assay at [8]) [8] 7.8 <2> (<2> assay at [13]) [13] 8 <1> (<1> assay at [11]) [11] Temperature optimum ( C) 25 <2> (<2> assay at [3,13]) [3, 13] 37 <1, 4, 5, 7, 8> (<1,4,5,7,8> assay at [6,7,8,11,12,14]) [6, 7, 8, 11, 12, 14]

4 Enzyme Structure Molecular weight 30000 <4> (<4> gel filtration [6]) [6] 41000 <7> (<7> recombinant enzyme, analytical ultracentrifugation, hydrodynamic studies [12]) [12] 44800 <2> (<2> recombinant enzyme, analytical ultracentrifugation at 37 C [5]) [5] 46300 <2> (<2> recombinant enzyme, hydrodynamic analysis, NMR structure study, analytical ultracentrifugation at 4 C, overview [5]) [5] 47000 <2, 5> [15] 51600 <5> (<5> recombinant enzyme, analytical ultrafiltration [8]) [8] Subunits ? <2, 3, 8> (<2> x * 23177, mass spectrometry [3]; <8> x * 23251, recombinant enzyme, mass spectrometry [14]; <3> x * 28000, SDS-PAGE, x * 22500, about, sequence calculation [9]) [3, 9, 14] dimer <2, 5, 6, 7> (<7> 2 * 22530, mass spectrometry, 2 * 22658, full-length enzyme, sequence calculation [12]; <2> 2 * 23000, SDS-PAGE, hydrodynamic analysis, NMR structure study, 2 * 23351, mass spectrometry [5]; <5> 2 * 25799, recombinant enzyme, mass spectrometry and SDS-PAGE [8]; <6> determination of the active sites in the homodimer, crystal structure, overview [4]; <5> enzyme mutant H147S, crystal structure [2]; <2,5> three-dimensional structure, structure-activity relationship study using NMR spectroscopy, residue-specific isotope labeling, and protein deuteration strategies, solution structure, overview [15]) [2, 4, 5, 8, 12, 15]

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monomer <4> (<4> 1 * 24000, SDS-PAGE [6]) [6] Additional information <2, 5, 7> (<2> hydrodynamic analysis, NMR structure study, isotope labeling, the homodimeric protein obeys strict C2 symmetry, overview [5]; <2> structure determination and analysis of functions of single residues, overview, dimer interface structure, localization of the active site, sequence and structure comparison [16]; <5> structure-function relationship, overview [10]; <7> the enzyme possesses an essential acidic active-site loop, active site structure, structure comparisons, overview [12]) [5, 10, 12, 16]

5 Isolation/Preparation/Mutation/Application Localization cytosol <3> (<3> mainly [9]) [9] mitochondrion <3> (<3> in the intermembrane space, possibly associated with the outer membrane, minor enzyme portion [9]) [9] Additional information <3> (<3> subcellular localization analysis, overview [9]) [9] Purification <2> (recombinant enzyme from strain Bl21(DE3) by hydrophobic interaction and anion exchange chromatography, and ultrafiltration) [3] <2> (recombinant enzyme from strain M15 by anion exchange chromatography and gel filtration) [5] <4> (native enzyme to homogeneity, by anion and cation exchange chromatography, hydroxyapatite chromatography, and gel filtration) [6] <5> (recombinant wild-type and mutant enzymes from Escherichia coli by hydrophobic interaction and hydroxyapatite chromatography, and ultrafiltration) [8] <6> (recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange and hydroxylapatite chromatography, followed by ultrafiltration) [1] <7> (recombinant enzyme, expressed from the synthetic gene in Escherichia coli, to homogeneity by hydrophobic interaction chromatography, ultrafiltration, and gel filtration) [12] <8> (native enzyme by anion and cation exchange chromatography, hydroxyapatite chromatography, and gel filtration, to homogeneity, recombinant enzyme from the overexpressing strain by anion exchange chromatography and gel filtration) [14] Crystallization <2> (purified recombinant enzyme, hanging drop vapour diffusion method at room temperature, 4-5 days, 0.0022 ml of protein solution containing 24 mg/ml protein in 50 mM Tris-HCl pH 7.5, is mixed with 0.0007 ml of precipitating well solution containing 3 M CsCl, 3 M Cs-formate, 20 mM Bis-Trispropane-NaOH, pH 6.9, or 6 M sodium formate, 25 mM HEPES-NaOH, pH 7.0, labeling with 1.5 mM Au(CN)2 , X-ray diffraction structure determination

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and analysis at 1.4-2.4 A resolution, multiwavelength anomalous diffraction) [16] <5> (purified enzyme mutant H147S in complex with substrate ribulose 5phosphate, monoclinic crystal form, X-ray diffraction structure determination and analysis at 1.55-1.7 A resolution) [2] <6> (purified recombinant enzyme, crystallization of different enzyme complexes: E-SO24-, E-SO24- Mg2+ , E-SO24- Mn2+ , E-SO24- Mn2+ -glycerol, and ESO24- Zn2+ complexes with X-ray diffraction structure determination and analysis at resolutions that extend to 1.55 A, 0.98 A, 1.60 A, 1.16 A, and 1.00 A, respectively, divalent cation-free enzyme from 24-30% PEG 5000 monomethyl ether, 0.2 M Li2 SO4, and 0.1 M MES-NaOH, pH 6.0-6.5, by the hanging drop vapor diffusion method, to prepare divalent cation-containing crystals, 200 mM MgCl2 , 200 mM MnCl2 , or 200 mM zinc acetate are added to the crystals for 8-16 h in soaking solutions of the well solutions omitting Li2 SO4 and 4% higher in the concentration of PEG 5000 monomethyl ether) [4] <6> (purified recombinnat enzyme divalent cation free, soaked in Zn2+ or soaked in Mg2+ , hanging drop vapor diffusion method, room temperature, 0.001 ml of 7 mg/ml protein in 50 mM Tris-HCl, pH 7.5, is mixed with 0.001 ml well solution containing 24-30% PEG monomethyl ether, 0.2 M Li2 SO4, and 0.1 M MES-NaOH, pH 6.0-6.5, 1 week-3 months, rectangular plates, Xray diffraction structure determination and analysis at 1.5 A, 1.0 A, and 1.8 A resolution, respectively) [1] <7> (purified recombinant apoenzyme in complex with the substrate ribulose 5-phosphate, sitting drop vapour diffusion method, 0.003 ml of 17-34 mg/ml protein in 50 mM Tris-HCl, pH 7.5, is mixed with 0.001 ml of reservoir solution containing 85 mM sodium citrate, pH 5.0, and 17% PEG 8000, with or without 5 mM EDTA, equilibration against 0.3 ml reservoir solution, 0.003 ml of the complex solution is mixed with 0.001 ml of 90 mM Mes/NaOH, pH 6.0, containing 18% PEG 8000, addition of 2 mM d-ribulose 5-phosphate, 20 C, X-ray diffraction structure determination and analysis at 1.6-1.7 A resolution, molecular replacement, modelling) [12] Cloning <1> (overexpression in strain 1012 by gene insertion in the sacB locus) [11] <2> [15] <2> (gene ribB, DNA determination and analysis, overexpression in strain M15) [5] <2> (subcloning in strain DH5a, expression in strain BL21(DE3)) [3] <3> (gene RIB3, expression of the wild-type gene restores the ability to respire in the aE280/U1 pet mutant A137T of Saccharomyces cerevisiae, which is partially deficient in cytochromes a, a3 , and cytochrome b) [9] <5> (DNA and amino acid sequence determination and analysis, expression of wild-type and mutant enzymes in Escherichia coli strains XL-1 Blue and M15) [8] <5> (expression in Escherichia coli) [15]

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<6> (cloning by functional complementation of an Escherichia coli DS knockout mutant, expression in Escherichia coli strain BL21(DE3)) [1] <6> (expression in Escherichia coli) [4] <7> (functional expression of the synthetic gene in Escherichia coli strains XL1-Blue and M15) [12] <8> (gene ribB, overexpression in an rib-defective mutant strain) [14] Engineering A137T <3> (<3> naturally occuring Rib3 mutant which is partially deficient in cytochromes a, a3 , and cytochrome b, the respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1. growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin, 2. the levels of flavin nucleotides are not significantly different in the mutant and wild type, phenotype, overview [9]) [9] C59A <7> (<7> site-directed mutagenesis, the mutant shows 70% of wildtype enzyme activity [12]) [12] D21E <5> (<5> site-directed mutagenesis, inactive mutant [8]) [8] D21N <5> (<5> site-directed mutagenesis, inactive mutant [8]) [8] D30E <5> (<5> site-directed mutagenesis, inactive mutant [8]) [8] D92A <7> (<7> site-directed mutagenesis, inactive mutant [12]) [12] E166A <7> (<7> site-directed mutagenesis, inactive mutant [12]) [12] E185D <5> (<5> site-directed mutagenesis, inactive mutant [8]) [8] E185X <5> (<5> site-directed mutagenesis, inactive mutant [10]) [10] E26D <5> (<5> site-directed mutagenesis, inactive mutant [8]) [8] E26Q <5> (<5> site-directed mutagenesis, inactive mutant [8]) [8] E28D <5> (<5> site-directed mutagenesis, inactive mutant [8]) [8] H147S <5> (<5> site-directed mutagenesis, the mutant enzyme shows about 10% of the wild-type enzyme activity [10]; <5> site-directed mutagenesis, the mutant shows about 10% of wild-type enzyme activity, crystal structure determination with bound substrate [2]) [2, 10] H164N <5> (<5> site-directed mutagenesis, inactive mutant [8]) [8] Q181R/Q183R <7> (<7> site-directed mutagenesis, construction of a synthetic gene, derived from orf 6.2440, with nucleotide exchanges at positions 414, 426, 477, 480, and 581 [12]) [12] Y87A <7> (<7> site-directed mutagenesis, the mutant shows 2% of wild-type enzyme activity [12]) [12] Additional information <1, 3> (<1> an additional single copy of the ribA gene introduced into the sacBlocus of the riboflavin production strain and constitutive expression from the medium strength vegI promoter leads to improved riboflavin titers and yields of riboflavin on glucose of up to 25%, strain VB2XL1, both enzymatic activities of RibA, the 3,4-dihydroxy-2-butanone 4-phosphate synthase activity located in the N-terminal half of the protein and the GTP cyclohydrolase II activity of the C-terminal domain, are necessary for the improved riboflavin productivity, method, overview [11]; <3> construction of RIB3 disruption mutants, restoration by riboflavin of growth of a rib3 deletion mutant on glucose but not glycerol/ethanol, phenotype, overview [9]) [9, 11]

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Application drug development <2, 7> (<2> the enzyme is a target in antimicrobial inhibitor development, structure-based inhibitor design [3]; <2> the enzyme is an attractive target for antibiotics, design of mechanism-based inhibitors [15]; <7> the enzyme is a potential anti-infective target in the pathogenic yeast [12]) [3, 12, 15] synthesis <1> (<1> the enzyme is essential for industrial riboflavin production by Bacillus subtilis overproducing strains, overview [11]) [11]

References [1] Liao, D.I.; Viitanen, P.V.; Jordan, D.B.: Cloning, expression, purification and crystallization of dihydroxybutanone phosphate synthase from Magnaporthe grisea. Acta Crystallogr. Sect. D, 56, 1495-1497 (2000) [2] Steinbacher, S.; Schiffmann, S.; Bacher, A.; Fischer, M.: Metal sites in 3,4dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with the substrate ribulose 5-phosphate. Acta Crystallogr. Sect. D, 60, 1338-1340 (2004) [3] Picollelli, M.A.; Viitanen, P.V.; Jordan, D.B.: Spectrophotometric determination of 3,4-dihydroxy-2-butanone-4-phosphate synthase activity. Anal. Biochem., 287, 347-349 (2000) [4] Liao, D.I.; Zheng, Y.J.; Viitanen, P.V.; Jordan, D.B.: Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone-4phosphate synthase. Biochemistry, 41, 1795-1806 (2002) [5] Richter, G.; Kelly, M.; Krieger, C.; Yu, Y.; Bermel, W.; Karlsson, G.; Bacher, A.; Oschkinat, H.: NMR studies on the 46-kDa dimeric protein, 3,4-dihydroxy-2-butanone 4-phosphate synthase, using 2 H, 13 C, and 15 N-labelling. Eur. J. Biochem., 261, 57-65 (1999) [6] Volk, R.; Bacher, A.: Studies on the 4-carbon precursor in the biosynthesis of riboflavin. Purification and properties of l-3,4-dihydroxy-2-butanone-4phosphate synthase. J. Biol. Chem., 265, 19479-19485 (1990) [7] Volk, R.; Bacher, A.: Biosynthesis of riboflavin. Studies on the mechanism of l-3,4-dihydroxy-2-butanone 4-phosphate synthase. J. Biol. Chem., 266, 20610-20618 (1991) [8] Fischer, M.; Romisch, W.; Schiffmann, S.; Kelly, M.; Oschkinat, H.; Steinbacher, S.; Huber, R.; Eisenreich, W.; Richter, G.; Bacher, A.: Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase of Methanococcus jannaschii. J. Biol. Chem., 277, 41410-41416 (2002) [9] Jin, C.; Barrientos, A.; Tzagoloff, A.: Yeast dihydroxybutanone phosphate synthase, an enzyme of the riboflavin biosynthetic pathway, has a second unrelated function in expression of mitochondrial respiration. J. Biol. Chem., 278, 14698-14703 (2003) [10] Steinbacher, S.; Schiffmann, S.; Richter, G.; Huber, R.; Bacher, A.; Fischer, M.: Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the

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[11]

[12]

[13] [14] [15]

[16]

3,4-Dihydroxy-2-butanone-4-phosphate synthase

substrate ribulose 5-phosphate: implications for the catalytic mechanism. J. Biol. Chem., 278, 42256-42265 (2003) Huembelin, M.; Griesser, V.; Keller, T.; Schurter, W.; Haiker, M.; Hohmann, H.P.; Ritz, H.; Richter, G.; Bacher, A.; Van Loon, A.P.G.M.: GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production. J. Indust. Microbiol. Biotechnol., 22, 1-7 (1999) Echt, S.; Bauer, S.; Steinbacher, S.; Huber, R.; Bacher, A.; Fischer, M.: Potential anti-infective targets in pathogenic yeasts: structure and properties of 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans. J. Mol. Biol., 341, 1085-1096 (2004) Goetze, E.; Kis, K.; Eisenreich, W.; Yamauchi, N.; Kakinuma, K.; Bacher, A.: Biosynthesis of riboflavin. Stereochemistry of the 3,4-dihydroxy-2-butanone 4-phosphate synthase reaction. J. Org. Chem., 63, 6456-6457 (1998) Richter, G.; Krieger, C.; Volk, R.; Kis, K.; Ritz, H.; Gotze, E.; Bacher, A.: Biosynthesis of riboflavin: 3,4-dihydroxy-2-butanone-4-phosphate synthase. Methods Enzymol., 280, 374-382 (1997) Kelly, M.J.; Ball, L.J.; Krieger, C.; Yu, Y.; Fischer, M.; Schiffmann, S.; Schmieder, P.; Kuhne, R.; Bermel, W.; Bacher, A.; Richter, G.; Oschkinat, H.: The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4phosphate synthase and ligand binding studies reveal the location of the active site. Proc. Natl. Acad. Sci. USA, 98, 13025-13030 (2001) Liao, D.I.; Calabrese, J.C.; Wawrzak, Z.; Viitanen, P.V.; Jordan, D.B.: Crystal structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase of riboflavin biosynthesis. Structure, 9, 11-18 (2001)

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Cyclohexa-1,5-dienecarbonyl-CoA hydratase

4.2.1.100

1 Nomenclature EC number 4.2.1.100 Systematic name 6-hydroxycyclohex-1-enecarbonyl-CoA-hydrolyase (cyclohexa-1,5-dienecarbonyl-CoA-forming) Recommended name cyclohexa-1,5-dienecarbonyl-CoA hydratase Synonyms cyclohexa-1,5-diene-1-carboxyl-CoA hydratase EC 4.2.1.102 cyclohexa-1,5-diene-1-carbonyl-CoA hydratase dienoyl-CoA hydratase CAS registry number 214355-79-0

2 Source Organism <1> Azoarcus evansii (no sequence specified) [1, 2] <2> Thauera aromatica (no sequence specified) [1, 3, 4]

3 Reaction and Specificity Catalyzed reaction 6-hydroxycyclohex-1-ene-1-carbonyl-CoA = cyclohex-1,5-dienecarbonyl-CoA + H2 O Reaction type addition Natural substrates and products S cyclohexa-1,5-diene-1-carbonyl-CoA + H2 O <1, 2> (Reversibility: ?) [2, 4] P 6-hydroxycyclohex-1-enecarbonyl-CoA Substrates and products S cyclohex-1-ene-1-carbonyl-CoA + H2 O <2> (Reversibility: ?) [3] P 2-hydroxycyclohexane-1-carbonyl-CoA <2> [3] S cyclohexa-1,5-diene-1-carbonyl-CoA + H2 O <1, 2> (Reversibility: r) [2, 4] P 6-hydroxycyclohex-1-ene-1-carbonyl-CoA <1, 2> [2, 4]

80

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Cyclohexa-1,5-dienecarbonyl-CoA hydratase

S cyclohexa-1,5-diene-1-carbonyl-CoA + H2 O <1, 2> (Reversibility: ?) [2, 4] P 6-hydroxycyclohex-1-enecarbonyl-CoA S cyclohexa-2,5-diene-1-carbonyl-CoA + H2 O <2> (<2> only when dithionite is used as an artificial electron donor [3]) (Reversibility: ?) [3] P 6-hydroxycyclohex-2-ene-1-carbonyl-CoA <2> [3] Turnover number (min–1) 51 <2> (cyclohexa-1,5-diene-1-carbonyl-CoA) [4] Specific activity (U/mg) 110 <2> (<2> reversible reaction with 6-hydroxycyclohex-1-ene-1-carboxylCoA as substrat [3]) [3] Km-Value (mM) 60 <2> (cyclohexa-1,5-diene-1-carbonyl-CoA) [4] pH-Optimum 7.4-7.6 <2> [4]

4 Enzyme Structure Molecular weight 55000 <2> (<2> gel filtration [4]) [4] Subunits dimer <2> (<2> a2 , 2 * 28000, SDS-PAGE [4]) [4]

5 Isolation/Preparation/Mutation/Application Purification <2> (DEAE-Sepharose, hydroxyapatite, Mono-Q, Blue-Sepharose) [4]

References [1] Harwood, C.S.; Gibson, J.: Shedding light on anaerobic benzene ring degradation: a process unique to prokaryotes?. J. Bacteriol., 179, 301-309 (1997) [2] Koch, J.; Eisenreich, W.; Bacher, A.; Fuchs, G.: Products of enzymatic reduction of benzoyl-CoA, a key reaction in anaerobic aromatic metabolism. Eur. J. Biochem., 211, 649-661 (1993) [3] Boll, M.; Laempe, D.; Eisenreich, W.; Bachers, A.; Mittelberger, T.; Heinze, J.; Fuchs, G.: Nonaromatic products from anoxic conversion of benzoyl-CoA with benzoyl-CoA reductase and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase. J. Biol. Chem., 275, 21889-21895 (2000) [4] Lampe, D.; Eisenreich, W.; Bacher, A.; Fuchs, G.: Cyclohexa-1,5-diene-1-carboxyl-CoA hydratase, an enzyme involved in anaerobic metabolism of benzoyl-CoA in the denitrifying bacterium Thauera aromatica. Eur. J. Biochem., 255, 618-627 (1998)

81

trans-Feruloyl-CoA hydratase

4.2.1.101

1 Nomenclature EC number 4.2.1.101 Systematic name 4-hydroxy-3-methoxyphenyl-b-hydroxypropanoyl-CoA hydro-lyase (feroylCoA-forming) Recommended name trans-feruloyl-CoA hydratase Synonyms 4-hydroxycinnamoyl-CoA hydratase/lyase <1, 2> (<1> enzyme has hydratase and lyase activity [2]) [1, 2, 3, 4, 5] 4-hydroxycinnamoyl-coenzyme A hydratase-lyase <1> [6] FCHL <1> [6] HCHL <1> [5, 6] enoyl-CoA hydratase/aldolase <2> [3] feruloyl-CoA hydratase-lyase <1> [6] CAS registry number 197462-62-7

2 Source Organism <1> Pseudomonas fluorescens (no sequence specified) [1, 2, 4, 5, 6] <2> Pseudomonas sp. (no sequence specified) [3]

3 Reaction and Specificity Catalyzed reaction 4-hydroxy-3-methoxyphenyl-b-hydroxypropionyl-CoA = trans-feruloyl-CoA + H2 O Reaction type cleavage of a C-O bond Natural substrates and products S 4-caffeoyl-CoA <1> (<1> i.e. 3,4-dihydroxycinnamoyl-CoA [5]) (Reversibility: ir) [5] P 3,4-dihydroxybenzaldehyde + acetyl-CoA <1> (<1> i.e. protocatechuic aldehyde [5]) [5]

82

4.2.1.101

trans-Feruloyl-CoA hydratase

S 4-coumaroyl-CoA <1> (<1> i.e. 4-hydroxycinnamoyl-CoA [5]) (Reversibility: ir) [5] P 4-hydroxybenzaldehyde + acetyl-CoA <1> [5] S 4-feruloyl-CoA <1> (<1> i.e. 4-hydroxy-3-methoxyphenyl-b-hydroxypropionyl-CoA [5]) (Reversibility: ir) [5] P 4-hydroxy-3-methoxybenzaldehyde + acetyl-CoA <1> (<1> i.e. vanillin [5]) [5] S 4-hydroxy-3-methoxyphenyl-b-hydroxypropionyl-CoA <1, 2> (Reversibility: ?) [1, 2, 3, 4] P 4-hydroxy-3-methoxybenzaldehyde + acetyl-CoA <1, 2> [1, 2, 3, 4] S trans-feruloyl-CoA + H2 O <1, 2> (Reversibility: ?) [1, 2, 3, 4, 6] P 4-hydroxy-3-methoxyphenyl-b-hydroxypropionyl-CoA <1, 2> [1, 2, 3, 4] Substrates and products S 3,4-dihydroxycinnamoyl-CoA + H2 O <1> (Reversibility: ?) [2] P 3,4-dihydroxybenzaldehyde + acetyl-CoA <1> [2] S 4-caffeoyl-CoA <1> (<1> i.e. 3,4-dihydroxycinnamoyl-CoA [5]) (Reversibility: ir) [5] P 3,4-dihydroxybenzaldehyde + acetyl-CoA <1> (<1> i.e. protocatechuic aldehyde [5]) [5] S 4-coumaroyl-CoA <1> (<1> i.e. 4-hydroxycinnamoyl-CoA [5]) (Reversibility: ir) [5] P 4-hydroxybenzaldehyde + acetyl-CoA <1> [5] S 4-feruloyl-CoA <1> (<1> i.e. 4-hydroxy-3-methoxyphenyl-b-hydroxypropionyl-CoA [5]) (Reversibility: ir) [5] P 4-hydroxy-3-methoxybenzaldehyde + acetyl-CoA <1> (<1> i.e. vanillin [5]) [5] S 4-hydroxy-3-methoxyphenyl-b-hydroxypropionyl-CoA <1, 2> (Reversibility: ?) [1, 2, 3, 4] P 4-hydroxy-3-methoxybenzaldehyde + acetyl-CoA <1, 2> [1, 2, 3, 4] S 4-hydroxycinnamoyl-CoA + H2 O <1> (Reversibility: ?) [2] P 4-hydroxybenzaldehyde + acetyl-CoA <1> [2] S 4-trans-feruloyl-CoA <1> (<1> i.e. 4-hydroxy-3-methoxyphenyl-b-hydroxypropionyl-CoA [5]) (Reversibility: ?) [5] P 4-hydroxy-3-methoxybenzaldehyde + acetyl-CoA <1> (<1> i.e. vanillin [5]) [5] S trans-feruloyl-CoA + H2 O <1, 2> (Reversibility: ?) [1, 2, 3, 4, 6] P 4-hydroxy-3-methoxyphenyl-b-hydroxypropionyl-CoA <1, 2> [1, 2, 3, 4] Turnover number (min–1) 2.3 <1> (4-coumaroyl-CoA, <1> at 30 C [2]) [2] Specific activity (U/mg) 0.003 <1> [2] Additional information <1> (<1> enzyme activity in several transgenic cell lines from hairy roots of Datura stramonium [5]) [5]

83

trans-Feruloyl-CoA hydratase

4.2.1.101

Km-Value (mM) 1.6 <1> (caffeoyl-CoA) [2] 2.4 <1> (feruloyl-CoA) [2] 5.2 <1> (4-coumaroyl-CoA) [2] pH-Optimum 8.5 <1> (<1> assay at [5]) [5] 8.5-9.5 <1> (<1> in Tris-HCl or borate buffer, activity declined to ca. 50% of its maximum at pH 6.5 [2]) [2] Temperature optimum ( C) 30 <1> (<1> assay at [5]) [5]

4 Enzyme Structure Molecular weight 63000 <1> (<1> gel filtration [2]) [2] 186000 <1> (<1> calculated from crystallization data [6]) [6] Subunits ? <1> (<1> x * 31000, SDS-PAGE [5]) [5] dimer <1> (<1> a, a, 2 * 31000, SDS-PAGE, deduced from gene sequence [2,4]) [2, 4] hexamer <1> (<1> 6 * 31007, crystallization, calculated from amino acid sequence [6]) [6]

5 Isolation/Preparation/Mutation/Application Purification <1> [5] <1> (Mono-Q, Mono-P, Phenyl-Superose) [2] <1> (recombinant protein from Escherichia coli) [6] Crystallization <1> (by the hanging-drop method of vapour diffusion using polyethylene glycol 20000 Da as the precipitant, 1.8 A resolution) [6] Cloning <1> (expressed in Escherichia coli) [2] <1> (expression in Escherichia coli strain BL21(DE3)) [6] <1> (functional expression in hairy root cells of Datura stramonium via infection with Agrobacterium rhizogenes, no accumulation of 4-hydroxybenzaldehyde derivatives occurs, but diminished feruloyl-CoA availability, leading to reduced lignin formation, and formation of new glucoconjugates of 4hydroxybenzaldehyde derivatives, e.g. 4-hydroxybenzyl alcohol-O-b-d-glucoside) [5]

84

4.2.1.101

trans-Feruloyl-CoA hydratase

6 Stability Storage stability <1>, -70 C, several months [2]

References [1] Narbad, A.; Gasson, M.J.: Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly isolated strain of Pseudomonas fluorescens. Microbiology, 144, 1397-1405 (1998) [2] Mitra, A.; Kitamura, Y.; Gasson, M.J.; Narbad, A.; Parr, A.J.; Pyne, J.; Rhodes, M.J.C.; Sewter, C.; Walton, N.J.: 4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL)-an enzyme of phenylpropanoid chain cleavage from Pseudomonas. Arch. Biochem. Biophys., 365, 10-16 (1999) [3] Overhage, J.; Priefert, H.; Steinbuchel, A.: Biochemical and genetic analysis of ferulic acid catabolism in Pseudomonas sp. strain HR199. Appl. Environ. Microbiol., 65, 4837-4847 (1999) [4] Gasson, M.J.; Kitamura, Y.; McLauchlan, W.R.; Narbad, A.; Parr, A.J.; Parsons, E.L.; Payne, J.; Rhodes, M.J.; Walton, N.J.: Metabolism of ferulic acid to vanillin. A bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of a hydroxycinnamic acid SCoA thioester. J. Biol. Chem., 273, 4163-4170 (1998) [5] Mitra, A.; Mayer, M.J.; Mellon, F.A.; Michael, A.J.; Narbad, A.; Parr, A.J.; Waldron, K.W.; Walton, N.J.: 4-Hydroxycinnamoyl-CoA hydratase/lyase, an enzyme of phenylpropanoid cleavage from Pseudomonas, causes formation of C(6)-C(1) acid and alcohol glucose conjugates when expressed in hairy roots of Datura stramonium L.. Planta, 215, 79-89 (2002) [6] Leonard, P.M.; Marshall, C.M.; Dodson, E.J.; Walton, N.J.; Grogan, G.: Purification, crystallization and preliminary x-ray crystallographic analysis of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), a crotonase homologue active in phenylpropanoid metabolism. Acta Crystallogr. Sect. D, D60, 2343-2345 (2004)

85

Cyclohexa-1,5-dienecarbonyl-CoA hydratase

1 Nomenclature EC number 4.2.1.102 (transferred to EC 4.2.1.100) Recommended name cyclohexa-1,5-dienecarbonyl-CoA hydratase

86

4.2.1.102

Cyclohexyl-isocyanide hydratase

4.2.1.103

1 Nomenclature EC number 4.2.1.103 Systematic name N-cyclohexylformamide hydro-lyase (cyclohexyl-isocyanide-forming) Recommended name cyclohexyl-isocyanide hydratase Synonyms N-cyclohexylformamide hydro-lyase cyclohexyl isocyanide hydratase isocyanide hydratase isonitrile hydratase <2> [2] CAS registry number 358974-06-8

2 Source Organism <1> Pseudomonas putida (no sequence specified) [1] <2> Pseudomonas putida (UNIPROT accession number: Q8G9F9) [2]

3 Reaction and Specificity Catalyzed reaction N-cyclohexylformamide = cyclohexyl isocyanide + H2 O (<2> the enzyme from Pseudomonas putida strain N19-2 can also catalyse the hydration of other isonitriles to the corresponding N-substituted formamides, active site Cys101 and T102 are essential for activity, while Glu79 and Glu81 are not [2]) Reaction type addition Natural substrates and products S cyclohexyl isocyanide + H2 O <1> (<1> enzyme is involved in nitrogencarbon triple bond cleavage [1]) (Reversibility: ?) [1] P N-cyclohexylformamide

87

Cyclohexyl-isocyanide hydratase

4.2.1.103

Substrates and products S benzyl isocyanide <1> (<1> 224% of the activity with cyclohexyl isocyanide [1]) (Reversibility: ?) [1] P N-phenylformamide S cyclohexyl isocyanide + H2 O <1, 2> (<1> enzyme is involved in nitrogencarbon triple bond cleavage [1]) (Reversibility: ?) [1, 2] P N-cyclohexylformamide <1, 2> [1, 2] S isocyanomethyl phosphonic acid diethyl ether + H2 O <1> (<1> 3% of the activity with cyclohexyl isocyanide [1]) (Reversibility: ?) [1] P formylaminomethyl-phosphonic acid diethyl ester S Additional information <2> (<2> the enzyme can also catalyse the hydration of other isonitriles to the corresponding N-substituted formamides [2]) (Reversibility: ?) [2] P ? <2> [2] Inhibitors AgNO3 <1> (<1> 1 mM, 99% inhibition [1]) [1] CdCl2 <1> (<1> 1 mM, 87% inhibition [1]) [1] CoCl2 <1> (<1> 1 mM, 98% inhibition [1]) [1] CuSO4 <1> (<1> 1 mM, 98% inhibition [1]) [1] diethyldicarbamate <1> (<1> 1 mM, 20% inhibition [1]) [1] FeSO4 <1> (<1> 1 mM, 50% inhibition [1]) [1] H2 O2 <1> (<1> 1 mM, 49% inhibition [1]) [1] HgCl2 <1> (<1> 1 mM, 99% inhibition [1]) [1] hydroxylamine <1> (<1> 1 mM, 10% inhibition [1]) [1] iodoacetate <1> (<1> 1 mM, 97% inhibition [1]) [1] KCN <1> (<1> 1 mM, 26% inhibition [1]) [1] NEM <1> (<1> 1 mM,51% inhibition [1]) [1] NiSO4 <1> (<1> 1 mM, 98% inhibition [1]) [1] PCMB <1> (<1> 1 mM, 50% inhibition [1]) [1] phenylmethanesulfonyl fluoride <1> (<1> 1 mM, 57% inhibition [1]) [1] SnCl2 <1> (<1> 1 mM, 16% inhibition [1]) [1] ZnCl2 <1> (<1> 1 mM, 34% inhibition [1]) [1] ammonium persulfate <1> (<1> 1 mM, 23% inhibition [1]) [1] Metals, ions Additional information <2> (<2> no metal requirements [2]) [2] Specific activity (U/mg) 1.4 <2> (<2> purified recombinant mutant T102A [2]) [2] 15.4 <2> (<2> purified recombinant mutant E81Q [2]) [2] 17.3 <2> (<2> purified recombinant wild-type enzyme [2]) [2] 18.7 <2> (<2> purified recombinant mutant E79Q [2]) [2] Additional information <1> [1] Km-Value (mM) 16.2 <1> (cyclohexyl isocyanide) [1]

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Cyclohexyl-isocyanide hydratase

pH-Optimum 6-6.5 <1> [1] pH-Range 5.5-9 <1> (<1> pH 5.5: about 45% of maximal activity, potassium phosphate buffer, pH 9.0: about 30% of maximal activity, NH4 OH/NH4 Cl buffer [1]) [1] Temperature optimum ( C) 35 <1> [1] Temperature range ( C) 20-45 <1> (<1> 20 C: about 55% of maximal activity, 45 C: about 55% of maximal activity [1]) [1]

4 Enzyme Structure Subunits ? <2> (<2> x * 24211, amino acid sequence calculation [2]) [2] dimer <1> (<1> 2 * 59000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> [1] <2> (recombinant wild-type and mutants from Escherichia coli) [2] Cloning <2> (gene inhA, DNA and amino acid sequence determination and analysis, overexpression of wild-type and mutant enzyme in Escherichia coli) [2] Engineering C101A <2> (<2> site-directed mutagenesis, inactive mutant [2]) [2] E79Q <2> (<2> site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme [2]) [2] E81Q <2> (<2> site-directed mutagenesis, slightly decreased activity compared to the wild-type enzyme [2]) [2] T102A <2> (<2> site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme [2]) [2]

6 Stability Temperature stability 10 <1> (<1> 30 min, 20 <1> (<1> 30 min, 30 <1> (<1> 30 min, 35 <1> (<1> 30 min,

10% 10% 10% 10%

v/v v/v v/v v/v

glycerol, glycerol, glycerol, glycerol,

3% loss of activity [1]) [1] 11% loss of activity [1]) [1] 16% loss of activity [1]) [1] 22% loss of activity [1]) [1]

89

Cyclohexyl-isocyanide hydratase

40 45 50 55

<1> (<1> 30 <1> (<1> 30 <1> (<1> 30 <1> (<1> 30

min, min, min, min,

10% 10% 10% 10%

4.2.1.103

v/v v/v v/v v/v

glycerol, glycerol, glycerol, glycerol,

31% 47% 67% 81%

loss loss loss loss

of activity of activity of activity of activity

[1]) [1]) [1]) [1])

[1] [1] [1] [1]

References [1] Goda, M.; Hashimoto, Y.; Shimizu, S.; Kobayashi, M.: Discovery of a novel enzyme, isonitrile hydratase, involved in nitrogen-carbon triple bond cleavage. J. Biol. Chem., 276, 23480-23485 (2001) [2] Goda, M.; Hashimoto, Y.; Takase, M.; Herai, S.; Iwahara, Y.; Higashibata, H.; Kobayashi, M.: Isonitrile hydratase from Pseudomonas putida N19-2. Cloning, sequencing, gene expression, and identification of its active acid residue. J. Biol. Chem., 277, 45860-45865 (2002)

90

Cyanase

4.2.1.104

1 Nomenclature EC number 4.2.1.104 Systematic name carbamate hydro-lyase Recommended name cyanase Synonyms cyanase <1, 3, 5> [13, 14] cyanate aminohydrolase cyanate hydrolase cyanate lyase EC 3.5.5.3 EC 4.3.99.1 <1, 3, 5> (<1,3,5> formerly [13,14]) [13, 14] hydrolase, cyanate cyanate C-N-lyase cyanate hydratase CAS registry number 37289-24-0

2 Source Organism <1> Escherichia coli (no sequence specified) (<1> a-subunit of Fdh3 [10]; <1> alternatively spliced variant 4, i.e. Dhcr7-AS-4 [1,2]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14] <2> Chromobacterium violaceum (no sequence specified) [16] <3> Synechococcus sp. (no sequence specified) [13] <4> Pseudomonas pseudoalcaligenes (no sequence specified) [15] <5> Synechocystis sp. (no sequence specified) [13]

3 Reaction and Specificity Catalyzed reaction cyanate + HCO-3 + 2 H+ = NH3 + 2 CO2 (<1> mechanism [3]; <1> rapid equilibrium random mechanism [7,11]; <3> the enzyme requires bicarbonate as a cofactor. Its mechanism is to catalyse the attack of bicarbonate on cya-

91

Cyanase

4.2.1.104

nate, with elimination of carbon dioxide, thus catalysing hydration of the cyanate to carbamate. The carbamate spontaneously hydrolyses to ammonia and carbon dioxide [13]; <1> the enzyme requires bicarbonate as a cofactor. Its mechanism is to catalyse the attack of bicarbonate on cyanate, with elimination of carbon dioxide, thus catalysing hydration of the cyanate to carbamate. The carbamate spontaneously hydrolyses to ammonia and carbon dioxide, the decamer shows half-side binding of substrates and substrate analogues, catalytic mechanism, catalytic site involves Arg96, Glu99, and Ser122 [14]; <5> the enzyme requires bicarbonate as a cofactor. Its mechanism is to catalyse the attack of bicarbonate on cyanate, with elimination of carbon dioxide, thus catalysing hydration of the cyanate to carbamate. The carbamate spontaneously hydrolyses to ammonia and carbon dioxide [13]) cyanate + HCO-3 + H+ = carbamate + CO2 carbamate + H+ = NH3 + CO2 Reaction type carbon-nitrogen lyase reaction elimination of CO2 Natural substrates and products S cyanate + bicarbonate <1> (<1> enzyme could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea, alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way [4]; <1> breakdown of the inhibitory substance [1]; <1> cyanasedeficient strains have increased sensitivity to cyanate and are not able to use cyanate as nitrogen source [10]) (Reversibility: ?) [1, 4, 10] P ? S Additional information <4> (<4> activity is induced during growth with cyanide or cyanate, but not with ammonium or nitrate as the nitrogen source [15]) (Reversibility: ?) [15] P ? Substrates and products S cyanate + bicarbonate <1> (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12] P CO2 + carbamate <1> (<1> initial product is carbamate or a related, unstable compound and/or carbamate precursor, which subsequently decomposes to ammonia and bicarbonate [2]; <1> ammonia + bicarbonate [8,9]) [2, 7, 8, 9] S cyanate + bicarbonate <1> (<1> enzyme could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea, alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way [4]; <1> breakdown of the inhibitory substance [1]; <1> cyanasedeficient strains have increased sensitivity to cyanate and are not able to use cyanate as nitrogen source [10]) (Reversibility: ?) [1, 4, 10] P ?

92

4.2.1.104

Cyanase

S NCO- + HCO3- <1, 3, 5> (Reversibility: ?) [13, 14] P NH+4 + CO2 <1, 3, 5> [13, 14] S Additional information <4> (<4> activity is induced during growth with cyanide or cyanate, but not with ammonium or nitrate as the nitrogen source [15]) (Reversibility: ?) [15] P ? Inhibitors 2-oxoglutarate <1> [11] 3-nitropropionate <1> [11] acetate <1> (<1> competitive to bicarbonate [7]) [7] Br- <1> [7] chloride <1> [14] Cl- <1> [7] EDTA <1> [11] formate <1> [7] fumarate <1> [11] glutarate <1> [11] Hg2+ <1> [5] hydroxymalonate <1> [11] malate <1> (<1> d- and l-isomer [11]) [11] maleate <1> [11] malonate <1> (<1> reversible inhibition which can be prevented by saturating concentrations of cyanate or bicarbonate [7]) [7, 11] methyl methanethiosulfonate <1> [5] methylmalonate <1> [11] N-ethylmaleimide <1> [5] N3- <1> (<1> competitive to cyanate [7]) [7] NH+4 <3, 5> (<3, 5> negative regulation, represses enzyme expression [13]) [13] NO2- <1> [7] NO-3 <1> [7] oxalate <1> (<1> reversible inhibition which can be prevented by saturating concentrations of cyanate or bicarbonate [7]) [7, 11, 14] oxaloacetate <1> (<1> reversible inhibition which can be prevented by saturating concentrations of cyanate or bicarbonate [7]) [7, 11] S2 O23- <1> [11] SO23- <1> [11] SO24- <1> [11] succinate <1> [11] sulfoacetate <1> [11] tetranitromethane <1> [5] Additional information <1> (<1> NaF [1]; <1> not: KCN [1]; <1> in native cyanase the sulfhydryl group per se is not required for catalytic acivity, but it may play a role in stabilizing octameric structure, and that octameric structure is required for catalytic activity [5]; <1> free sulfhydryl groups are not required for catalytic activity, the catalytic activity may be dependent upon oligomeric structure [8]) [1, 5, 8]

93

Cyanase

4.2.1.104

Activating compounds cyanate <1> (<1> extracellular, enzyme induction [14]) [14] NtcA <3, 5> (<3,5> transcription factor, global nitrogen regulator of cyanobacteria, required for expression [13]) [13] bicarbonate <1, 3, 5> [13, 14] Specific activity (U/mg) 0.047 <3> (<3> complemented deficient Escherichia coli cells [13]) [13] Additional information <1> [1] Km-Value (mM) 0.6 <1> (cyanate) [2] 29 <1> (cyanate) [1] pH-Optimum 7 <1> [1] 7.4 <1> [2] Temperature optimum ( C) 37 <1> (<1> assay at [2,4]) [2, 4]

4 Enzyme Structure Molecular weight 150000 <1> (<1> Escherichia coli, sucrose density gradient centrifugation, gel filtration [2]) [2] 170000 <1> (<1> crystal structure [14]) [14] Subunits ? <3> (<3> x * 16362, amino acid sequence calculation [13]) [13] decamer <1> (<1> decameric structure required for activity [6]; <1> 10 * 17000, SDS-PAGE [14]) [6, 14] octamer <1> (<1> 8 * 17000, octameric structure required for activity [5]; <1> 8 * 17008, crystallographic data [9]) [5, 9] oligomer <1> (<1> x * 15200, SDS-PAGE [2]; <1> 8 or 10 * 16350 (4 or 5 disulfide linked dimers), amino acid sequence determination, [12]; <1> x * 14661, minimum molecular weight calculated from amino acid composition [2]) [2, 12] Additional information <1> (<1> the monomers are composed of 2 domains, subunit arrangement, decamer is formed by 5 dimers assembled into a pentamer, model [14]) [14]

5 Isolation/Preparation/Mutation/Application Purification <1> (B) [1, 2] <1> (partial) [1]

94

4.2.1.104

Cyanase

Renaturation <1> (more than 85% renaturation after urea denaturation) [8] Crystallization <1> [9] <1> (selenomethionine-labeled purified recombinant enzyme, also crystals of enzyme with complexed inhibitors oxalate and chloride, 2 mM dithiothreitol, sitting drop vapour diffusion method, from 2.1 M ammonium sulfate, 50 mM Na2 KPO4, pH 7.3, microseeding with large sitting drops at 18 C with wildtype crystals, 5-7 days, X-ray diffraction structure determinzation and analysis at 1.65 A resolution by multiwavelength anomalous diffraction MAD method) [14] Cloning <1> [10] <3> (gene cynS, DNA and amino acid sequence determination and analysis, tightly clustered with 2 genes located upstream, which encode proteins similar to the subunits of nitrate-nitrite transporter, complementation of deficient Escherichia coli strain) [13] <5> (gene cynS, tightly clustered with 4 putative molybdenum cofactor biosynthesis genes located downstream) [13] Application environmental protection <2> (<2> potential biotechnological application in environmental detoxification [16]) [16]

6 Stability Temperature stability 100 <1> (<1> 1 min, complete loss of activity [1]) [1] Storage stability <1>, -20 C, stable for weeks [1]

References [1] Taussig, A.: The synthesis of the induced enzyme cyanase in E. coli. Biochim. Biophys. Acta, 44, 510-519 (1960) [2] Anderson, P.M.: Purification and properties of the inducible enzyme cyanase. Biochemistry, 19, 2882-2888 (1980) [3] Johnson, W.V.; Anderson, P.M.: Bicarbonate is a recycling substrate for cyanase. J. Biol. Chem., 262, 9021-9025 (1987) [4] Guilloton, M.; Karst, F.: Isolation and characterization of Escherichia coli mutants lacking inducible cyanase. J. Gen. Microbiol., 133, 645-653 (1987) [5] Anderson, P.M.; Johnson, W.V.; Korte, J.J.; Xiong, X.; Sung, Y.C.; Fuchs, J.A.: Reversible dissociation of active octamer of cyanase to inactive dimer pro-

95

Cyanase

[6]

[7] [8] [9] [10] [11] [12] [13]

[14]

[15]

[16]

96

4.2.1.104

moted by alteration of the sulfhydryl group. J. Biol. Chem., 263, 5674-5680 (1988) Anderson, P.M.; Korte, J.J.; Holcomb, T.A.; Cho, Y.; Son, C.; Sung, Y.: Formation of intersubunit disulfide bonds and properties of the single histidine and cysteine residues in each subunit relative to the decameric structure of cyanase. J. Biol. Chem., 269, 15036-15045 (1994) Anderson, P.M.; Little, R.M.: Kinetic properties of cyanase. Biochemistry, 25, 1621-1626 (1986) Little, R.M.; Anderson, P.M.: Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity. J. Biol. Chem., 262, 10120-10126 (1987) Kim, K.H.; Honzatko, R.B.; Little, R.M.; Anderson, P.M.: Preliminary X-ray crystallographic study of cyanase from Escherichia coli. J. Mol. Biol., 198, 137-138 (1987) Sung, Y.C.; Parsell, D.; Anderson, P.M.; Fuchs, J.A.: Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12. J. Bacteriol., 169, 2639-2642 (1987) Anderson, P.M.; Johnson, W.V.; Endrizzi, J.A.; Little, R.M.; Korte, J.J.: Interaction of mono- and dianions with cyanase: evidence for apparent half-site binding. Biochemistry, 26, 3938-3943 (1987) Chin, C.C.Q.; Anderson, P.M.; Wold, F.: The amino acid sequence of Escherichia coli cyanase. J. Biol. Chem., 258, 276-282 (1983) Harano, Y.; Suzuki, I.; Maeda, S.; Kaneko, T.; Tabata, S.; Omata, T.: Identification and nitrogen regulation of the cyanase gene from the cyanobacteria Synechocystis sp. strain PCC 6803 and Synechococcus sp. strain PCC 7942. J. Bacteriol., 179, 5744-5750 (1997) Walsh, M.A.; Otwinowski, Z.; Perrakis, A.; Anderson, P.M.; Joachimiak, A.: Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site. Structure Fold Des., 8, 505-514 (2000) Luque-Almagro, V.M.; Huertas, M.J.; Martinez-Luque, M.; Moreno-Vivian, C.; Roldan, M.D.; Garcia-Gil, L.J.; Castillo, F.; Blasco, R.: Bacterial degradation of cyanide and its metal complexes under alkaline conditions. Appl. Environ. Microbiol., 71, 940-947 (2005) Carepo, M.S.; Azevedo, J.S.; Porto, J.I.; Bentes-Sousa, A.R.; Batista Jda, S.; Silva, A.L.; Schneider, M.P.: Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification. Genet. Mol. Res., 3, 181-194 (2004)

2-Hydroxyisoflavanone dehydratase

4.2.1.105

1 Nomenclature EC number 4.2.1.105 Systematic name 2,7,4’-trihydroxyisoflavanone hydro-lyase (daidzein-forming) Recommended name 2-hydroxyisoflavanone dehydratase Synonyms 2,7,4’-trihydroxyisoflavanone dehydratase <1> [1] CAS registry number 166800-10-8

2 Source Organism <1> Pueraria lobata (no sequence specified) [1] <2> Glycyrrhiza echinata (UNIPROT accession number: Q5NUF4) (<2> isoenzyme IA [2]) [2] <3> Glycine max (UNIPROT accession number: Q5NUF3) (<3> isoenzyme IA [2]) [2]

3 Reaction and Specificity Catalyzed reaction 2,7,4’-trihydroxyisoflavanone = daidzein + H2 O Substrates and products S 2,5,7,4’-tetrahydroxyisoflavanone <2, 3> (<2> activity is 0.5% of the activity with 2,7-dihydroxy-4-methoxyisoflavanone [2]) (Reversibility: ?) [2] P genistein + H2 O S 2,7,4’-trihydroxyisoflavanone <1, 2, 3> (<2> activity is 0.5% of the activity with 2,7-dihydroxy-4-methoxyisoflavanone [2]; <3> activity is 40% of the activity with 2,5,7,4-tetrahydroxyisoflavanone [2]) (Reversibility: ?) [1, 2] P daidzein + H2 O S 2,7-dihydroxy-4’-methoxyisoflavanone <2, 3> (<3> activity is 15% of the activity with 2,5,7,4-tetrahydroxyisoflavanone [2]) (Reversibility: ?) [2]

97

2-Hydroxyisoflavanone dehydratase

4.2.1.105

P formononetin + H2 O S p-nitrophenyl butyrate <3> (<3> activity is 0.6% of the activity with 2,5,7,4-tetrahydroxyisoflavanone [2]) (Reversibility: ?) [2] P ? Inhibitors 2,3-butadione <1> (<1> 0.1 mM, complete inhibition [1]) [1] diethyldicarbonate <1> (<1> 0.5 mM, 69% inhibition [1]) [1] iodoacetamide <1> (<1> 50 mM, 56% inhibition [1]) [1] iodoacetic acid <1> (<1> 50 mM, 43% inhibition [1]) [1] NEM <1> (<1> 1.0 mM, 71% inhibition [1]) [1] PCMB <1> (<1> 0.01 mM, 91% inhibition [1]) [1] Cofactors/prosthetic groups Additional information <1> (<1> no cofactor required [1]) [1] Turnover number (min–1) 0.12 <2> (2,7,4’-trihydroxyisoflavanone, <2> pH 7.5, 30 C [2]) [2] 0.19 <2> (2,5,7,4’-tetrahydroxyisoflavanone, <2> pH 7.5, 30 C [2]) [2] 1.6 <3> (2,7-dihydroxy-4’-methoxyisoflavanone, <3> pH 7.5, 30 C [2]) [2] 5.3 <3> (2,7,4’-trihydroxyisoflavanone, <3> pH 7.5, 30 C [2]) [2] 9.8 <2> (2,7-dihydroxy-4’-methoxyisoflavanone, <2> pH 7.5, 30 C [2]) [2] 18.1 <3> (2,5,7,4’-tetrahydroxyisoflavanone, <3> pH 7.5, 30 C [2]) [2] Specific activity (U/mg) 3.408 <1> [1] Km-Value (mM) 0.029 <3> (2,7-dihydroxy-4’-methoxyisoflavanone, <3> pH 7.5, 30 C [2]) [2] 0.058 <2> (2,7-dihydroxy-4’-methoxyisoflavanone, <2> pH 7.5, 30 C [2]) [2] 0.114 <3> (2,7,4’-trihydroxyisoflavanone, <3> pH 7.5, 30 C [2]) [2] 0.17 <3> (2,5,7,4’-tetrahydroxyisoflavanone, <3> pH 7.5, 30 C [2]) [2] 0.21 <2> (2,7,4’-trihydroxyisoflavanone, <2> pH 7.5, 30 C [2]) [2] 0.304 <2> (2,5,7,4’-tetrahydroxyisoflavanone, <2> pH 7.5, 30 C [2]) [2] 7 <1> (2,7,4’-trihydroxyisoflavanone) [1] pH-Optimum 6.8 <1> [1] pH-Range 6-7.9 <1> (<1> half-maximal activity at pH 6.0 and pH 7.9 in potassium phosphate buffer [1]) [1]

4 Enzyme Structure Molecular weight 35000 <1> (<1> gel filtration [1]) [1] Subunits monomer <1> (<1> 1 * 38000, SDS-PAGE [1]) [1]

98

4.2.1.105

2-Hydroxyisoflavanone dehydratase

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <1> [1] Localization microsome <1> [1] Purification <1> [1] Cloning <2> [2] <3> [2]

References [1] Hakamatsuka, T.; Mori, K.; Ishida, S.; Ebizuka, Y.; Sankawa, U.: Purification of 2-hydroxyisoflavanone dehydratase from the cell cultures of Pueraria lobata. Phytochemistry, 49, 497-505 (1998) [2] Akashi, T.; Aoki, T.; Ayabe, S.: Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis. Plant Physiol., 137, 882-891 (2005)

99

Bile-acid 7a-dehydratase

4.2.1.106

1 Nomenclature EC number 4.2.1.106 Systematic name 7a,12a-dihydroxy-3-oxochol-4-enoate hydro-lyase (12a-hydroxy-3-oxochola4,6-dienoate-forming) Recommended name bile-acid 7a-dehydratase CAS registry number 85130-33-2

2 Source Organism <1> Eubacterium sp. (no sequence specified) (<1> a-subunit OST1 [3,4]) [3, 4] <2> Clostridium sp. (no sequence specified) [1] <3> Clostridium hiranonis (no sequence specified) [2]

3 Reaction and Specificity Catalyzed reaction 7a,12a-dihydroxy-3-oxochol-4-enoate = 12a-hydroxy-3-oxochola-4,6-dienoate + H2 O Natural substrates and products S 7a,12a-dihydroxy-3-oxochol-4-enoate <1, 2, 3> (Reversibility: ?) [1, 2, 3, 4] P 12a-hydroxy-3-oxocholate-4,6-dienoate Substrates and products S 7a,12a-dihydroxy-3-oxochol-4-enoate <1, 2, 3> (Reversibility: ?) [1, 2, 3, 4] P 12a-hydroxy-3-oxocholate-4,6-dienoate S 7a-hydroxy-3-oxochol-4-enoate <1> (<1> 84% of activity with 7a,12adihydroxy-3-oxochol-4-enoate [4]) (Reversibility: ?) [4] P 3-oxocholate-4,6-dienoate Km-Value (mM) 0.16 <1> (7a,12a-dihydroxy-3-oxochol-4-enoate, <1> pH 7.5 [4]) [4]

100

4.2.1.106

Bile-acid 7a-dehydratase

4 Enzyme Structure Molecular weight 36000 <1> (<1> gel filtration [4]) [4] 114000 <1> (<1> gel filtration [3]) [3] Subunits dimer <1> (<1> 2 * 23000, SDS-PAGE [4]; <1> 2 * 19500, deduced from nucleotide sequence [4]) [4]

5 Isolation/Preparation/Mutation/Application Purification <1> (DE-52, Phenyl-Sepharose, HPLC-DEAE, gel filtration) [4] Cloning <1> (expression in Escherichia coli) [4] <2> (cloning of bai operon containing BaiE gene coding for bile acid 7adehydratase) [1]

References [1] Wells, J.E.; Hylemon, P.B.: Identification and characterization of a bile acid 7a-dehydroxylation operon in Clostridium sp. strain TO-931, a highly active 7a-dehydroxylating strain isolated from human feces. Appl. Environ. Microbiol., 66, 1107-1113 (2000) [2] Kitahara, M.; Takamine, F.; Imamura, T.; Benno, Y.: Clostridium hiranonis sp. nov., a human intestinal bacterium with bile acid 7a-dehydroxylating activity. Int. J. System. Evol. Microbiol., 51, 39-44 (2001) [3] Coleman, J.P.; White, W.B.; Egestad, B.; Sjoevall, J.; Hylemon, P.B.: Biosynthesis of a novel bile acid nucleotide and mechanism of 7a-dehydroxylation by an intestinal Eubacterium species. J. Biol. Chem., 262, 4701-4707 (1987) [4] Dawson, J.A.; Mallonee, D.H.; Bjorkhem, I.; Hylemon, P.B.: Expression and characterization of a C24 bile acid 7a-dehydratase from Eubacterium sp. strain VPI 12708 in Escherichia coli. J. Lipid Res., 37, 1258-1267 (1996)

101

3a,7a,12a-Trihydroxy-5b-cholest-24-enoylCoA hydratase

4.2.1.107

1 Nomenclature EC number 4.2.1.107 Systematic name (24R,25R)-3a,7a,12a,24-tetrahydroxy-5b-cholestanoyl-CoA hydro-lyase [(24E)-3a,7a12a-trihydroxy-5b-cholest-24-enoyl-CoA-forming] Recommended name 3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA hydratase Synonyms 46 kDa hydratase <2> [1]

2 Source Organism <1> Homo sapiens (no sequence specified) [3] <2> Rattus norvegicus (no sequence specified) [1, 2, 4, 5]

3 Reaction and Specificity Catalyzed reaction (24R,25R)-3a,7a,12a,24-tetrahydroxy-5b-cholestanoyl-CoA = (24E)3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA + H2 O Reaction type elimination Natural substrates and products S (24E)-3a,7a,12a-trihydroxy-5b-cholest-24-en-26-oyl-CoA + H2 O <1> (<1> reaction in cholic acid synthesis is catalyzed by peroxisomal multifunctional enzyme type 2. Human perMFE-2 is induced by transcription factors AP2a and AP2g and attenuated by Sp1 binding to recognition sequence located within and -151/-142 region [3]) (Reversibility: ?) [3] P (24R,25R)-3a,7a,12a,24-tetrahydroxy-5b-cholestan-26-oyl-CoA Substrates and products S (24E)-3a,7a,12a-trihydroxy-5b-cholest-24-en-26-oyl-CoA + H2 O <1, 2> (<1> reaction in cholic acid synthesis is catalyzed by peroxisomal multifunctional enzyme type 2. Human perMFE-2 is induced by transcription

102

4.2.1.107

P S P S P

3a,7a,12a-Trihydroxy-5b-cholest-24-enoyl-CoA hydratase

factors AP2a and AP2g and attenuated by Sp1 binding to recognition sequence located within and -151/-142 region [3]) (Reversibility: ?) [1, 3] (24R,25R)-3a,7a,12a,24-tetrahydroxy-5b-cholestan-26-oyl-CoA (24E)-3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA + H2 O <2> (Reversibility: ?) [2] (24R,25R)-3a,7a,12a,24-tetrahydroxy-5b-cholestanoyl-CoA 3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA + H2 O <2> (Reversibility: ?) [4, 5] 3a,7a,12a,24-tetrahydroxy-5b-cholestanoyl-CoA

5 Isolation/Preparation/Mutation/Application Source/tissue liver <2> [1, 2, 4, 5] Localization mitochondrion <2> [5] peroxisome <1, 2> [1, 2, 3]

References [1] Kurosawa, T.; Sato, M.; Inoue, K.; Yoshimura, T.; Tohma, M.; Jiang, L.L.; Hashimoto, T.: Separation of stereoisomers of C27-bile acid CoA esters by liquid chromatography and its application to the study of the stereospecificities of d- and l-bifunctional protein in bile acid biosynthesis. Anal. Chim. Acta, 365, 249-257 (1998) [2] Xu, R.; Cuebas, D.A.: The reactions catalyzed by the inducible bifunctional enzyme of rat liver peroxisomes cannot lead to the formation of bile acids. Biochem. Biophys. Res. Commun., 221, 271-278 (1996) [3] Novikov, D.K.; Kamps, M.E.: Characterization of the promoter region of the human peroxisomal multifunctional enzyme type 2 gene. Biochem. Biophys. Res. Commun., 284, 226-231 (2001) [4] Kinoshita, T.; Miyata, M.; Ismail, S.M.; Fujimoto, Y.; Kakinuma, K.; Ikekawa, N.; Morisaki, M.: Synthesis and determination of stereochemistry of four diastereoisomers at the C-24 and C-25 position of 3a,7a,12a,24-tetrahydroxy-5b-cholestan-26-oic acid. Chem. Pharm. Bull., 36, 134-141 (1988) [5] Fujimoto, Y.; Kinoshita, T.M Oya, I.; Kakinuma, K.; Ikekawa, N.; Sonoda, Y.; Sato, Y.; Morisaki, M.: Non-stereoselective conversion of the four diastereomers at the C-24 and c-25 positions of 3a,7a,12a,24-tetrahydroxy-5b-cholestan-26-oic acid into cholic acid. Chem. Pharm. Bull., 36, 142-145 (1988)

103

Ectoine synthase

4.2.1.108

1 Nomenclature EC number 4.2.1.108 Systematic name N4 -acetyl-l-2,4-diaminobutanoate hydro-lyase (l-ectoine-forming) Recommended name ectoine synthase Synonyms EctC <18> [4] CAS registry number 130457-09-9

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11> <12> <13> <14> <15> <16> <17> <18>

no activity in Bacillus subtilis [3] Bacillus alcalophilus (no sequence specified) [3] no activity in Nicotiana tabacum [6] Ectothiorhodospira halochloris [1] no activity in Bacillus cereus [3] no activity in Bacillus megaterium [3] no activity in Bacillus licheniformis [3] no activity in Bacillus circulans [3] Halomonas elongata (no sequence specified) [1,2,6] Sporosarcina psychrophilus (no sequence specified) [3] Bacillus pasteurii (UNIPROT accession number: Q9AP33) [3] Salibacillus salexigens (no sequence specified) [3] Virgibacillus pantothenticus (no sequence specified) [3] no activity in Bacillus thuringiensis [3] no activity in Aneuribacillus aneurinilyticus [3] no activity in Paenibacillus polymyxa [3] Marinococcus halophilus (UNIPROT accession number: O06061) [5] Methylomicrobium alcaliphilum (UNIPROT accession number: Q4JQJ3) [4] <19> Chromohalobacter salexigens (UNIPROT accession number: Q9ZEU6) [3]

104

4.2.1.108

Ectoine synthase

3 Reaction and Specificity Catalyzed reaction N4 -acetyl-l-2,4-diaminobutanoate = l-ectoine + H2 O Natural substrates and products S N4 -acetyl-l-2,4-diaminobutanoate <2, 4, 9, 10, 11, 12, 13, 17, 18, 19> (<2, 10, 11, 12, 13, 19> final step in l-ectoine biosynthesis, l-ectoine acts as an osmoprotectant [3]; <9> final step in l-ectoine biosynthesis, l-ectoine is the compatible solute in the cell [6]; <4,9,17,18> final step in l-ectoine biosynthesis, pathway overview [1, 2, 4, 5]; <9> final step in l-ectoine biosynthesis, pathway overview, l-ectoine is the main compatible solute in the organism with a cytoplasmic concentration of 1-2 M [1]) (Reversibility: r) [1, 2, 3, 4, 5, 6] P l-ectoine + H2 O S Additional information <2, 10, 11, 12, 13, 17, 19> (<2, 10, 11, 12, 13, 19> phylogenetic analysis [3]; <17> the enzyme is osmoregulated in response to medium salinity allowing the organism to live in environments with high concentration of osmolytes [5]) (Reversibility: ?) [3, 5] P ? Substrates and products S N4 -acetyl-l-2,4-diaminobutanoate <2, 4, 9, 10, 11, 12, 13, 17, 18, 19> (<2, 10, 11, 12, 13, 19> final step in l-ectoine biosynthesis, l-ectoine acts as an osmoprotectant [3]; <9> final step in l-ectoine biosynthesis, l-ectoine is the compatible solute in the cell [6]; <4, 9, 17, 18> final step in l-ectoine biosynthesis, pathway overview [1, 2, 4, 5]; <9> final step in l-ectoine biosynthesis, pathway overview, l-ectoine is the main compatible solute in the organism with a cytoplasmic concentration of 1-2 M [1]; <2, 4, 9, 10, 11, 12, 13, 17, 18, 19> i.e. 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid [1, 3, 4, 5, 6]; <9> i.e. 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid, circularization, l-ectoine as an effective osmoprotectant, the enzyme shows absolute substrate specificity, reversibility of the reaction is not detected but might be below detection range [2]) (Reversibility: r) [1, 2, 3, 4, 5, 6] P l-ectoine + H2 O (<9> NMR and thin layer chromatography product identification [1]; <4> NMR and TLC product identification [1]; <10, 11> NMR identification [3]; <2, 12, 13, 18, 19> NMR product identification [3, 4]) S Additional information <2, 10, 11, 12, 13, 17, 19> (<2, 10, 11, 12, 13, 19> phylogenetic analysis [3]; <17> the enzyme is osmoregulated in response to medium salinity allowing the organism to live in environments with high concentration of osmolytes [5]) (Reversibility: ?) [3, 5] P ? Activating compounds Additional information <2, 10, 11, 12, 13, 17, 18, 19> (<18> enzyme expression is 12fold induced by NaCl at 1 M [4]; <2, 10, 11, 12, 13, 19> high osmo-

105

Ectoine synthase

4.2.1.108

larity induces the enzyme expression, requirements [3]; <17> high salinity/ osmolarity of the medium induces enzyme expression [5]) [3, 4, 5] Metals, ions NaCl <9, 11, 18> (<11,18> required for activity [3,4]; <9> required for activity, maximal activity at 0.5 M [2]) [2, 3, 4] Additional information <9> (<9> the enzyme is not affected by Mg2+ , Ca2+ , Mn2+ , Li+ , K+ , and NH+4 [2]) [2] Specific activity (U/mg) 16 <9> (<9> purified enzyme [2]) [2] Km-Value (mM) 8.4 <9> (N4 -acetyl-l-2,4-diaminobutanoate, <9> pH 9.5, 15 C, in presence of 0.77 M NaCl [2]) [2] 11 <9> (N4 -acetyl-l-2,4-diaminobutanoate, <9> pH 9.5, 15 C, in presence of 0.05 M NaCl [2]) [2] pH-Optimum 8.5 <18> (<18> assay at [4]) [4] 8.5-9 <9> [2] 9 <4, 9> (<4,9> assay at [1]) [1] Temperature optimum ( C) 0-10 <9> (<9> in presence of 0.05 M NaCl [2]) [2] 15 <9> (<9> in presence of 0.77 M NaCl [2]) [2] 20 <18> (<18> assay at [4]) [4] 30 <9> (<9> in presence of 3.0 M NaCl [2]) [2] 37 <4, 9> (<4,9> assay at [1]) [1] Temperature range ( C) 0-30 <9> (<9> the temperature optimum shifts from 0 C to 30 C with increasing NaCl concentration [2]) [2]

4 Enzyme Structure Molecular weight 35000 <9> (<9> gel filtration in presence of 2 M NaCl [2]) [2] Subunits ? <9> (<9> x * 19000, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <4> (<4> photosynthetically grown cells [1]) [1] culture condition:glucose-grown cell <9> [1]

106

4.2.1.108

Ectoine synthase

Additional information <18> (<18> cells are grown in an atmosphere of airmethane 1:1 in 1 M NaCl [4]) [4] Purification <9> (native enzyme by ammonium sulfate fractionation, hydrophobic interaction chromatography, ultrafiltration, and hydroxylapatite chromatography, in presence of 1 mM l-2,4-diaminobutanoate and over 2 M NaCl to homogeneity) [2] Cloning <9> (functional expression in transgenic Nicotiana tabaccum BY2 cells using the Agrobacterium tumefaciens infection system) [6] <11> (gene ectC, encoded in the ectABC gene cluster, sequence determination and analysis, phylogenetic analysis, DNA sequence comparison, expression in Escherichia coli) [3] <17> (gene ectC, DNA and amino acid sequence determination and analysis, genetic mapping and organization, functional expression in Escherichia coli, the recombinant enzyme expression is osmoregulated in response to medium salinity in Escherichia coli) [5] <18> (gene ectC located in the singel operon ectABCask, DNA sequence determination and analysis, genetic organization, expression in Escherichia coli strain X1-Blue) [4] <19> (gene ectC encoded in the ectABC gene cluster, functional expression in Escherichia coli) [3] Engineering Additional information <9> (<9> expression in transgenic Nicotiana tabacum BY2 cells increases the osmotolerance and growth rate of the cells under hyperosmotic conditions [6]) [6] Application biotechnology <17> (<17> inducible recombinant enzyme expression in Escherichia coli confers the capability to grow on high concentration of osmolytes by increasing the osmotolerance via l-ectoine production [5]) [5]

6 Stability Temperature stability 30 <9> (<9> the enzyme is quite stable below 30 C in presence of 2 M NaCl [2]) [2] General stability information <9>, NaCl stabilizes the enzyme at above 2 M [2]

107

Ectoine synthase

4.2.1.108

References [1] Peters, P.; Galinski, E.A.; Trper, H.G.: The biosynthesis of ectoine. FEMS Microbiol. Lett., 71, 157-162 (1990) [2] Ono, H.; Sawada, K.; Khunajakr, N.; Tao, T.; Yamamoto, M.; Hiramoto, M.; Shinmyo, A.; Takano, M.; Murooka, Y.: Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. J. Bacteriol., 181, 91-99 (1999) [3] Kuhlmann, A.U.; Bremer, E.: Osmotically regulated synthesis of the compatible solute ectoine in Bacillus pasteurii and related Bacillus spp.. Appl. Environ. Microbiol., 68, 772-783 (2002) [4] Reshetnikov, A.S.; Khmelenina, V.N.; Trotsenko, Y.A.: Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z“. Arch. Microbiol., 184, 286-297 (2006) [5] Louis, P.; Galinski, E.A.: Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology, 143 (Pt 4), 1141-1149 (1997) [6] Nakayama, H.; Yoshida, K.; Ono, H.; Murooka, Y.; Shinmyo, A.: Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. Plant Physiol., 122, 1239-1247 (2000)

108

Methylthioribulose 1-phosphate dehydratase

4.2.1.109

1 Nomenclature EC number 4.2.1.109 Systematic name S-methyl-5-thio-d-ribulose-1-phosphate 4-hydro-lyase [5-(methylthio)-2,3dioxopentyl-phosphate-forming] Recommended name methylthioribulose 1-phosphate dehydratase Synonyms 1-PMT-ribulose dehydratase <2> [1]

2 Source Organism <1> Rattus norvegicus (no sequence specified) [2] <2> Klebsiella pneumoniae (no sequence specified) [1, 2]

3 Reaction and Specificity Catalyzed reaction S-methyl-5-thio-d-ribulose 1-phosphate = 5-(methylthio)-2,3-dioxopentyl phosphate + H2 O Natural substrates and products S S-methyl-5-thio-d-ribulose 1-phosphate <1, 2> (<1,2> the enzyme forms part of the methionine-salvage pathway [2]) (Reversibility: ?) [2] P 5-(methylthio)-2,3-dioxopentyl phosphate + H2 O Substrates and products S S-methyl-5-thio-d-ribulose 1-phosphate <1, 2> (<1,2> the enzyme forms part of the methionine-salvage pathway [2]) (Reversibility: ?) [1, 2] P 5-(methylthio)-2,3-dioxopentyl phosphate + H2 O

5 Isolation/Preparation/Mutation/Application Source/tissue liver <1> [2]

109

Methylthioribulose 1-phosphate dehydratase

4.2.1.109

Purification <2> (partial) [1]

References [1] Furfine, E.S.; Abeles, R.H.: Intermediates in the conversion of 5’-Smethylthioadenosine to methionine in Klebsiella pneumoniae. J. Biol. Chem., 263, 9598-9606 (1988) [2] Wray, J.W.; Abeles, R.B.: The methionine salvage pathway in Klebsiella pneumoniae and rat liver. J. Biol. Chem., 270, 3147-3153 (1995)

110

Aldos-2-ulose dehydratase

4.2.1.110

1 Nomenclature EC number 4.2.1.110 Systematic name 1,5-anhydro-d-fructose hydro-lyase (microthecin-forming) Recommended name aldos-2-ulose dehydratase Synonyms 1,5-anhydro-d-fructose dehydratase (microthecin-forming) <1, 2, 3, 4, 5, 6, 7> [2] AUDH <1, 2, 3, 4, 5, 6, 7, 8> [1, 2] pyranosone dehydratase <1, 2, 3, 4, 5, 6, 7> [2] CAS registry number 101920-80-3 145266-93-9

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8>

Gracilariopsis lemaneiformis (no sequence specified) [2, 3] Morchella costata (no sequence specified) [2] Morchella vulgaris (no sequence specified) [2] Phanerochaete chrysosporium (no sequence specified) [2] Spongipellis unicolor (no sequence specified) [2] Microthecium compressum (no sequence specified) [2] Microthecium sobelii (no sequence specified) [2] Phanerochaete chrososperium (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction 1,5-anhydro-d-fructose = 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose + H2 O 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one 1,5-anhydro-d-fructose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)one + H2 O (overall reaction)

111

Aldos-2-ulose dehydratase

4.2.1.110

Reaction type elimination Natural substrates and products S 1,5-anhydro-d-fructose <1, 2, 3, 4, 5, 6, 7, 8> (<5> 1,5-anhydro-d-fructose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one + H2 O (overall reaction), (1a) 1,5-anhydro-d-fructose = 1,5-anhydro-4-deoxy-d-glycerohex-3-en-2-ulose + H2 O, (1b) 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2ulose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one. This enzyme catalyses two of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydro-d-fructose [1,2]. The other enzymes involved in this pathway are EC 4.2.1.111 (1,5-anhydro-d-fructose dehydratase), EC 4.2.2.13 (exo-(1,4)-a-d-glucan lyase) and EC 5.3.3.15 (ascopyrone tautomerase). This is a bifunctional enzyme that acts as both a lyase and as an isomerase. Differs from EC 4.2.1.111, which can carry out only reaction 1a [2]; <1, 2, 3, 4, 6, 7, 8> 1,5-anhydro-dfructose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one + H2 O (overall reaction), (1a) 1,5-anhydro-d-fructose = 1,5-anhydro-4-deoxy-dglycero-hex-3-en-2-ulose + H2 O, (1b) 1,5-anhydro-4-deoxy-d-glycerohex-3-en-2-ulose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one. This enzyme catalyses two of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydrod-fructose. The other enzymes involved in this pathway are EC 4.2.1.111 (1,5-anhydro-d-fructose dehydratase), EC 4.2.2.13 (exo-(1,4)-a-d-glucan lyase) and EC 5.3.3.15 (ascopyrone tautomerase). This is a bifunctional enzyme that acts as both a lyase and as an isomerase. Differs from EC 4.2.1.111, which can carry out only reaction 1a [1,2,3]) (Reversibility: ir) [1, 2, 3] P 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one + H2 O (<1, 2, 3, 4, 5, 6, 7, 8> i.e. microthecin [1, 2, 3]) Substrates and products S 1,5-anhydro-d-fructose <1, 2, 3, 4, 5, 6, 7, 8> (<5> 1,5-anhydro-d-fructose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one + H2 O (overall reaction), (1a) 1,5-anhydro-d-fructose = 1,5-anhydro-4-deoxy-d-glycerohex-3-en-2-ulose + H2 O, (1b) 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2ulose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one. This enzyme catalyses two of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydro-d-fructose [1,2]. The other enzymes involved in this pathway are EC 4.2.1.111 (1,5-anhydro-d-fructose dehydratase), EC 4.2.2.13 (exo-(1,4)-a-d-glucan lyase) and EC 5.3.3.15 (ascopyrone tautomerase). This is a bifunctional enzyme that acts as both a lyase and as an isomerase. Differs from EC 4.2.1.111, which can carry out only reaction 1a [2]; <1, 2, 3, 4, 6, 7, 8> 1,5-anhydro-dfructose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one + H2 O (overall reaction), (1a) 1,5-anhydro-d-fructose = 1,5-anhydro-4-deoxy-dglycero-hex-3-en-2-ulose + H2 O, (1b) 1,5-anhydro-4-deoxy-d-glycerohex-3-en-2-ulose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one.

112

4.2.1.110

Aldos-2-ulose dehydratase

This enzyme catalyses two of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydrod-fructose. The other enzymes involved in this pathway are EC 4.2.1.111 (1,5-anhydro-d-fructose dehydratase), EC 4.2.2.13 (exo-(1,4)-a-d-glucan lyase) and EC 5.3.3.15 (ascopyrone tautomerase). This is a bifunctional enzyme that acts as both a lyase and as an isomerase. Differs from EC 4.2.1.111, which can carry out only reaction 1a [1, 2, 3]; <8> formation of microthecin is irreversible. 1,5-anhydro-d-fructose = 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one + H2 O (overall reaction), (1a) 1,5-anhydro-d-fructose = 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose + H2 O, (1b) 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose = 2-hydroxy2-(hydroxymethyl)-2H-pyran-3(6H)-one. This enzyme catalyses two of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydro-d-fructose. The other enzymes involved in this pathway are EC 4.2.1.111 (1,5-anhydro-d-fructose dehydratase), EC 4.2.2.13 (exo-(1,4)-a-d-glucan lyase) and EC 5.3.3.15 (ascopyrone tautomerase). This is a bifunctional enzyme that acts as both a lyase and as an isomerase. Differs from EC 4.2.1.111, which can carry out only reaction 1a [1]) (Reversibility: ir) [1, 2, 3] P 2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one + H2 O (<1, 2, 3, 4, 5, 6, 7, 8> i.e. microthecin [1, 2, 3]) S glucosone <4, 5, 8> (<8> 20% of the activity with 1,5-anhydro-d-fructose [1]) (Reversibility: ?) [1, 2] P cortalcerone + H2 O Inhibitors 1,5-anhydro-4-deoxy-d-glycero-hexo-2,3-diulose dihydrate <8> (<8> 4 mM, 50% inhibition when 1,5-anhydro-d-fructose is used as the substrate at 5 mM and pH 5.8 [1]) [1] pH-Optimum 5.8 <8> (<8> formation of 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose [1]) [1] 6.8 <8> (<8> formation of microthecin [1]) [1]

4 Enzyme Structure Subunits ? <8> (<8> x * 97400, SDS-PAGE [1]) [1] dimer <4> [2]

5 Isolation/Preparation/Mutation/Application Purification <8> [1]

113

Aldos-2-ulose dehydratase

4.2.1.110

References [1] Yu, S.: Enzymatic description of the anhydrofructose pathway of glycogen degradation II. Gene identification and characterization of the reactions catalyzed by aldos-2-ulose dehydratase that converts 1,5-anhydro-d-fructose to microthecin with ascopyrone M as the intermediate. Biochim. Biophys. Acta, 1723, 63-73 (2005) [2] Yu, S.; Fiskesund, R.: The anhydrofructose pathway and its possible role in stress response and signaling. Biochim. Biophys. Acta, 1760, 1314-1322 (2006) [3] Broberg, A.; Kenne, L.; Pedersen, M.: Presence of microthecin in the red alga Gracilariopsis lemaneiformis and its formation from 1,5-anhydro-d-fructose. Phytochemistry, 41, 151-154 (1996)

114

1,5-Anhydro-D-fructose dehydratase

4.2.1.111

1 Nomenclature EC number 4.2.1.111 Systematic name 1,5-anhydro-d-fructose hydro-lyase (ascopyrone-M-forming) Recommended name 1,5-anhydro-d-fructose dehydratase Synonyms 1,5-anhydro-d-arabino-hex-2-ulose dehydratase <1> [1] 1,5-anhydro-d-fructose 4-dehydratase <1> [1] 1,5-anhydro-d-fructose hydro-lyase <1> [1] 1,5-anhydro-d-fructose hydrolyase <1> [1] AF dehydratase <1, 2, 3> [1, 2] AFDH <1, 2, 3> [1, 2] CAS registry number 9044-86-4

2 Source Organism <1> Anthracobia melaloma (no sequence specified) [1, 2] <2> Plicaria anthracina (no sequence specified) [2] <3> Plicaria leiocarpa (no sequence specified) [2]

3 Reaction and Specificity Catalyzed reaction 1,5-anhydro-d-fructose = 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose + H2 O Natural substrates and products S 1,5-anhydro-d-fructose <1> (<1> i.e. 1,5-anhydro-d-arabino-hex-2-ulose. This enzyme catalyses one of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydrod-fructose [1]) (Reversibility: ir) [1] P 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose + H2 O (<1> i.e.ascopyrone M [1])

115

1,5-Anhydro-D-fructose dehydratase

4.2.1.111

Substrates and products S 1,5-anhydro-d-fructose <1, 2, 3> (<1> i.e. 1,5-anhydro-d-arabino-hex-2ulose. This enzyme catalyses one of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydro-d-fructose [1]; <1,2,3> i.e. 1,5-anhydro-d-arabino-hex-2-ulose [1,2]) (Reversibility: ir) [1, 2] P 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose + H2 O (<1,2,3> i.e.ascopyrone M [1,2]) S Additional information <1> (<1> the enzyme is inactive towards: glucosone, glucose, galactose, mannose, fructose, ribose and g-gluconolactone [2]) (Reversibility: ?) [2] P ? Inhibitors EDTA <1> (<1> 50 mM, 84% inhibition [2]) [2] Metals, ions CaCl2 <1> (<1> requirement of alkaline earth metal ion for activity. 10 mM, around 2fold increase in activity [2]) [2] MgCl2 <1> (<1> requirement of alkaline earth metal ion for activity. 25 mM, around 2fold increase in activity [2]) [2] NaCl <1> (<1> 0.1 mM, around 2fold increase in activity [2]) [2] Specific activity (U/mg) 1870 <1> [1] pH-Optimum 6.5 <1> (<1> optimal pH-range is 5.9-7.0 [2]) [2] pH-Range 5.9-7 <1> (<1> optimal pH-range [2]) [2]

4 Enzyme Structure Molecular weight 140000 <1> (<1> gel filtration [1]) [1] 230000 <1> (<1> gel filtration [2]) [2] Subunits ? <1> (<1> x * 98000, SDS-PAGE [1]) [1] dimer <1> (<1> 2 * 98000, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Purification <1> [1]

116

4.2.1.111

1,5-Anhydro-D-fructose dehydratase

References [1] Yu, S.; Refdahl, C.; Lundt, I.: Enzymatic description of the anhydrofructose pathway of glycogen degradation; I. Identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and a-1,4-glucan lyase in the fungus Anthracobia melaloma. Biochim. Biophys. Acta, 1672, 120-129 (2004) [2] Yu, S.; Fiskesund, R.: The anhydrofructose pathway and its possible role in stress response and signaling. Biochim. Biophys. Acta, 1760, 1314-1322 (2006)

117

Acetylene hydratase

4.2.1.112

1 Nomenclature EC number 4.2.1.112 Systematic name acetaldehyde hydro-lyase (acetylene-forming) Recommended name acetylene hydratase Synonyms AH <3> [4] tungsten-dependent acetylene hydratase <3> [1] Additional information <3> (<3> the enzyme structurally belongs to the dimethyl sulfoxide reductase family of enzymes [6]) [6] CAS registry number 75788-81-7

2 Source Organism <1> <2> <3> <4>

Rhodococcus ruber (no sequence specified) [3] Rhodococcus opacus (no sequence specified) [3] Pelobacter acetylenicus (no sequence specified) [1, 2, 4, 5, 6] Gordona sp. (no sequence specified) [3]

3 Reaction and Specificity Catalyzed reaction acetaldehyde = acetylene + H2 O (<3> active site channel structure and detailed catalytic mechanism [6]; <3> reaction mechanism involving molybdenum-cofactor and tungsten/iron-sulfur cluster, structure-function modeling, overview [4]) Natural substrates and products S acetylene + H2 O <1, 2, 3, 4> (Reversibility: r) [1, 2, 3, 4, 5, 6] P acetaldehyde

118

4.2.1.112

Acetylene hydratase

Substrates and products S acetylene + H2 O <1, 2, 3, 4> (<3> a non-redox reaction [6]; <3> the hydration reaction is unique for a tungsten and molybdenum containing enzyme, which are usually redox enzymes [4]) (Reversibility: r) [1, 2, 3, 4, 5, 6] P acetaldehyde (<3> the subsequent disproportionation of acetaldehyde yields acetate and ethanol [1]) S Additional information <3> (<3> no activity with ethylene, methylene blue, or anthraquinone disulfonate, the purified enzyme has no coenzyme A-acetylating aldehyde dehydrogenase activity [5]) (Reversibility: ?) [5] P ? Inhibitors CO <3> (<3> 90% inhibition at 0.8 mM [5]) [5] HgCl2 <3> (<3> reduces enzyme activity by 40% at 0.01 mM, 80% at 0.1 mM, and 98% at 0.2 mM [5]) [5] KCN <3> (<3> 20% inhibition at 1 mM, 40% inhibition at 5-10 mM [5]) [5] molybdate <3> [5] nitric oxide <3> (<3> complete inhibition at 3 mM [5]) [5] Additional information <3> (<3> no inhibition by citrate, ascorbate, tiron, ferrocene, and EDTA, no inhibition by ethylene at 8 mM [5]) [5] Cofactors/prosthetic groups molybdopterin guanine dinucleotide <3> (<3> 1.3 mol of cofactor per mol of enzyme [2]; <3> structure-function analysis [4]) [2,4] Activating compounds dithionite <3> (<3> a strong reductant is required for activity [1,5]) [1, 5] Ti(III)citrate <3> (<3> a strong reductant is required for activity [1,5]) [1, 5] Additional information <1, 3, 4> (<3> no activation by tungstate at 5 mM [5]; <1,4> the enzyme does not require reductive additives [3]; <3> the enzyme is inducible by acetylene feeding [2]) [2, 3, 5] Metals, ions Fe2+ <3> (<3> dependent on, the enzyme is a tungsten/iron-sulfur protein with [3Fe-4S], 2.0 gAV, low potential [4Fe-4S] cluster which is highly sensitive to oxidation, 4.8 mol of iron per mol of enzyme, 3.9 mol of acid-labile sulfur per mol of enzyme [2]; <3> dependent on, the enzyme is a tungsten/ironsulfur protein with [4Fe-4S], N-terminal binding cluster Cys-Xaa-Cys-XaaCys [1]) [1, 2] iron <3> (<3> non-redox-active tungsten/[4Fe-4S] enzyme, a cubane-type [4Fe:4S] cluster, structure analysis, overview [6]; <3> the enzyme is a tungsten/iron-sulfur protein [4]) [4, 6] iron-sulfur cluster <3> (<3> dependent on, the enzyme is a tungsten/ironsulfur protein, 4.8 mol of iron per mol of enzyme and 3.9 mol od acid-labile sulfur per mol of enzyme [5]) [5] molybdenum <1, 2, 3, 4> (<3> 1.3 mol of molybdopterin-guanine dinucleotide per mol of enzyme [2]; <1,2,4> dependent on, enzyme expression also

119

Acetylene hydratase

4.2.1.112

requires molybdenum [3]; <3> molybdopterin-guanine dinucleotide cofactor [4]) [2, 3, 4] Ti(III)citrate <2> (<2> a strong reductant is absolutely required for activity [3]) [3] tungsten <3> (<3> a non-redox-active tungsten/[4Fe-4S] enzyme, contains two molybdopterin guanine dinucleotide cofactors, MGD, designated P and Q, structure analysis, bis-molybdopterin guanine dinucleotide-ligated tungsten atom, overview, the tungsten center binds a water molecule that is activated by an adjacent aspartate residue, enabling to attack acetylene bound in a distinct, hydrophobic pocket, this mechanism requires a strong shift of pKa of the aspartate, caused by nearby low-potential [4Fe:4S] cluster, overview [6]; <3> dependent on, the enzyme is a tungsten/iron-sulfur protein [1]; <3> dependent on, the enzyme is a tungsten/iron-sulfur protein, 0.4 mol of tungsten per mol of enzyme [5]; <3> dependent on, the enzyme is a tungsten/iron-sulfur protein, 0.5 mol of tungsten per mol of enzyme [2]; <3> dependent on, tungstoenzyme [4]) [1, 2, 4, 5, 6] Additional information <1, 3, 4> (<3> no molybdenum detectable [5]; <1,4> the enzyme does not require reductive additives [3]) [3, 5] Specific activity (U/mg) 10.7 <3> (<3> purified native enzyme [1]) [1] 13 <3> (<3> crude cell extract of cells grown in presence of acetylene [2]) [2] 26.5 <3> (<3> purified native enzyme [2]) [2] 69.2 <3> (<3> purified enzyme [5]) [5] Additional information <1, 2, 4> [3] Km-Value (mM) 0.014 <3> (acetylene, <3> pH 7.0, 30 C [5]) [5] Additional information <3> (<3> the enzyme activity depends on the redox status, overview [1,2,5]) [1, 2, 5] pH-Optimum 6-6.5 <3> [5] 6-7 <3> [1] 7 <3> (<3> assay at [2]) [2] pH-Range 4-9 <3> [5] Temperature optimum ( C) 30 <3> (<3> assay at [2]) [2] 50 <3> [1, 5] Temperature range ( C) 22-80 <3> [5]

120

4.2.1.112

Acetylene hydratase

4 Enzyme Structure Molecular weight 60000 <3> (<3> gel filtration [5]) [5] 83550 <3> (<3> MALDI mass spectrometry [2]) [2] 85000 <3> (<3> mass spectrometry [1]) [1] Subunits monomer <3> (<3> 1 * 73000, SDS-PAGE [2,5]; <3> 1 * 73000 [4]; <3> 1 * 85000, MALDI mass spectrometry [1]) [1, 2, 4, 5] Additional information <3> (<3> four domain structure with active site access pathway, active site architecture, overview [6]) [6]

5 Isolation/Preparation/Mutation/Application Source/tissue culture condition:acetylene-grown cell <1, 2, 3, 4> [3, 5] culture condition:molybdate-grown cell <3> [5] culture condition:tungstate-grown cell <3> [5] Additional information <3> (<3> strictly anaerobic growth conditions [6]; <3> the organism is grown anaerobically in tungstate-supplemented fresh water medium [1]; <3> the organism is grown in carbonate-buffered, sulfide-reduced, and tungstate-supplemented fresh water medium, optimally at pH 6.8-7.0 [2]) [1, 2, 6] Purification <3> (native enzyme 29.7fold by ammonium sulfate fractionation, ion exchange chromatography, and gel filtration to homogeneity) [1] <3> (native enzyme 7.6fold by ammonium sulfate fractionation, ion exchange chromatography, and gel filtration to homogeneity) [2] <3> (under air at room temperature, native enzyme 240-fold by ammonium sulfate fractionation, anion exchange chromatography, gel filtration, and a second anion exchange chromatography step to homogeneity) [5] Crystallization <3> (purified enzyme, the mother liquor contains plus 15% (v/v) 2-methyl2,5-pentanediol, X-ray diffraction structure determination and analysis at 1.26-1.95 A resolution, modeling) [6] <3> (purified native enzyme, sitting drop vapour diffusion method in a 95%N2/5%H2 atmosphere, 10 mg/ml protein in 5 mM HEPES-NaOH, pH 7.5, and 3 mM dithionite or Ti(III)citrate, mixing with an equal volume of 0.002 ml of precipitant solution equilibrated against 0.3 ml of reservoir, 20 C, 3 weeks, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement) [1]

121

Acetylene hydratase

4.2.1.112

6 Stability Oxidation stability <3>, the enzyme is oxygen-sensitive [1,2] Storage stability <3>, enzyme activity is stable even after prolonged storage of the cell extract or of the purified protein under air [5]

References [1] Einsle, O.; Niessen, H.; Abt, D.J.; Seiffert, G.; Schink, B.; Huber, R.; Messerschmidt, A.; Kroneck, P.M.H.: Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicus. Acta Crystallogr. Sect. F, F61, 299-301 (2005) [2] Meckenstock, R.U.; Krieger, R.; Ensign, S.; Kroneck, P.M.H.; Schink, B.: Acetylene hydratase of Pelobacter acetylenicus. Molecular and spectroscopic properties of the tungsten iron-sulfur enzyme. Eur. J. Biochem., 264, 176182 (1999) [3] Rosner, B.M.; Rainey, F.A.; Kroppenstedt, R.M.; Schink, B.: Acetylene degradation by new isolates of aerobic bacteria and comparison of acetylene hydratase enzymes. FEMS Microbiol. Lett., 148, 175-180 (1997) [4] Yadav, J.; Das, S.K.; Sarkar, S.: A functional mimic of the new class of tungstoenzyme acetylene hydratase. J. Am. Chem. Soc., 119, 4315-4316 (1997) [5] Rosner, B.M.; Schink, B.: Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein. J. Bacteriol., 177, 5767-5772 (1995) [6] Seiffert, G.B.; Ullmann, G.M.; Messerschmidt, A.; Schink, B.; Kroneck, P.M.H.; Einsle, O.: Structure of the non-redox-active tungsten/[4Fe:4S] enzyme acetylene hydratase. Proc. Natl. Acad. Sci. USA, 104, 3073-3077 (2007)

122

o-succinylbenzoate synthase

4.2.1.113

1 Nomenclature EC number 4.2.1.113 Systematic name (1R,6R)-6-hydroxy-2-succinylcyclohexa-2,4-diene-1-carboxylate hydrolyase (2-succinylbenzoate-forming) Recommended name o-succinylbenzoate synthase Synonyms OSB synthase <1> [3] OSBS <1, 3> [1, 2] o-succinylbenzoic acid synthase <1> [3] CAS registry number 97089-83-3

2 Source Organism <1> Escherichia coli (no sequence specified) [2, 3] <2> Amycolatopsis sp. (no sequence specified) [4, 5] <3> Escherichia coli (UNIPROT accession number: P29208) [1]

3 Reaction and Specificity Catalyzed reaction (1R,6R)-6-hydroxy-2-succinylcyclohexa-2,4-diene-1-carboxylate = 2-succinylbenzoate + H2 O Reaction type dehydration Natural substrates and products S (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate <2> (<2> reaction in menaquinone biosynthetic pathway [4]) (Reversibility: ?) [4] P 4-(2-carboxyphenyl)-4-oxobutanoic acid + H2 O

123

o-succinylbenzoate synthase

4.2.1.113

S 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid <1, 3> (Reversibility: ir) [1, 2] P 4-(2-carboxyphenyl)-4-oxobutyrate + H2 O <1, 3> [1, 2] S Additional information <1, 3> (<1,3> involved in the menaquinone biosynthetic pathway [1,2]) (Reversibility: ?) [1, 2] P ? <1, 3> [1, 2] Substrates and products S (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate <2> (<2> reaction in menaquinone biosynthetic pathway [4]) (Reversibility: ?) [4] P 4-(2-carboxyphenyl)-4-oxobutanoic acid + H2 O S 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid <1, 3> (Reversibility: ir) [1, 2] P 4-(2-carboxyphenyl)-4-oxobutyrate + H2 O <1, 3> [1, 2] S Additional information <1, 2, 3> (<1,3> involved in the menaquinone biosynthetic pathway [1,2]; <2> the enzyme also shows N-acylamino acid racemase activity, with the optimal substrate being the enantiomers of Nacetyl methionine [4]) (Reversibility: ?) [1, 2, 4] P ? <1, 3> [1, 2] Inhibitors EDTA <1> (<1> loses all activity during treatment with EDTA, activity is most efficiently restored by Mn2+ [3]) [3] Cofactors/prosthetic groups thiamine diphosphate <1> (<1> tightly bound to the enzyme [3]) [3] Metals, ions Mg2+ <1, 3> (<3> essential for activity [1]; <1> required for activity [2]) [1, 2] Mn2+ <1> (<1> restores activity of enzyme inactivated by EDTA [3]) [3] Turnover number (min–1) 120 <2> ((1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate) [4] Specific activity (U/mg) Additional information <1> [3] Km-Value (mM) 0.48 <2> ((1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate) [4] pH-Optimum 8.2 <1> [3] pH-Range 7.5-9 <1> (<1> pH 7.5: about 60% of maximal activity, pH 9.0: about 60% of maximal activity [3]) [3]

124

4.2.1.113

o-succinylbenzoate synthase

Temperature optimum ( C) 50 <1> [3] Temperature range ( C) 37-60 <1> (<1> about 30% of maximal activity at 37 C and at 60 C [3]) [3]

4 Enzyme Structure Molecular weight 35760 <3> (<3> mass spectrometry [1]) [1] 37740 <1> (<1> mass spectrometry [2]) [2] 195500 <1> (<1> gel filtration [3]) [3] Subunits ? <1> (<1> x * 66500, SDS-PAGE [3]) [3]

5 Isolation/Preparation/Mutation/Application Purification <1> [3] <1> (purification of a recombinant His-tagged enzyme by affinity chromatography followed by tag cleavage and anion-exchange chromatography) [1] <2> [5] Crystallization <1> (co-crystallization of the K133R mutant with 2-succinyl-6-hydroxy-2,4cyclohexadiene-1-carboxylic acid) [2] <1> (crystallization of the apoenzyme and a complex with Mg2+ and 4-(2carboxyphenyl)-4-oxobutyrate) [1] <2> (hanging-drop vapor diffusion method, the best crystals are observed growing at room temperature from poly(ethylene glycol) 8000 at pH 8.0, soace group R32 with unit cell dimensions of a = b = 216.0 A and c = 261.0 A and contains 4 subunits in the asymmetric unit. Three-dimensional structures of liganded complexes of the enzyme with o-succinylbenzoate, N-acetylmethionine, N-succinylmethionine, succinylphenylglycine to 2.2, 2.3, 2.1, and 1.9 A resolution, respectively) [5] Cloning <1> (expression in Escherichia coli of several recombinant mutant enzymes) [2] <1> (expression of a recombinant enzyme in Escherichia coli) [1] Engineering K133A <1> (<1> inactive mutant [2]) [2] K133R <1> (<1> inactive mutant [2]) [2] K133S <1> (<1> inactive mutant [2]) [2]

125

o-succinylbenzoate synthase

4.2.1.113

K163R <2> (<2> no o-succinylbenzoate synthase activity and no N-acylamino acid racemase activity, mutant enzyme catalyzes the stereospecific exchange of the a-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen [4]) [4] K163S <2> (<2> no o-succinylbenzoate synthase activity and no N-acylamino acid racemase activity, mutant enzyme catalyzes the stereospecific exchange of the a-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen [4]) [4] K235A <1> (<1> inactive mutant [2]) [2] K235R <1> (<1> inactive mutant [2]) [2] K235S <1> (<1> inactive mutant [2]) [2] K263R <2> (<2> no o-succinylbenzoate synthase activity and no N-acylamino acid racemase activity, mutant enzyme catalyzes the stereospecific exchange of the a-hydrogen of N-succinyl-(R)-phenylglycine with solvent hydrogen [4]) [4] K263S <2> (<2> no o-succinylbenzoate synthase activity and no N-acylamino acid racemase activity, mutant enzyme catalyzes the stereospecific exchange of the a-hydrogen of N-succinyl-(R)-phenylglycine with solvent hydrogen [4]) [4]

References [1] Thompson, T.B.; Garrett, J.B.; Taylor, E.A.; Meganathan, R.; Gerlt, J.A.; Rayment, I.: Evolution of enzymatic activity in the enolase superfamily: structure of o-succinylbenzoate synthase from Escherichia coli in complex with Mg2+ and o-succinylbenzoate. Biochemistry, 39, 10662-10676 (2000) [2] Klenchin, V.A.; Taylor Ringia, E.A.; Gerlt, J.A.; Rayment, I.: Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli. Biochemistry, 42, 14427-14433 (2003) [3] Weische, A.; Garvert, W.; Leistner, E.: Biosynthesis of o-succinylbenzoic acid. II.: Properties of o-succinylbenzoic acid synthase, an enzyme involved in vitamin K2 biosynthesis. Arch. Biochem. Biophys., 256, 223-231 (1987) [4] Taylor Ringia, E.A.; Garrett, J.B.; Thoden, J.B.; Holden, H.M.; Rayment, I.; Gerlt, J.A.: Evolution of enzymatic activity in the enolase superfamily: functional studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis. Biochemistry, 43, 224-229 (2004) [5] Thoden, J.B.; Taylor Ringia, E.A.; Garrett, J.B.; Gerlt, J.A.; Holden, H.M.; Rayment, I.: Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis. Biochemistry, 43, 5716-5727 (2004)

126

Glucuronan lyase

4.2.2.14

1 Nomenclature EC number 4.2.2.14 Systematic name (1!4)-b-d-glucuronan lyase Recommended name glucuronan lyase Synonyms (1,4)-b-d-glucuronan lyase <2> [2] (1!4)-b-d-glucuronan lyase GL2 glucuronan lyase <3> [5] endopolyglucuronate lyase <2> [2] CAS registry number 193766-71-1

2 Source Organism <1> <2> <3> <4>

Rhizobium meliloti (no sequence specified) [1] Sinorhizobium meliloti (no sequence specified) [2] Trichoderma sp. (no sequence specified) [3, 5] Trichoderma sp. GL2 (no sequence specified) [4]

3 Reaction and Specificity Catalyzed reaction Eliminative cleavage of (1!4)-b-d-glucuronans to give oligosaccharides with 4-deoxy-b-d-gluc-4-enuronosyl groups at their non-reducing ends. Complete degradation of glucuronans results in the formation of tetrasaccharides (<1> complete degradation of glucuronans results in the formation of tetrasaccharides [1]) Reaction type elimination Natural substrates and products S acetylated (1!4)-b-d-glucuronan <1> (Reversibility: ?) [1] P 4-deoxy-b-d-gluc-4-enuronosyl (1!4)-b-d-glucuronan <1> [1]

127

Glucuronan lyase

4.2.2.14

Substrates and products S 3-O-acetylated glucuronan <2> (<2> b(1-4)-linked [2]) (Reversibility: ?) [2] P ? <2> [2] S acetylated (1!4)-b-d-glucuronan <1> (Reversibility: ?) [1] P 4-deoxy-b-d-gluc-4-enuronosyl (1!4)-b-d-glucuronan <1> [1] S deacetylated (1!4)-b-d-glucuronan <1> (Reversibility: ?) [1] P 4-deoxy-b-d-gluc-4-enuronosyl (1!4)-b-d-glucuronan <1> [1] S deacetylated polyglucuronate <2> (<2> b-(1-4)-linked, best substrate, enzyme shows specific endopolyglucuronate lyase activity [2]) (Reversibility: ?) [2] P 4,5-unsaturated oligoglucuronates <2> [2] S glucuronan <4> (Reversibility: ?) [4] P 4-deoxy-b-d-hex-4-enopyranosylglucuronate-(1-4)-O-b-d-glucuropyranosyluronate-(1-4)-O-b-d-glucuropyranosyluronate + ? S glucuronan <3> (Reversibility: ?) [5] P oligoglucuronan S Additional information <2> (<2> no activity with polyglucuronate methyl ester a-(1-4)-linked, hyaluronate b-(1-3), b(1-4)-linked, glucogluronan b-(1-3), b(1-4)-linked, a-l-guluronate blocks a-(1-4)-linked, b-dmannuronate blocks b(1-4)-linked, 2,3-di-O-acetylated glucuronan b(14)-linked, glucuronan b(1-4)-linked [2]) (Reversibility: ?) [2] P ? <2> [2] Inhibitors Ag+ <2> (<2> 100% reduced activity at 1 mM [2]) [2] Ca2+ <2> (<2> 21-30% reduced activity at 1 mM [2]) [2] Cd2+ <2> (<2> 26-29% reduced activity at 1 mM [2]) [2] Cu2+ <2, 3> (<3> 1 mM, 81% inhibition [3]; <2> 85% reduced activity at 1 mM [2]) [2, 3] EDTA <2> (<2> 21-30% reduced activity at 1 mM [2]) [2] Fe2+ <3> (<3> 1 mM, 19% inhibition [3]) [3] Hg2+ <2> (<2> 94% reduced activity at 1 mM [2]) [2] Li+ <2> (<2> 26-29% reduced activity at 1 mM [2]) [2] Mn2+ <2> (<2> 21-30% reduced activity at 1 mM [2]) [2] NaN3 <2> (<2> 26-29% reduced activity at 1 mM [2]) [2] Ni2+ <3> (<3> 1 mM, 60% inhibition [3]) [3] Rb2+ <3> (<3> 1 mM, 10% inhibition [3]) [3] Zn2+ <2> (<2> 59% reduced activity at 1 mM [2]) [2] Additional information <2> (<2> no inhibition by Mg2+ [2]) [2] Metals, ions Ca2+ <3> (<3> 1 mM, enhances activity 4.56fold [3]) [3] Co2+ <3> (<3> 1 mM, enhances activity 1.5fold [3]) [3] Li+ <3> (<3> 1 mM, enhances activity 4.4fold [3]) [3] Mg2+ <3> (<3> 1 mM, enhances activity 2.5fold [3]) [3] Mn2+ <3> (<3> 1 mM, enhances activity 2.2fold [3]) [3]

128

4.2.2.14

Glucuronan lyase

Additional information <3> (<3> enzyme appears not to be really affected by ionic strength variations. The optimum is 300 mM concentration of potassium acetate buffer [3]) [3] Specific activity (U/mg) 162 <3> [3] pH-Optimum 5.5 <3> [3] 6.5 <2> [2] Temperature optimum ( C) 50 <2> [2] 55 <3> [3]

4 Enzyme Structure Molecular weight 20000 <2> (<2> gel filtration [2]) [2] Subunits ? <3> (<3> x * 27000, SDS-PAGE [3]) [3] monomer <2> (<2> 1 * 20000, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Localization extracellular <3> [3] Purification <2> (to homogeneity, 394fold) [2] <3> [3] Application synthesis <3, 4> (<3> production of glucuronan oligosaccharides [5]; <4> production of oligoglucuronans by enzymatic depolymerization of nascent glucuronan [4]) [4, 5]

6 Stability pH-Stability 4-8 <3> (<3> 30 min, stable [3]) [3] Temperature stability 35 <3> (<3> 1 h, stable below [3]) [3] 40 <3> (<3> 1 h, 90% loss of activity [3]) [3] 52 <2> (<2> 50% of maximal activity [2]) [2]

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Glucuronan lyase

4.2.2.14

60 <3> (<3> 1 h, complete inactivation [3]) [3] 67 <2> (<2> complete inactivation [2]) [2] Storage stability <2>, -80 C, 50 mM KCl, purified enzyme, stable for several weeks [2]

References [1] Michaud, P.; Pheulpin, P.; Petit, E.; Seguin, J.P.; Barboutin, J.N.; Heyraud, A.; Courtois, B.; Courtois, J.: Identification of glucuronan lyase from a mutant strain of Rhizobium meliloti. Int. J. Biol. Macromol., 21, 3-9 (1997) [2] Da Costa, A.; Michaud, P.; Petit, E.; Heyraud, A.; Colin-Morel, P.; Courtois, B.; Courtois, J.: Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472). Appl. Environ. Microbiol., 67, 5197-5203 (2001) [3] Delattre, C.; Michaud, P.; Keller, C.; Elboutachfaiti, R.; Beven, L.; Courtois, B.; Courtois, J.: Purification and characterization of a novel glucuronan lyase from Trichoderma sp. GL2. Appl. Microbiol. Biotechnol., 70, 437-443 (2006) [4] Delattre, C.; Michaud, P.; Keller, C.; Courtois, B.; Courtois, J.: Production of oligoglucuronans by enzymatic depolymerization of nascent glucuronan. Biotechnol. Prog., 21, 1775-1781 (2005) [5] Delattre, C.; Michaud, P.; Lion, J.M.; Courtois, B.; Courtois, J.: Production of glucuronan oligosaccharides using a new glucuronan lyase activity from a Trichoderma sp. strain. J. Biotechnol., 118, 448-457 (2005)

130

Anhydrosialidase

4.2.2.15

1 Nomenclature EC number 4.2.2.15 Systematic name glycoconjugate siayl-lyase (2,7-cyclizing) Recommended name anhydrosialidase Synonyms EC 3.2.1.138 (formerly) a-(2,3)-specific trans-salidase anhydroneuraminidase neuraminidase, anhydrosialglycoconjugate N-acylneuraminylhydrolase (2,7-cyclizing) sialidase L Additional information CAS registry number 157857-11-9

2 Source Organism <1> Trypanosoma cruzi (no sequence specified) [4] <2> Macrobdella decora (no sequence specified) [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction elimination of a-sialyl groups in N-acetylneuraminic acid glycosides, releasing 2,7-anhydro-a-N-acetylneuraminate Reaction type hydrolysis of O-glycosyl bond Natural substrates and products S 4-methylumbelliferyl-a-N-acetylneuraminic acid glycoproteins or ganglioside + H2 O <2> (Reversibility: ?) [1, 2, 3] P 2,7-anhydro-a-N-acetylneuraminic acid <2> [1, 2]

131

Anhydrosialidase

4.2.2.15

Substrates and products S 4-methylumbelliferyl-a-N-acetylneuraminic acid + H2 O <2> (<2> only sialoglycoconjugates, not free a-N-acetylneuraminic acid [2]; <2> strict specificity towards the hydrolysis of NeuAca(2-3)Gal-linkages [1,3]) (Reversibility: ?) [1, 2, 3] P 4-methylumbelliferone + 2,7-anhydro-a-N-acetylneuraminic acid <2> [1, 2] S 4-methylumbelliferyl-a-N-acetylneuraminic acid glycoproteins or ganglioside + H2 O <2> (Reversibility: ?) [1, 2, 3] P 2,7-anhydro-a-N-acetylneuraminic acid <2> [1, 2] S a-(2,3)-sialyl oligosacharides <1> (Reversibility: ?) [4] P ? Inhibitors Additional information <2> (<2> not: 2-deoxy-2,3-dehydroacetylneuraminic acid, EDTA, Ca2+ , Mg2+ , Mn2+ [1]) [1] Specific activity (U/mg) Additional information <2> [1] Km-Value (mM) 0.68 <2> (4-methylumbelliferyl-a-N-acetylneuraminic acid, <2> pH 5.5, 37 C [1]) [1] pH-Optimum 5.5-7 <2> [1] Temperature optimum ( C) 37 <2> (<2> assay at [1]) [1]

4 Enzyme Structure Subunits ? <2> (<2> x * 84000, SDS-PAGE [1]) [1] monomer <2> (<2> 1* 83000 [3]) [3]

5 Isolation/Preparation/Mutation/Application Purification <2> [1] <2> (partial) [2] Crystallization <2> [3] Cloning <2> (expression in Escherichia coli) [3]

132

4.2.2.15

Anhydrosialidase

Application synthesis <1> (<1> synthesis of sialyl oligosaccharides using enzyme, since it is difficult to obtain them from natural sources [4]) [4]

6 Stability General stability information <2>, 50% loss of activity after one freeze and thawing cycle, in presence of 10% glycerol stable to several freeze and thawing cycles [1] Storage stability <2>, -20 C, 10% glycerol, stable for more than 4 months [1]

References [1] Chou, M.Y.; Li, S.C.; Kiso, M.; Hasegawa, A.; Li, Y.T.: Purification and characterization of sialidase L, a NeuAc a(2-3)Gal-specific sialidase. J. Biol. Chem., 269, 18821-18826 (1994) [2] Li, Y.T.; Nakagawa, H.; Ross, S.A.; Hansson, G.C.; Li, S.C.: A novel sialidase which releases 2,7-anhydro-a-N-acetylneuraminic acid from sialoglycoconjugates. J. Biol. Chem., 265, 21629-21633 (1990) [3] Luo, Y.; Chou, M.; Li, S.; Li, Y.; Luo, M.: Crystallization and preliminary Xray studies of sialidase L from the leech Macrobdella decora. Acta Crystallogr. Sect. D, 54, 111-113 (1998) [4] Takahashi, N.; Lee, K.B.; Nakagawa, H.; Tsukamoto, Y.; Kawamura, Y.; Li, Y.T.; Lee, Y.C.: Enzymatic sialylation of N-linked oligosaccharides using an a(2,3)-specific trans-sialidase from Trypanosoma cruzi: structural identification using a three-dimensional elution mapping technique. Anal. Biochem., 230, 333-342 (1995)

133

Levan fructotransferase (DFA-IV-forming)

4.2.2.16

1 Nomenclature EC number 4.2.2.16 Systematic name 2,6-b-d-fructan lyase (di-b-d-fructofuranose-2,6’:2’,6-dianhydride-forming) Recommended name levan fructotransferase (DFA-IV-forming) Synonyms 2,6-b-d-fructan d-fructosyl-d-fructosyltransferase (forming di-b-d-fructofuranose 2,6’:2’,6-dianhydride) <2> [4] 2,6-etab-d-fructan d-fructosyl-d-fructosyltransferase (forming di-b-d-fructofuranose 2,6’:2’,6-dianhydride) FTF <1> [12] LFTase <2, 3, 4> [2, 10, 11] LftM <6> [8] fructotransferase, levan <2> [4] levan fructotransferase <2, 4> [4, 11] CAS registry number 88593-15-1

2 Source Organism <1> <2> <3> <4> <5> <6>

Bacillus subtilis (no sequence specified) [12] Arthrobacter ureafaciens (no sequence specified) [1, 4, 5, 9, 10, 13] Arthrobacter oxydans (no sequence specified) [2] Microbacterium sp. (no sequence specified) [11] Arthrobacter nicotinovorans (no sequence specified) [3,6,7] Microbacterium sp. (UNIPROT accession number: Q9EVQ9) [8]

3 Reaction and Specificity Catalyzed reaction produces di-b-d-fructofuranose 2,6’:2’,6-dianhydride (DFA IV) by successively eliminating the diminishing (2!6)-b-d-fructan (levan) chain from the terminal d-fructosyl-d-fructosyl disaccharide

134

4.2.2.16

Levan fructotransferase (DFA-IV-forming)

Reaction type elimination Natural substrates and products S levan <5> (<5> the enzyme is specifically induced by levan [6]) (Reversibility: ?) [6] P di-d-fructose-2,6’:2’,6 dianhydride <5> [6] Substrates and products S levan <2> (Reversibility: ?) [1] P di-d-fructofuranose-2,6’:2’,6 dianhydride <2> (<2> and small amounts of several oligosaccharides and free fructose [1]) [1] S levan <2> (Reversibility: ?) [13] P di-d-fructose-2,6’:2’,6 dianhydride + fructose + limited levan (<2> di-dfructose-2,6:2,6 dianhydride (49.1%) + fructose (3.5%) + limited levan (16.2%) [13]) S levan <2, 3, 4, 5, 6> (<3> levan from Zymomonas mobilis, conversion yield is 35% [2]; <4> levan from Zymomonas mobilis: conversion yield 28.4% after 24 h. Levan from Serratia: conversion yield: 28.4% after 24 h [11]; <2> the enzyme degrades Bacillus mesentericus levan up to 40% leaving the levan fragment at the limit of the enzymatic degradation, formation of 5% reducing sugars as d-fructose [4]; <5> the enzyme is specifically induced by levan [6]) (Reversibility: ?) [2, 3, 4, 5, 6, 8, 9, 11] P di-d-fructose-2,6’:2’,6 dianhydride <2, 3, 4, 5, 6> (<2> and two or three minor fructooligosaccharides [4]; <5> di-d-fructose-2,6:2,6 dianhydride is the main product, 75%. Fructose, levanbiose, and two unidentified oligosaccharides are minor products [6]) [2, 3, 4, 5, 6, 8, 9, 11] S phlein <2> (<2> while it degrades Phleum pratense phlein more than 90%, formation of 6% reducing sugars as d-fructose [4]) (Reversibility: ?) [4] P di-d-fructose-2,6’:2’,6 dianhydride <2> (<2> and two or three minor fructooligosaccharides [4]) [4] S raffinose + d-galactopyranose <1> (<1> transfer of the b-fructofuranosyl residue from donor substrate to the 1-C of the acceptor galactopyranose, equilibrium towards galactosyl-fructoside [12]) (Reversibility: r) [12] P ? S stachyose + d-galactopyranose <1> (<1> transfer of the b-fructofuranosyl residue from donor substrate to the 1-C of the acceptor galactopyranose, equilibrium towards galactosyl-fructoside [12]) (Reversibility: r) [12] P ? S sucrose + d-galactopyranose <1> (<1> transfer of the b-fructofuranosyl residue from donor substrate to the 1-C of the acceptor galactopyranose, equilibrium towards galactosyl-fructoside [12]) (Reversibility: r) [12] P ? S Additional information <2, 3, 4, 6> (<4> no activity with dextran, soluble starch, inulin, maltose, sucrose, raffinose, 1-kestose, nystose and 1F- fructofuranosyl nystose [11]; <3> no activity with inulin, levanbiose, su-

135

Levan fructotransferase (DFA-IV-forming)

4.2.2.16

crose, dextran or starch [2]; <2> no formation of fructose, levanbiose, nor difructose anhydride IV from bifurcose and [O-b-d-Fru(f)-(2-1)]-O-[b-dFru(f)-(2-6)]2-O-b-d-Fru(f)-(2-1)-a-d-G(p) [5]; <6> the enzyme never hydrolyzes b-2,1-fructosyl-linked fructans, such as inulin, as well as oligosaccharides such as 1-kestose and nystose [8]) (Reversibility: ?) [2, 5, 8, 11] P ? <2, 3, 4, 6> [2, 5, 8, 11] Inhibitors 2-mercaptoethanol <4> (<4> 10 mM, 81% inhibition [11]) [11] Ag2SO4 <3> (<3> 1 mM, complete inhibition [2]) [2] AgNO3 <4> (<4> 10 mM, 62% inhibition [11]) [11] CoCl2 <3> (<3> 1 mM, 16% inhibition [2]) [2] CuSO4 <3> (<3> 1 mM, 36% inhibition [2]) [2] EDTA <4> (<4> 10 mM, 17% inhibition [11]) [11] FeCl2 <2, 4> (<2> 1 mM, 96% inhibition [1]; <4> 10 mM, 61% inhibition [11]) [1, 11] FeSO4 <3> (<3> 1 mM, 99% inhibition [2]) [2] HgCl2 <2> (<2> 1 mM, complete inhibition [1]) [1] HgSO4 <3> (<3> 1 mM, 84% inhibition [2]) [2] KCl <2> (<2> 1 mM, 11% inhibition [1]) [1] KMNO4 <4> (<4> 10 mM, 52% inhibition [11]) [11] LiCl <2> (<2> 1 mM, 8% inhibition [1]) [1] MgCl2 <2> (<2> 1 mM, 37% inhibition [1]) [1] MnCl2 <2, 3> (<2> 1 mM, 91% inhibition [1]; <3> 1 mM, 44% inhibition [2]) [1, 2] NH4 Cl <2> (<2> 1 mM, 54% inhibition [1]) [1] NaCl <3> (<3> 1 mM, 10% inhibition [2]) [2] NiCl2 <2, 3> (<2> 1 mM, 26% inhibition [1]; <3> 1 mM, 29% inhibition [2]) [1, 2] SDS <2> (<2> 10 mM, 68% inhibition [1]) [1] ZnCl2 <2> (<2> 1 mM, 76% inhibition [1]) [1] Metals, ions CaCl2 <2, 3> (<3> 1 mM, activation to 136% of control [2]; <2> 1 mM, activation to 156% of control. 10 mM, activation to 181% of control [1]) [1, 2] MgCl2 <4> (<4> 10 mM, activation to 127% of control [11]) [11] NaCl <2> (<2> 1 mM, activation to 125% of control. 10 mM, activation to 142% of control [1]) [1] Specific activity (U/mg) 145.2 <4> [8] 1432 <3> [2] 2269 <2> [1] Additional information <4> [11] Km-Value (mM) Additional information <6> (<6> Km for levan: 2 mg/ml at pH 7.0 and 40 C [8]) [8]

136

4.2.2.16

Levan fructotransferase (DFA-IV-forming)

pH-Optimum 4 <2> (<2> enzyme immobilized to Diaion WA30 [9]) [9] 4-5.5 <2> (<2> enzyme immobilized on Chitopearl BCW2501 beads [9]) [9] 5.8 <2> [1] 6 <2, 5> (<2> free enzyme [9]) [6, 9] 6.5 <3> [2] 6.5-7.5 <6> [8] 7 <4> [11] pH-Range 4.5-9 <6> (<6> about 50% of maximal activity at pH 4.5 and pH 9.0 [8]) [8] 5-7.5 <3> (<3> 5.0: about 35% of maximal activity, pH 7.5: about 40% of maximal activity [2]) [2] Temperature optimum ( C) 40 <4> [11] 45 <3> (<3> 30 min, enzyme retains 87% of initial activity [2]) [2] 50 <1, 5> [6, 12] 55 <2> (<2> free enzyme [9]) [9] 60 <2> (<2> enzyme immobilized on Chitopearl BCW2501 [9]) [9] Additional information <2> [1] Temperature range ( C) 30-50 <3> (<3> 30 C: about 35% of maximal activity, 50 C: about 80% of maximal activity, 60 C: about 15% of maximal activity [2]) [2]

4 Enzyme Structure Molecular weight 46000 <4> (<4> gel filtration [11]) [11] 48000 <5> (<5> gel filtration [6]) [6] 96000 <2> (<2> gel filtration [1]) [1] Subunits ? <6> (<6> x * 54000, SDS-PAGE [8]) [8] dimer <2> (<2> 2 * 51000, SDS-PAGE [1]) [1] monomer <4, 5> (<4> 1 * 46000, SDS-PAGE [11]; <5> 1 * 52000, SDS-PAGE [6]) [6, 11]

5 Isolation/Preparation/Mutation/Application Source/tissue culture medium <2> [1]

137

Levan fructotransferase (DFA-IV-forming)

4.2.2.16

Localization cytoplasm <6> [8] extracellular <2, 5> (<2> recombinant enzyme expressed in Escherichia coli [10]) [1, 6, 10] Purification <2> [1] <3> [2] <4> [11] <4> (recombinant enzyme) [8] <5> [3, 6] Cloning <2> (expressed with N-terminal fusion of a LacZ-derived secretion motif TMITNSSSVP using lac promoter system in recombinant Escherichia coli JM109[pUDF-A8]) [10] <2> (overexpression in Escherichia coli) [13] <4> (expression in Escherichia coli) [11] <4> (subcloned into a high-expression vector, pET-29b, and the recombinant enzyme with a tag of six histidine is overexpressed in Escherichia coli) [8] <5> (expression in Escherichia coli) [3] <5> (expression in Escherichia coli. An Escherichia coli transformant carrying the plasmid pLFT-BB1 expresses six times as much activity of levan fructotransferase as that of the original strain Arthrobacter nicotinovorans GS-9) [7] Application biotechnology <2> (<2> production of Arthrobacter levan fructotransferase from recombinant Escherichia coli at high levels via secretion directed by a novel N-terminal motif, TMITNSSSVP. A large amount of extracellular recombinant levan fructotransferase can be produced with high productivity through cost-effective processes [10]) [10] medicine <5> (<5> the product of the enzymatic reaction di-d-fructose2,6:2,6 dianhydride stimulates calcium absorption mainly in small intestine of rats and has a practical physiological property in preventing osteoporosis [3]) [3] synthesis <2> (<2> synthesis of di-d-fructose-2,6:2,6 dianhydride [13]) [13]

6 Stability Temperature stability 40 <4, 5> (<4> stable up to [11]; <5> 20 min, 90% of the initial activity remains [6]) [6, 11] 50 <2, 4> (<2> 30 min, enzyme retains 87% of its activity [1]; <4> activity diminishes quickly above [11]) [1, 11]

138

4.2.2.16

Levan fructotransferase (DFA-IV-forming)

55 <2> (<2> 60 min, 45% loss of activity of enzyme immobilized on Chitopearl BCW2501 beads, 95% loss of activity of native enzyme. Native enzyme loses about 80% of its initial activity after 30 min [9]) [9] General stability information <1>, the enzyme is immobilized on Eupergit C250 L and Trisospor-amino without loss of enzymatic activity [12] <2>, enzyme immobilized on Chitopearl BCW2501 beads retains about 60% of its initial activity after being used for 20 cycles [9]

References [1] Song, K.B.; Bae, K.S.; Lee, Y.B.; Lee, K.Y.; Rhee, S.K.: Characteristics of levan fructotransferase from Arthrobacter ureafaciens K2032 and difructose anhydride IV formation from levan. Enzyme Microb. Technol., 27, 212-218 (2000) [2] Jang, K.H.; Ryu, E.J.; Park, B.S.; Song, K.B.; Kang, S.A.; Kim, C.H.; Uhm, T.B.; Park, Y.I.; Rhee, S.K.: Levan fructotransferase from Arthrobacter oxydans, J17-21 catalyzes the formation of the di-d-fructose dianhydride IV from levan. J. Agric. Food Chem., 51, 2632-2636 (2003) [3] Saito, K.; Tomita, F.: Difructose anhydrides: Their mass-production and physiological functions. Biosci. Biotechnol. Biochem., 64, 1321-1327 (2000) [4] Tanaka, K.; Yamaguchi, F.; Kusui, S.: Action of levan fructotransferase of Arthrobacter ureafaciens on levan and phlein. Agric. Biol. Chem., 53, 1203-1211 (1989) [5] Tanaka, K.; Akai, T.: Action of levan fructotransferase of Arthrobacter ureafaciens on a mixture of branched levanpentasaccharides. Biosci. Biotechnol. Biochem., 56, 814-815 (1992) [6] Saito, K.; Goto, H.; Yokota, A.; Tomita, F.: Purification of levan fructotransferase from Arthrobacter nicotinovorans GS-9 and production of DFA IV from levan by the enzyme. Biosci. Biotechnol. Biochem., 61, 1705-1709 (1997) [7] Saito, K.; Yokota, A.; Tomita, F.: Molecular cloning of levan fructotransferase gene from Arthrobacter nicotinovorans GS-9 and its expression in Escherichia coli. Biosci. Biotechnol. Biochem., 61, 2076-2079 (1997) [8] Yang, S.J.; Park, N.H.; Lee, T.H.; Cha, J.: Expression, purification and characterization of a recombinant levan fructotransferase. Biotechnol. Appl. Biochem., 35, 199-203 (2002) [9] Lim, S.; Lee, K.-Y.; Lee, Y.-B.; Song, K.-B.: Immobilization of levan fructotransferase for the production of di-fructose anhydride from levan. Biotechnol. Lett., 23, 1335-1339 (2001) [10] Lee, J.; Saraswat, V.; Koh, I.; Song, K.B.; Park, Y.H.; Rhee, S.K.: Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli. FEMS Microbiol. Lett., 195, 127-132 (2001)

139

Levan fructotransferase (DFA-IV-forming)

4.2.2.16

[11] Cha, J.; Park, N.H.; Yang, S.J.; Lee, T.H.: Molecular and enzymatic characterization of a levan fructotransferase from Microbacterium sp. AL-210. J. Biotechnol., 91, 49-61 (2001) [12] Baciu, I.E.; Joerdening, H.J.; Seibel, J.; Buchholz, K.: Investigations of the transfructosylation reaction by fructosyltransferase from B. subtilis NCIMB 11871 for the synthesis of the sucrose analogue galactosyl-fructoside. J. Biotechnol., 116, 347-357 (2004) [13] Kim, C.H.; Jang, E.K.; Kim, S.H.; Jang, K.H.; Kang, S.A.; Song, K.B.; Kwon, O.S.; Rhee, S.K.: Molecular cloning of levan fructotransferase gene from Arthrobacter ureafaciens K2032 and its expression in Escherichia coli for the production of difructose dianhydride IV. Lett. Appl. Microbiol., 40, 228234 (2005)

140

Inulin fructotransferase (DFA-I-forming)

4.2.2.17

1 Nomenclature EC number 4.2.2.17 Systematic name 2,1-b-d-fructan lyase (a-d-fructofuranose-b-d-fructofuranose-1,2’:2,1’-dianhydride-forming) Recommended name inulin fructotransferase (DFA-I-forming) Synonyms EC 2.4.1.200 (formerly) inulin d-fructosyl-d-fructosyltransferase (1,2’:2’,1-dianhydride-forming) inulin fructotransferase (DFA I-producing) <2> [6] inulin fructotransferase (DFA-I-producing) inulin fructotransferase (depolymerizing, difructofuranose-1,2’:2’,1-dianhydride-forming) Additional information CAS registry number 125008-19-7

2 Source Organism <1> Arthrobacter globiformis (no sequence specified) [1, 2, 4] <2> Arthrobacter ureafaciens (no sequence specified) [5, 6] <3> Arthrobacter (no sequence specified) [3]

3 Reaction and Specificity Catalyzed reaction produces a-d-fructofuranose b-d-fructofuranose 1,2’:2,1’-dianhydride (DFA I) by successively eliminating the diminishing (2!1)-b-d-fructan (inulin) chain from the terminal d-fructosyl-d-fructosyl disaccharide Reaction type d-fructosyl-d-fructofuranosyl group transfer

141

Inulin fructotransferase (DFA-I-forming)

4.2.2.17

Natural substrates and products S inulin <1, 3> (Reversibility: ?) [1, 2, 3, 4] P di-d-fructofuranose-1,2’:2,1’-dianhydride <1, 3> (<1, 3> i.e. DFA I [1, 2, 3, 4]) [1, 2, 3, 4] Substrates and products S inulin <1, 2, 3> (Reversibility: ?) [1, 2, 3, 4, 5, 6] P di-d-fructofuranose-1,2’:2,1’-dianhydride <1, 3> (<1, 3> i.e. DFA I [1, 2, 3, 4]) [1, 2, 3, 4] Inhibitors Co2+ <1> (<1> weak [4]) [4] Cu2+ <1> (<1> weak [4]) [4] Fe3+ <1> (<1> 1 mM, strong [4]) [4] Hg2+ <1> (<1> 1 mM, strong [4]) [4] Ni2+ <1> (<1> weak [4]) [4] Activating compounds EDTA <1> (<1> slight stimulation [4]) [4] Specific activity (U/mg) 127 <2> [6] 172 <2> [5] Km-Value (mM) 4 <2> (inulin, <2> pH 5.5, 45 C [5]) [5] pH-Optimum 5.5 <2> [5, 6] 6 <1, 3> [3, 4] pH-Range 3.5-8 <1> (<1> 10% of maximal activity at pH 3.5, 25% of maximal activity at pH 8.0 [4]) [4] 4.5-7 <2> (<2> pH 4.5: about 50% of maximal activity, pH 7.0: about 50% of maximal activity [6]) [6] Temperature optimum ( C) 40 <1> [4] 45 <2> [5, 6] 50 <3> [3] Temperature range ( C) 30 <1> (<1> 80% of maximal activity at 30 C, 20% of maximal activity at 65 C [4]) [4] 30-60 <2> (<2> 30 C: about 70% of maximal activity, 60 C: about 45% of maximal activity [6]) [6] 30-65 <2> (<2> 30 C: about 80% of maximal activity [5]) [5]

142

4.2.2.17

Inulin fructotransferase (DFA-I-forming)

4 Enzyme Structure Molecular weight 39000 <1> (<1> SDS-PAGE [2]) [2] 40000 <3> (<3> SDS-PAGE and gel filtration [3]) [3] 41600 <1> (<1> amino acid analysis [2]) [2] 46000 <1> (<1> gel filtration [2,4]) [2, 4] 60000 <2> (<2> gel filtration [6]) [6] 61000 <2> (<2> gel filtration [5]) [5] Subunits dimer <2> (<2> 2 * 38000, SDS-PAGE [5]; <2> 2 * 37000, SDS-PAGE [6]) [5, 6] monomer <1, 3> (<1> 1 * 46000, gel filtration [4]; <3> 1 * 40000, gel filtration [3]) [3, 4]

5 Isolation/Preparation/Mutation/Application Source/tissue culture supernatant <2> [6] Localization extracellular <1> [2] Purification <1> (partial) [4] <2> [5, 6] Cloning <1> [1] <1> (expression in Escherichia coli MV1184) [2] Application nutrition <3> (<3> di-d-fructofuranose-1,2:2,1-dianhydride is expected to be useful as a low-calorie sweetener [3]) [3]

6 Stability pH-Stability 3 <1> (<1> stable at 25 C [4]) [4] 4 <3> (<3> 30 C, 30 min., stable [3]) [3] Temperature stability 30 <1> (<1> stable, rapid inactivation above 80 C [4]) [4] 70 <3> (<3> pH 6, 30 min., 80% activity [3]) [3] 75 <2> (<2> pH 5.5, 1 h, stable up to [5]; <2> pH 5.5, 20 min, stable up to [6]) [5, 6]

143

Inulin fructotransferase (DFA-I-forming)

4.2.2.17

80 <2, 3> (<3> 30 min., complete activity loss [3]; <2> pH 5.5, 1 h, about 20% loss of activity [5]; <2> pH 5.5, 20 min, complete inactivation [6]) [3, 5, 6] 85 <2> (<2> pH 5.5, 1 h, complete inactivation [5]) [5]

References [1] Haraguchi, K.; Hayashi, K.; Yanagimoto, M.: Cloning of the gene of DFA I oligosaccharide-producing enzyme. Jpn. Agric. Res. Q, 32, 1-5 (1998) [2] Haraguchi, K.; Seki, K.; Kishimoto, M.; Nagata, T.; Kasumi, T.; Kainuma, K.; Kobayashi, S.: Cloning and nucleotide sequence of the inulin fructotransferase (DFA I-producing) gene of Arthrobacter globiformis S14-3. Biosci. Biotechnol. Biochem., 59, 1809-1812 (1995) [3] Ueda, M.; Sashida, R.; Morimoto, Y.; Ohkishi, H.: Purification of inulin fructotransferase (DFA I-producing) from Arthrobacter sp. MCI2493 and production of DFA I from inulin by the enzyme. Biosci. Biotechnol. Biochem., 58, 574-575 (1994) [4] Seki, K.; Haraguchi, K.; Kishimoto, M.; Koboyashi, S.; Kainuma, K.: Purification and properties of a novel inulin fructotransferase (DFA I-producing) from Arthrobacter globiformis S14-3. Agric. Biol. Chem., 53, 2089-2094 (1989) [5] Haraguchi, K.; Yoshida, M.; Ohtsubo, K.-i.: Purification and properties of a heat-stable inulin fructotransferase from Arthrobacter ureafaciens. Biotechnol. Lett., 25, 1049-1053 (2003) [6] Haraguchi, K.; Yoshida, M.; Yamanaka, T.; Ohtsubo, K.-i.: Purification and characterization of a heat stable inulin fructotransferase (DFA I-producing) from Arthrobacter pascens a62-1. Carbohydr. Polym., 53, 501-505 (2003)

144

Inulin fructotransferase (DFA-III-forming)

4.2.2.18

1 Nomenclature EC number 4.2.2.18 Systematic name 2,1-b-d-fructan lyase (a-d-fructofuranose-b-d-fructofuranose-1,2’:2,3’-dianhydride-forming) Recommended name inulin fructotransferase (DFA-III-forming) Synonyms EC 2.4.1.93 IFTase <3> [2] IFTaseIII <2> [14] fructotransferase, inulin (depolymerizing) inulase II inulin d-fructosyl-d-fructosyltransferase (1,2’:2,3’-dianhydride-forming) inulin fructotransferase (DFA III-producing) <3> [15] inulin fructotransferase (DFA-III-producing) inulin fructotransferase (DFAIII-producing) <2> [14] inulin fructotransferase (depolymerizing) inulin fructotransferase (depolymerizing, difructofuranose-1,2’:2,3’-dianhydride-forming) inulinase II Additional information CAS registry number 50936-42-0

2 Source Organism <1> <2> <3> <4> <5> <6> <7>

Bacillus sp. (no sequence specified) [5] Arthrobacter globiformis (no sequence specified) [1, 3, 10, 14] Arthrobacter sp. (no sequence specified) [1, 2, 3, 4, 6, 8, 15] Streptomyces sp. (no sequence specified) [1, 13] Arthrobacter ureafaciens (no sequence specified) [7,11] Flavobacterium sp. (no sequence specified) [12] Arthrobacter ilicis (no sequence specified) [9]

145

Inulin fructotransferase (DFA-III-forming)

4.2.2.18

3 Reaction and Specificity Catalyzed reaction produces a-d-fructofuranose b-d-fructofuranose 1,2’:2,3’-dianhydride (DFA III) by successively eliminating the diminishing (2!1)-b-d-fructan (inulin) chain from the terminal d-fructosyl-d-fructosyl disaccharide Reaction type hexosyl group transfer Natural substrates and products S inulin <2, 3, 6, 7> (<2,7> inulin decomposition [9,10]) (Reversibility: ?) [4, 9, 10, 12] P di-d-fructofuranose 1,2’:2,3’ dianhydride <2, 3, 6, 7> [4, 9, 10, 12] Substrates and products S 2,1-b-linked fructose oligosaccharides <5> (<5> with the exception of inulobiose [11]) (Reversibility: ?) [11] P ? S inulin <2, 3, 4> (<2, 3, 4> cf. EC 2.4.1.200 [1, 3, 13]) (Reversibility: ?) [1, 3, 13, 15] P di-d-fructofuranose 1,2’:2,1’ dianhydride <2, 3, 4> (<2, 3, 4> i.e. DFA I [1, 3, 13]; <3> other products are: nystose and 1-F-fructofuranosyl-nystose [1]) [1, 3, 13] S inulin <1, 2, 3, 5, 6, 7> (<5> the enzyme attacks 2,1-b-linked fructan molecules from the nonreducing fructose ends and requires the presence of at least 2 adjacent 2,1-b-fructofuranosyl linkages [11]; <3> other substrates: sucrose, 1-kestose, nystose, 1-F-fructofuranosyl-nystose, raffinose, melibiose, melezitose, stachyose, bacterial levans [8]; <2,7> inulin decomposition [9,10]) (Reversibility: ?) [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12] P di-d-fructofuranose 1,2’:2,3’ dianhydride <1, 2, 3, 5, 6, 7> (<3> after exhaustive digestion of inulin nystose and 1-F-fructofuranosyl-nystose are produced in addition to di-d-fructofuranose 1,2:2,3 dianhydride [8]; <5> fructose-glucose oligosaccharides: O-b-d-fructofuranosyl-2,1-O-b-d-fructofuranosyl a-d-glucopyranoside (1-kestose) [11]; <1, 3, 6> i.e. DFA III [2, 3, 4, 5, 6, 12]; <1> other products through exhaustive enzymic digestion of inulin: 1-kestose, 1-nystose, and 1-F-fructofuranosyl-nystose [5]; <5> O-b-d-fructofuranosyl-[2,1-O-b-d-fructofuranosyl]2 a-d-glucopyranoside [11]; <5> O-b-d-fructofuranosyl-[2,1-O-b-d-fructofuranosyl]3 ad-glucopyranoside [11]; <3, 5, 6> + oligosaccharides, small amount [3, 6, 7, 8, 11, 12]) [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12] S Additional information <5> (<5> 2,6-b-fructans are no substrates [11]) (Reversibility: ?) [11] P ? Inhibitors Ba2+ <1> (<1> slight inhibition [5]) [5] Cu2+ <1, 4, 5, 7> (<1> strong inhibition at 1 mM [5]) [5, 7, 9, 13]

146

4.2.2.18

Inulin fructotransferase (DFA-III-forming)

Fe2+ <1> (<1> strong inhibition at 1 mM [5]) [5] Hg2+ <1, 5, 7> (<1,5> complete inhibition at 1 mM [5,7]) [5, 7, 9] Ni2+ <1> (<1> slight inhibition [5]) [5] Pb2+ <5> (<5> weak [7]) [7] Additional information <1, 5> (<1,5> no effect of EDTA and cysteine [5,7]) [5, 7] Activating compounds Additional information <5> (<5> no effect of EDTA and cysteine [7]) [7] Metals, ions Additional information <1, 4, 5, 7> (<1> no effect of Mg2+ and Co2+ [5]; <4,7> no or little affected by various cations [9,13]; <5> no effect of Ca2+ , Co2+, Mg2+ , Mn2+ , Zn2+ [7]) [5, 7, 9, 13] Specific activity (U/mg) 45.8 <1> (<1> purified enzyme [5]) [5] 81.5 <2> (<2> purified enzyme [1]) [1] 256 <4> (<4> purified enzyme [1,13]) [1, 13] 294 <2> (<2> purified enzyme [10]) [10] 384 <3> (<3> purified enzyme [1]) [1] 604 <3> (<3> purified enzyme [8]) [8] 740 <3> (<3> purified recombinant enzyme [2]) [2] 853 <7> (<7> purified enzyme [9]) [9] 933 <3> [15] Additional information <5, 6> [7, 12] Km-Value (mM) 0.8 <3> (inulin, <3> with MW of inulin taken as 5000 Da [8]) [8] 5.4 <1> (inulin, <1> with MW of inulin taken as 5000 Da [5]) [5] 10 <3> (inulin, <3> pH 5.5, 55 C [15]) [15] pH-Optimum 5 <2> [10] 5.5 <3, 7> [8, 9] 5.5-6 <3> [15] 6 <1, 2, 3, 4, 5> (<3,5> assay at [2,11]) [1, 2, 5, 6, 7, 11, 13] Additional information <2, 3> (<2> pI: 5.1 [10]; <3> strain A-6, pI: 4.6 [6]; <3> strain H65-7, pI: 4.7 [8]) [6, 8, 10] pH-Range 3-8 <2> [10] 3-9 <4> [13] 4-8 <3> (<3> about 50% of activity maximum at pH 4.0 and pH 8.0 [8]; <3> pH 4.0: about 40% of maximal activity, pH 8.0: about 50% of maximal activity [15]) [8, 15] 4-9 <1> [5] 6-8 <5> [7]

147

Inulin fructotransferase (DFA-III-forming)

4.2.2.18

Temperature optimum ( C) 37 <5> (<5> assay at [11]) [11] 40 <1, 2> [1, 5] 50 <3, 5> [1, 7] 55 <2, 3, 4> [10, 13, 15] 60 <3, 7> [8, 9] 65 <4> [1] 70 <3> (<3> recombinant enzyme [2]) [2, 6] Temperature range ( C) 30 <1> (<1> 80% of maximal activity at 30 C and 60 C [5]) [5] 30-70 <3> (<3> 30 C: about 30% of maximal activity, 70 C: about 60% of maximal activity [15]) [15] 40 <3> (<3> about 50% of activity maximum at 40 C and 75 C [8]) [8]

4 Enzyme Structure Molecular weight 40000 <3> (<3> gel filtration [1]) [1] 45000 <2> (<2> SDS-PAGE [10]) [10] 46000 <2> (<2> gel filtration [1]) [1] 50000 <2, 7> (<2,7> gel filtration [9,10]) [9, 10] 70000 <4> (<4> gel filtration [1,13]) [1, 13] 73000 <3> (<3> gel filtration [15]) [15] 100000 <3> (<3> gel filtration [8]) [8] 145000 <3> (<3> gel filtration [6]) [6] Additional information <1, 2, 3> (<1> amino acid composition [5]; <1> Nterminal amino acid sequence [5]; <2,3> amino acid sequence, comparison [3]; <3> strain A-6, N-terminal amino acid sequence [6]) [3, 5, 6] Subunits ? <1, 3> (<1> x * 62000, SDS-PAGE [5]; <3> 2 * 43000, SDS-PAGE [15]; <3> x * 46500, SDS-PAGE [2]; <3> x * 49000, SDS-PAGE [3]) [2, 3, 5, 15] dimer <3, 4, 7> (<7> 2 * 27000, SDS-PAGE [9]; <3> 2 * 49000, SDS-PAGE [8]; <4> 2 * 36000, SDS-PAGE [1,13]) [1, 8, 9, 13] monomer <2, 3> (<3> 1 * 40000, SDS-PAGE [1]; <2> 1 * 45000, SDS-PAGE [10]; <2> 1 * 46000, SDS-PAGE [1]) [1, 10] trimer <2, 3> (<2> 3 * 43000, SDS-PAGE [14]; <3> 3 * 49000, SDS-PAGE [6]) [6, 14]

5 Isolation/Preparation/Mutation/Application Source/tissue culture medium <1, 3, 4, 5, 6, 7> [1, 3, 5, 6, 7, 8, 9, 12, 13] culture supernatant <3> [15]

148

4.2.2.18

Inulin fructotransferase (DFA-III-forming)

Localization extracellular <1, 3, 4, 5, 6, 7> [1, 3, 5, 6, 7, 8, 9, 12, 13] Purification <1> [5] <2> [1, 10] <2> (recombinant) [14] <3> [15] <3> (enzyme from strain A-6 recombinant in Escherichia coli) [2] <3> (from strain H65-7) [3, 8] <3> (from strain MCI-2493) [1] <3> (large scale, flocculation from fermenter broth after disruption of recombinant Escherichia coli cells, expressing the enzyme intracellularly) [4] <3> (strain A-6) [6] <4> [1, 13] <5> [7] <7> [9] Crystallization <2> (crystals are obtained at 20 C by hanging drop vapour-diffusion method using 0.1 M Na HEPES pH 7.5 buffer containing 1.5 M lithium sulfate as a precipitant. Crystals of the recombinant wild-type enzyme diffract to better than 1.5 A at -173 C. The crystals belong to space group R32, with unit-cell parameters a = b = 90.02 A, c = 229.82 A in the hexagonal axes. Selenomethionine-derivative crystals belong to a different space group, C2, with unit-cell parameters a = 159.32, cb = 91.92, c = 92.58 A, b = 125.06. The C2 selenomethionine-derived crystal contains three molecules per asymmetric unit) [14] Cloning <2> (overexpression in Escherichia coli) [14] <3> (functional expression of gene ift, strain H65-7, in Escherichia coli strain JM109, DNA sequence determination and analysis, most of the enzyme activity is intracellular) [3] <3> (overexpression in Escherichia coli XL-1 blue) [4] <3> (strain A-6, functional expression in Escherichia coli DH5a/pDFE) [2] Engineering G221R <3> (<3> point-mutation introduced via error-prone PCR, increase in activity of 35% [4]) [4] Additional information <3> (<3> immobilization of enzyme on alginate beads lowered the activity [4]; <3> genetic engineering of the enzyme for enlarged thermotolerance in large scale production of DFA III, recombinant expression in Escherichia coli, elimination of sequence coding for a signalpeptide: 18% secretion, 82% intracellular localisation of the enzyme in Escherichia coli [4]) [4]

149

Inulin fructotransferase (DFA-III-forming)

4.2.2.18

Application biotechnology <3> (<3> improved enzyme for large scale production of lowcalorie sweet food additive [1]; <3> large scale production of sweet food additive DFA III via recombinant expression in E. coli, genetic engineering of the enzyme for enlarged thermotolerance, immobilization of the enzyme on alginate beads [4]) [1, 4]

6 Stability pH-Stability 4 <1, 2, 3, 5, 7> (<5,7> stable [7,9]; <2> 25 C, 24 h, stable [10]; <3> 30 C, 30 min, stable [1]; <1> 80% of maximal activity at pH 4.0 and pH 7.0 [5]) [1, 5, 7, 9, 10] 4.5 <3, 4> (<3,4> stable [8,13]) [8, 13] Temperature stability 50 <5> (<5> stable below [7]) [7] 60 <1, 5> (<1> stable up to [5]; <5> and above, rapidly inactivated [7]) [5, 7] 65 <4> (<4> stable up to [13]) [13] 70 <3, 7> (<3> stable up to [8]; <7> pH 7.0, 30 min, stable up to [9]; <3> more than 80% activity remains after 30 min, pH 6.0 [1]; <3> recombinant enzyme, stable for 5 h [2]) [1, 2, 8, 9] 75 <2> (<2> pH 5.0, 20 min, stable [10]) [10] 80 <2, 3> (<3> most of the activity is lost after 30 min [1]; <2> pH 5.0, rapid inactivation [10]; <3> recombinant enzyme, 65% remaining activity after 30 min [2]; <3> 1 h, pH 5.5, stable up to [15]) [1, 2, 10, 15] 85 <3> (<3> 1 h, pH 5.5, about 90% loss of activity [15]) [15] Storage stability <5>, refrigerator, pH 6.0, under toluene, stable for a few months [7]

References [1] Ueda, M.; Sashida, R.; Morimoto, Y.; Ohkishi, H.: Purification of inulin fructotransferase (DFA I-producing) from Arthrobacter sp. MCI2493 and production of DFA I from inulin by the enzyme. Biosci. Biotechnol. Biochem., 58, 574-575 (1994) [2] Kim, H.Y.; Kim, I.H.; Choi, Y.J.: An efficient purification with a high recovery of the inulin fructotransferase of Arthrobacter sp. A-6 from recombinant Escherichia coli. Biotechnol. Lett., 22, 291-293 (2000) [3] Sakurai, H.; Yokota, A.; Tomita, F.: Molecular cloning of an inulin fructotransferase (depolymerizing) gene from Arthrobacter sp. H65-7 and its expression in Escherichia coli. Biosci. Biotechnol. Biochem., 61, 87-92 (1997) [4] Jahnz, U.; Schubert, M.; Baars-Hibbe, H.; Vorlop, K.D.: Process for producing the potential food ingredient DFA III from inulin: screening, genetic

150

4.2.2.18

[5] [6] [7] [8] [9] [10]

[11] [12] [13]

[14]

[15]

Inulin fructotransferase (DFA-III-forming)

engineering, fermentation and immobilisation of inulase II. Int. J. Pharm., 256, 199-206 (2003) Kang, S.I.; Kim, W.P.; Chang, Y.J.; Kim, S.I.: Purification and properties of inulin fructotransferase (DFA III-producing) from Bacillus sp. snu-7. Biosci. Biotechnol. Biochem., 62, 628-631 (1998) Park, J.B.; Choi, Y.J.: Purification and characterization of inulin fructotransferase (depolymerizing) from Arthrobacter sp. A-6. J. Microbiol. Biotechnol., 6, 402-406 (1996) Uchiyama, T.; Niwa, S.; Tanaka, K.: Purification and properties of Arthrobacter ureafaciens inulase II. Biochim. Biophys. Acta, 315, 412-420 (1973) Yokota, A.; Enomoto, K.; Tomita, F.: Purification and properties of inulin fructotransferase (depolymerizing) from Arhrobacter sp. H65-7. J. Ferment. Bioeng., 72, 262-265 (1991) Kawamura, M.; Takahashi, S.; Uchiyama, T.: Purification and some properties of inulin fructotransferase (depolymerizing) from Arhrobacter ilicis. Agric. Biol. Chem., 52, 3209-3210 (1988) Haraguchi, K.; Kishimoto, M.; Seki, K.; Hayashi, K.; Kobayashi, S.; Kainuma, K.: Purification and properties of inulin fructotransferase (depolymerizing) from Arhrobacter globiformis C11-1. Agric. Biol. Chem., 52, 291-292 (1988) Uchiyama, T.: Action of Arthrobacter ureafaciens inulinase II on several oligofructans and bacterial levans. Biochim. Biophys. Acta, 397, 153-163 (1975) Cho, C.M.; Lim, Y.S.; Kang, S.K.; Jang, K.L.; Lee, T.H.: Production of inulin fructotransferase(depolymerizing) from Flavobacterium sp. LC-413. J. Food Sci. Nutr., 1, 121-126 (1996) Kushibe, S.; Sashida, R.; Morimoto, Y.; Ohkishi, H.: Purification and characterization of a di-d-fructofuranose 2’,1;2,1’-dianhydride producing enzyme from Streptomyces sp. MCI-2524. Biosci. Biotechnol. Biochem., 57, 2054-2058 (1993) Momma, M.; Fujimoto, Z.; Maita, N.; Haraguchi, K.; Mizuno, H.: Expression, crystallization and preliminary X-ray crystallographic studies of Arthrobacter globiformis inulin fructotransferase. Acta Crystallogr. Sect. D, D59, 2286-2288 (2003) Haraguchi, K.; Yoshida, M.; Ohtsubo, K.i.: Thermostable inulin fructotransferase (DFA III-producing) from Arthrobacter sp. L68-1. Carbohydr. Polym., 59, 411-416 (2005)

151

Chondroitin B lyase

4.2.2.19

1 Nomenclature EC number 4.2.2.19 Systematic name chondroitin B lyase Recommended name chondroitin B lyase Synonyms ChnB <2> [3] ChonB <4> [8] chondroitinase B <1, 2, 4> [1, 2, 3, 4, 5, 6, 7, 8, 10] lyase, chondroitin B <2> [7] CAS registry number 52227-83-5

2 Source Organism <1> Mammalia (no sequence specified) [6] <2> Flavobacterium heparinum (no sequence specified) [1, 5] <3> Flavobacterium heparinum (UNIPROT accession number: Q46079) [2, 3, 4, 7, 9, 10] <4> Pedobacter heparinus (UNIPROT accession number: Q46079) [8]

3 Reaction and Specificity Catalyzed reaction eliminative cleavage of dermatan sulfate containing 1,4-b-d-hexosaminyl and 1,3-b-d-glucurosonyl or 1,3-a-l-iduronosyl linkages to disaccharides containing 4-deoxy-b-d-gluc-4-enuronosyl groups to yield a 4,5-unsaturated dermatan-sulfate disaccharide (DUA-GalNAC-4S) (<3> acts on dermatan sulfate as sole substrate, active site structure involving Lys250, Arg271, His272, and Glu333, substrate binding mechanism involving Arg363 and Arg364 [7]; <3> acts on dermatan sulfate as sole substrate, catalytic mechanism, Glu333 is involved, substrate binding mechanism involving Arg318 and Arg364 interacting with the sulfate group [10]; <4> acts on dermatan sulfate as sole sub-

152

4.2.2.19

Chondroitin B lyase

strate, enzyme cleaves the b(1,4)-linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides, calcium-dependent catalytic mechanism, active site structure [8]) Reaction type elimination (<3> C-O bond cleavage [7]) Natural substrates and products S dermatan sulfate <1> (<1> substrate is involved in activities related to metastasis [6]) (Reversibility: ?) [6] P ? <1> [6] S Additional information <1> (<1> enzyme inhibits endothelial and melanoma proliferation and invasion, but to a lesser extent than chondroitin AC lyase, EC 4.2.2.5, enzyme has no effect on gelatinase excretion by melanoma cells, enzyme does not activate caspase-3 activity and apoptosis in melanoma and endothelial cells [6]) (Reversibility: ?) [6] P ? <1> [6] Substrates and products S dermatan sulfate <2> (Reversibility: ?) [1] P oligosaccharides <2> [1] S dermatan sulfate <2> (Reversibility: ?) [10] P disaccharides <2> (<2> structures [10]) [10] S dermatan sulfate <2> (Reversibility: ?) [2] P unsaturated oligosaccharides <2> [2] S dermatan sulfate <1, 2, 4> (<4> strictly specific for [8]; <2> analysis of interactions between enzyme and substrate [7]; <1> substrate is involved in activities related to metastasis [6]) (Reversibility: ?) [3, 4, 5, 6, 7, 8, 9] P ? <1, 2, 4> [3, 4, 5, 6, 7, 8, 9] S Additional information <1> (<1> enzyme inhibits endothelial and melanoma proliferation and invasion, but to a lesser extent than chondroitin AC lyase, EC 4.2.2.5, enzyme has no effect on gelatinase excretion by melanoma cells, enzyme does not activate caspase-3 activity and apoptosis in melanoma and endothelial cells [6]) (Reversibility: ?) [6] P ? <1> [6] Inhibitors EGTA <4> (<4> complete inhibition [8]) [8] glucose <2> (<2> represses enzyme induction in cells by chondroitin sulfate A [3]) [3] lauric acid <2> [9] linoleic acid <2> (<2> strong inhibition [9]) [9] linolenic acid <2> (<2> strong inhibition [9]) [9] myristic acid <2> [9] oleic acid <2> (<2> strong inhibition [9]) [9] palmitic acid <2> [9] palmitoleic acid <2> [9] arachidic acid <2> [9] behenic acid <2> [9]

153

Chondroitin B lyase

4.2.2.19

capric acid <2> [9] docosenoic acid <2> (<2> strong inhibition [9]) [9] eicosadienoic acid <2> [9] eicosanoic acid <2> (<2> strong inhibition [9]) [9] eicosapentaenoic acid <2> [9] eicosatetraenoic acid <2> [9] eicosatrienoic acid <2> (<2> noncompetitive [9]) [9] elaidic acid <2> (<2> strong inhibition [9]) [9] methyloleate <2> [9] myristoleic acid <2> [9] nervonic acid <2> (<2> strong inhibition [9]) [9] petroselinic acid <2> (<2> strong inhibition [9]) [9] ricinoleic acid <2> [9] stearic acid <2> [9] vaccenic acid <2> (<2> strong inhibition [9]) [9] Additional information <2> (<2> degree of inhibition by fatty acids depends on the chain length and the number of unsaturated bonds as well as the stereochemistry, overview [9]) [9] Activating compounds chondroitin sulfate A <3> (<3> induces enzyme expression in cells [3]) [3] Metals, ions Ca2+ <4> (<4> dependent on, required for catalysis, activates, protein-Ca2+ oligosaccharide complex [8]) [8] Additional information <3> (<3> no bound Ca2+ [7]) [7] Turnover number (min–1) 4.6 <3> (dermatan sulfate, <3> mutant E333A, pH 8.0, 30 C [7]) [7] 29 <3> (dermatan sulfate, <3> mutant H272A, pH 8.0, 30 C [7]) [7] 87-190 <3> (dermatan sulfate, <3> recombinant enzyme, pH 8.0, 30 C, dependent on expression system [4]) [4] 190 <3> (dermatan sulfate, <3> wild-type enzyme, pH 8.0, 30 C [7]) [7] 210 <4> (dermatan sulfate, <4> recombinant wild-type enzyme, pH 8.0, 30 C, in presence of 0.01 mM Ca2+ [8]) [8] 404 <3> (dermatan sulfate, <3> mutant R363A, pH 8.0, 30 C [7]) [7] 410 <4> (dermatan sulfate, <4> recombinant wild-type enzyme, pH 8.0, 30 C, in presence of 5 mM Ca2+ [8]) [8] Specific activity (U/mg) 80.8 <3> (<3> purified recombinant enzyme [4]) [4] Additional information <4> [8] Km-Value (mM) 0.0012 <4> (dermatan sulfate, <4> recombinant wild-type enzyme, pH 8.0, 30 C, in presence of 5 mM Ca2+ [8]) [8] 0.0027 <2> (dermatan sulfate, <2> mutant H272A, pH 8.0, 30 C [7]) [7] 0.0028 <2> (dermatan sulfate, <2> mutant E333A, pH 8.0, 30 C [7]) [7]

154

4.2.2.19

Chondroitin B lyase

0.0028-0.0046 <2> (dermatan sulfate, <2> recombinant enzyme, pH 8.0, 30 C, dependent on expression system [4]) [4] 0.0043 <4> (dermatan sulfate, <4> recombinant wild-type enzyme, pH 8.0, 30 C, in presence of 0.01 mM Ca2+ [8]) [8] 0.0046 <2> (dermatan sulfate, <2> mutant R363A, pH 8.0, 30 C [7]; <2> wild-type enzyme, pH 8.0, 30 C [7]) [7] 0.0163 <2> (dermatan sulfate, <2> pH 8.0, 37 C [9]) [9] Additional information <2> (<2> kinetics [1]; <2> kinetics of wild-type and mutant enzymes [7]) [1, 7] Ki-Value (mM) 0.037 <3> (eicosatrienoic acid, <3> pH 8.0, 37 C [9]) [9] Additional information <3> [9] pH-Optimum 6.8-8 <3> (<3> broad optimum [10]) [10] 8 <3, 4> (<3,4> assay at [4,7,8]; <3> assay at, Tris-HCl [9]) [4, 7, 8, 9] Temperature optimum ( C) 30 <3, 4> (<3,4> assay at [4,7,8,9]) [4, 7, 8, 9]

4 Enzyme Structure Molecular weight 54070 <3> (<3> mass spectrometry [4]) [4] Subunits ? <2> (<2> x * 55000, SDS-PAGE, recombinant and native enzyme [3]; <2> x * 55200, SDS-PAGE [1]) [1, 3] Additional information <2> (<2> structural analysis [7]) [7] Posttranslational modification glycoprotein <3> (<3> a heptasaccharide glycosylation site attached to Ser234 [10]) [10]

5 Isolation/Preparation/Mutation/Application Source/tissue SK-MEL cell <1> (<1> melanoma cell line [6]) [6] endothelial cell <1> [6] Localization periplasm <3> [10] Purification <2> (purification based on affinity chromatography) [5] <2> (recombinant His-tagged enzyme from Escherichia coli, His-tag is removed, purified to homogeneity, 69fold) [4]

155

Chondroitin B lyase

4.2.2.19

<2> (recombinant enzyme comprising residues 25-506 overexpressed in Flavobacterium heparinum cells, to homogeneity) [2] <2> (to homogeneity) [1] Crystallization <3> (6-8 mg/ml purified recombinant enzyme, residues 25-506, in 20 mM Tris-HCl, pH 8.0, 1 mM sodium phosphate, pH 7.0, 3 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 0.001 mg/ml aprotinin, 0.001 mg/ml leupeptin, 0.001 mg/ml E64, hanging drop vapour diffusion method, 292 K, mixed with a double volume of reservoir solution containing 19% w/v PEG 8000, 100 mM bicine buffer, pH 9.0, or 100 mM Tris-HCl, pH 8.8, 0.15 M ammonium acetate, 15% v/v 2-methyl-2,4-pentanediol, drops are suspended over 1 ml of reservoir solution, crystals appear overnight, seeds are introduced into drops conisting of 0.002 ml protein solution, 6.1 mg/ml protein, and 0.004 ml reservoir solution containing 16.5% w/v PEG 8000, 0.1 M Tris-HCl, pH 8.8, 15% v/v 2methyl-2,4-pentanediol, 0.25 M ammonium acetate, 292 K, 2-3 weeks, X-ray diffraction structure determination and analysis at 2.20-2.28 A resolution) [2] <3> (purified recombinant enzyme, residues 25-506 corresponding to the full size mature enzyme, labeling and complexing of enzyme in crystals via soaking in cryoprotectant solution containing the heavy atom or disaccharide product in 22.5% w/v PEG 8000, 0.1 M Tris-HCl, pH 8.7, 15% v/v 2-methyl2,4-pentanediol, and 0.25 M ammonium acetate, equilibration over 1 ml reservoir solution, complexed with disaccharide product, X-ray diffraction structure determination and analysis at 1.7 A resolution, structure modeling of the right-handed parallel b-helix protein) [10] <4> (purified recombinant wild-type enzyme complexed with dermatan sulfate pentasaccharide and hexasaccharide, or chondroitin-4-sulfate tetrasacharide, X-ray diffraction structure determination and analysis at 1.7-1.8 A resolution, modeling of substrate binding in the active site groove) [8] Cloning <2> (expression in Escherichia coli as N-terminally His-tagged enzyme, 2 different expression systems) [4] <2> (gene cslB, expression in Escherichia coli without the N-terminal signal sequence, different expression plasmids constructed, expression in soluble and insoluble form) [3] <2> (overexpression of enzyme, residues 25-506, in Flavobacterium heparinum cells) [2] Engineering E243A <4> (<4> site-directed mutagenesis, Ca2+ -binding residue, about 3.6fold reduced activity compared to the wild-type enzyme [8]) [8] E243A/E245A <4> (<4> site-directed mutagenesis, Ca2+ -binding residue, about 4fold reduced activity compared to the wild-type enzyme [8]) [8] E245A <4> (<4> site-directed mutagenesis, Ca2+ -binding residue, about 6fold reduced activity compared to the wild-type enzyme [8]) [8] E333A <3> (<3> PCR site-directed mutagenesis, E333 is a key residue in catalysis, reduced activity [7]) [7]

156

4.2.2.19

Chondroitin B lyase

H272A <3> (<3> PCR site-directed mutagenesis, H272 is a key residue in catalysis, reduced activity [7]) [7] K250A <3> (<3> PCR site-directed mutagenesis, inactive mutant [7]) [7] N213Q <4> (<4> site-directed mutagenesis, Ca2+ -binding residue, about 6fold reduced activity compared to the wild-type enzyme [8]) [8] R271A <3> (<3> PCR site-directed mutagenesis, R271 is a key residue in catalysis [7]) [7] R271E <4> (<4> site-directed mutagenesis, active site mutant, catalytically inactive [8]) [8] R271K <4> (<4> site-directed mutagenesis, active site mutant, about 10fold reduced activity compared to the wild-type enzyme [8]) [8] R363A <3> (<3> PCR site-directed mutagenesis, R363 is a key residue in catalysis, 2fold increased activity [7]) [7] R364A <3> (<3> PCR site-directed mutagenesis, highly reduced activity, altered product profil, residue is involved in determination of substrate specificity [7]) [7]

6 Stability Temperature stability 37 <3> (<3> recombinant and native enzyme are completely stable [3]) [3] General stability information <3>, protease inhibitors like aprotinin, leupeptin or E64 are required for stability of the enzyme during storage at 277 K [2]

References [1] Gu, K.; Linhardt, R.L.; Laliberte, M.; Gu, K.; Zimmermann, J.: Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum. Biochem. J., 312, 569-577 (1995) [2] Li, Y.; Matte, A.; Su, H.; Cygler, M.: Crystallization and preliminary X-ray analysis of chondroitinase B from Flavobacterium heparinum. Acta Crystallogr. Sect. D, 55, 1055-1057 (1999) [3] Tkalec, A.L.; Fink, D.; Blain, F.; Zhang-Sun, G.; Laliberte, M.; Bennett, D.C.; Gu, K.; Zimmermann, J.J.F.; Su, H.: Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum. Appl. Environ. Microbiol., 66, 29-35 (2000) [4] Pojasek, K.; Shriver, Z.; Kiley, P.; Venkataraman, G.; Sasisekharan, R.: Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum. Biochem. Biophys. Res. Commun., 286, 343-351 (2001)

157

Chondroitin B lyase

4.2.2.19

[5] Ototani, N.; Yosizawa, Z.: Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B. Carbohydr. Res., 70, 295-306 (1979) [6] Denholm, E.M.; Lin, Y.Q.; Silver, P.J.: Anti-tumor activities of chondroitinase AC and chondroitinase B: inhibition of angiogenesis, proliferation and invasion. Eur. J. Pharmacol., 416, 213-221 (2001) [7] Pojasek, K.; Raman, R.; Kiley, P.; Venkataraman, G.; Sasisekharan, R.: Biochemical characterization of the chondroitinase B active site. J. Biol. Chem., 277, 31179-31186 (2002) [8] Michel, G.; Pojasek, K.; Li, Y.; Sulea, T.; Linhardt, R.J.; Raman, R.; Prabhakar, V.; Sasisekharan, R.; Cygler, M.: The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calciumdependent catalytic machinery. J. Biol. Chem., 279, 32882-32896 (2004) [9] Suzuki, K.; Terasaki, Y.; Uyeda, M.: Inhibition of hyaluronidases and chondroitinases by fatty acids. J. Enzyme Inhib. Med. Chem., 17, 183-186 (2002) [10] Huang, W.; Matte, A.; Li, Y.; Kim, Y.S.; Linhardt, R.J.; Su, H.; Cygler, M.: Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution. J. Mol. Biol., 294, 1257-1269 (1999)

158

Chondroitin-sulfate-ABC endolyase

4.2.2.20

1 Nomenclature EC number 4.2.2.20 Systematic name chondroitin-sulfate-ABC endolyase Recommended name chondroitin-sulfate-ABC endolyase Synonyms ChS ABC lyase I <3> [2] EC 4.2.2.4 (formerly) CAS registry number 9024-13-9

2 Source Organism <1> Proteus vulgaris (no sequence specified) [2] <2> Flavobacterium heparinum (no sequence specified) [1] <3> Proteus vulgaris NCTC 4636 (no sequence specified) [2]

3 Reaction and Specificity Catalyzed reaction endolytic cleavage of (1!4)-b-galactosaminic bonds between N-acetylgalactosamine and either d-glucuronic acid or l-iduronic acid to produce a mixture of D4 -unsaturated oligosaccharides of different sizes that are ultimately degraded to D4 -unsaturated tetra- and disaccharides (<1> related enzyme EC 4.2.2.21 [2]) Substrates and products S chondroitin <3> (<3> 32% of the rate with chondroitin-6-sulfate [2]) (Reversibility: ?) [2] P ? S chondroitin 4-sulfate <3> (<3> 121% of the rate with chondroitin-6-sulfate [2]) (Reversibility: ?) [2] P ?

159

Chondroitin-sulfate-ABC endolyase

4.2.2.20

S chondroitin sulfate D <3> (<3> 70% of the rate with chondroitin-6-sulfate [2]) (Reversibility: ?) [2] P ? S chondroitin sulfate E <3> (<3> 23% of the rate with chondroitin-6-sulfate [2]) (Reversibility: ?) [2] P ? S chondroitin sulfate proteoglycan <3> (<3> 70% of the rate with chondroitin-6-sulfate [2]) (Reversibility: ?) [2] P ? S chondroitin-6-sulfate <3> (Reversibility: ?) [2] P unsaturated sulfated disaccharides S dermatan sulfate <2> (Reversibility: ?) [1] P unsaturated sulfated disaccharide + oligosaccharides S dermatan sulfate <3> (<3> 55% of the rate with chondroitin-6-sulfate [2]) (Reversibility: ?) [2] P ? S hyaluronan <3> (<3> 1% of the rate with chondroitin-6-sulfate [2]) (Reversibility: ?) [2] P ? S Additional information <3> (<3> no substrate: keratan sulfate, heparin, heparan sulfate. Enzyme produces a wide range of differently sized oligosaccharides. After exhaustive degradation, end products are tetra- and disaccharides [2]) (Reversibility: ?) [2] P ? Inhibitors Cu2+ <3> (<3> 1 mM, 80% inhibition [2]) [2] Fe2+ <3> (<3> 1 mM, 80% inhibition [2]) [2] Ni2+ <3> (<3> 1 mM, 80% inhibition [2]) [2] Zn2+ <3> (<3> 1 mM, complete inhibition [2]) [2] Additional information <2, 3> (<2> not inhibitory: ammonium sulfate [1]; <3> not inhibitory: Ca2+ , Mg2+ , Ba2+ , Mn2+ [2]) [1, 2] Specific activity (U/mg) 310 <3> (<3> pH 8.0, 37 C [2]) [2] Km-Value (mM) 0.066 <3> (chondroitin-6-sulfate, <3> pH 8.0, 37 C [2]) [2] pH-Optimum 8 <3> [2] Temperature optimum ( C) 37 <3> [2]

160

4.2.2.20

Chondroitin-sulfate-ABC endolyase

5 Isolation/Preparation/Mutation/Application Purification <2> (isoform chondroitinase B, cell disruption by ultrasound, under mild conditions, solubilization of most of enzyme activity) [1] <3> [2]

6 Stability Temperature stability 40 <3> (<3> 30 min, 47% loss of activty [2]) [2]

References [1] Aguiarand, J.A.K.; Michelacci, Y.M.: Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography. Braz. J. Med. Biol. Res., 32, 545-550 (1999) [2] Hamai, A.; Hashimoto, N.; Mochizuki, H.; Kato, F.; Makiguchi, Y.; Horie, K.; Suzuki, S.: Two distinct chondroitin sulfate ABC lyases. An endoeliminase yielding tetrasaccharides and an exoeliminase preferentially acting on oligosaccharides. J. Biol. Chem., 272, 9123-9130 (1997)

161

Chondroitin-sulfate-ABC exolyase

4.2.2.21

1 Nomenclature EC number 4.2.2.21 Systematic name chondroitin-sulfate-ABC exolyase Recommended name chondroitin-sulfate-ABC exolyase Synonyms ChS ABC lyase II <1> [8] chondroitinase ABC <1> [5] chondroitinase AC <2> [5] chondroitinase-ABC <1> [5]

2 Source Organism <1> <2> <3> <4>

Proteus vulgaris (no sequence specified) [1, 3, 4, 5, 8, 9, 10, 11, 12] Flavobacterium heparinum (no sequence specified) [2, 5, 7, 11] Bacteroides thetaiotaomicron (no sequence specified) [6] Falvobacterium heparinum (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction exolytic cleavage of disaccharide residues from the non-reducing ends of both polymeric chondroitin sulfates and their oligosaccharide fragments (<1> related enzyme EC 4.2.2.20 [8]) Substrates and products S bovine nasal cartilage proteoglycan <1, 4> (Reversibility: ?) [1] P 2-acetamido-2-deoxy-3-0-(3-d-gluco-4-enepyranosyluronic acid)-4-0-sulfo-d-galactose + bovine nasal cartilage proteoglycan core S chondroitin <1, 2> (<1> 96% of the rate with chondroitin-6-sulfate [8]; <1> reaction is less efficient than for chondroitin sulfates [5]) (Reversibility: ?) [5, 8, 9] P ? S chondroitin 4-sulfate <2> (Reversibility: ?) [2] P unsaturated disaccharide

162

4.2.2.21

Chondroitin-sulfate-ABC exolyase

S chondroitin 4-sulfate <1> (<1> 83% of the rate with chondroitin-6-sulfate [8]) (Reversibility: ?) [8] P ? S chondroitin 6-sulfate <1, 2> (Reversibility: ?) [2, 9, 10] P unsaturated disaccharide S chondroitin sulfate <2> (Reversibility: ?) [7] P unsaturated sulfated disaccharide S chondroitin sulfate A <1> (Reversibility: ?) [12] P unsaturated sulfated disaccharide S chondroitin sulfate A <1, 2> (Reversibility: ?) [5] P D4;5 -unsaturated disaccharide S chondroitin sulfate A <3> (Reversibility: ?) [6] P ? S chondroitin sulfate B <1> (Reversibility: ?) [12] P unsaturated sulfated disaccharide (<1> contains 2-acetamido-2-deoxy-3O-(P-n-gluco-4-enepyranosyluronic acid)-4-O-sulfo-n-galactose [12]) S chondroitin sulfate B <1> (Reversibility: ?) [5] P D4;5 -unsaturated disaccharide S chondroitin sulfate C <1> (Reversibility: ?) [12] P unsaturated sulfated disaccharide S chondroitin sulfate C <1, 2> (Reversibility: ?) [5] P D4;5 -unsaturated disaccharide S chondroitin sulfate C <3> (Reversibility: ?) [6] P ? S chondroitin sulfate D <1> (Reversibility: ?) [12] P unsaturated sulfated disaccharide (<1> contains 2-acetamido-2-deoxy-3O-(P-n-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose [12]) S chondroitin sulfate D <1> (<1> 93% of the rate with chondroitin-6-sulfate [8]) (Reversibility: ?) [8, 9] P ? S chondroitin sulfate E <1> (Reversibility: ?) [12] P unsaturated sulfated disaccharide S chondroitin sulfate E <1> (<1> 90% of the rate with chondroitin-6-sulfate [8]) (Reversibility: ?) [8, 9] P ? S chondroitin sulfate proteoglycan <1> (<1> 67% of the rate with chondroitin-6-sulfate [8]) (Reversibility: ?) [8] P ? S chondroitin-4-sulfate <1, 4> (Reversibility: ?) [1] P 2-acetamido-2-deoxy-3-0-(3-d-gluco-4-enepyranosyluronic acid)-4-0-sulfo-d-galactose S chondroitin-6-sulfate <1> (Reversibility: ?) [8] P unsaturated sulfated disaccharides S dermatan sulfate <2> (Reversibility: ?) [2] P unsaturated disaccharide S dermatan sulfate <1> (<1> 77% of the rate with chondroitin-6-sulfate [8]) (Reversibility: ?) [8, 9, 10]

163

Chondroitin-sulfate-ABC exolyase

4.2.2.21

P ? S hyaluronan <1> (<1> 17% of the rate with chondroitin-6-sulfate [8]) (Reversibility: ?) [8, 9] P ? S hyaluronic acid <1, 2> (<1> bad substrate [5]) (Reversibility: ?) [5] P ? S Additional information <1, 2> (<2> no substrate: chondroitin sulfate B, keratosulfate, heparin, or heparitin sulfate [5]; <1> no substrate: keratan sulfate, heparin, heparan sulfate. Activity of enzyme increases with decreasing of chain size from poly-, hexa-, to tetrasaccharide substrates [8]; <1> no substrate: keratosulfate, heparin, or heparitin sulfate [5]) (Reversibility: ?) [5, 8] P ? Inhibitors Ca 2+ <2> (<2> 10 mM, partial inhibition [2]) [2] Cu2+ <2> [5] Fe 3+ <2> (<2> 10 mM, complete inhibition [2]) [2] Fe2+ <2> [5] heparin <2, 3> (<3> 50% inhibition at 0.050 mg/ml for enzyme I and 0.005 mg/ml for enzyme II [6]) [5, 6] Mn2+ <2> (<2> 10 mM, partial inhibition [2]) [2] N-desulfated heparin <3> (<3> 50% inhibition at 0.150 mg/ml for enzyme I and 0.025 mg/ml for enzyme II [6]) [6] NaCl <1> (<1> optimal concentration 62.5 mM for substrate chondroitin 6sulfate, 50% inhibition at 125 mM, optimal concentration 125 mM for substrate dermatan sulfate, 50% inhibition at 250 mM [9]) [9] phosphate <2> (<2> 10 mM, 10% residual activity [2]) [2] Zn2+ <1, 2> (<1> 1 mM, complete inhibition [8]) [5, 8] heparitin sulfate <2> [5] keratosulfate <2> [5] Additional information <1, 2> (<2> not inhibitory: ammonium sulfate [7]; <1> not inhibitory: Ni2+ , Fe2+ , Cu2+ , Ca2+ , Mg2+ , Ba2+ , Mn2+ [8]) [7, 8] Activating compounds NaCl <1> (<1> optimal concentration 62.5 mM for substrate chondroitin 6sulfate, inhibitory above 125 mM, optimal concentration 125 mM for substrate dermatan sulfate, inhibitory above 250 mM [9]) [9] phosphate <3> (<3> activity in 20 mM phosphate buffer is less than half of the activity in 50 mM phosphate buffer, maximum activity for isoenzyme I in 50 mM, isoenzyme II in 50-100 mM phosphate [6]) [6] sodium acetate <1> (<1> required [9]) [9] Metals, ions Ca2+ <2> [5] Mg2+ <2> [5] Mn2+ <2> [5]

164

4.2.2.21

Chondroitin-sulfate-ABC exolyase

Turnover number (min–1) 0.5 <1> (chondroitin 6-sulfate, <1> mutant Y508F, pH 8.0, 37 C [10]) [10] 1.8 <1> (dermatan sulfate, <1> mutant Y508F, pH 8.0, 37 C [10]) [10] 2.7 <1> (dermatan sulfate, <1> mutant R500A, pH 8.0 [9]; <1> mutant R500A, pH 8.0, 37 C [10]) [9, 10] 6.8 <1> (chondroitin 6-sulfate, <1> mutant R500A, pH 8.0, 37 C [10]) [10] 7 <1> (chondroitin 6-sulfate, <1> mutant R500A, pH 8.0 [9]) [9] 10 <1> (dermatan sulfate, <1> mutant H712A, pH 8.0 [9]) [9] 19 <1> (chondroitin 6-sulfate, <1> mutant H712A, pH 8.0 [9]) [9] 26.7 <1> (chondroitin 6-sulfate, <1> mutant E653Q, pH 8.0, 37 C [10]) [10] 66 <1> (dermatan sulfate, <1> mutant H561A, pH 8.0 [9]) [9] 87 <1> (dermatan sulfate, <1> mutant E653Q, pH 8.0, 37 C [10]) [10] 450 <1> (dermatan sulfate, <1> wild-type, pH 8.0 [9]; <1> wild-type, pH 8.0, 37 C [10]) [9, 10] 617 <1> (chondroitin 6-sulfate, <1> wild-type, pH 8.0 [9]; <1> wild-type, pH 8.0, 37 C [10]) [9, 10] 652 <1> (chondroitin 6-sulfate, <1> mutant H561A, pH 8.0 [9]) [9] 867 <1> (chondroitin 4-sulfate, <1> wild-type, pH 8.0 [9]) [9] Specific activity (U/mg) 1.45 <2> (<2> 30 C, pH 7.5 [2]) [2] 14.8 <1> (<1> substrate hyaluronan, pH 8.0 [9]) [9] 34 <1> (<1> pH 8.0, 37 C [8]) [8] 34.8 <1> (<1> substrate chondroitin sulfate E, pH 8.0 [9]) [9] 37.5 <1> (<1> pH 8.0, 37 C [5]) [5] 43 <2> (<2> pH 8.0, 37 C [5]) [5] 54.2 <1> (<1> substrate chondroitin sulfate D, pH 8.0 [9]) [9] 69.8 <1> (<1> substrate chondroitin, pH 8.0 [9]) [9] 122.3 <1> (<1> substrate dermatan sulfate, pH 8.0 [9]) [9] 174.6 <1> (<1> substrate chondroitin 6-sulfate, pH 8.0 [9]) [9] 290.6 <1> (<1> substrate chondroitin 4-sulfate, pH 8.0 [9]) [9] Additional information <1, 2, 4> (<1,4> colorimetric assay for enzyme activity [1]; <1,2> micromethods for the measurement of as little as 3 pg of chondroitin sulfate A, B, or C in mixtures with other mucopolysaccharides or in urine [11]) [1, 11] Km-Value (mM) 0.0012 <1> (chondroitin 6-sulfate, <1> wild-type, pH 8.0, 37 C [10]; <1> wild-type, pH 8.0 [9]) [9, 10] 0.0015 <1> (chondroitin 4-sulfate, <1> wild-type, pH 8.0 [9]) [9] 0.0025 <1> (dermatan sulfate, <1> wild-type, pH 8.0, 37 C [10]; <1> wildtype, pH 8.0 [9]) [9, 10] 0.0042 <1> (dermatan sulfate, <1> mutant E653Q, pH 8.0, 37 C [10]) [10] 0.0051 <1> (dermatan sulfate, <1> mutant H712A, pH 8.0 [9]) [9] 0.0061 <1> (chondroitin 6-sulfate, <1> mutant E653Q, pH 8.0, 37 C [10]) [10] 0.0069 <1> (dermatan sulfate, <1> mutant H561A, pH 8.0 [9]) [9] 0.0086 <1> (chondroitin 6-sulfate, <1> mutant H712A, pH 8.0 [9]) [9]

165

Chondroitin-sulfate-ABC exolyase

4.2.2.21

0.015 <1> (chondroitin 6-sulfate, <1> mutant H561A, pH 8.0 [9]) [9] 0.02 <1> (chondroitin 6-sulfate, <1> mutant R500A, pH 8.0 [9]; <1> mutant R500A, pH 8.0, 37 C [10]) [9, 10] 0.036 <1> (chondroitin 6-sulfate, <1> mutant Y508F, pH 8.0, 37 C [10]) [10] 0.036 <1> (dermatan sulfate, <1> mutant R500A, pH 8.0 [9]; <1> mutant R500A, pH 8.0, 37 C [10]) [9, 10] 0.049 <1> (dermatan sulfate, <1> mutant Y508F, pH 8.0, 37 C [10]) [10] 0.08 <1> (chondroitin-6-sulfate, <1> pH 8.0, 37 C [8]) [8] pH-Optimum 5 <2> (<2> substrate hyaluronic acid [5]) [5] 6 <2> (<2> substrate chondroitin [5]) [5] 6-7.5 <2> [2] 6.2 <1> (<1> substrate chondroitin [5]) [5] 6.6 <2> (<2> substrate chondroitin sulfate A, or C [5]) [5] 6.8 <1> (<1> substrate hyaluronic acid [5]) [5] 7.1 <3> (<3> isoenzyme I [6]) [6] 7.6 <3> (<3> isoenzyme II [6]) [6] 8 <1> (<1> substrate chondroitin sulfate A, B or C [5]) [5, 8, 9] 9 <1> (<1> inactive [9]) [9] Temperature optimum ( C) 30 <1, 2> (<1> and up to 37 C, substrate dermatan sulfate [9]) [2, 9] 37 <1, 2> (<1> substrate chondroitin 6-sulfate [9]) [5, 9] 40 <1> [8] Temperature range ( C) 37 <2> (<2> 15% residual activity [2]) [2]

4 Enzyme Structure Molecular weight 120000 <1> (<1> gel filtration [4]) [4] 150000 <1, 2> (<1,2> or slightly lower, gel filtration [5]) [5] Subunits ? <1, 3> (<3> x * 108000, isoenzyme I, x * 104000, isoenzyme II, SDS-PAGE [6]; <1> x * 110000, SDS-PAGE, x * 112614, calculated [9]) [6, 9] heterodimer <1> (<1> 1 * 86000 + 1 * 32000, SDS-PAGE [4]) [4] monomer <1, 2> (<1,2> 1 * 150000 or slightly lower, SDS-PAGE [5]) [5]

5 Isolation/Preparation/Mutation/Application Purification <1> [4, 5, 8] <2> [5]

166

4.2.2.21

Chondroitin-sulfate-ABC exolyase

<2> (isoform chondroitinase AC, cell disruption by ultrasound, under mild conditions, solubilization of most of enzyme activity) [7] Engineering E653A <1> (<1> no enzymic activity [9,10]) [9, 10] E653D <1> (<1> no enzymic activity [10]) [10] E653Q <1> (<1> 5fold increase in Km -value [10]) [10] H501A <1> (<1> no enzymic activity [9,10]) [9, 10] H501K <1> (<1> no enzymic activity [10]) [10] H501R <1> (<1> no enzymic activity [10]) [10] H561A <1> (<1> increase in Km -values [9]) [9] H712A <1> (<1> increase in Km -values [9]) [9] R500A <1> (<1> 20fold increase in Km -value [10]; <1> increase in Km -values, large decrease in kcat values [9]) [9, 10] R560A <1> (<1> no enzymic activity [9,10]) [9, 10] Y508A <1> (<1> no enzymic activity [9,10]) [9, 10] Y508F <1> (<1> 30fold increase in Km -value [10]) [10] Application analysis <1, 2> (<1,2> micromethods for the measurement of as little as 3 pg of chondroitin sulfate A, B, or C in mixtures with other mucopolysaccharides or in urine [11]) [11] synthesis <1> (<1> Production of enzyme for industrial use. Induction of enzyme by addition of chondroitin sulfate C to growth medium. Optimal conditions are pH 8.0, 25 C, plus the addition of yeast extract, peptone and casamino acid. Induction is inhibited by chloramphenicol and actinomycin D [3]) [3]

6 Stability Temperature stability 40 <1> (<1> 30 min, 23% loss of activity [8]) [8] 45 <1> (<1> dramatic loss of activity [9]) [9] General stability information <3>, sensitive to lyophilization and freezing and thawing, both isoenzyme I and II [6] Storage stability <2>, -20 C, 12 months, no loss of activity [2] <3>, 4 C, two weeks, 70-80% residual activity, both isoenzyme I and II [6]

References [1] Hascall, V.C.; Riolo, R.L.; Hayward, J.; Reynolds, C.C.: Treatment of bovine nasal cartilage proteoglycan with chondroitinases from Flavobacterium heparinum and Proteus vulgaris. J. Biol. Chem., 247, 4521-4528 (1972)

167

Chondroitin-sulfate-ABC exolyase

4.2.2.21

[2] Michelacci, Y.M.; Horton, D.S.P.Q.; Poblacion, C.A.: Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum. Biochim. Biophys. Acta, 923, 291-301 (1987) [3] Sato, N.; Murata, K.; Kimura, A.: Cultural conditions for the induction of chondroitinase ABC in Proteus vulgaris cells. J. Ferment. Technol., 64, 155159 (1986) [4] Sato, N.; Murata, K.; Kimura, A.: Subunit structure of chondroitinase ABC from Proteus vulgaris. Agric. Biol. Chem., 50, 1057-1059 (1986) [5] Yamagata, T.; Saito, H.; Habuchi, O.; Suzuki, S.: Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem., 243, 1523-1535 (1968) [6] Linn, S.; Chan, T.; Lipeski, L.; Salyers, A.A.: Isolation and characterization of two chondroitin lyases from Bacteroides thetaiotaomicron. J. Bacteriol., 156, 859-866 (1983) [7] Aguiarand, J.A.K.; Michelacci, Y.M.: Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography. Braz. J. Med. Biol. Res., 32, 545-550 (1999) [8] Hamai, A.; Hashimoto, N.; Mochizuki, H.; Kato, F.; Makiguchi, Y.; Horie, K.; Suzuki, S.: Two distinct chondroitin sulfate ABC lyases. An endoeliminase yielding tetrasaccharides and an exoeliminase preferentially acting on oligosaccharides. J. Biol. Chem., 272, 9123-9130 (1997) [9] Prabhakar, V.; Capila, I.; Bosques, C.J.; Pojasek, K.; Sasisekharan, R.: Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification. Biochem. J., 386, 103-112 (2005) [10] Prabhakar, V.; Raman, R.; Capila, I.; Bosques, C.J.; Pojasek, K.; Sasisekharan, R.: Biochemical characterization of the chondroitinase ABC I active site. Biochem. J., 390, 395-405 (2005) [11] Saito, H.; Yamagata, T.; Suzuki, S.: Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem., 243, 1536-1542 (1968) [12] Suzuki, S.; Saito, H.; Yamagata, T.; Anno, K.; Seno, N.; Kawai, Y.; Furuhashi, T.: Formation of three types of disulfated disaccharides from chondroitin sulfates by chondroitinase digestion. J. Biol. Chem., 243, 1543-1550 (1968)

168

Pectate trisaccharide-lyase

4.2.2.22

1 Nomenclature EC number 4.2.2.22 Systematic name (1-4)-a-d-galacturonan reducing-end-trisaccharide-lyase Recommended name pectate trisaccharide-lyase Synonyms PelA <1, 2> [1, 2] exopectate-lyase <1> [1] pectate lyase A <1, 2> (<2> is a subunit of the extracellular multienzyme complex, called the cellulosome [2]) [1, 2] CAS registry number 9015-75-2 (cf. EC 4.2.2.2)

2 Source Organism <1> Thermotoga maritima (no sequence specified) [1] <2> Clostridium cellulovorans (UNIPROT accession number: Q9AQF3) [2] <3> Bacillus licheniformis (UNIPROT accession number: Q8GCB2) [3]

3 Reaction and Specificity Catalyzed reaction eliminative cleavage of unsaturated trigalacturonate as the major product from the reducing end of polygalacturonic acid/pectate Natural substrates and products S pectin <1> (<1> the ability of Thermotoga maritima to grow on pectin as sole carbon source coincides with the secretion of a pectate lyase A [1]) (Reversibility: ?) [1] P ? S polygalacturonic acid <2> (<2> pectate lyase A is a subunit of the extracellular multienzyme complex, called the cellulosome, which is involved in plant cell wall degradation [2]) (Reversibility: ?) [2] P trigalacturonic acid + digalacturonic acid

169

Pectate trisaccharide-lyase

4.2.2.22

Substrates and products S pectin <1> (<1> pectins with an increasing degree of methylation are degraded at a decreasing rate [1]) (Reversibility: ?) [1] P unsaturated trigalacturonate S pectin <1> (<1> the ability of Thermotoga maritima to grow on pectin as sole carbon source coincides with the secretion of a pectate lyase A [1]) (Reversibility: ?) [1] P ? S polygalacturonic acid <1, 3> (<1> the enzyme attacks from the reducing end, since only unsaturated trigalacturonic acid is formed, followed by slight formation of unsaturated digalacturonate [1]) (Reversibility: ?) [1, 3] P unsaturated trigalacturonate (<3> main product [3]) S polygalacturonic acid <2> (<2> pectate lyase A is a subunit of the extracellular multienzyme complex, called the cellulosome, which is involved in plant cell wall degradation [2]) (Reversibility: ?) [2] P trigalacturonic acid + digalacturonic acid Inhibitors CaCl2 <2> (<2> 1 mM reduces activity to less than that without addition of CaCl2 [2]) [2] EDTA <2> (<2> 1 mM [2]) [2] EGTA <1> (<1> 1 mM, complete inhibition [1]) [1] polygalacturonic acid <3> (<3> recombinant fusion protein containing a Cterminal His-tag is inhibited by excess substrate [3]) [3] Metals, ions Ca2+ <1> (<1> dependent on [1]) [1] CaCl2 <2> (<2> optimal activity at 0.05-0.5 mM [2]) [2] NaCl <1> (<1> highest activity at 200 mM [1]) [1] Km-Value (mM) 0.06 <1> (polygalacturonic acid) [1] Additional information <3> (<3> Km -value for polygalacturonic acid is 0.56 g/l, recombinant fusion protein containing a C-terminal His-tag [3]) [3] Ki-Value (mM) Additional information <3> (<3> Ki -value for polygalacturonic acid is 1.08 g/l [3]) [3] pH-Optimum 8 <2> [2] 9 <1> [1] pH-Range 7-8 <2> (<2> pH 7.0: less than 30% of maximal activity, pH 8.0: optimum [2]) [2]

170

4.2.2.22

Pectate trisaccharide-lyase

Temperature optimum ( C) 50 <2> [2] 65 <3> (<3> recombinant fusion protein containing a C-terminal His-tag [3]) [3] 90 <1> [1] Temperature range ( C) 40-60 <2> (<2> more than 50% of maximal activity at 40 C and at 60 C [2]) [2]

4 Enzyme Structure Molecular weight 152000 <1> (<1> gel filtration [1]) [1] Subunits ? <2, 3> (<2> x * 42000, SDS-PAGE [2]; <3> x * 36000, SDS-PAGE [3]; <3> x * 33451, calculated from sequence [3]) [2, 3] tetramer <1> (<1> 4 * 40000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Localization extracellular <1, 2> [1, 2] Purification <1> [1] <2> (recombinant enzyme) [2] <3> (recombinant) [3] Cloning <1> (overexpression in Escherichia coli) [1] <2> [2] <3> (expression in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag) [3]

6 Stability Temperature stability 95 <1> (<1> half-life: 110 min [1]) [1] 100 <1> (<1> 2 min, 50% loss of activity [1]) [1] 103 <1> (<1> apparent melting temperature [1]) [1] General stability information <1>, Ca2+ stabilizes [1]

171

Pectate trisaccharide-lyase

4.2.2.22

References [1] Kluskens, L.D.; van Alebeek, G.J.; Voragen, A.G.; de Vos, W.M.; van der Oost, J.: Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima. Biochem. J., 370, 651-659 (2003) [2] Tamaru, Y.; Doi, R.H.: Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome. Proc. Natl. Acad. Sci. USA, 27, 4125-4129 (2001) [3] Berensmeier, S.; Singh, S.A.; Meens, J.; Buchholz, K.: Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase. Appl. Microbiol. Biotechnol., 64, 560-567 (2004)

172

Threonine synthase

4.2.3.1

1 Nomenclature EC number 4.2.3.1 Systematic name O-phospho-l-homoserine phospho-lyase (adding water; threonine-forming) Recommended name threonine synthase Synonyms EC 4.2.99.2 TS <9> [24] ThrS <3> [23] synthase, threonine threonine synthase <4, 9, 10> [25, 26, 27] threonine synthetase CAS registry number 9023-97-6

2 Source Organism <1> Beta vulgaris (no sequence specified) [4] <2> Bacillus subtilis (no sequence specified) [2, 3, 9, 11] <3> Thermus thermophilus (no sequence specified) (<3> extracellular isozyme [23]) [23] <4> Escherichia coli (no sequence specified) [6, 8, 16, 17, 19, 25] <5> Saccharomyces cerevisiae (no sequence specified) [22] <6> Neurospora crassa (no sequence specified) [1] <7> Pisum sativum (no sequence specified) [5] <8> Glycine max (no sequence specified) [15] <9> Arabidopsis thaliana (no sequence specified) [12,14,18,20,21,24,26] <10> Arabidopsis sp. (no sequence specified) [27] <11> Brevibacterium lactofermentum (no sequence specified) [13] <12> Raphanus sativus (no sequence specified) [4] <13> Lemna paucicostata (no sequence specified) [7, 10]

173

Threonine synthase

4.2.3.1

3 Reaction and Specificity Catalyzed reaction O-phospho-l-homoserine + H2 O = l-threonine + phosphate (<2, 4, 9, 13> mechanism [7, 9, 17, 18]; <6> reaction proceeds via phospate removal and isomerization from primary to secondary alcohol [1]; <2> mechanism of phosphohomoserine dephosphorylation/deamination activity [3]; <4> inhibitor studies concerning mechanism, model of stepwise catalytic mechanism [16]; <4, 9, 10> reaction proceeds via phosphate removal and isomerization from primary to secondary alcohol [25, 26, 27]) Reaction type elimination Natural substrates and products S O-phospho-l-homoserine <1, 4> (<4> studies on regulatory properties [19]; <1> involved in allocation of phosphohomoserine between cystathione and threonine pathways [4]) (Reversibility: ?) [4, 19] P ? S O-phospho-l-homoserine + H2 O <4, 5, 9, 10> (<9> enzyme at the metabolic branch point between methionine and threonine biosynthesis [21]; <5> final step of threonine biosynthesis [22]; <9> last reaction in the synthesis of threonine from aspartate [24]; <4, 9, 10> threonine synthesis in eukaryotes [25, 26, 27]) (Reversibility: ir) [21, 22, 24, 25, 26, 27] P l-threonine + phosphate <5, 9> [21, 22, 24] Substrates and products S dl-3-chloroalanine <4> (<4> b-elimination [17]) (Reversibility: ir) [17] P pyruvate + NH3 + HCl <4> [17] S dl-vinylglycine + H2 O <4> (Reversibility: ir) [17] P l-threonine <4> [17] S l-allo-threonine + H2 O <4> (<4> b-elimination [17]) (Reversibility: ir) [17] P 2-oxobutyrate + NH3 <4> [17] S l-serine + H2 O <4> (<4> b-elimination [17]) (Reversibility: ir) [17] P pyruvate + NH3 <4> [17] S O-phospho-l-homoserine <1, 4> (<4> studies on regulatory properties [19]; <1> involved in allocation of phosphohomoserine between cystathione and threonine pathways [4]) (Reversibility: ?) [4, 19] P ? S O-phospho-l-homoserine + H2 O <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13> (<9> enzyme at the metabolic branch point between methionine and threonine biosynthesis [21]; <5> final step of threonine biosynthesis [22]; <9> last reaction in the synthesis of threonine from aspartate [24]; <4,9,10> threonine synthesis in eukaryotes [25, 26, 27]) (Reversibility: ir) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 21, 22, 23, 24, 25, 26, 27] P l-threonine + phosphate <1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13> [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 21, 22, 23, 24]

174

4.2.3.1

Threonine synthase

S phosphohomoserine <2> (<2> bypass of threonine in isoleucine biosynthesis [3]) (Reversibility: ir) [3] P 2-oxobutyrate + phosphate + ? <2> [3] S threonine <2, 4> (<4> b-elimination [17]; <2> genetic evidence for identity of protein with both activities [2]) (Reversibility: ir) [2, 3, 17] P 2-oxobutyrate + NH3 <2, 4> [2, 3, 17] S Additional information <2, 4> (<4> half-transamination reactions [17]) (Reversibility: ?) [11, 17] P ? Inhibitors AMP <9, 13> (<13> 50% inhibition at 40 mM, reversible, depends on phosphohomoserine concentration [10]; <9> same binding site as S-adenosylmethionine [14]) [10, 14, 21] AMP-derivatives <13> [10] dl-2-amino-3[(phosphonomethyl)thio]propionic acid <4> (<4> Ki : 0.057 mM, kinact: 1.44 min-1 [17]) [17] dl-E-2-amino-5-phosphono-4-pentenoic acid <4> (<4> Ki : 0.54 mM [17]) [17] GMP <13> (<13> 25% inhibition at 67 mM [10]) [10] IMP <13> (<13> 23% inhibition at 67 mM [10]) [10] KCN <6> (<6> 22% inhibition at 1 mM [1]) [1] l-2,3-methanohomoserine phosphate <4> (<4> Ki : 0.01 mM [17]) [17] l-2-amino-3[(phosphonomethyl)thio]propionic acid <4> (<4> Ki : 0.00011 mM, “slow, tight“ inhibition kinetics [16]; <4> Ki : 0.011 mM [17]) [16, 17] l-3-hydroxyhomoserine <4> (<4> Ki : 0.05 mM [17]) [17] l-cysteine <1, 2, 6, 7, 8, 13> (<6> 85% inhibition at 35 mM [1]; <7> 80% inhibition at 1 mM in presence of S-adenosylmethionine [5]; <1> stereospecific, inhibits S-adenosylmethionine activation [4]; <8> strong inhibition at 0.5 mM [15]; <13> 26% inhibition at 1 mM, reversible [7]) [1, 2, 4, 5, 7, 15] l-threo-3-hydroxyhomoserine <4> (<4> marked inhibition at 5 mM, abolished by 60 mM Mg2+ [8]; <4> Ki : 0.006 mM [16]) [8, 16] NH2 OH <6> (<6> 65% inhibition at 1 mM [1]) [1] O-phospho-l-serine <13> [7] O-phospho-dl-threonine <13> [7] phosphate <6, 13> (<6> 40% inhibition at 50 mM [1]; <13> 50% inhibition at 1 mM, competitive, reversible, depends on phosphohomoserine concentration [10]) [1, 10] sulfate <2> [2] vinylglycine <13> (<13> 22% inhibition at 10 mM [7]) [7] phospho-threonine <6> (<6> 35% inhibition at 10 mM [1]) [1] phosphonovaleric acid <4> (<4> Ki : 0.031 mM [16]) [16] Cofactors/prosthetic groups pyridoxal 5’-phosphate <2, 6, 7, 11> (<7> maximal activity at 0.4-1.0 mM [5]; <6,7> required cofactor [1,5]) [1, 2, 5, 13]

175

Threonine synthase

4.2.3.1

Activating compounds S-Adenosyl-l-methionine <1, 7, 9, 13> (<9> activates [21]); <7> allosteric activator, 4fold increase of Vmax, 3.3fold decrease of substrate Km at 0.4 mM [5]; <9> affects both Km and Vmax by allosteric and cooperative transition of enzyme, two mol per mol enzyme, 25fold decrease of Km at 0.06 mM [18]; <13> allosteric activator, maximal activation at 0.1-0.2 mM [7]; <1> allosteric activator, 14fold increase of Vmax, 8% decrease of substrate Km at 0.5 mM [4]; <9> 3-20fold increase of specific activity in various recombinant enzymes at 0.2 mM [12]; <9> same binding site as inhibitor AMP, 85fold increase of activity, maximum activity at 0.06-0.25 mM [14]) [4, 5, 7, 12, 14, 18, 21, 24] Turnover number (min–1) 0.0333 <9> (O-phosphohomoserine, <9> without S-adenosylmethionine [14]) [14] 0.4 <9> (O-phosphohomoserine, <9> without S-adenosylmethionine [18]) [18] 0.86 <9> (O-phosphohomoserine, <9> in presence of S-adenosylmethionine [14]) [14] 3.5 <9> (O-phosphohomoserine, <9> at 0.06 mM S-adenosylmethionine [18]) [18] 7.33 <4> (O-phosphohomoserine) [17] Specific activity (U/mg) 0.00098 <9> (<9> purified enzyme from cloned gene [12]) [12] 0.012 <7> [5] 0.014 <2> (<2> mutation Ile-3 [2]) [2] 0.018 <2> (<2> wild type [2]) [2] 0.0261 <13> [7] 1.73 <6> [1] 3.6 <11> [13] 3.8 <9> [18] 7.7 <4> (<4> purified enzyme from cloned gene [6]) [6] 8.8 <2> (<2> mutation SprA-44 [2]) [2] Additional information <4, 8, 10> (<8> activity with addition of several amino acids to several mutant cell lines [15]; <4> homozygous line from plant 829-2 exhibits 10.1fold and 2.1fold higher activity without and with 0.2 mmol/l S-adenosyl methionine compared to wild type. Homozygous line from plant 829-9 exhibits 25.2fold and 3.8fold higher activity without and with 0.2 mmol/l S-adenosyl methionine compared to wild type. Homozygous line from plant 829-14 exhibits 6.5fold and 1.5fold higher activity without and with 0.2 mmol/l S-adenosyl methionine compared to wild type [25]; <10> threonine synthase is significantly increased in recombinant overexpressing cells compared to expression in wild-type plants, ranging from 4.3fold in cell line S4, 5.1fold in cell line S3 and 7.1fold in cell line S1 [27]) [15, 25, 27]

176

4.2.3.1

Threonine synthase

Km-Value (mM) 0.002-0.007 <13> (O-phospohomoserine, <13> at saturating S-adenosylmethionine concentrations [7]) [7] 0.03 <9> (O-phosphohomoserine, <9> in presence of S-adenosylmethionine [14]) [14] 0.12 <9> (O-phosphohomoserine, <9> without S-adenosylmethionine [14]) [14] 0.5 <4> (O-phosphohomoserine) [17] 1.3-2.7 <1, 7, 12> (O-phosphohomoserine) [4, 5] Ki-Value (mM) 0.006 <4> (l-threo-3-hydroxyhomoserine) [16] 0.01 <4> (l-2,3-methanohomoserine phosphate) [17] 0.011 <4> (l-2-amino-3[(phosphonomethyl)thio]propionic acid) [17] 0.031 <4> (phosphonovaleric acid) [16] 0.05 <4> (l-3-hydroxyhomoserine) [17] 0.057 <4> (dl-2-amino-3[(phosphonomethyl)thio]propionic acid, <4> kinact: 1.44 min-1 [17]) [17] 0.54 <4> (dl-E-2-amino-5-phosphono-4-pentenoic acid) [17] pH-Optimum 7.3 <1, 6, 8> (<1,6,8> enzyme assay at [1,4,15]) [1, 4, 15] 7.4 <7> [5] 7.5 <2, 4> (<2,4> enzyme assay at [2,16]) [2, 16] 7.8 <4> (<4> enzyme assay at, recombinant enzyme [19]) [19] 8 <2, 9, 13> (<2,9> enzyme assay at [3,18]) [3, 7, 18] 8.5 <9> (<9> enyzme assay at [14]) [14] pH-Range 6.8-8.4 <7> [5] 7-8.5 <13> [7] Temperature optimum ( C) 20 <9> (<9> enzyme assay at [14]) [14] 30 <1, 6, 8, 9> (<1,6,8,9> enzyme assay at [1,4,15,18]) [1, 4, 15, 18] 35 <2> (<2> enzyme assay at [2]) [2] 37 <4> (<4> enzyme assay at, recombinant enzyme [19]) [19]

4 Enzyme Structure Molecular weight 46000-48000 <4> (<4> gel filtration [6]) [6] 53000 <11> (<11> gel filtration [13]) [13] 110000 <9> (<9> gel filtration [12,14,18]) [12, 14, 18] 190000 <7> (<7> gel filtration [5]) [5]

177

Threonine synthase

4.2.3.1

Subunits dimer <9> (<9> 2 * 58000, comparison of nucleotide sequence and molecular mass of native enzyme [12,14]; <9> N-terminal amino acids involved in dimerization [14]; <9> four-domain dimer with a two-stranded b-sheet arm protruding from one monomer onto the other [24]) [12, 14, 18, 24] monomer <11> (<11> 1 * 52800, SDS-PAGE [13]) [13] Additional information <4, 9, 11, 13> (<13> amino acid analysis [7]; <4> determination of N-terminal amino acid sequence [6]; <9> identification of active site amino acids [12]; <11> tertiary structure [13]) [6, 7, 12, 13]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <4, 8> [15, 19] cell suspension culture <4, 8> (<4> suspension culture of Nicotiana tabacum cells expressing Escherichia coli enzyme [19]) [15, 19] leaf <1, 9, 10, 12> (<10> rosette leaves [27]) [4, 12, 27] seedling <7> [5] Localization chloroplast <8, 9, 13> (<8> not exclusively in stroma [15]) [10, 14, 15] Purification <2> [3] <2> (partial) [2] <4> [6, 8] <6> [1] <7> (partial) [5] <9> [14] <9> (native and recombinant enzymes) [12] <9> (recombinant enzyme) [18] <11> (native enzyme) [13] <13> [7] Crystallization <3> (hanging drop vapour diffusion method at 293 K, unligated enzyme form and complex with substrate analogue 2-amino-5-phosphonopentanoic acid, structure determined at 2.15 A and 2.0 A resolution) [23] <5> (sitting-drop vapour-diffusion method, crystal structure at 2.7 A resolution) [22] <9> (hanging drop method, 4 C, pH 6.5, resolution of 2.6 A) [26] <9> (hanging-drop vapour-diffusion method at 293 K. Selenomethioninesubstituted apo threonine synthase, 14 Met residues in 58000 Da) [20] <9> (hanging-drop vapour-diffusion method, crystal structure of apo threonine synthase as solved at 2.25 A resolution from triclinic crystals) [24] <9> (X-ray diffraction studies) [14]

178

4.2.3.1

Threonine synthase

Cloning <2> (expressed in Escherichia coli) [9] <4> [6] <4> (expressed in Nicotiana tabacum) [19] <4> (expression in transgenic Arabidopsis) [25] <9> (expressed in Escherichia coli) [12, 14, 18] <10> (overexpression under the control of the 35S cauliflower mosaic virus promotor in Arabidopsis sp. plants) [27] <11> (expressed in Escherichia coli) [13] Application agriculture <9> (<9> possible herbicide target [14,18]; <9> as one of the few enzymes that are cross-activated by the product of another pathway, S-adenosyl-l-methionine, it has a potential application as a target for herbicides [24]) [14, 18, 24] medicine <4> (<4> possibly involved in formation of bacteriocidal antimetabolites [8]) [8] nutrition <9> (<9> interesting with respect to attempts to obtain transgenic plants with elevated levels of essential amino acids Met, Lys, Thr [18]) [18]

6 Stability pH-Stability 5.2 <6> (<6> inactivation and precipitation below [1]) [1] General stability information <13>, bovine serum albumine stabilizes [7] Storage stability <6>, -15 C, glycylglycine, several months [1] <8>, -80 C, MOPS-buffer, pH 7.5, EDTA, dithioerythritol, 2-benzothiazolethiol, glycerol, polyvinylpyrrolidone, several months, no loss of activity [15] <9>, -80 C, Na-HEPES buffer, pH 7.5, several months, no loss of activity [18] <13>, -80 C, pH 7.8, several months [7]

References [1] Flavin, M.; Slaughter, C.: Purification and properties of threonine synthetase of Neurospora. J. Biol. Chem., 235, 1103-1108 (1960) [2] Skarstedt, M.T.; Greer, S.B.: Threonine synthetase of Bacillus subtilis. The nature of an associated dehydratase activity. J. Biol. Chem., 248, 1032-1044 (1973) [3] Schildkraut, I.; Greer, S.: Threonine synthetase-catalyzed conversion of phosphohomoserine to a-ketobutyrate in Bacillus subtilis. J. Bacteriol., 115, 777-785 (1973)

179

Threonine synthase

4.2.3.1

[4] Madison, J.T.; Thompson, J.F.: Threonine synthetase from higher plants: stimulation by S-adenosylmethionine and inhibition by cysteine. Biochem. Biophys. Res. Commun., 71, 684-691 (1976) [5] Thoen, A.; Rognes, S.E.; Aarnes, H.: Biosynthesis of threonine from homoserine in pea seedlings. II. Threonine synthase. Plant Sci. Lett., 13, 113-119 (1978) [6] Parsot, C.; Cossart, P.; Saint-Girons, I.; Cohen, G.N.: Nucleotide sequence of thrC and of the transcription termination region of the threonine operon in Escherichia coli K12. Nucleic Acids Res., 11, 7331-7345 (1983) [7] Giovanelli, J.; Veluthambi, K.; Thompson, G.A.; Mudd, S.H.; Datko, A.H.: Threonine synthase of Lemna paucicostata Hegelm. 6746. Plant Physiol., 76, 285-292 (1984) [8] Shames, S.L.; Ash, D.E.; Wedler, F.C.; Villafranca, J.J.: Interaction of aspartate and aspartate-derived antimetabolites with the enzymes of the threonine biosynthetic pathway of Escherichia coli. J. Biol. Chem., 259, 1533115339 (1984) [9] Parsot, C.: Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and d-serine dehydratase. EMBO J., 5, 3013-3019 (1986) [10] Giovanelli, J.; Mudd, S.H.; Datko, A.H.; Thompson, G.A.: Effects of orthophosphate and adenosine 5’-phosphate on threonine synthase and cystathionine g-synthase of Lemna paucicostata Hegelm. 6746. Plant Physiol., 81, 577-583 (1986) [11] Parsot, C.: A common origin for enzymes involved in the terminal step of the threonine and tryptophan biosynthetic pathways. Proc. Natl. Acad. Sci. USA, 84, 5207-5210 (1987) [12] Curien, G.; Dumas, R.; Ravanel, S.; Douce, R.: Characterization of an Arabidopsis thaliana cDNA encoding an S-adenosylmethionine-sensitive threonine synthase. Threonine synthase from higher plants. FEBS Lett., 390, 8590 (1996) [13] Malumbres, M.; Mateos, L.M.; Lumbreras, M.A.; Guerrero, C.; Martin, J.F.: Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase. Appl. Environ. Microbiol., 60, 2209-2219 (1994) [14] Laber, B.; Maurer, W.; Hanke, C.; Graefe, S.; Ehlert, S.; Messerschmidt, A.; Clausen, T.: Characterization of recombinant Arabidopsis thaliana threonine synthase. Eur. J. Biochem., 263, 212-221 (1999) [15] Greenberg, J.M.; Thompson, J.F.; Madison, J.T.: Homoserine kinase and threonine synthase in methionine-overproducing soybean tissue cultures. Plant Cell Rep., 7, 477-480 (1988) [16] Farrington, G.K.; Kumar, A.; Shames, S.L.; Ewaskiewicz, J.I.; Ash, D.A.; Wedler, F.C.: Threonine synthase of Escherichia coli: inhibition by classical and slow-binding analogues of homoserine phosphate. Arch. Biochem. Biophys., 307, 165-174 (1993) [17] Laber, B.; Gerbling, K.P.; Harde, C.; Neff, K.H.; Nordhoff, E.; Pohlenz, H.D.: Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors. Biochemistry, 33, 3413-3423 (1994)

180

4.2.3.1

Threonine synthase

[18] Curien, G.; Job, D.; Douce, R.; Dumas, R.: Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine. Biochemistry, 37, 1321213221 (1998) [19] Muhitch, M.J.: Effects of expressing E. coli threonine synthase in tobacco (Nicotiana tabacum L.) suspension culture cells on free amino acid levels, aspartate pathway enzyme activities and uptake of aspartate into the cells. Plant Physiol., 150, 16-22 (1997) [20] Thomazeau, K.; Curien, G.; Thompson, A.; Dumas, R.; Biou, V.: MAD on threonine synthase: the phasing power of oxidized selenomethionine. Acta Crystallogr. Sect. D, 57, 1337-1340 (2001) [21] Curien, G.; Ravanel, S.; Dumas, R.: A kinetic model of the branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana. Eur. J. Biochem., 270, 4615-4627 (2003) [22] Garrido-Franco, M.; Ehlert, S.; Messerschmidt, A.; Marinkovic, S.; Huber, R.; Laber, B.; Bourenkov, G.P.; Clausen, T.: Structure and function of threonine synthase from yeast. J. Biol. Chem., 277, 12396-12405 (2002) [23] Omi, R.; Goto, M.; Miyahara, I.; Mizuguchi, H.; Hayashi, H.; Kagamiyama, H.; Hirotsu, K.: Crystal structures of threonine synthase from Thermus thermophilus HB8: conformational change, substrate recognition, and mechanism. J. Biol. Chem., 278, 46035-46045 (2003) [24] Thomazeau, K.; Curien, G.; Dumas, R.; Biou, V.: Crystal structure of threonine synthase from Arabidopsis thaliana. Protein Sci., 10, 638-648 (2001) [25] Lee, M.; Martin, M.N.; Hudson, A.O.; Lee, J.; Muhitch, M.J.; Leustek, T.: Methionine and threonine synthesis are limited by homoserine availability and not the activity of homoserine kinase in Arabidopsis thaliana. Plant J., 41, 685-696 (2005) [26] Mas-Droux, C.; Biou, V.; Dumas, R.: Allosteric threonine synthase. Reorganization of the pyridoxal phosphate site upon asymmetric activation through S-adenosylmethionine binding to a novel site. J. Biol. Chem., 281, 5188-5196 (2006) [27] Avraham, T.; Amir, R.: The expression level of threonine synthase and cystathionine-g-synthase is influenced by the level of both threonine and methionine in Arabidopsis plants. Transgenic Res., 14, 299-311 (2005)

181

Ethanolamine-phosphate phospho-lyase

4.2.3.2

1 Nomenclature EC number 4.2.3.2 Systematic name ethanolamine-phosphate phospho-lyase (deaminating; acetaldehyde-forming) Recommended name ethanolamine-phosphate phospho-lyase Synonyms EC 4.2.99.7 (formerly) O-phosphoethanolamine-phospholyase O-phosphorylethanol-amine phospho-lyase amino alcohol O-phosphate phospholyase phospho-lyase, ethanolamine phosphate CAS registry number 37290-88-3

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8>

Rattus norvegicus (no sequence specified) [1] Oryctolagus cuniculus (no sequence specified) [1] Achromobacter sp. (no sequence specified) [3] Erwinia carotovora (no sequence specified) [2] Flavobacterium arborescens (no sequence specified) [3] Erwinia ananas (no sequence specified) [2] Erwinia milletiae (no sequence specified) [2] Flavobacterium rhenanum (no sequence specified) [3]

3 Reaction and Specificity Catalyzed reaction ethanolamine phosphate + H2 O = acetaldehyde + NH3 + phosphate Reaction type elimination

182

4.2.3.2

Ethanolamine-phosphate phospho-lyase

Natural substrates and products S ethanolamine phosphate <4, 6, 7> (<4, 6, 7> role in deamination process necessary for N-assimilation [2]) (Reversibility: ?) [2] P ? Substrates and products S ethanolamine phosphate <4, 6, 7> (<4, 6, 7> role in deamination process necessary for N-assimilation [2]) (Reversibility: ?) [2] P ? S ethanolamine phosphate + H2 O <1, 2, 3, 4, 5, 6, 7, 8> (Reversibility: ?) [1, 2, 3] P acetaldehyde + NH3 + phosphate <1, 2, 3, 4, 5, 6, 7, 8> [1, 2, 3] Inhibitors Ag+ <1, 2> (<1,2> 0.1 mM, 92% inhibition [1]) [1] CN- <1, 2> (<1,2> 1 mM, 88% inhibition [1]) [1] Cd2+ <1, 2> (<1,2> 0.1 mM, 100% inhibition [1]) [1] Co2+ <1, 2> (<1,2> 0.1 mM, 25% inhibition [1]) [1] Cu2+ <1, 2> (<1,2> 0.1 mM, 94% inhibition [1]) [1] hydroxylamine <3, 5, 8> [3] NaBH4 <2> [1] Pb2+ <1, 2> (<1,2> 0.1 mM, 75% inhibition [1]) [1] SO24- <1, 2> (<1,2> 10 mM, 49% inhibition [1]) [1] Zn2+ <1, 2> (<1,2> 0.1 mM, 91% inhibition [1]) [1] p-chloromercuribenzoate <1, 2> (<1,2> 0.1 mM, 98.4% inhibition [1]) [1] Cofactors/prosthetic groups pyridoxal 5’-phosphate <1, 2> (<1,2> required, Km : 0.00027 mM [1]) [1] Specific activity (U/mg) 0.00014 <6> [2] 0.000156 <7> [2] 0.00022 <4> [2] 0.073 <1> [1] 0.914 <2> [1] Km-Value (mM) 0.28 <3> (ethanolamine phosphate) [3] 0.32 <8> (ethanolamine phosphate) [3] 0.45 <5> (ethanolamine phosphate) [3] 0.61 <1> (ethanolamine phosphate) [1] 0.63 <2> (ethanolamine phosphate) [1] pH-Optimum 7.5-8 <1, 2> [1] pH-Range 6.7-8.6 <1, 2> (<1,2> less than 50% of maximal activity above and below [1]) [1]

183

Ethanolamine-phosphate phospho-lyase

4.2.3.2

4 Enzyme Structure Molecular weight 168000 <1> (<1> gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue liver <1, 2> [1] Purification <1> [1] <2> [1]

References [1] Fleshood, H.L.; Pitot, H.C.: The metabolism of O-phosphorylethanolamine in animal tissues. I. O-phosphorylethanolamine phospho-lyase: partial purification and characterization. J. Biol. Chem., 245, 4414-4420 (1970) [2] Jones, A.; Faulkner, A.; Turner, J.M.: Microbial metabolism of amino alcohols. Metabolism of ethanolamine and 1-aminopropan-2-ol in species of Erwinia and the roles of amino alcohol kinase and amino alcohol O-phosphate phospho-lyase in aldehyde formation. Biochem. J., 134, 959-968 (1973) [3] Faulkner, A.; Turner, J.M.: Phosphorylation of ethanolamine in catabolism. Biodegradative adenosine triphosphate-ethanolamine phosphotransferase and related enzymes in bacteria. Biochem. Soc. Trans., 2, 133-136 (1974)

184

Methylglyoxal synthase

4.2.3.3

1 Nomenclature EC number 4.2.3.3 Systematic name glycerone-phosphate phospho-lyase (methylglyoxal-forming) Recommended name methylglyoxal synthase Synonyms EC 4.2.99.11 (formerly) MGS methylglyoxal synthetase synthase, methylglyoxal CAS registry number 37279-01-9

2 Source Organism <1> Salmonella typhimurium (no sequence specified) [1] <2> Escherichia coli (no sequence specified) [1, 2, 7, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26] <3> Saccharomyces cerevisiae (no sequence specified) [13] <4> Aerobacter aerogenes (no sequence specified) [1] <5> Clostridium sphenoides (no sequence specified) [12] <6> Serratia marcescens (no sequence specified) [1] <7> Proteus mirabilis (no sequence specified) [15] <8> Proteus vulgaris (no sequence specified) [1,5,6,8,11,15] <9> Clostridium pasteurianum (no sequence specified) [4] <10> Capra hircus (no sequence specified) [9] <11> Pseudomonas saccharophila (no sequence specified) [3, 4] <12> Clostridium tetanomorphum (no sequence specified) [4] <13> Morganella morganii (no sequence specified) [15] <14> Clostridium acetobutylicum (no sequence specified) [16] <15> Aeromonas formicans (no sequence specified) [4] <16> Aeromonas hydrophila (no sequence specified) [15] <17> Klebsiella sp. (no sequence specified) [15] <18> Salmonella sp. (no sequence specified) [15]

185

Methylglyoxal synthase

<19> <20> <21> <22> <23>

4.2.3.3

Proteus rettgeri (no sequence specified) [1] Erwinia uredovora (no sequence specified) [1] Obesumbacterium proteus (no sequence specified) [4] Pseudotsuga menziesii (no sequence specified) [10] Shigella sp. (no sequence specified) [15]

3 Reaction and Specificity Catalyzed reaction glycerone phosphate = methylglyoxal + phosphate (<2> mechanism [21, 22, 23, 25]; <8> stereospecific deprotonation of the pro-S hydrogen at C-3 of dihydroxyacetone, the true product of the enzymatic reaction is the enol form of methylglyoxal which is ketonized in solution [8]; <8> since the enzyme accepts the trans-enediol-phosphate as a substrate, it is likely that it or the trans-enediolate is an intermediate in the catalytic reaction with dihydroxyacetone phosphate [11]; <2> catalytic mechanism [24]) Reaction type elimination Natural substrates and products S dihydroxyacetone phosphate <2, 5, 11> (<2> first enzyme in the reaction sequence for the conversion of dihydroxyacetone phosphate to pyruvate, may play a role in the control of glycolysis [1]; <5> the enzyme is involved in the methylglyoxal by-pass [12]; <2> during elevated metabolism, the synthesis of methylglyoxal from dihydroxyacetone phosphate temporarily relieves the cells from stress caused by phosphorylated intermediates and allows the cells to grow for a limited time. If during this period the environment changes, e.g. the level of the carbon source is reduced, the cells are not only able to survive but are also able to colonize their environment [14,19]; <2> the enzyme plays an important role in the catabolism of the triose phosphates [2]; <11> constitutive enzyme, may be involved in by-pass sequence for part of the Embden-Meyerhof glycolytic pathway [4]) (Reversibility: ?) [1, 2, 4, 12, 14, 19] P ? S glycerone phosphate <2> (<2> the unregulated production of methylglyoxal appears to be due to a rapid increase in the glycolysis intermediates from ribose degradation. Such a metabolic burden may result in methylglyoxal production by methylglyoxal synthase [26]) (Reversibility: ?) [26] P methylglyoxal + phosphate Substrates and products S dihydroxyacetone phosphate <2, 5, 11> (<2> first enzyme in the reaction sequence for the conversion of dihydroxyacetone phosphate to pyruvate, may play a role in the control of glycolysis [1]; <5> the enzyme is involved in the methylglyoxal by-pass [12]; <2> during elevated metabo-

186

4.2.3.3

P S

P

S

P S

P

Methylglyoxal synthase

lism, the synthesis of methylglyoxal from dihydroxyacetone phosphate temporarily relieves the cells from stress caused by phosphorylated intermediates and allows the cells to grow for a limited time. If during this period the environment changes, e.g. the level of the carbon source is reduced, the cells are not only able to survive but are also able to colonize their environment [14,19]; <2> the enzyme plays an important role in the catabolism of the triose phosphates [2]; <11> constitutive enzyme, may be involved in by-pass sequence for part of the Embden-Meyerhof glycolytic pathway [4]) (Reversibility: ?) [1, 2, 4, 12, 14, 19] ? dihydroxyacetone phosphate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23> (<3, 8, 10> strictly specific for [5,9,13]; <8> since the enzyme accepts the trans-enediol-phosphate as a substrate, it is likely that it or the trans-enediolate is an intermediate in the catalytic reaction with dihydroxyacetone phosphate [11]; <2> catalyzes the elimination reaction of DHAP, which leads to phosphate and the enol of methylglyoxal, which is subsequently tautomerized to methylglyoxal in solution [24]; <2> converts dihydrocyacetone phosphate to a cis-enediolic intermediate, then catalyses the elimination of phosphate to form the enol of methylglyoxal [21]; <2> converts dihydrocyacetone phosphate to enol pyruvaldehyde, this enol then tautomerizes to methylglyoxal in solution [22]; <2> intermediate is enol pyruvaldehyde is stereospecifically formed [23]) (Reversibility: ir) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25] methylglyoxal + phosphate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23> (<2> the true product of the enzymatic reaction is the enol form of methylglyoxal which is ketonized in solution [7]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25] glycerone phosphate <2> (<2> the unregulated production of methylglyoxal appears to be due to a rapid increase in the glycolysis intermediates from ribose degradation. Such a metabolic burden may result in methylglyoxal production by methylglyoxal synthase [26]) (Reversibility: ?) [26] methylglyoxal + phosphate Additional information <2> (<2> enzyme dos no catalyze the elimination reaction with glyceraldehyde phosphate [24]; <2> enzyme is not capable of abstracting the C2 proton from glyceraldehyde phosphate, which is an absolute requirement for the function of MGS [20]) (Reversibility: ?) [20, 24] ? <2> [20, 24]

Inhibitors 1,3-diphosphoglycerate <3> [13] 2-mercaptoethanol <3> [13] 2-phosphoglycerate <2, 3> [13, 17] 2-phosphoglycolate <2> (<2> inhibits [20]) [20] 3-phosphoglycerate <2, 3, 11> [1, 2, 3, 4, 13]

187

Methylglyoxal synthase

4.2.3.3

3-bromoacetol phosphate <8> (<8> irreversible inactivation by reacting with a nucleophilic group located in the active center, dihydroxyacetone phosphate or phosphate protects [6]) [6] 3-chloroacetol phosphate <8> (<8> competitive [6]) [6] 3-iodoacetol phosphate <8> (<8> irreversible inactivation by reacting with a nucleophilic group located in the active center, dihydroxyacetone phosphate or phosphate protects [6]) [6] ADP <3, 10, 14> [9, 13, 16] AMP <3> [13] ATP <3, 14> [13, 16] arsenate <2> [2] DTT <3> [13] diphosphate <2, 10, 11, 14> [1, 2, 3, 4, 9, 16] fructose 1,6-diphosphate <3, 14> [13, 16] glucose 1-phosphate <3> [13] glucose 6-phosphate <3> [13] glutathione <3> [13] glyceraldehyde 3-phosphate <3> [13] l-Cys <3> [13] methylglyoxal <8> (<8> linear noncompetitive inhibitor [8]) [8] phosphate <2, 3, 5, 8, 10, 11, 14> (<2> acts as competitive inhibitor on the H98Q variant and as an allosteric-type inhibitor on the wild-type enzyme [22]; <2> alosteric inhibitor [25]) [1, 2, 3, 5, 9, 12, 13, 16, 22, 25] phosphoenolpyruvate <2, 3, 10, 11, 14> [1, 2, 3, 4, 9, 13, 16] phosphoglycolate <2> (<2> inhibits wild-type enzyme and activates H98Q variant [22]) [22] phosphoglycolohydroxamic acid <2> (<2> tight binding inhibitor, neither a strictly competitive, non-competitive, nor uncompetitive mechanism [21]) [21] Additional information <8> (<8> no inactivation by iodoacetate or p-mercuribenzoate [6]) [6] Activating compounds phosphoglycolate <2> (<2> activates H98Q variant [22]) [22] Turnover number (min–1) 0.96 <2> (dihydroxyacetone phosphate, <2> pH 7, 25 C, H98Q mutant, absence of phosphoglycolate [22]) [22] 4.4 <2> (dihydroxyacetone phosphate, <2> pH 7, 25 C, H98N mutant [22]) [22] 6.08 <2> (dihydroxyacetone phosphate, <2> pH 7, 25 C, H98Q mutant, absence of phosphoglycolate [22]) [22] 17 <2> (dihydroxyacetone phosphate, <2> pH 7, 25 C, H98Q mutant, presence of phosphoglycolate [22]) [22] 179 <2> (dihydroxyacetone phosphate, <2> pH 6, 25 C, wild-type [22]) [22] 220 <2> (dihydroxyacetone phosphate, <2> wild-type enzyme [17]; <2> pH 7, 25 C, wild-type [22]) [17, 22]

188

4.2.3.3

Methylglyoxal synthase

Specific activity (U/mg) 5.57 <11> [3] 9.43 <8> [5] Additional information <2, 10, 11, 14> [1, 4, 9, 16] Km-Value (mM) 0.09 <11> (dihydroxyacetone phosphate) [3, 4] 0.19 <8> (dihydroxyacetone phosphate, <8> enzyme form II [5]) [5] 0.2 <2> (dihydroxyacetone phosphate, <2> wild-type enzyme [17]; <2> pH 7, 25 C, wild-type [22]) [17, 22] 0.22 <8> (dihydroxyacetone phosphate) [6] 0.23 <8> (dihydroxyacetone phosphate, <8> enzyme form I [5]) [5] 0.26 <2> (dihydroxyacetone phosphate, <2> pH 7, 25 C, H98N mutant [22]) [22] 0.47 <2> (dihydroxyacetone phosphate) [2] 0.5 <2> (dihydroxyacetone phosphate) [1] 0.53 <14> (dihydroxyacetone phosphate) [16] 0.6 <2> (dihydroxyacetone phosphate, <2> pH 7, 25 C, H98Q mutant, presence of phosphoglycolate [22]) [22] 0.62 <2> (dihydroxyacetone phosphate, <2> pH 6, 25 C, wild-type [22]) [22] 0.76 <10> (dihydroxyacetone phosphate) [9] 1.3 <2> (dihydroxyacetone phosphate, <2> pH 7, 25 C, H98Q mutant, absence of phosphoglycolate [22]) [22] 3 <3> (dihydroxyacetone phosphate) [13] Ki-Value (mM) 0.0000058 <2> (phosphoglycolate, <2> pH 7, 25 C, H98N variant [22]) [22] 0.000093 <2> (phosphate, <2> pH 7, 25 C, H98Q variant [22]) [22] 0.000093 <2> (phosphoglycolohydroxamic acid) [21] pH-Optimum 6.3 <2> (<2> H98Q variant [22]) [22] 6.8 <2> (<2> H98N variant [22]) [22] 7.2 <10> [9] 7.5 <2, 14> (<2> wild-type enzyme [22]) [2, 16, 22] 7.7 <8> [5] 8.2 <11> [3, 4] 9.5-10.5 <3> [13] pH-Range 5.6-8.3 <8> (<8> 50% of maximal activity at pH 5.6 and at pH 8.3 [5]) [5] 6.6-7.6 <10> (<10> pH 6.6: about 50% of maximal activity, pH 7.6: about 55% of maximal activity [9]) [9] 8-10.5 <3> (<3> pH 8: about 50% of maximal activity, pH 9.5-10.5: optimum [13]) [13]

189

Methylglyoxal synthase

4.2.3.3

4 Enzyme Structure Molecular weight 53000 <3> (<3> gel filtration [13]) [13] 60000 <14> (<14> gel filtration [16]) [16] 67000 <2, 11> (<2,11> gel filtration [2,3,4,17]) [2, 3, 4, 17] 135000 <8> (<8> gel filtration [5]) [5] Subunits dimer <3, 8> (<8> 2 * 66000, SDS-PAGE [5]; <3> x * 26000, SDS-PAGE [13]) [5, 13] hexamer <2> (<2> homohexamer, crystallization experiments [21]) [21] tetramer <2, 14> (<14> 4 * 15000, SDS-PAGE [16]; <2> 4 * 17000, SDS-PAGE [17]) [16, 17]

5 Isolation/Preparation/Mutation/Application Source/tissue liver <10> [9] needle <22> (<22> of various ages [10]) [10] Purification <2> [2] <2> (95% pure) [22] <2> (partial) [1, 19] <3> [13] <10> (partial) [9] <11> [3] <14> [16] Crystallization <2> (bound to formate and substiochiometric amounts of phosphate, hanging drop vapor diffusion method) [25] <2> (complexed with 2-phosphoglycolate, sitting drop vapor diffusion method) [20] <2> (hanging drop vapor diffusion method) [22] <2> (sitting drop vapor diffusion method) [21] <8> [5] Cloning <2> [17, 22, 25] <14> (overexpression in Escherichia coli) [16] Engineering D101E <2> (<2> reduced ratio of turnover-numer:Km -value by about 10000fold compared to the wild-type enzyme [17]) [17] D101N <2> (<2> reduced ratio of turnover-numer:Km -value by about 10000fold compared to the wild-type enzyme [17]) [17]

190

4.2.3.3

Methylglyoxal synthase

D71E <2> (<2> reduced ratio of turnover-numer:Km -value by about 1000fold compared to the wild-type enzyme [17]) [17] D71N <2> (<2> reduced ratio of turnover-numer:Km -value by about 1000fold compared to the wild-type enzyme [17]; <2> 2500fold decrease in kcat [21]) [17, 21] H98N <2> (<2> 50fold decrease in kcat [22]) [22] H98Q <2> (<2> 250fold lower catalytic activity than wild-type enzyme, change in conformation [22]) [22] Application synthesis <2> (<2> strains in which the genes for glycerol dehydrogenase, methylglyoxal synthase or both are overexpressed produce 1,2-propanediol as a fermentation product of glucose [18]) [18]

6 Stability Temperature stability 100 <2> (<2> 1 min, complete inactivation [2]) [2] Additional information <8> (<8> phosphate stabilizes towards both cold-induced inactivation and against heat-induced inactivation [8]) [8] General stability information <2>, D71N mutant enzyme rapidly becomes inactive in the absence of phosphate [21] <2>, dihydroxyacetone phosphate or bovine serum albumin stabilizes [2] <8>, phosphate stabilizes towards both cold-induced inactivation and against heat-induced inactivation [8] <11>, removal of phosphate from the purified enzyme leads to rapid loss of activity, which is more pronounced at 0 C than at 22 C. Dihydroxyacetone phosphate and bovine serum albumin overcome the inactivation to some extent [4] Storage stability <8>, 4 C, 10 mM imidazole buffer, pH 7.0, 1 mM EDTA, 1 mM KH2 PO4, 0.05% v/v 2-mercaptoethanol, 0.1-0.5 mg/ml protein, stable [8] <14>, -20 C, stable for at least 1 month [16]

References [1] Hopper, D.J.; Cooper, R.A.: The regulation of Escherichia coli methylglyoxal synthase a new control site in glycolysis. FEBS Lett., 13, 213-216 (1971) [2] Hopper, D.J.; Cooper, R.A.: The purification and properties of Escherichia coli methylglyoxal synthase. Biochem. J., 128, 321-329 (1972) [3] Cooper, R.A.: Methylglyoxal formation during glucose catabolism by Pseudomonas saccharophila. Identification of methylglyoxal synthase. Eur. J. Biochem., 44, 81-86 (1974)

191

Methylglyoxal synthase

4.2.3.3

[4] Cooper, R.A.: Methylglyoxal synthase. Methods Enzymol., 41B, 502-508 (1975) [5] Tsai, P.K.; Gracy, R.W.: Isolation and characterization of crystalline methylglyoxal synthetase from Proteus vulgaris. J. Biol. Chem., 251, 364-367 (1976) [6] Yuan, P.M.; Gracy, R.W.; Hartman, F.C.: Haloacetol phosphates as affinity labels for methylglyoxal synthase. Biochem. Biophys. Res. Commun., 74, 1007-1013 (1977) [7] Summers, M.C.; Rose, I.A.: Proton transfer reactions of methylglyoxal synthase. J. Am. Chem. Soc., 99, 4475-4478 (1977) [8] Yuan, P.M.; Gracy, R.W.: The conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate by methylglyoxal synthase. Arch. Biochem. Biophys., 183, 1-6 (1977) [9] Ray, S.; Ray, M.: Isolation of methylglyoxal synthase from goat liver. J. Biol. Chem., 256, 6230-6233 (1981) [10] Smits, M.M.; Johnson, M.A.: Methylglyoxal: enzyme distributions relative to its presence in Douglas-fir needles and absence in Douglas-fir needle callus. Arch. Biochem. Biophys., 208, 431-439 (1981) [11] Iyengar, R.; Rose, I.A.: Methylglyoxal synthase uses the trans isomer or triose-1,2-enediol 3-phosphate. J. Am. Chem. Soc., 105, 3301-3303 (1983) [12] Tran-Din, K.; Gottschalk, G.: Formation of d(-)-1,2-propanediol and d(-)lactate from glucose by Clostridium sphenoides under phosphate limitation. Arch. Microbiol., 142, 87-92 (1985) [13] Murata, K.; Fukuda, Y.; Watanabe, K.; Saikusa, T.; Shimosaka, M.; Kimura, A.: Characterization of methylglyoxal synthase in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun., 131, 190-198 (1985) [14] Ferguson, G.P.; Tçtemeyer, S.; MacLean, M.J.; Booth, I.R.: Methylglyoxal production in bacteria: suicide or survival. Arch. Microbiol., 170, 209-219 (1998) [15] Baskaran, S.; Rajan, D.P.; Balasubramanian, K.A.: Formation of methylglyoxal by bacteria isolated from human faeces. J. Med. Microbiol., 28, 211-215 (1989) [16] Huang, K.X.; Rudolph, F.B.; Bennett, G.N.: Characterization of methylglyoxal synthase from Clostridium acetobutylicum ATCC 824 and its use in the formation of 1,2-propanediol. Appl. Environ. Microbiol., 65, 3244-3247 (1999) [17] Saadat, D.; Harrison, D.H.T.: Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues. Biochemistry, 37, 10074-10086 (1998) [18] Altaras, N.E.; Cameron, D.C.: Metabolic engineering of a 1,2-propanediol pathway in Escherichia coli. Appl. Environ. Microbiol., 65, 1180-1185 (1999) [19] Tçtemeyer, S.; Booth, N.A.; Nichols, W.W.; Dunbar, B.; Booth, I.R.: From famine to feast: the role of methylglyoxal production in Escherichia coli. Mol. Microbiol., 27, 553-562 (1998)

192

4.2.3.3

Methylglyoxal synthase

[20] Saadat, D., Harrison, D.H.: Mirroring perfection: the structure of methylglyoxal synthase complexed with the competitive inhibitor 2-phosphoglycolate. Biochemistry, 39, 2950-2960 (2000) [21] Marks, G.T., Harris, T.K., Massiah, M.A., Mildvan, A.S., Harrison, D.H.: Mechanistic implications of methylglyoxal synthase complexed with phosphoglycolohydroxamic acid as observed by X-ray crystallography and NMR spectroscopy. Biochemistry, 40, 6805-6818 (2001) [22] Marks, G.T., Susler, M., Harrison, D.H.: Mutagenic studies on histidine 98 of methylglyoxal synthase: effects on mechanism and conformational change. Biochemistry, 43, 3802-3813 (2004) [23] Rose, I.A., Nowick, J.S.: Methylglyoxal synthetase, enol-pyruvaldehyde, glutathione and the glyoxylase system. J. Am. Chem. Soc., 124, 13047-13052 (2002) [24] Zhang, X., Harrison, D.H., Cui, Q.: Functional specificities of methylglyoxal synthase and triosephosphate isomerase: a combined QM/MM analysis. J. Am. Chem. Soc., 124, 14871-14878 (2002) [25] Saadat, D., Harrison, D.H.: The crystal structure of methylglyoxal synthase from Escherichia coli. Structure Fold. Des., 7, 309-317 (1999) [26] Kim, I.; Kim, E.; Yoo, S.; Shin, D.; Min, B.; Song, J.; Park, C.: Ribose utilization with an excess of mutarotase causes cell death due to accumulation of methylglyoxal. J. Bacteriol., 186, 7229-7235 (2004)

193

3-Dehydroquinate synthase

4.2.3.4

1 Nomenclature EC number 4.2.3.4 Systematic name 3-deoxy-arabino-heptulosonate-7-phosphate phosphate-lyase (cyclizing; 3dehydroquinate-forming) Recommended name 3-dehydroquinate synthase Synonyms 3-dehydroquinate synthetase 3-dehydroquinic acid synthetase 5-dehydroquinate synthase 5-dehydroquinic acid synthetase DHQS <7> [27, 28] EC 4.6.1.3 (formerly) dehydroquinate synthase <1, 4, 7> [29, 30, 31] dehydroquinate synthetase synthase, 5-dehydroquinate CAS registry number 37211-77-1

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11> <12> <13>

194

Staphylococcus aureus (no sequence specified) [29] Bacillus subtilis (no sequence specified) [19] Candida utilis (no sequence specified) [2] Thermus thermophilus (no sequence specified) (<4> extracellular isozyme [31]) [31] Escherichia coli (no sequence specified) [5, 9, 10, 12, 14, 15, 17, 18, 23, 26] Saccharomyces cerevisiae (no sequence specified) [2,10] Aspergillus nidulans (no sequence specified) [10,11,24,25,27,28,29,30] Neurospora crassa (no sequence specified) [2,10,13,21,22] Pisum sativum (no sequence specified) (<9> hoxH, b-subunit [3,6]) [3,6] Camellia sinensis (no sequence specified) [1] Helicobacter pylori (no sequence specified) [8] Brevibacterium lactofermentum (no sequence specified) [7] Sorghum sp. (no sequence specified) [20]

4.2.3.4

<14> <15> <16> <17> <18> <19> <20> <21> <22> <23> <24>

3-Dehydroquinate synthase

Hansenula henricii (no sequence specified) [2] Hansenula fabianii (no sequence specified) [2] Hansenula anomala (no sequence specified) [2] Pichia guilliermondii (no sequence specified) [2] Lodderomyces elongisporus (no sequence specified) [2] Saccharomycopsis lipolytica (no sequence specified) [2] Rhodotorula rubra (no sequence specified) [2] Rhodosporidium sphaerocarpum (no sequence specified) [2] Rhodosporidium toruloides (no sequence specified) [2] Amycolatopsis methanolica (no sequence specified) [4] Phaseolus mungo (no sequence specified) [16]

3 Reaction and Specificity Catalyzed reaction 3-deoxy-d-arabino-hept-2-ulosonate 7-phosphate = 3-dehydroquinate + phosphate Reaction type elimination Natural substrates and products S 3-deoxy-d-arabino-heptulosonic acid 7-phosphate <5, 8, 9> (Reversibility: ?) [5, 6, 9, 10] P ? Substrates and products S 3-deoxy-d-arabino-heptulosonic acid 7-phosphate <5, 8, 9> (Reversibility: ?) [5, 6, 9, 10] P ? S 3-deoxy-d-arabino-heptulosonic acid 7-phosphate <5, 8> (Reversibility: ir) [5, 9, 10, 13] P 3-dehydroquinate + phosphate <5, 8> [5, 9, 10, 13] S 3-deoxy-arabino-heptulonate 7-phosphate <7> (Reversibility: ?) [30] P 3-dehydroquinate + phosphate Inhibitors (-)-epicatechin gallate <10> [1] (-)-epigallocatechin gallate <10> [1] 1,10-phenanthroline <8> [13] 3-deoxy-d-arabino-heoptulosonic acid <5> [18] 3-deoxy-d-arabino-heptulosonic acid 7-homophosphate <5> [17] 3-deoxy-d-arabino-heptulosonic acid 7-phosphonate <5, 9> [5, 6] 3-deoxy-d-arabino-heptulosonic acid-2-O-methylglycoside 7-phosphate <5> [18] Cd2+ <2> [19] Cu2+ <2, 8> [13, 19] d-2-cis-hydroquinic acid <5> [18]

195

3-Dehydroquinate synthase

4.2.3.4

d-gluco-3-heptulosonate 7-phosphate <5> [17] d-gluco-heptulosonate 7-homophosphonate <5> [17] d-gluco-heptulosonate 7-phosphonate <5> [17] diethyl dicarbonate <7> [24] dithiothreitol <8> [13] EDTA <2, 7, 8, 13, 24> [13, 16, 19, 20, 24] EGTA <5> [9] Fe2+ <8> [13] gallic acid <10> [1] NADH <2, 8, 13> [13, 19, 20] Ni2+ <8> [13] a-(2,6-anhydro-3-deoxy-d-arabino-heptulopyranosid)onate 7-phosphate <5, 9> [5, 6] a-(2,6-anhydro-3-deoxy-d-arabino-heptulopyranosid)onate 7-phosphonate <5> [5] b-(2,6-anhydro-3-deoxy-d-arabino-heptulopyranosid)onate 7-phosphate <5> [5] bistrispropane <8> [13] carabaphosphonate <7> [25] epoxyshikimic acid <5> [18] Cofactors/prosthetic groups NAD+ <1, 2, 5, 7, 8, 10, 13, 23> (<5> 1 mol of NAD+ per mol of protein [9]) [1, 4, 9, 10, 13, 14, 17, 18, 19, 20, 24, 25, 29] NADH <7> [27] Activating compounds NAD+ <4> (<4> binding on the N-terminal a/b domain [31]) [31] Metals, ions Co2+ <1, 2, 5, 7, 10, 13> [1, 18, 19, 20, 23, 29] Cu2+ <24> [16] Mn2+ <2> [19] Zn2+ <4, 7, 8> (<4> binding on the C-terminal a-helical domain [31]) [13, 24, 25, 31] Turnover number (min–1) 6.8 <7> (3-deoxy-d-arabino-heptulosonate 7-phosphate) [30] 19 <8> (3-deoxy-d-arabino-heptulosonic acid 7-phosphate) [13] Specific activity (U/mg) 0.07 <7> (<7> mutant R130K [30]) [30] 0.59 <9> [6] 9.5 <7> [30] 14.2 <5> [18] 14.6 <2> [19] 14.9 <7> [24] 44 <5> [9, 14] Additional information <5, 8, 9, 10, 13, 24> [1, 3, 13, 16, 20, 21, 23]

196

4.2.3.4

3-Dehydroquinate synthase

Km-Value (mM) 0.0014 <8> (3-deoxy-d-arabino-heptulosonic acid 7-phosphate) [13] 0.0019 <7> (NAD+ ) [30] 0.0022 <7> (3-deoxy-d-arabino-heptulosonate phosphate) [24] 0.003 <7> (NAD+ ) [24] 0.014 <7> (NAD+, <7> mutant R130K [30]) [30] 0.021 <7> (3-deoxy-d-arabino-heptulosonate 7-phosphate) [30] 0.033-0.055 <2, 5> (3-deoxy-d-arabino-heptulosonic acid 7-phosphate) [9, 17, 18, 19] 0.055 <2> (NAD+ ) [19] 0.228 <7> (3-deoxy-d-arabino-heptulosonate 7-phosphate, <7> mutant R130K [30]) [30] pH-Optimum 7-7.5 <13> [20] 7.4-8.4 <5> [23] pH-Range 6.8-8 <13> [20]

4 Enzyme Structure Molecular weight 24000 <2> (<2> enzyme complex with chorismate synthase, SDS-PAGE [19]) [19] 38880 <5> (<5> amino acid sequence [12]; <5> HPLC [9]; <5> denaturing gel electrophoresis [9]; <5> comparison of values from bacteria + yeasts [10]) [9, 10, 12] 40000-44000 <5> (<5> HPLC [14]) [14] 43000 <7, 24> (<7> gel filtration [24]; <24> SDS-PAGE [16]) [16, 24] 44000 <5> (<5> HPLC [9]) [9] 57000 <5> (<5> gel filtration [18]) [18] 66000 <9> (<9> gel filtration [6]) [6] 67000 <24> (<24> gel filtration [16]) [16] 78400 <4> (<4> dynamic light scattering method, bimodal analysis [31]) [31] 80000 <3, 14, 15, 16, 17, 18> (<3,14,15,16,17,18> gel filtration [2]) [2] 290000-330000 <3, 6, 8, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22> (<6, 18, 20, 21, 22> gel filtration [2]; <8> sedimentation equilibrum centrifugation [22]; <8> gel electrophoresis after cross-linkage with dimethyl suberimidate [21]; <8> arom-multienzyme complex, gel filtration [2]) [2, 20, 21, 22] Subunits dimer <8> (<8> 2 * 165000, SDS-electrophoresis [21]) [21] homodimer <4> (<4> dynamic light scattering with 1 mg/ml protein at pH 8.0, 18 C in 20 mM Tris-HCl buffer [31]) [31] monomer <5, 7> (<5> 1 * 40000-44000, HPLC [14]; <5> 1 * 57000, gel filtration [18]; <7> 1 * 43000, gel filtration [24]) [14, 18, 24]

197

3-Dehydroquinate synthase

4.2.3.4

5 Isolation/Preparation/Mutation/Application Source/tissue seedling <9, 13> [3, 6, 20] shoot <10> [1] Localization chloroplast <9> [3] Purification <2> (together with chorismate synthase) [19] <5> [9, 14, 18] <7> [11, 24] <8> (arom-multienzyme complex) [13, 21, 22] <9> [6] <13> (partial) [20] <23> (partial) [4] <24> [16] Crystallization <1> (sitting-drop vapour-diffusion crystallisation at 4 C utilising microbridges, crystals of unliganded enzyme as well as in complexes with NAD, the substrate analogue [1R-(1a,3b,4a,5b)]-5-phosphonomethyl-1,3,4-trihydroxycyclohexane-1-carboxylic acid and together with both, NAD and [1R(1a,3b,4a,5b)]-5-phosphonomethyl-1,3,4-trihydroxycyclohexane-1-carboxylic acid) [29] <4> (oil microbatch method, homodimer 1.8 A resolution) [31] <7> [25] <7> (sitting-drop vapour-diffusion crystallisation at 4 C utilising microbridges, crystals of unliganded enzyme as well as in complexes with NAD+, the substrate analogue [1R-(1a,3b,4a,5b)]-5-phosphonomethyl-1,3,4-trihydroxycyclohexane-1-carboxylic acid and together with both, NAD+ and [1R(1a,3b,4a,5b)]-5-phosphonomethyl-1,3,4-trihydroxycyclohexane-1-carboxylic acid) [29] <7> (sitting-drop vapour-diffusion, crystals of unliganded enzyme, binary complexes with either the substrate analogue, carbaphosphonate or the cofactor NADH, as well as the ternary enzyme-carbaphosphonate-cofactor complex) [27] <7> (sitting-drop vapour-diffusion, structure at 1.7 A resolution) [28] Cloning <4> (expression in Escherichia coli BL21) [31] <5> [12, 15] <5> (overexpression in Escherichia coli) [9, 14] <7> (expression in Escherichia coli) [30] <7> (overexpression of the dehydroquinate synthase domain of the pentafunctional AROM protein in Escherichia coli) [11] <11> (expression in Escherichia coli) [8] <12> [7]

198

4.2.3.4

3-Dehydroquinate synthase

6 Stability Temperature stability -20 <2> (<2> inactivation [19]) [19] 4 <2> (<2> 48 h, 65% activity [19]) [19] 45 <5> (<5> 3-deoxy-d-arabino-heptulosonate protects against inactivation [17]) [17] 55 <2> (<2> inactivation [19]) [19] General stability information <24>, freezing and thawing , inactivation [16] Storage stability <5>, -70 C, more than 1 year [9] <8>, -20 C, more than 1 year [13] <8>, 50% glycerol [21] <9>, -80 C [6] <24>, 1 month [16]

References [1] Saijo, R.; Takeo, T.: Some properties of the initial four enzymes involved in shikimic acid biosynthesis in tea plant. Agric. Biol. Chem., 43, 1427-1432 (1979) [2] Bode, R.; Birnbaum, D.: Aggregation und Trennbarkeit der Enzyme des Shikimat-Pathway bei Hefen. Z. Allg. Mikrobiol., 21, 417-422 (1981) [3] Mousdale, D.M.; Coggins, J.R.: Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.. Planta, 163, 241-249 (1985) [4] Euverink, G.J.W.; Hessels, G.I.; Vrijbloed, J.W.; Coggins, J.R.; Dijkhuizen, L.: Purification and characterization of a dual function 3-dehydroquinate dehydratase from Amycolatopsis methanolica. J. Gen. Microbiol., 138, 24492457 (1992) [5] Myrvold, S.; Reimer, L.M.; Pompliano,D.L.; Frost, J. W.: Chemical inhibition of dehydroquinate synthase. J. Am. Chem. Soc., 111, 1861-1866 (1989) [6] Pompliano, D.L.; Reimer, L.M.; Myrvold, S.; Frost, J. W.: Probing lethal metabolic perturbations in plants with chemical inhibition of dehydroquinate synthase. J. Am. Chem. Soc., 111, 1866-1871 (1989) [7] Matsui, K.; Miwa, K.; Sano, K.: Cloning of a gene cluster of aroB, aroE and aroL for aromatic amino acid biosynthesis in Brevibacterium lactofermentum, a glutamic acid-producing bacterium. Agric. Biol. Chem., 52, 525-531 (1988) [8] Bereswill, S; Fassbinder, F.; Voelzing, C.; Haas, R.; Reuter, K.; Ficner, R.; Kist, M.: Cloning and functional characterization of the genes encoding 3dehydroquinate synthase (aroB) and tRNA-guanine transglycosylase (tgt) from Helicobacter pylori. Med. Microbiol. Immunol., 186, 125-134 (1997)

199

3-Dehydroquinate synthase

4.2.3.4

[9] Mehdi, S.; Frost, J.W.; Knowles, J.R.: Dehydroquinate synthase from Escherichia coli, and its substrate 3-deoxy-d-arabino-heptulosonic acid 7-phosphate. Methods Enzymol., 142, 306-314 (1987) [10] Coggins, J.R.; Duncan, K.; Anton, I.A.; Boocock, M.R.; Chaudhuri, S.; Lambert, J.M.; Lewendon, A.; Millar, G.; Mousdale, D.M.; Smith, D.D.S.: The anatomy of a multifunctional enzyme. Biochem. Soc. Trans., 15, 754-759 (1987) [11] Van den Hombergh, J.P.T.W.; Moore, J.D.; Charles, I.G.: Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein. Biochem. J., 284, 861-867 (1992) [12] Millar, G.; Coggins, J.R.: The complete amino acid sequence of 3-dehydroquinate synthase of Escherichia coli K12. FEBS Lett., 200, 11-17 (1986) [13] Lambert, J.M.; Boocock, M.R.; Coggins, J.R.: The 3-dehydroquinate synthase activity of the pentafunctional arom enzyme complex of Neurospora crassa is Zn2+ -dependent. Biochem. J., 226, 817-829 (1985) [14] Frost, J.W.; Bender, J.L.; Kadonaga, J.T.; Knowles, J.R.: Dehydroquinate synthase from Escherichia coli: Purification, cloning and construction of overproducers of the enzyme. Biochemistry, 23, 4470-4475 (1984) [15] Duncan, K.; Coggins, J.R.: Subcloning of the Escherichia coli genes aro A (5-enolyruvylshikimate 3-phosphate synthase) and aro B (3-dehydroquinate synthase). Biochem. Soc. Trans., 12, 274-275 (1984) [16] Yamamoto, E.: Purification and metal requirement of 3-dehydroquinate synthase from Phaseolus mungo seedlings. Phytochemistry, 19, 779-781 (1980) [17] LeMarechal, P.; Froussios, C.; Level, M.; Azerad, R.: The interaction of phosphonate and homophosphonate analogues of 3-deoxy-d-arabino heptulosonate 7-phosphate with 3-dehydroquinate synthetase from Escherichia coli. Biochem. Biophys. Res. Commun., 92, 1104-1109 (1980) [18] Maitra, U.S.; Sprinson, D.B.: 5-Dehydro-3-deoxy-d-arabino-heptulosonic acid 7-phosphate. J. Biol. Chem., 253, 55426-5430 (1978) [19] Hasan, N.; Nester, E.W.: Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase. J. Biol. Chem., 253, 4999-5004 (1978) [20] Saijo, R.; Kosuge, T.: The conversion of 3-deoxyarabinoheptulosonate 7phosphate to 3-dehydroquinate by Sorghum seedling preparations. Phytochemistry, 17, 223-225 (1978) [21] Lumsden, J.; Coggins, J.R.: The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain. Biochem. J., 161, 599-607 (1977) [22] Gaertner, F.H.: Purification of two multienzyme complexes in the aromatictryptophan pathway of Neurospora crassa. Arch. Biochem. Biophys., 151, 277-284 (1972) [23] Srinivasan, P.R.; Rothschild, J.; Sprinson, D.B.: The enzymic conversion of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate to 5-dehydroquinate. J. Biol. Chem., 238, 3176-3182 (1963) [24] Moore, J.D.; Coggins, J.R.; Virden, R.; Hawkins, A.R.: Efficient independent activity of a monomeric, monofunctional dehydroquinate synthase derived

200

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[25] [26] [27] [28]

[29]

[30]

[31]

3-Dehydroquinate synthase

from the N-terminus of the pentafunctional AROM protein of Aspergillus nidulans. Biochem. J., 301, 297-304 (1994) Carpenter, E.P.; Hawkins, A.R.; Frost, J.W.; Browns, K.A.: Structure of dehydroquinate synthase reveals an active site capable of multistep catalysis. Nature, 394, 299-302 (1998) Frost, J.W.; Piehler, L.T.; Montchamp, J.L.: In vitro and in vivo inhibition of dehydroquinate synthase. Biosynthesis and molecular regulation of amino acids in plants (Singh, B.K.; Flores, H.E.; Shannon, J.C eds.), 163-173 (1992) Nichols, C.E.; Ren, J.; Lamb, H.; Haldane, F.; Hawkins, A.R.; Stammers, D.K.: Identification of many crystal forms of Aspergillus nidulans dehydroquinate synthase. Acta Crystallogr. Sect. D, 57, 306-309 (2001) Nichols, C.E.; Hawkins, A.R.; Stammers, D.K.: Structure of the ’open’ form of Aspergillus nidulans 3-dehydroquinate synthase at 1.7 A resolution from crystals grown following enzyme turnover. Acta Crystallogr. Sect. D, 60, 971-973 (2004) Nichols, C.E.; Ren, J.; Leslie, K.; Dhaliwal, B.; Lockyer, M.; Charles, I.; Hawkins, A.R.; Stammers, D.K.: Comparison of ligand-induced conformational changes and domain closure mechanisms, between prokaryotic and eukaryotic dehydroquinate synthases. J. Mol. Biol., 343, 533-546 (2004) Park, A.; Lamb, H.K.; Nichols, C.; Moore, J.D.; Brown, K.A.; Cooper, A.; Charles, I.G.; Stammers, D.K.; Hawkins, A.R.: Biophysical and kinetic analysis of wild-type and site-directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein. Protein Sci., 13, 21082119 (2004) Sugahara, M.; Nodake, Y.; Sugahara, M.; Kunishima, N.: Crystal structure of dehydroquinate synthase from Thermus thermophilus HB8 showing functional importance of the dimeric state. Proteins, 58, 249-252 (2004)

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Chorismate synthase

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1 Nomenclature EC number 4.2.3.5 Systematic name 5-O-(1-carboxyvinyl)-3-phosphoshikimate forming)

phosphate-lyase

(chorismate-

Recommended name chorismate synthase Synonyms 5-enolpyruvylshikimate-3-phosphate phospholyase CS <6> [29] CS1 <10> [32] CS2 <10> [32] EC 4.6.1.4 VEG216 vegetative protein 216 chorismate synthetase CAS registry number 9077-07-0

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11> <12> <13> <14> <15>

202

Staphylococcus aureus (no sequence specified) [17, 32] Bacillus subtilis (no sequence specified) [1, 4, 7, 32] Escherichia coli (no sequence specified) [2, 4, 6, 10, 11, 12, 13, 21, 24, 25, 32] Saccharomyces cerevisiae (no sequence specified) [15, 28, 32] Euglena gracilis (no sequence specified) [19, 32] Neurospora crassa (no sequence specified) [2,3,4,8,9,14,24,27,29,32] Pisum sativum (no sequence specified) (<7> hoxH, b-subunit [5]) [5] Streptococcus pneumoniae (no sequence specified) [34] Mycobacterium tuberculosis (no sequence specified) [35] Lycopersicon esculentum (no sequence specified) (<10> orf7, isozyme oppA1 [16,20]) [16, 20, 32] Helicobacter pylori (no sequence specified) [23, 30] Plasmodium falciparum (no sequence specified) [31] Corydalis sempervirens (no sequence specified) [18, 22, 32] Aquifex aeolicus (no sequence specified) [33] Thermotoga maritima (UNIPROT accession number: Q9WYI2) [26]

4.2.3.5

Chorismate synthase

3 Reaction and Specificity Catalyzed reaction 5-O-(1-carboxyvinyl)-3-phosphoshikimate = chorismate + phosphate (<3> mechanism [21]; <3> stereochemistry [10]; <2, 4, 5, 6> combination with flavin reductase activity [7, 14, 15, 19]; <6> combination with diaphorase activity [3,8]) Reaction type 1,4-trans elimination b-elimination elimination Natural substrates and products S O5 -(1-carboxyvinyl)-3-phosphoshikimate <3, 4, 6, 7> (Reversibility: ?) [3, 5, 12, 13, 15, 21] P ? S O5 -(1-carboxyvinyl)-3-phosphoshikimate <1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 15> (<3, 6, 15> enzyme of the shikimate pathway [24, 26]; <12> enzyme is involved in shikimate pathway [31]; <6> last step in shikimate pathway [29]; <11> last step of shikimate pathway [23]; <1, 2, 3, 4, 5, 6, 10, 13> seventh enzyme in shikimate pathway [32]; <4> seventh enzyme of shikimate pathway [28]; <8> the enzyme catalyzes the final step of shikimate pathway [34]) (Reversibility: ir) [23, 24, 26, 28, 29, 30, 31, 32, 34] P chorismate + phosphate <1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 15> [23, 24, 26, 28, 29, 30, 31, 32, 34] Substrates and products S (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate <3> (<3> ? [13]) (Reversibility: ?) [13] P 6-fluorochorismate + phosphate <3> [13] S FMN + NADPH <6> (<6> recombinant enzyme has a diaphorase activity. NADPH binds in or near the substrate (O5 -(1-carboxyvinyl)-3-phosphoshikimate) binding site, suggesting that NADPH binding to the enzyme is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity [27]) (Reversibility: ?) [27] P ? <6> [27] S O5 -(1-carboxyvinyl)-3-phosphoshikimate <3, 4, 6, 7> (Reversibility: ?) [3, 5, 12, 13, 15, 21] P ? S O5 -(1-carboxyvinyl)-3-phosphoshikimate <1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15> (<3, 4, 6, 7> 5-enolpyruvylshikimate 3-phosphate [3, 5, 12, 13, 15, 21]; <3> evidence for a radical mechanism [25]; <3, 6> secondary b deuterium kinetic isotope effect [24]; <3, 6, 15> enzyme of the shikimate pathway [24, 26]; <12> enzyme is involved in shikimate pathway [31]; <6> last step in shikimate pathway [29]; <11> last step of shikimate pathway [23]; <1, 2, 3, 4, 5, 6, 10, 13> seventh enzyme in shikimate pathway

203

Chorismate synthase

4.2.3.5

[32]; <4> seventh enzyme of shikimate pathway [28]; <8> the enzyme catalyzes the final step of shikimate pathway [34]) (Reversibility: ir) [3, 5, 12, 13, 15, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34] P chorismate + phosphate <1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15> [3, 5, 12, 13, 15, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34] S Additional information <1, 2, 3, 4, 5, 6, 10, 12, 13, 15> (<2> enzyme has flavin reductase activity [32]; <5,6> enzyme shows flavin reductase activity [32]; <4> enzyme shows flavin reductase activity [32]; <15> monofunctional enzyme that does not have an intrinsic ability to reduce the FMN cofactor [26]; <12> monofunctional enzyme that does not possess an intrinsic flavin reductase activity [31]; <1,3,10,13> no flavin reductase activity [32]; <6> the enzyme also shows FMN:NADPH oxidoreductase activity [29]) (Reversibility: ?) [26, 29, 31, 32] P ? <1, 2, 3, 4, 5, 6, 10, 12, 13, 15> [26, 29, 31, 32] Inhibitors (6R)-6-fluoro-5-enoylpyruvylshikimate 3-phosphate <3> (<3> IC50 is 0.0005 mM when the enzyme is preincubated with the inhibitor, the IC50 is 0.25 mM when the enzyme is not preincubated with the inhibitor, inhibition is not absolutely irreversible [25]) [25] bathophenanthroline <3> [11] diethyl dicarbonate <6> [29] Fe2+ <3> [11] Cofactors/prosthetic groups FMN <3, 15> (<3,15> cofactor [25,26]) [25, 26] FMNH2 <1, 2, 4, 5, 6, 10, 11, 13> (<4,10> absolute requirement [28, 32]; <6> absolute requirement for FMNH2 , which is not consumed during the reaction [29]; <1> absolute requirement, Km : 0.0048 mM [32]; <2> absolute requirement, Km : 0.0125 mM [32]; <13> absolute requirement, Km : 37 nM [32]; <4> absolute requirement, Km : 42 nM [32]; <6> absolute requirement, Km : 66 nM [32]; <5> absolute requirement, Km : 76 nM [32]; <11> required. The unique FMN-binding site is formed largely by a single subunit, with a small contribution from a neighboring subunit. The isoalloxazine ring of the bound FMN is significantly non-planar [30]) [28, 29, 30, 32] flavins <1, 2, 3, 4, 5, 6, 10, 13> (<1, 2, 3, 4, 5, 6, 10, 13> required, reduced forms e.g. FMN, FAD [3, 7, 8, 11, 12, 15, 17, 19, 20, 21, 22]) [3, 7, 8, 11, 12, 15, 17, 19, 20, 21, 22] NADPH <2, 4, 6> (<2, 4, 6> required [3, 7, 8, 15]) [3, 7, 8, 15] Additional information <3> (<3> no activity with 5-deaza-FMN [25]) [25] Activating compounds light <5> (<5> stimulation [19]) [19] Metals, ions Mg2+ <2> [7]

204

4.2.3.5

Chorismate synthase

Specific activity (U/mg) 0.92 <2> [7] 4 <1> [17] 14.8 <3> [2] 32.1 <6> [2] Additional information <2, 3, 6, 7> [3, 4, 5, 8, 11, 14] Km-Value (mM) 0.0013-0.0022 <3> (O5 -(1-carboxyvinyl)-3-phosphoshikimate) [32] 0.0027-0.007 <6> (O5 -(1-carboxyvinyl)-3-phosphoshikimate) [32] 0.0097 <4> (O5 -(1-carboxyvinyl)-3-phosphoshikimate) [32] 0.011 <10> (5-enolpyruvylshikimate 3-phosphate, <10> isozyme MatCS1 [20]) [20] 0.011 <10> (O5 -(1-carboxyvinyl)-3-phosphoshikimate, <10> enzyme form CS1 [32]) [32] 0.0127 <1> (5-enolpyruvylshikimate 3-phosphate) [17] 0.0127 <1> (O5 -(1-carboxyvinyl)-3-phosphoshikimate) [32] 0.027 <5> (5-enolpyruvylshikimate 3-phosphate) [19] 0.027 <5> (O5 -(1-carboxyvinyl)-3-phosphoshikimate) [32] 0.043 <6> (NADPH, <6> FMN:NADPH oxidoreductase activity, wild-type enzyme [29]) [29] 0.047 <13> (5-enolpyruvylshikimate 3-phosphate) [18] 0.053 <13> (O5 -(1-carboxyvinyl)-3-phosphoshikimate) [32] 0.08 <10> (5-enolpyruvylshikimate 3-phosphate, <10> isozyme MatCS2 [20]) [20] 0.08 <10> (O5 -(1-carboxyvinyl)-3-phosphoshikimate, <10> enzyme form CS2 [32]) [32] 0.11 <6> (NADPH, <6> FMN:NADPH oxidoreductase activity, mutant enzyme H17A [29]) [29] 0.28 <6> (NADPH, <6> FMN:NADPH oxidoreductase activity, mutant enzyme H106A [29]) [29] pH-Optimum 6-8 <10> (<10> enzyme form CS1 [32]; <10> enzyme form CS2 [32]) [32] 6.5-8.5 <3> [32] 7-8 <6> [9] 7-8.5 <6> [32] 8 <13> [32] 8.2 <5> [19, 32]

4 Enzyme Structure Molecular weight 80100 <13> (<13> gel filtration [22]) [22] 110000 <6> (<6> sucrose gradient density centrifugation [8]) [8] 110000-138000 <5> (<5> gel filtration [19]) [19]

205

Chorismate synthase

4.2.3.5

144000 <3> (<3> gel filtration after cross-linkage with dimethyl suberimidate [2]) [2] 186800 <1> (<1> gel filtration [17]) [17] 190000 <3> (<3> nondenaturing PAGE [12]) [12] 198000 <6, 11> (<6> gel filtration after cross-linkage with dimethyl suberimidate [2]; <11> dynamic light scattering analysis [23]) [2, 23] 200000 <15> (<15> non-denaturing PAGE in presence of O5 -(1-carboxyvinyl)-3-phosphoshikimate and FMN [26]) [26] Subunits ? <5> (<5> x * 41700 [32]) [32] dimer <4, 6, 9, 10, 13> (<6> 2 * 55000, SDS-PAGE [3,8]; <13> 2 * 41900, SDSPAGE [22]; <4> 2 * 40800, dimer or tetramer [32]; <10> 2 * 41605, dimer or tetramer, enzyme form CS2 [32]; <13> 2 * 41771 [32]; <10> 2 * 41902, dimer or tetramer, enzyme form CS1 [32]; <6> 2 * 46400, dimer or tetramer [32]; <9> analytical ultracentrifugation, low equilibrium between dimer and tetramer [35]) [3, 8, 22, 32, 35] tetramer <1, 3, 4, 6, 8, 9, 10, 15> (<15> 4 * 42000, SDS-PAGE [26]; <6> 4 * 50000, SDS-PAGE [2]; <3> 4 * 38000, SDS-PAGE [2]; <1> 4 * 43024, electrospray mass spectrometry [17]; <3> 4 * 39138 [32]; <4> 4 * 40800, dimer or tetramer [32]; <10> 4 * 41605, dimer or tetramer, enzyme form CS2 [32]; <15> 4 * 41754, calculation from amino acid sequence [26]; <10> 4 * 41902, dimer or tetramer, enzyme form CS1 [32]; <1> 4 * 43026 [32]; <6> 4 * 46400, dimer or tetramer [32]; <9> crystallization data, low equilibrium between dimer and tetramer [35]) [2, 17, 26, 32, 34, 35] trimer <2> (<2> heterotrimer [32]) [32] Additional information <2, 3, 5> (<3> x * 40000, SDS-PAGE [6]; <2> x * 24000, SDS-PAGE [1,7]; <5> x * 41700, SDS-PAGE [19]) [1, 6, 7, 19] Posttranslational modification proteolytic modification <10, 13> (<10> the precursor protein has a MW of 46871, enzyme form CS2 [32]; <13> the precursor protein has a MW of 47722 Da [32]; <10> the precursor protein has a MW of 47722, enzyme form CS1 [32]) [32]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture <13> [22] flower <10> [16] root <10> [16] seedling <7> [5] Localization chloroplast <7> [5] cytosol <12> (<12> parasite cytosol [31]) [31]

206

4.2.3.5

Chorismate synthase

Purification <2> (together with NADPH dependent flavin reductase) [1, 7] <3> [2, 11] <5> [19] <6> [2, 3, 8, 27] <6> (expressed in Escherichia coli) [14] <10> (expressed in Escherichia coli) [20] <11> (recombinant enzyme fused with an eight-residue C-terminal tag) [23] <13> [22] <13> (expressed in Escherichia coli) [18] <15> (recombinant enzyme) [26] Crystallization <4> (hanging drop vapour diffusion method, three-dimensional X-ray structure from selenomethionine-labeled crystals at 2.2 A resolution. The structure shows a novel b,a,b,a fold consisting of an alternate tight packing of two a-helical and two b-sheet layers. The molecule is arranged as a tight tetramer with D2 symmetry) [28] <8> (hanging-drop vapour diffusion method. Crystal streucture solved at 1.0 A) [34] <9> (b-a-b sandwich structure) [35] <11> (crystal structure of chorismate synthase in both FMN-bound and FMN-free form. It is a tetrameric enzyme, with each monomer possessing a novel b-a-b sandwich fold) [30] <11> (hanging-drop vapour-diffusion method, crystallization at 296 K using polyethylene glycol 400 as precipitant, recombinant enzyme fused with an eight-residue C-terminal tag) [23] <14> (crystal structure of the enzyme reveals a novel b a b sandwich topology) [33] Cloning <1> (overexpression in Escherichia coli) [17] <3> (overexpression in Escherichia coli) [6] <6> (expression in Escherichia coli) [14, 27] <10> (expression in Escherichia coli) [20] <11> (overexpression in Escherichia coli, fused with an eight-residue Cterminal tag) [23] <12> [31] <13> (expression in Escherichia coli) [18] <15> (expression in Escherichia coli) [26] Engineering H106A <6> (<6> mutant enzyme with 20fold reduced activity [29]) [29] H17A <6> (<6> mutant enzyme with 10fold reduced activity [29]) [29]

207

Chorismate synthase

4.2.3.5

6 Stability Temperature stability 92 <15> (<15> melting temperature above [26]) [26] General stability information <6>, bovine serum albumin stabilizes [3, 8] Storage stability <2>, -20 C, 0.05 mM Tris-HCl, pH 7.5, 0.01 mM FMN, 10% glycerol, 3 mM mercaptoethanol, 4 months [7] <2>, 4 C, 2 weeks [7] <3>, -15 C, several months [11] <6>, -15 C, 0.1 M potassium phosphate buffer, pH 7.0, 0.1 mM EDTA, 0.2 mM dithiothreitol, more than 6 months [8] <6>, liquid nitrogen [3] <3, 6>, -20 C, 50 mM Tris-HCl buffer, pH 7.5, 0.4 mM dithiothreitol, 50% glycerol [2]

References [1] Hasan, N.; Nester, E.W.: Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase. J. Biol. Chem., 253, 4999-5004 (1978) [2] White, P.J.; Millar, G.; Coggins, J.R.: The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa. Biochem. J., 251, 313-322 (1988) [3] Gaertner, F.H.: Chorismate synthase: A bifunctional enzyme in Neurospora crassa. Methods Enzymol., 142, 362-366 (1987) [4] White, P.J.; Mousdale, D.M.; Coggins, J.R.: A simple anaerobic assay for chorismate synthase. Biochem. Soc. Trans., 15, 144-145 (1978) [5] Mousdale, D.M.; Coggins, J.R.: Detection and subcellular localization of a higher plant chorismate synthase. FEBS Lett., 205, 328-332 (1986) [6] Millar, G.; Anton, I.A.; Mousdale, D.M.; White, P.J.; Coggins, J.R.: Cloning and overexpression of the Escherichia coli aroC gene encoding the enzyme chorismate synthase. Biochem. Soc. Trans., 14, 262-263 (1986) [7] Hasan, N.; Nester, E.W.: Purification and properties of chorismate synthase from Bacillus subtilis. J. Biol. Chem., 253, 4993-4998 (1978) [8] Welch, G.R.; Cole, K.W.; Gaertner, F.H.: Chorismate synthase of Neurospora crassa: A flavoprotein. Arch. Biochem. Biophys., 165, 505-518 (1974) [9] Gaertner, F.H.; Cole, K.W.: Properties of chorismate synthase in Neurospora crassa. J. Biol. Chem., 248, 4602-4609 (1973) [10] Floss, H.G.; Onderka, D.K.; Carrol, M.: Stereochemistry of the 3-deoxy-darabino-heptulosonate 7-phosphate synthetase reaction and the chorismate synthetase reaction. J. Biol. Chem., 247, 736-744 (1972)

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[11] Morell, H.; Clark, M.J.; Knowles, P.F.; Sprinson, D. B.: The enzymic synthesis of chorismic and prephenic acids from 3-enolpyruvylshikimic acid 5phosphate. J. Biol. Chem., 242, 82-90 (1967) [12] Macheroux, P.; Schoenbrunn, E.; Svergun, D.I.; Volkov, V.V.; Koch, M.H.J.; Bornemann, S.; Thorneley, R.N.F.: Evidence for a major structural change in Escherichia coli chorismate synthase induced by flavin and substrate binding. Biochem. J., 335, 319-327 (1998) [13] Bornemann, S.; Ramjees, M.K.; Balasurbramanian, S.; Abell, C.; Coggins, J.R.; Lowe, D.J.; Thorneley, R.N.F.: Escherichia coli chorismate synthase catalyzes the conversion of (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate to 6-fluorochorismate. Implications for the enzyme mechanism and the antimicrobial action of (6S)-6-fluoroshikimate. J. Biol. Chem., 270, 2281122815 (1995) [14] Henstrand, J.M.; Amrhein, N.; Schmid, J.: Cloning and characterization of a heterologously expressed bifunctional chorismate synthase/flavin reductase from Neurospora crassa. J. Biol. Chem., 270, 20447-20452 (1995) [15] Henstrand, J.M.; Schaller, A.; Braun, M.; Amrhein, N.; Schmid, J.: Saccharomyces cerevisiae chorismate synthase has a flavin reductase activity. Mol. Microbiol., 22, 859-866 (1996) [16] Goerlach, J.; Schmid, J.; Amrhein, N.: Differential expression of tomato (Lycopersicon esculentum L.) genes encoding shikimate pathway isoenzymes. II. Chorismate synthase. Plant Mol. Biol., 23, 707-716 (1993) [17] Horsburgh, M.J.; Foster, T.J.; Barth, P.T.; Coggins, J.R.: Chorismate synthase from Staphylococcus aureus. Microbiology, 142, 2943-2950 (1996) [18] Henstrand, J.M.; Schmid, J.; Amrhein, N.: Only the mature form of the plastidic chorismate synthase is enzymatically active. Plant Physiol., 108, 11271132 (1995) [19] Schaller, A.; van Afferden, M.; Windhofer, V.; Buelow, S.; Abel, G.; Schmid, J.; Amrhein, N.: Purification and characterization of chorismate synthase from Euglena gracilis. Plant Physiol., 97, 1271-1279 (1991) [20] Braun, M.; Henstrand, J.M.; Goerlach, J.; Amrhein, N.; Schmid, J.: Enzymatic properties of chorismate synthase isozymes of tomato (Lycopersicon esculentum Mill.). Planta, 200, 64-70 (1996) [21] Bornemann, S.; Lowe, D.J.; Thorneley, R.N.F.: The transient kinetics of Escherichia coli chorismate synthase: Substrate consumption, product formation, phosphate dissociation, and characterization of a flavin intermediate. Biochemistry, 55, 9907-9916 (1996) [22] Schaller, A.; Windhofer, V.; Amrhein, N.: Purification of chorismate synthase from a cell culture of the higher plant Corydalis sempervirens Pers. Arch. Biochem. Biophys., 282, 437-442 (1990) [23] Ahn, H.J.; Yang, J.K.; Lee, B.I.; Yoon, H.J.; Kim, H.W.; Suh, S.W.: Crystallization and preliminary X-ray crystallographic studies of chorismate synthase from Helicobacter pylori. Acta Crystallogr. Sect. D, 59, 569-571 (2003) [24] Bornemann, S.; Theoclitou, M.E.; Brune, M.; Webb, M.R.; Thorneley, R.N.; Abell, C.: A secondary b deuterium kinetic isotope effect in the chorismate synthase reaction. Bioorg. Chem., 28, 191-204 (2000)

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Chorismate synthase

4.2.3.5

[25] Osborne, A.; Thorneley, R.N.; Abell, C.; Bornemann, S.: Studies with substrate and cofactor analogues provide evidence for a radical mechanism in the chorismate synthase reaction. J. Biol. Chem., 275, 35825-35830 (2000) [26] Fitzpatrick, T.B.; Killer, P.; Thomas, R.M.; Jelesarov, I.; Amrhein, N.; Macheroux, P.: Chorismate synthase from the hyperthermophile Thermotoga maritima combines thermostability and increased rigidity with catalytic and spectral properties similar to mesophilic counterparts. J. Biol. Chem., 276, 18052-18059 (2001) [27] Kitzing, K.; Macheroux, P.; Amrhein, N.: Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH. J. Biol. Chem., 276, 42658-42666 (2001) [28] Quevillon-Cheruel, S.; Leulliot, N.; Meyer, P.; Graille, M.; Bremang, M.; Blondeau, K.; Sorel, I.; Poupon, A.; Janin, J.; van Tilbeurgh, H.: Crystal structure of the bifunctional chorismate synthase from Saccharomyces cerevisiae. J. Biol. Chem., 279, 619-625 (2004) [29] Kitzing, K.; Auweter, S.; Amrhein, N.; Macheroux, P.: Mechanism of chorismate synthase. Role of the two invariant histidine residues in the active site. J. Biol. Chem., 279, 9451-9461 (2004) [30] Ahn, H.J.; Yoon, H.J.; Lee, B., 2nd; Suh, S.W.: Crystal structure of chorismate synthase: a novel FMN-binding protein fold and functional insights. J. Mol. Biol., 336, 903-915 (2004) [31] Fitzpatrick, T.; Ricken, S.; Lanzer, M.; Amrhein, N.; Macheroux, P.; Kappes, B.: Subcellular localization and characterization of chorismate synthase in the apicomplexan Plasmodium falciparum. Mol. Microbiol., 40, 65-75 (2001) [32] Macheroux, P.; Schmid, J.; Amrhein, N.; Schaller, A.: A unique reaction in a common pathway. Mechanism and function of chorismate synthase in the shikimate pathway. Planta, 207, 325-334 (1999) [33] Viola, C.M.; Saridakis, V.; Christendat, D.: Crystal structure of chorismate synthase from Aquifex aeolicus reveals a novel b a b sandwich topology. Proteins Struct. Funct. Bioinform., 54, 166-169 (2003) [34] Maclean, J.; Ali, S.: The structure of chorismate synthase reveals a novel flavin binding site fundamental to a unique chemical reaction. Structure, 11, 1499-1511 (2003) [35] Dias, M.V.; Borges, J.C.; Ely, F.; Pereira, J.H.; Canduri, F.; Ramos, C.H.; Frazzon, J.; Palma, M.S.; Basso, L.A.; Santos, D.S.; de Azevedo, W.F., Jr.: Structure of chorismate synthase from Mycobacterium tuberculosis. J. Struct. Biol., 154, 130-143 (2006)

210

Pentalenene synthase

4.2.3.7

1 Nomenclature EC number 4.2.3.7 Systematic name 2-trans,6-trans-farnesyldiphosphate diphosphate-lyase (cyclizing, pentalenene-forming) Recommended name pentalenene synthase Synonyms EC 4.6.1.5 (formerly) pentalenene synthetase synthetase, pentalenene CAS registry number 90597-46-9

2 Source Organism <1> Streptomyces sp. (no sequence specified) [1, 2, 3, 4, 6, 7] <2> Streptomyces sp. (UNIPROT accession number: Q55012) [5, 8, 9]

3 Reaction and Specificity Catalyzed reaction 2-trans,6-trans-farnesyl diphosphate = pentalenene + diphosphate (<1> mechanism [4,7]; <1> stereochemistry [3,4]; <1> stereochemistry, SE’ reaction type [6]) Reaction type P-O bond cleavage Natural substrates and products S farnesyl diphosphate <1> (Reversibility: ?) [1, 2, 3, 4] P pentalenene + diphosphate <1> [1, 2, 3, 4]

211

Pentalenene synthase

4.2.3.7

Substrates and products S farnesyl diphosphate <1> (<1> the initial step in the reaction is probably a cyclization of farnesyl diphosphate to form humulene [3]; <1> trans,trans [2,4]) (Reversibility: ?) [1, 2, 3, 4] P pentalenene + diphosphate <1> [1, 2, 3, 4] Inhibitors diphosphate <1, 2> (<1> plus pentalene: no inhibition alone, both products bind cooperatively at the active site [2]) [2, 5] Mn2+ <1> (<1> divalent metal required, Mg2+ or Mn2+ , inhibition above 2.5 mM [2]) [2] pentalenene plus diphosphate <1> (<1> no inhibition alone, both products bind cooperatively at the active site [2]) [2] Metals, ions Mg2+ <1, 2> (<2> absolute requirement [5]; <1> divalent metal required, Mg2+ or Mn2+ [2]) [2, 5] Mn2+ <1> (<1> divalent metal required, Mg2+ or Mn2+ , inhibition above 2.5 mM [2]) [2] Specific activity (U/mg) 0.287 <1> (<1> 30 C, pH 8.4 [2]) [2] 0.324 <2> (<2> 30 C, pH 8.2 [5]) [5] Km-Value (mM) 0.0003 <2> (farnesyl diphosphate, <2> 30 C, pH 8.2 [8]) [8] 0.00031 <2> (farnesyl diphosphate, <2> 30 C, pH 8.2 [5]) [5] 0.00077 <1> ((E,E)-farnesyl diphosphate, <1> 30 C, pH 8.4 [2]) [2] Ki-Value (mM) 0.0032 <2> (diphosphate) [5] pH-Optimum 8.2-8.4 <2> [5] 8.4 <1> [2] Temperature optimum ( C) 30 <1> (<1> assay at [2]) [2]

4 Enzyme Structure Molecular weight 57000 <1> (<1> gel filtration [1]) [1, 2] Subunits ? <2> (<2> x * 41000, SDS-PAGE [5]) [5]

212

4.2.3.7

Pentalenene synthase

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <1> (<1> from a 60 h cell culture [1]) [1] Purification <1> (partial) [2] <2> [5] Crystallization <1> [7] Cloning <2> [5] Engineering F77Y <2> (<2> compared to wild type, kcat /Km value is 20fold lower [9]) [9] H309A <2> (<2> retains enzymatic activity, minor increase in Km -value [8]) [8] H309C <2> (<2> retains enzymatic activity, minor increase in Km -value [8]) [8] H309F <2> (<2> retains enzymatic activity, minor increase in Km -value [8]; <2> reaction products are pentalene plus varying proportions of (+)-germacrene A and protoilludene [9]) [8, 9] H309S <2> (<2> retains enzymatic activity, minor increase in Km -value [8]) [8] N219A <2> (<2> catalytically inactive [9]) [9] N219D <2> (<2> compared to wild type, kcat /Km value is 3300fold lower, reaction products are pentalene plus b-caryophyllene [9]) [9] N219L <2> (<2> catalytically inactive [9]) [9] W308F <2> (<2> reaction products are pentalene plus varying proportions of (+)-germacrene A [9]) [9] W308F/H309F <2> (<2> reaction products are pentalene plus varying proportions of (+)-germacrene A [9]) [9]

6 Stability Storage stability <1>, 4 C, crude enzyme stable for up to 6 days [2]

References [1] Cane, D.E.: Cell-free studies of monoterpene and sesquiterpene biosynthesis. Biochem. Soc. Trans., 11, 510-515 (1983) [2] Cane, D.E.; Pargellis, C.: Partial purification and characterization of pentalenene synthase. Arch. Biochem. Biophys., 254, 421-429 (1987)

213

Pentalenene synthase

4.2.3.7

[3] Cane, D.E.; Oliver, J.S.; Harrison, P.H.M.; Abell, C.; Hubbard, B.R.; Kane, C.T.; Lattman, R.: The biosynthesis of pentalenene and pentalenolactone. J. Am. Chem. Soc., 112, 4513-4524 (1990) [4] Cane, D.E.; Abell, C.; Lattman, R.; Kane, C.T.; Hubbard, B.R.; Harrison, P.H.M.: Pentalenene biosynthesis and the enzymatic cyclisation of farnesyl pyrophosphate. Anti stereochemistry in a biological SE reaction. J. Am. Chem. Soc., 110, 4081-4082 (1988) [5] Cane, D.E.; Sohng, J.K.; Lamberson, C.R.; Rudnicki, S.M.; Wu, Z.; Lloyd, M.D.; Oliver, J.S.; Hubbard, B.R.: Pentalenene synthase. Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319. Biochemistry, 33, 5846-5857 (1994) [6] Cane, D.E.; Weiner, S.W.: Cyclization of farnesyl diphosphate to pentalenene. Orthogonal stereochemistry in an enzyme-catalyzed SE’ reaction. Can. J. Chem., 72, 118-127 (1994) [7] Lesburg, C.A.; Zhai, G.; Cane, D.E.; Christianson, D.W.: Crystal structure of pentalenene synthase: mechanistic insights on terpenoid cyclization reactions in biology. Science, 277, 1820-1824 (1997) [8] Seemann, M.; Zhai, G.; Umezawa, K.; Cane, D.: Pentalenene synthase. Histidine-309 is not required for catalytic activity. J. Am. Chem. Soc., 121, 591592 (1999) [9] Seemann, M.; Zhai, G.; de Kraker, J.W.; Paschall, C.M.; Christianson, D.W.; Cane, D.E.: Pentalenene synthase. Analysis of active site residues by site-directed mutagenesis. J. Am. Chem. Soc., 124, 7681-7689 (2002)

214

Casbene synthase

4.2.3.8

1 Nomenclature EC number 4.2.3.8 Systematic name geranylgeranyl-diphosphate diphosphate-lyase (cyclizing, casbene-forming) Recommended name casbene synthase Synonyms EC 4.6.1.7 (formerly) casbene synthetase CAS registry number 69106-45-2

2 Source Organism <1> Ricinus communis (no sequence specified) [1, 2, 3, 4, 5, 6, 7, 8] <2> Ricinus communis (UNIPROT accession number: P59287) [5]

3 Reaction and Specificity Catalyzed reaction geranylgeranyl diphosphate = casbene + diphosphate Reaction type P-O bond cleavage Natural substrates and products S geranylgeranyl diphosphate <1> (<1> regulation of phytoalexin biosynthesis, enzyme produces the antifungal diterpene casbene, a stress metabolite [1,2,3,4,5,6,7,8]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] P casbene + diphosphate <1> (<1> diterpene phytoalexin, product of a single enzyme-catalyzed transformation [1,8]) [1, 2, 3, 4, 5, 6, 7, 8] Substrates and products S geranylgeranyl diphosphate <1> (<1> regulation of phytoalexin biosynthesis, enzyme produces the antifungal diterpene casbene, a stress metabolite [1,2,3,4,5,6,7,8]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8]

215

Casbene synthase

4.2.3.8

P casbene + diphosphate <1> (<1> diterpene phytoalexin, product of a single enzyme-catalyzed transformation [1,8]) [1, 2, 3, 4, 5, 6, 7, 8] Inhibitors Mn2+ <1> (<1> above 0.2 mM [1]) [1] N-ethylmaleimide <1> (<1> activity is inhibited by 50% [1]) [1] tributyl-2,4-dichlorobenzylphosphonium chloride <1> (<1> phosphonD, growth retardant, inhibits the activity by 55% [1]) [1] Additional information <1> (<1> 10 mM iodoacetamide is not inhibitory, SKF525A, N,N-dimethylaminoethyl-2,2-diphenylpentanoate and the growth retardant Amo-1618, 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenyl piperidine-1-carboxylate are ineffective inhibitors [1]) [1] Activating compounds Rhizpus stolonifer polygalcturonase <1> (<1> elicitor, stimulates germinating seedlings to produce casbene synthetase activity [2,3]) [2, 3] Metals, ions Mg2+ <1> (<1> highly dependent on, maximal stimulation of the activity above 5 mM, Mn2+ is much less effective [1]) [1] Turnover number (min–1) 0.03 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutant Q355E [7]) [7] 0.04 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutant Q356E [7]) [7] 0.2 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutants Q359E and H294Q [7]) [7] 0.21 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutant H131Q [7]) [7] 0.23 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, wild-type and mutant C519W [7]) [7] 0.24 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutants H171Q [7]) [7] Specific activity (U/mg) 0.0026 <1> (<1> recombinant enzyme [6]) [6] 0.0437 <1> [1] 0.25 <1> [4] 0.359 <1> (<1> recombinant enzyme [7]) [7] Km-Value (mM) 0.00176 <1> (geranylgeranyl diphosphate, <1> pH 7.5, 30 C [6]) [6] 0.0019 <1> (geranylgeranyl diphosphate, <1> pH 9.0, 30 C [1]) [1] 0.0031 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, wild-type [7]) [7] 0.0032 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutants Q359E and H131Q [7]) [7] 0.0033 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutants H171Q and C519W [7]) [7]

216

4.2.3.8

Casbene synthase

0.0034 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutant H294Q [7]) [7] 0.0125 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutant Q356E [7]) [7] 0.032 <1> (geranylgeranyl diphosphate, <1> pH 8.5, 30 C, mutant Q355E [7]) [7] pH-Optimum 7.5-9 <1> (<1> half-maximal activity at pH 6.0 and 9.8 [1]) [1] pH-Range 6-9.8 <1> (<1> half-maximal activity at pH 6.0 and 9.8 [1]) [1]

4 Enzyme Structure Molecular weight 53000 <1> (<1> native enzyme, gel filtration [1,4]) [1, 4] 68960 <1> (<1> predicted from cDNA sequence [5]) [5] Subunits monomer <1> (<1> 1 * 59000, SDS-PAGE [4,6]) [4, 6]

5 Isolation/Preparation/Mutation/Application Source/tissue seedling <1> (<1> infected with the fungus Rhizpus stolonifer, no detectable levels of casbene synthetase mRNA in healthy seedlings [1,4]) [1, 2, 3, 4, 5, 6, 8] Localization cytoplasm <1> [1] proplastid <1> [6] Purification <1> [4] <1> (partial) [1] <1> (recombinant enzyme) [6, 7] Cloning <1> (cDNA clone pCS7) [5] <1> (cloned, expressed and overproduced in Escherichia coli) [6, 7] <2> [5] Engineering C519W <1> (<1> site-directed mutagenesis [7]) [7] H131Q <1> (<1> site-directed mutagenesis [7]) [7] H171Q <1> (<1> site-directed mutagenesis [7]) [7] H294Q <1> (<1> site-directed mutagenesis [7]) [7]

217

Casbene synthase

4.2.3.8

Q355E <1> (<1> site-directed mutagenesis [7]) [7] Q356E <1> (<1> site-directed mutagenesis [7]) [7] Q359E <1> (<1> site-directed mutagenesis [7]) [7] Application agriculture <1> (<1> plant disease resistance macrocyclic diterpene hydrocarbons formed in response to fungal attack, antibiotic properties of phytoalexins as hypersensitive response of plants to invasion by potentially pathogenic microorganisms [2]) [2, 8]

References [1] Dueber, M.T.; Adolf, W.; West, C.A.: Biosynthesis of diterpene phytoalexin casbene: partial purification and characterization of casbene synthetase from Rhizinus communis. Plant Physiol., 62, 598-603 (1978) [2] Lee, S.-C.; West, C.A.: Polygalacturonase from Rhizopus stolonifer, an elicitor of casbene synthetase activity in castor bean (Ricinus communis L.) seedlings. Plant Physiol., 67, 633-639 (1981) [3] Lee, S.-C.; West, C.A.: Properties of Rhizopus stolonifer polygalacturonase, an elicitor of casbene synthetase activity in castor bean (Ricinus communis L.) seedlings. Plant Physiol., 67, 640-645 (1981) [4] Moesta, P.; West, C.A.: Casbene synthetase: regulation of phytoalexin biosynthesis in Ricinus communis L. seedlings. Purification of casbene synthetase and regulation of its biosynthesis during elicitation. Arch. Biochem. Biophys., 238, 325-333 (1985) [5] Mau, C.J.D.; West, C.A.: Cloning of casbene synthase cDNA: evidence for conserved structural features among terpenoid cyclases in plants. Proc. Natl. Acad. Sci. USA, 91, 8497-8501 (1994) [6] Hill, A.M.; Cane, D.E.; Mau, C.J.; West, C.A.: High level expression of Ricinus communis casbene synthase in Escherichia coli and characterization of the recombinant enzyme. Arch. Biochem. Biophys., 336, 283-289 (1996) [7] Huang, K.; Huang, Q.; Scott, A.I.: Overexpression, single-step purification, and site-directed mutagenetic analysis of casbene synthase. Arch. Biochem. Biophys., 352, 144-152 (1998) [8] Huang, Q.; Huang, K.; Scott, A.I.: Enzymic syntheses of 13 C-enriched geranylgeranyl diphosphate and casbene from 13 C-labeled isopentenyl diphosphate. Tetrahedron Lett., 39, 2033-2036 (1998)

218

Aristolochene synthase

4.2.3.9

1 Nomenclature EC number 4.2.3.9 Systematic name trans,trans-farnesyl-diphosphate diphosphate-lyase (cyclizing, aristolocheneforming) Recommended name aristolochene synthase Synonyms 5-epi-aristolochene synthase <1> [10, 13] AS EC 2.5.1.40 (formerly) EC 4.1.99.7 (formerly) FPP-carbocyclase TEAS <1> [10] cyclase, farnesyl pyrophosphate farnesylpyrophosphate cyclase sesquiterpene cyclase synthase, aristolochene CAS registry number 94185-89-4

2 Source Organism <1> Nicotiana tabacum (no sequence specified) [10, 13] <2> Aspergillus terreus (no sequence specified) [1, 2, 9, 12] <3> Penicillium roquefortii (no sequence specified) [3, 4, 5, 6, 7, 8, 9, 11, 12]

3 Reaction and Specificity Catalyzed reaction 2-trans,6-trans-farnesyl diphosphate = aristolochene + diphosphate Reaction type internal cyclization

219

Aristolochene synthase

4.2.3.9

Natural substrates and products S trans,trans-farnesyl diphosphate <3> (<3> the enzyme appears to be transcriptionally regulated [4]) (Reversibility: ?) [4] P aristolochene + diphosphate Substrates and products S farnesyl diphosphate <1> (Reversibility: ?) [10] P (+)-5-epi-aristolochene + diphosphate (<1> 78.9% (+)-5-epi-aristolochene + 6.2% 4-epi-eremophilene + 3.6% (+)-germacrene A [10]) S trans,trans-farnesyl diphosphate <3> (Reversibility: ?) [11] P aristolochene + diphosphate + germacrene (<3> wild-type enzyme produces 92% aristolochene, 8% germacrene and a small amount of valencene [11]) S trans,trans-farnesyl diphosphate <2, 3> (Reversibility: ?) [12] P (+)-aristolochene + diphosphate (<3> (+)-aristolochene + minor amounts of (S)-(-)-germacrene A and (-)-valencene in a 94:4:2 ratio [12]; <2> (+)-aristolochene is the only product [12]) S trans,trans-farnesyl diphosphate <2, 3> (<2> cyclization of trans,transfarnesyl diphosphate is proceeding with inversion of configuration at C-1 of farnesyl diphosphate [1]; <2> H8si is lost in the formation of the 9,10double bond of aristolochene [2]; <3> metal-triggered carbocation formation initiates the cyclization cascade, which procedes through multiple complex intermediates to yield one exclusive structural stereochemical isomer of aristolochene [5]; <3> the steric bulk of residue 92 is central in binding of farnesyl diphosphate to the active site of the enzyme in a quasi-cyclic conformation, thereby facilitating attack of C1 by the C10 -C11 double bond to produce the cis-fused decalin S-germacrene A. The cyclization of farnesyl diphosphate to germacrene A in aristolochene synthase proceeds in a stepwise fashion through farnesyl cation [8]; <3> the enzyme appeears to be transcriptionally regulated [4]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9] P aristolochene + diphosphate <2, 3> (<3> 7.5% of the total amount of products are released from wild-type aristolochene synthase. The mutant enzyme Y92F releases significant amounts of germacrene A and also produces various amounts of a further five hydrocarbons of molecular weight 204, valencene, b-(E)-farnesene, a-selinene, b-selinene and selina-4,11diene [7]; <3> the mutant Y92A produces almost 80% of the alicyclic sesquiterpenes (E)-b-farnesene and (E,E)-a-farnesene. The mutant also produces small amounts of additional hydrocarbons with a molecular weight of 204: a-selinene, b-selinene, selina-4,11-diene, (E,Z)-a-farnesene, and b-bisabolene [8]) [1, 2, 3, 4, 5, 6, 7, 8, 9] Inhibitors Cu2+ <2> [9] Mn2+ <2, 3> (<3> above 0.01 mM, activation below [3]; <2> above 1.0 mM [9]) [3, 9] Additional information <3> (<3> no effect: phosphate up to concentrations of 5.0 mM [3]) [3]

220

4.2.3.9

Aristolochene synthase

Metals, ions Co2+ <2> (<2> can substitute for Mg2+ over the concentration range of 0.16 -5.0 mM [9]) [9] Mg2+ <2, 3> (<2> divalent cation required, 5 mM Mg2+ preferred [9]; <3> required, maximal activity at 3 mM [3]) [3, 9] Mn2+ <2, 3> (<3> 0.01 mM, can partially replace Mg2+ , inhibition above [3]; <2> Mn2+ can replace Mg2+ at 0.1-0.2 mM [9]) [3, 9] Turnover number (min–1) 0.00012 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme N244D [12]) [12] 0.00022 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme E252Q [12]) [12] 0.0004 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme Y92F [12]) [12] 0.000491 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5, mutant enzyme Y92C [8]) [8] 0.001 <3> (trans,trans-farnesyl diphosphate, <3> wild-type enzyme, mutant enzyme F178Y [11]) [11] 0.0013 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme D116E [12]; <3> pH 8.0, 30 C, mutant enzyme S248A [12]) [12] 0.00137 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5, mutant enzyme Y92A [8]) [8] 0.0016 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, mutant enzyme E227D [12]) [12] 0.0022 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme D116N [12]; <3> pH 8.0, 30 C, mutant enzyme E252D [12]) [12] 0.0023 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5, 30 C, mutant enzyme Y92F [7]) [7] 0.0048 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme E119Q [12]) [12] 0.0097 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme E119D [12]) [12] 0.014 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, native enzyme [9]) [9] 0.015 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, recombinant enzyme [9]) [9] 0.0173 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, wild-type enzyme [12]) [12] 0.03 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5, 30 C, recombinant wild-type enzyme [7]) [7] 0.043 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, recombinant enzyme [9]; <3> pH 8.0, 30 C, wild-type enzyme [12]) [9, 12] Specific activity (U/mg) 0.0236 <2> (<2> native enzyme [9]) [9] 0.07 <3> [3]

221

Aristolochene synthase

4.2.3.9

Km-Value (mM) 0.000012 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme Y92F [12]) [12] 0.0000135 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, native enzyme [9]) [9] 0.0000148 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, recombinant enzyme [9]) [9] 0.00015 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme E119Q [12]) [12] 0.00032 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme E119D [12]) [12] 0.00052 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, recombinant enzyme [9]) [9] 0.00055 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5 [3]) [3] 0.0006 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, wild-type enzyme [12]) [12] 0.0011 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme D116E [12]) [12] 0.0013 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, wild-type enzyme [12]) [12] 0.0014 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, mutant enzyme E227D [12]) [12] 0.0015 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme D116N [12]) [12] 0.0023 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5, 30 C, recombinant wild-type enzyme [7]) [7] 0.0027 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme D115E [12]) [12] 0.0033 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme N244D [12]) [12] 0.0034 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme E252D [12]) [12] 0.0039 <2> (trans,trans-farnesyl diphosphate, <2> pH 8.0, 30 C, mutant enzyme N219D [12]) [12] 0.0051 <3> (trans,trans-farnesyl diphosphate, <3> wild-type enzyme, mutant enzyme F178Y [11]) [11] 0.0055 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme S248A [12]) [12] 0.0101 <3> (trans,trans-farnesyl diphosphate, <3> pH 8.0, 30 C, mutant enzyme E252Q [12]) [12] 0.0112 <3> (trans,trans-farnesyl diphosphate, <3> wild-type enzyme, mutant enzyme F178V [11]) [11] 0.05027 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5, mutant enzyme Y92C [8]) [8] 0.0834 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5, mutant enzyme Y92A [8]) [8]

222

4.2.3.9

Aristolochene synthase

0.1887 <3> (trans,trans-farnesyl diphosphate, <3> pH 7.5, 30 C, mutant enzyme Y92F [7]) [7] pH-Optimum 6.3-7.5 <3> [3] 8 <2> (<2> recombinant enzyme, HEPES buffer [9]) [9]

4 Enzyme Structure Molecular weight 48000 <3> (<3> gel filtration [3]) [3] Subunits ? <2, 3> (<2> x * 39000, SDS-PAGE [9]; <2> x * 36480, calculation from nucleotide sequence [9]; <3> x * 39200, calculation from nucleotide sequence [4]) [4, 9] monomer <3> (<3> 1 * 37000, SDS-PAGE [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue mycelium <2> [1] stationary phase culture <3> [4] Purification <2> (native and recombinant enzyme) [9] <3> [3] <3> (mutant enzyme Y92C and Y92A) [8] <3> (recombinant enzyme) [5] <3> (wild-type and mutant enzymes) [7] Crystallization <3> (2.5-A resolution crystal structure of recombinant enzyme, hanging drop method) [5] Cloning <1> (expression in Escherichia coli) [10] <2> (high-level expression in Escherichia coli) [9] <3> (expression of the fusion protein proteinA/aristolochene synthase in Escherichia coli) [4] <3> (mutant enzymes Y92C and Y92A) [8] <3> (wild-type and mutant enzyme Y92F, expression in Escherichia coli) [7] Engineering D115E <3> (<3> kcat /KM is 12fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 75:6:119 [12]) [12] D115N <3> (<3> inactive mutant enzyme [12]) [12]

223

Aristolochene synthase

4.2.3.9

D116E <3> (<3> kcat /KM is 60fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 62:3:35 [12]) [12] D116N <3> (<3> kcat /KM is 48fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 63:2:35 [12]) [12] E119D <3> (<3> kcat /KM is 2.4fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 94:2:4 [12]) [12] E119Q <3> (<3> kcat /KM is 2.3fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 84:2:14 [12]) [12] E227D <2> (<2> kcat /KM is 1182fold higher than wild-type value, in contrast to wild-type enzyme that exclusively produces aristolochene from trans,trans-farnesyl diphosphate, the mutant enzyme produces 26% aristolochene and 74% germacrene A [12]) [12] E227Q <2> (<2> inactive mutant enzyme [12]) [12] E252D <3> (<3> kcat /KM is 111fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 19:0:81 [12]) [12] E252Q <3> (<3> mutant enzyme produces only (-)-germacrene A [12]) [12] F178V <3> (<3> wild-type enzyme produces 92% aristolochene, 8% germacrene and a small amount of valencene. Mutant enzyme produces 10.8% aristolochene, 54.1% germacrene, 5.2% valencene, 5.7% a-selinene, 9.1% b-selinine, 9.2% (E)-b-farnesene and 2.7% (E,E)-a-farnesene. kcat is 1429fold lower than wild-type value, Km -value is 4.9fold higher than wild-type value [11]) [11] F178Y <3> (<3> wild-type enzyme produces 92% aristolochene, 8% germacrene and a small amount of valencene. Mutant enzyme produces 86.4% aristolochene, 10.7% germacrene, and 2.7% valencene. kcat is 30fold lower than wild-type value, Km -value is 2.2fold higher than wild-type value [11]) [11] N219D <2> (<2> kcat /KM is 6667fold higher than wild-type value, in contrast to wild-type enzyme that exclusively produces aristolochene from trans,trans-farnesyl diphosphate, the mutant enzyme produces 44% aristolochene and 56% germacrene A [12]) [12] N244D <3> (<3> kcat /KM is 1978fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 19:0:81 [12]) [12] N244L <3> (<3> inactive mutant enzyme [12]) [12] S248A <3> (<3> kcat /KM is 300fold lower than wild-type value. Wild-type enzyme produces (+)-aristolochene, (-)-valencene and (S)-(-)-germacrene A in the ratio 93:2:4, the ratio of the mutant enzyme is 21:0:79 [12]) [12] S248A/E252D <3> (<3> inactive mutant enzyme [12]) [12] Y92A <3> (<3> turnover number is approximately 2 orders of magnitude lower than the value observed for the wild-type enzyme,the mutant enzyme produces almost 80% of the alicyclic sesquiterpenes (E)-b-farnesene and (E,E)-a-farnesene. The mutant also produces small amounts of additional hy-

224

4.2.3.9

Aristolochene synthase

drocarbons with a molecular weight of 204: a-selinene, b-selinene, selina4,11-diene, (E,Z)-a-farnesene, and b-bisabolene. Km -value for trans, transfarnesyl diphosphate is 0.0834 mM compared to 0.0023 mM for the wild-type enzyme [8]) [8] Y92C <3> (<3> turnover number is approximately 2 orders of magnitude lower than the value observed for the wild-type enzyme. Km -value for trans, trans-farnesyl diphosphate is 0.05027 mM compared to 0.0023 mM for the wild-type enzyme [8]) [8] Y92F <3> (<3> the mutant enzyme is approximately 0.1% as active as the nonmutated recombinant enzyme, the mutant releases significant amounts of germacrene A and also produces various amounts of a further five hydrocarbons of molecular weight 204, valencene, b-(E)-farnesene, a-selinene, bselinene and selina-4,11-diene. The CD spectrum of the mutant enzyme is very similar to that of the wild-type enzyme [7]; <3> 100fold reduction in kcat , 50fold decrease in KM , resulting in 2fold decrease in kcat /Km . The mutant enzyme produces (+)-aristolochene as 81% of the product, 7% (-)-valencene and 12% (S)-(-)-germacrene A [12]) [7, 12] Y92V <3> (<3> the mutant produces the alicyclic b-(E)-farnesene as the major product [6]) [6]

References [1] Cane, D.E.; Prabhakaran, P.C.; Oliver, J.S.; McIlwaine, D.B.: Aristolochene biosynthesis. Stereochemistry of the deprotonation steps in the enzymatic cyclization of farnesyl pyrophosphate. J. Am. Chem. Soc., 112, 3209-3210 (1990) [2] Cane, D.E.; Prabhakaran, P.C.; Salaski, E.J.; Harrison, P.M.H.; Noguchi, H.; Rawlings, B.J.: Aristolochene biosynthesis and enzymatic cyclization of farnesyl pyrophosphate. J. Am. Chem. Soc., 111, 8914-8916 (1989) [3] Hohn, T.M.; Plattner, R.D.: Purification and characterization of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti. Arch. Biochem. Biophys., 272, 137-143 (1989) [4] Proctor, R.H.; Hohn, T.M.: Aristolochene synthase. Isolation, characterization, and bacterial expression of a sesquiterpenoid biosynthetic gene (Ari1) from Penicillium roqueforti. J. Biol. Chem., 268, 4543-4548 (1993) [5] Caruthers, J.M.; Kang, I.; Rynkiewicz, M.J.; Cane, D.E.; Christianson, D.W.: Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti. J. Biol. Chem., 275, 25533-25539 (2000) [6] Calvert, M.J.; Taylor, S.E.; Allemann, R.K.: Tyrosine 92 of aristolochene synthase directs cyclisation of farnesyl pyrophosphate. Chem. Commun., 2002, 2384-2385 (2002) [7] Calvert, M.J.; Ashton, P.R.; Allemann, R.K.: Germacrene A is a product of the aristolochene synthase-mediated conversion of farnesylpyrophosphate to aristolochene. J. Am. Chem. Soc., 124, 11636-11641 (2002)

225

Aristolochene synthase

4.2.3.9

[8] Deligeorgopoulou, A.; Allemann, R.K.: Evidence for differential folding of farnesyl pyrophosphate in the active site of aristolochene synthase: a single-point mutation converts aristolochene synthase into an (E)-b-farnesene synthase. Biochemistry, 42, 7741-7747 (2003) [9] Cane, D.E.; Kang, I.: Aristolochene synthase: purification, molecular cloning, high-level expression in Escherichia coli, and characterization of the Aspergillus terreus cyclase. Arch. Biochem. Biophys., 376, 354-364 (2000) [10] O’Maille, P.E.; Chappell, J.; Noel, J.P.: Biosynthetic potential of sesquiterpene synthase: alternative product of tobacco 5-epi-aristolochene synthase. Arch. Biochem. Biophys., 448, 73-82 (2006) [11] Forcat, S.; Allemann, R.K.: Dual role for phenylalanine 178 during catalysis by aristolochene synthase. Chem. Commun., 2004, 2094-2095 (2004) [12] Felicetti, B.; Cane, D.E.: Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis. J. Am. Chem. Soc., 126, 7212-7221 (2004) [13] Wu, S.; Schoenbeck, M.A.; Greenhagen, B.T.; Takahashi, S.; Lee, S.; Coates, R.M.; Chappell, J.: Surrogate splicing for functional analysis of sesquiterpene synthase genes. Plant Physiol., 138, 1322-1333 (2005)

226

(-)-Endo-fenchol synthase

4.2.3.10

1 Nomenclature EC number 4.2.3.10 Systematic name geranyl-diphosphate diphosphate-lyase [cyclizing, (-)-endo-fenchol-forming] Recommended name (-)-endo-fenchol synthase Synonyms (-)-endo-fenchol cyclase EC 4.6.1.8 (formerly) cyclase, (-)-endo-fenchol geranyl pyrophosphate:(-)-endo-fenchol cyclase CAS registry number 117758-41-5

2 Source Organism <1> Foeniculum vulgare (no sequence specified) [1, 2, 3, 4]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate + H2 O = (-)-endo-fenchol + diphosphate (<1> reaction mechanism [2,3]; <1> (3R)-linalyl diphosphate is an intermediate in the reaction [2,3]) Reaction type P-O bond cleavage Natural substrates and products S geranyl diphosphate <1> (<1> biosynthesis of monoterpenes [4]) (Reversibility: ?) [2, 4] P (-)-endo-fenchol + diphosphate <1> [2, 4] S Additional information <1> (<1> monoterpene biosynthesis [1]) (Reversibility: ?) [1] P ?

227

(-)-Endo-fenchol synthase

4.2.3.10

Substrates and products S 2,3-cyclopropylgeranyl diphosphate <1> (<1> at 53% of the rate of conversion of geranyl diphosphate, formation of ether-soluble products [2]) (Reversibility: ?) [2] P ? S 6,7-dihydrogeranyl diphosphate <1> (<1> at 35% of the rate of conversion of geranyl diphosphate, formation of ether-soluble products [2]) (Reversibility: ?) [2] P ? S dimethylallyl diphosphate <1> (<1> at the same rate as the cyclization of geranyl diphosphate [2]) (Reversibility: ?) [2] P dimethylallyl alcohol + diphosphate <1> [2] S farnesyl diphosphate <1> (<1> at 25% of the rate of conversion of geranyl diphosphate, formation of ether-soluble products [2]) (Reversibility: ?) [2] P ? S geranyl diphosphate <1> (<1> mechanism of the isomerization-cyclization reaction [2,3]; <1> stereochemistry, syn-isomerization of substrate to (3R)-linalyl diphosphate and cyclization of the latter via the antiendo-conformer [3]; <1> (3R)-linalyl diphosphate is an intermediate in the reaction [2,3]; <1> biosynthesis of monoterpenes [4]) (Reversibility: ?) [2, 3, 4] P (-)-endo-fenchol + diphosphate <1> [2, 3, 4] S Additional information <1> (<1> monoterpene biosynthesis [1]) (Reversibility: ?) [1] P ? Inhibitors 2,3-cyclopropylgeranyl diphosphate <1> (<1> competitive inhibitor [2]) [2] 2-fluorogeranyl diphosphate <1> (<1> effective competitive inhibitor [2]) [2] 2-fluorolinalyl diphosphate <1> (<1> effective competitive inhibitor [2]) [2] 6,7-dihydrogeranyl diphosphate <1> (<1> competitive inhibitor [2]) [2] diethyl dicarbonate <1> (<1> inhibition is reversed by hydroxylamine [1]) [1] Additional information <1> (<1> synergistic effect of sulfonium analog inorganic diphosphate combinations in the inhibition of fenchol cyclase [2]; <1> not inhibited by N-acetylsuccinimide, up to 5 mM [4]) [2, 4] Metals, ions Mn2+ <1> (<1> Mn2+ -dependent formation of ether-soluble products from 6,7-dihydrogeranyl diphosphate [2]; <1> Mn2+ -dependent [3]) [2, 3] Additional information <1> (<1> metal ion-dependent [1]) [1] Km-Value (mM) 0.006 <1> (geranyl diphosphate) [2] 0.02 <1> (2,3-cyclopropylgeranyl diphosphate, <1> pH 6.5, 30 C [2]) [2] 0.025 <1> (6,7-dihydrogeranyl diphosphate, <1> pH 6.5, 30 C [2]) [2]

228

4.2.3.10

(-)-Endo-fenchol synthase

Ki-Value (mM) 0.01 <1> (2-fluorogeranyl diphosphate, <1> pH 6.5, 30 C [2]) [2] 0.013 <1> (2-fluorolinalyl diphosphate, <1> pH 6.5, 30 C [2]) [2] 0.035 <1> (2,3-cyclopropylgeranyl diphosphate, <1> pH 6.5, 30 C [2]) [2] 0.04 <1> (6,7-dihydrogeranyl diphosphate, <1> pH 6.5, 30 C [2]) [2] Additional information <1> [2] pH-Optimum 6.5 <1> (<1> assay at [2]) [2] Temperature optimum ( C) 30 <1> (<1> assay at [2,3,4]) [2, 3, 4]

5 Isolation/Preparation/Mutation/Application Source/tissue fruit <1> (<1> schizocarps [3]) [3] leaf <1> (<1> young emerging leaves from 30-day-old plants [2]) [2, 4] Purification <1> (partial) [3, 4]

6 Stability Temperature stability Additional information <1> (<1> no significant difference in the kinetics of inactivation up to 50 C of free and immobilized enzyme [4]) [4] General stability information <1>, immobilized enzyme form is markedly more stable than the free form [4] Storage stability <1>, 4 C, immobilized enzyme on CH-activated Sepharose, 1 month, retains over 30% of the original activity, the activity of the free enzyme is negligible [4] <1>, 4 C, immobilized enzyme on CH-activated Sepharose, in presence of 1 mM sodium diphosphate as substrate analog, 1 month, retains over 60% of the original activity [4]

References [1] Rajaonarivony, J.I.M.; Gershenzon, J.; Miyazaki, J.; Croteau, R.: Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases. Arch. Biochem. Biophys., 299, 77-82 (1992)

229

(-)-Endo-fenchol synthase

4.2.3.10

[2] Croteau, R.; Miyazaki, J.H.; Wheeler, C.J.: Monoterpene biosynthesis: mechanistic evaluation of the geranyl pyrophosphate:(-)-endo-fenchol cyclase from fennel (Foeniculum vulgare). Arch. Biochem. Biophys., 269, 507-516 (1989) [3] Croteau, R.; Satterwhite, D.M.; Wheeler, C.J.; Felton, N.M.: Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (-)-endo-fenchol. J. Biol. Chem., 263, 15449-15453 (1988) [4] Miyazaki, J.H.; Croteau, R.: Immobilization of cyclase enzymes for the production of monoterpenes and sesquiterpenes. Enzyme Microb. Technol., 12, 841-845 (1990)

230

Sabinene-hydrate synthase

4.2.3.11

1 Nomenclature EC number 4.2.3.11 Systematic name geranyl-diphosphate diphosphate-lyase (cyclizing, sabinene-hydrate-forming) Recommended name sabinene-hydrate synthase Synonyms EC 4.6.1.9 cyclase, sabinene hydrate sabinene hydrate cyclase CAS registry number 117164-95-1

2 Source Organism <1> Majorana hortensis (no sequence specified) [1, 2] <2> Origanum majorana (no sequence specified) [3] <3> Origanum microphyllum (no sequence specified) [3]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate + H2 O = sabinene hydrate + diphosphate (<1> both cisand trans-isomers of sabinene hydrate are formed. (3R)-Linalyl diphosphate is an intermediate in the reaction, mechanism and stereochemistry [1]) Reaction type P-O bond cleavage Substrates and products S 2,3-methanogeranyl diphosphate + H2 O <1> (<1> at 1.5% of the rate of conversion of geranyl diphosphate to sabinene hydrate, noncyclizable substrate analog of geranyl diphosphate [1]) (Reversibility: ?) [1] P ?

231

Sabinene-hydrate synthase

4.2.3.11

S 6,7-dihydrogeranyl diphosphate + H2 O <1> (<1> noncyclizable substrate analog of geranyl diphosphate [1]) (Reversibility: ?) [1] P 6,7-dihydromyrcene + ? S geranyl diphosphate <1, 2, 3> (Reversibility: ?) [1, 2, 3] P sabinene hydrate + diphosphate <1, 2, 3> (<2> no free intermediate detectable in the conversion of geranyl diphosphate to sabinene hydrate [3]; <1> the configuration at c1 of geranyl diphosphate is retained in the enzymatic conversion to (+)-cis and (+)-trans-sabinene hydrate [1]; <2> constant ratio of (+)-cis-sabinene hydrate to (+)-trans-sabinene hydrate is 20:1 [3]; <3> the ratio of (+)-cis-sabinene hydrate to (+)-trans-sabinene hydrate is not constant [3]; <1> both cis- and trans-isomers of sabinene hydrate are formed [1,2]) [1, 2, 3] S linalyl diphosphate <1> (<1> (3R)- and (3S)-configuration [1]) (Reversibility: ?) [1] P sabinene hydrate + diphosphate <1> (<1> cis- and trans-sabinene hydrate [1]) [1] S neryl diphosphate <1> (Reversibility: ?) [1, 2] P sabinene hydrate + diphosphate <1> (<1> cis- and trans-sabinene hydrate [2]) [2] Inhibitors (RS)-dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide <1> [1] (RS)-methyl-(4-methylpent-3-en-1-yl)vinyl sulfonium perchlorate <1> [1] 6,7-dihydrogeranyl diphosphate <1> (<1> competitive [1]) [1] diphosphate <1> (<1> 0.5 mM: 80% inhibition [2]) [2] EDTA <1> [2] NEM <1> (<1> IC50: 0.08 mM [2]) [2] NH4 VO3 <1> (<1> 10 mM, 45% inhibition [2]) [2] NaWO4 <1> (<1> 10 mM, 45% inhibition [2]) [2] PCMB <1> (<1> IC50: 0.02 mM [2]) [2] Activating compounds diphosphate <1> (<1> 0.1 mM: 30% stimulation [2]) [2] Metals, ions Mg2+ <1> (<1> absolute requirement for divalent cation in the reaction with 6,7-dihydrogeranyl diphosphate, preference for Mn2+ over Mg2+ [1]; <1> Mg2+ or Mn2+ required, Km : 2.4 mM [2]) [1, 2] Mn2+ <1> (<1> absolute requirement for divalent cation in the reaction with 6,7-dihydrogeranyl diphosphate, preference for Mn2+ over Mg2+ [1]; <1> Mn2+ or Mg2+ required, Km : 0.3 mM [2]) [1, 2] Km-Value (mM) 0.001 <1> ((3S)-linalyl diphosphate, <1> pH 7.0, 30 C [1]) [1] 0.0019 <1> (geranyl diphosphate, <1> pH 7.0, 30 C [1,2]) [1, 2] 0.0027 <1> ((3R)-linalyl diphosphate, <1> pH 7.0, 30 C [1]) [1] 0.0045 <1> (6,7-dihydrogeranyl diphosphate, <1> pH 7.0, 30 C [1]) [1] 0.0105 <1> (neryl diphosphate, <1> pH 7.0, 30 C [1,2]) [1, 2]

232

4.2.3.11

Sabinene-hydrate synthase

Ki-Value (mM) 0.0003 <1> ((RS)-methyl-(4-methylpent-3-en-1-yl)vinyl sulfonium perchlorate, <1> pH 7.0, 30 C [1]) [1] 0.0028 <1> ((RS)-dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide, <1> pH 7.0, 30 C [1]) [1] 0.0042 <1> (6,7-dihydrogeranyl diphosphate, <1> pH 7.0, 30 C [1]) [1] pH-Optimum 7 <1> [2] pH-Range 6.3-8 <1> (<1> 50% of maximal activity at pH 6.3 and 8.0 [2]) [2]

4 Enzyme Structure Molecular weight 56000 <1> (<1> gel filtration [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [1, 2] Localization soluble <1> [2] Purification <1> (partial) [1]

6 Stability Temperature stability 45 <1> (<1> complete inactivation after 20 min [2]) [2] General stability information <1>, proteolytic degradation by 0.01 mg/ml trypsin, folowed by addition of soybean trypsin inhibitor: all cyclase activities are destroyed within 10 min, t1=2 is 3 min [2]

References [1] Hallahan, T.W.; Croteau, R.: Monoterpene biosynthesis: mechanism and stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate. Arch. Biochem. Biophys., 269, 313326 (1989)

233

Sabinene-hydrate synthase

4.2.3.11

[2] Hallahan, T.W.; Croteau, R.: Monoterpene biosynthesis: demonstration of a geranyl pyrophosphate:sabinene hydrate cyclase in soluble enzyme preparations from sweet marjoram (Majorana hortensis). Arch. Biochem. Biophys., 264, 618-631 (1988) [3] Novak, J.; Bitsch, C.; Langbehn, J.; Pank, F.; Skoula, M.; Gotsiou, Y.; C, M.F.: Ratios of cis- and trans-sabinene sydrate in Origanum majorana L. and Origanum microphyllum (Bentham) Vogel. Biochem. Syst. Ecol., 28, 697-704 (2000)

234

6-Pyruvoyltetrahydropterin synthase

4.2.3.12

1 Nomenclature EC number 4.2.3.12 Systematic name 7,8-dihydrone opterin 3’-triphosphate triphosphate-lyase (6-pyruvoyl-5,6,7tetrahydropterin-forming) Recommended name 6-pyruvoyltetrahydropterin synthase Synonyms 2-amino-4-oxo-6-(erythro-1’,2’,3’-trihydroxypropyl)-7,8-dihydroxypterdine triphosphate lyase 6-(1,2-dioxopropyl)tetrahydropterin synthase 6-pyruvoyl-tetrahydropterin synthase <2, 4, 5, 8> [25] 6-pyruvoyltetrahydropterin synthase <4> [26, 27] 6-pyruvoyltetrahydropterin synthase [16-cysteine] (human clone lambda HSY2 gene PCBD subunit) 6-pyruvoyltetrahydropterin synthase [25-glutamine] (human clone lambdaHSY2 gene PCBD subunit) 6-pyruvoyltetrahydropterin synthase [de-57-valine] (human clone lambdaHSY2 gene PCBD subunit) EC 4.6.1.10 (formerly) PPH4 synthase PPH4S PTP synthase PTPS <4> [25, 26] protein purple pyruvoyltetrahydropterin synthase synthase, 6-pyruvoyltetrahydropterin synthase, 6-pyruvoyltetrahydropterin [16-cysteine] (human clone lamda HSY2 gene PCBD subunit) synthase, 6-pyruvoyltetrahydropterin [25-glutamine] (human clone lamdaHSY2 gene PCBD subunit) synthase, 6-pyruvoyltetrahydropterin [87-leucine] (human clone lamdaHSY2 gene PCBD subunit) synthase, 6-pyruvoyltetrahydropterin [de-57-valine] (human clone lambdaHSY2 gene PCBD subunit)

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CAS registry number 171716-27-1 (synthase, 6-pyruvoyltetrahydropterin [16-cysteine] (human clone lambda HSY2 gene PCBD subunit) /6-pyruvoyltetrahydropterin synthase [16-cysteine] (human clone lambda HSY2 gene PCBD subunit)) 171716-28-2 (synthase, 6-pyruvoyltetrahydropterin [25-glutamine] (human clone lambdaHSY2 gene PCBD subunit) /6-pyruvoyltetrahydropterin synthase [25-glutamine] (human clone lambdaHSY2 gene PCBD subunit)) 171716-29-3 (synthase, 6-pyruvoyltetrahydropterin [de-57-valine] (human clone lambdaHSY2 gene PCBD subunit) /6-pyruvoyltetrahydropterin synthase [de-57-valine] (human clone lambdaHSY2 gene PCBD subunit)) 171716-30-6 (synthase, 6-pyruvoyltetrahydropterin [87-leucine] (human clone lamdaHSY2 gene PCBD subunit)) 97089-82-2

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11> <12>

Drosophila melanogaster (no sequence specified) [3, 8, 15, 16] Mus musculus (no sequence specified) [25] Escherichia coli (no sequence specified) [16, 19] Homo sapiens (no sequence specified) [1, 3, 4, 8, 10, 11, 13, 16, 17, 18, 20, 21, 23, 24, 25, 26, 27] Rattus norvegicus (no sequence specified) [1,2,10,22,25] Bos taurus (no sequence specified) [5,8] Bombyx mori (no sequence specified) [8,9] Cercopithecus aethiops (no sequence specified) [25] Synechocystis sp. (no sequence specified) [16] Salmo salar (no sequence specified) [3, 6, 14] Homo sapiens (UNIPROT accession number: Q03393) [7] Rattus norvegicus (UNIPROT accession number: P27213) [8, 12]

3 Reaction and Specificity Catalyzed reaction 6-[(1S,2R)-1,2-dihydroxy-3-triphosphooxypropyl]-7,8-dihydropterin = 6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate (<6> mechanism [5]; <4> the enzyme catalyzes the elimination of triphosphate as well as a series of tautomerization reactions. Incorporation of protons into positions C6 and C3 of the product [20]) Reaction type P-O bond cleavage cleavage of triphosphate bond intramolecular redox reaction triphosphate elimination

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Natural substrates and products S 6-(L-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin <3, 4, 5, 10> (<10> one of the enzymes involved in tetrahydrobiopterin biosynthesis [14]; <4> 23 bp exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression in human macrophages and related cell types. The exon lacking leads to a premature stop codon encoding for a shorter protein instead of the full-length functional enzyme [18]; <4> rate-limiting enzyme in the synthesis of human tetrahydrobiopterin [10]; <4> the enzyme catalyzes the first step in the conversion of 7,8-dihydroneopterin triphosphate to tetrahydrobiopterin [13]; <4,5> the enzyme is involved in tetrahydrobiopterin biosynthesis [2,17]; <3,4,5> the enzyme catalyzes the second step in the tetrahydrobiopterin biosynthetic pathway [1,16,18,21,22]; <5> the enzyme has six active sites located at the interface of three subunits. The enzyme contains an intersubunit catalytic triad motif composed of the amino acid residues Cys A42, His B89 and Asp B88 which is involved in the abstraction of protons from the substrate side-chain carbons. The g and b phosphates of the substrate are essential for substrate binding and enhance the catalytic efficiency [22]; <4> the enzyme is posttranslationally phosphorylated in several Ser residues by protein kinases. The enzyme requires the phosphoserine 19 residue for maximal activity under in vivo conditions. Mutant enzymes with alterations in the protein kinase recognition site, phosphoserine 19 residue, are not phosphorylated by protein kinase and have reduced activity [17]) (Reversibility: ?) [1, 2, 10, 13, 14, 16, 17, 18, 19, 21, 22] P 6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin + triphosphate <3, 4, 5, 10> [1, 2, 10, 13, 14, 16, 17, 18, 19, 21, 22] S 7,8-dihydroneopterin triphosphate <4> (Reversibility: ?) [26, 27] P 6-pyruvoyl-5,6,7,8-tetrahydropterin triphosphate S 7,8-dihydroneopterin triphosphate <1, 4, 5, 10, 11, 12> (<1, 4, 5, 10, 11, 12> assay at [2, 3, 4, 7, 12, 13, 14, 15]) (Reversibility: ?) [2, 3, 4, 7, 12, 13, 14, 15] P 6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate <1, 4, 5, 10, 11, 12> [2, 3, 4, 7, 12, 13, 14, 15] S Additional information <1> (<1> the sequence of reaction steps include redox transfer between atoms N5, C6 and C1 and unusual triphosphate elimination at the C2-C3 bond in the side chain [15]) (Reversibility: ?) [15] P ? Substrates and products S 1’-3 H-dihydroneopterin triphosphate <6> (<6> tritium released is not incorporated in the product [5]) (Reversibility: ?) [5] P 6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate <6> [5] S 2’-3 H-dihydroneopterin triphosphate <6> (<6> tritium released is not incorporated in the product [5]) (Reversibility: ?) [5] P 6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate <6> [5]

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S 6-(l-erythro-1,2-dihydroxypropyl 3-monophosphate)-7,8-dihydropterin <5> (<5> the binding constant is 225fold increased with respect to the natural substrate [22]) (Reversibility: ?) [22] P 6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin + monophosphate <5> [22] S 6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin <3, 4, 5, 10> (<10> one of the enzymes involved in tetrahydrobiopterin biosynthesis [14]; <4> 23 bp exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression in human macrophages and related cell types. The exon lacking leads to a premature stop codon encoding for a shorter protein instead of the full-length functional enzyme [18]; <4> rate-limiting enzyme in the synthesis of human tetrahydrobiopterin [10]; <4> the enzyme catalyzes the first step in the conversion of 7,8-dihydroneopterin triphosphate to tetrahydrobiopterin [13]; <4,5> the enzyme is involved in tetrahydrobiopterin biosynthesis [2,17]; <3,4,5> the enzyme catalyzes the second step in the tetrahydrobiopterin biosynthetic pathway [1,16,18,21,22]; <5> the enzyme has six active sites located at the interface of three subunits. The enzyme contains an intersubunit catalytic triad motif composed of the amino acid residues Cys A42, His B89 and Asp B88 which is involved in the abstraction of protons from the substrate side-chain carbons. The g and b phosphates of the substrate are essential for substrate binding and enhance the catalytic efficiency [22]; <4> the enzyme is posttranslationally phosphorylated in several Ser residues by protein kinases. The enzyme requires the phosphoserine 19 residue for maximal activity under in vivo conditions. Mutant enzymes with alterations in the protein kinase recognition site, phosphoserine 19 residue, are not phosphorylated by protein kinase and have reduced activity [17]) (Reversibility: ?) [1, 2, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22] P 6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin + triphosphate <3, 4, 5, 10> [1, 2, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22] S 7,8-dihydroneopterin triphosphate <4> (Reversibility: ?) [26, 27] P 6-pyruvoyl-5,6,7,8-tetrahydropterin triphosphate S 7,8-dihydroneopterin triphosphate <1, 4, 5, 6, 10, 11, 12> (<1, 4, 5, 10, 11, 12> assay at [2, 3, 4, 7, 12, 13, 14, 15]) (Reversibility: ?) [2, 3, 4, 5, 7, 12, 13, 14, 15] P 6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate <1, 4, 5, 6, 10, 11, 12> [2, 3, 4, 5, 7, 12, 13, 14, 15] S sepiapterin <3> (<3> the enzyme cleaves the C6 side chain of sepiapterin, sepiapterin side chain releasing activity, SSCR activity [16]) (Reversibility: ?) [16, 19] P 7,8-dihydropterin <3> [16, 19] S Additional information <1> (<1> the sequence of reaction steps include redox transfer between atoms N5, C6 and C1 and unusual triphosphate elimination at the C2-C3 bond in the side chain [15]) (Reversibility: ?) [15] P ?

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Inhibitors 4-vinylpyridine <4, 5> (<4> 95% inactivation under non-denaturating and non-reducing conditions [10]; <5> 80% inactivation under non-denaturating and non-reducing conditions [10]) [10] ammonium sulfate <12> (<12> 50% inactivation at 400 mM [12]) [12] Ca2+ <3> (<3> 10 mM, 40-100% inhibition [16]) [16] Co2+ <3> (<3> 10 mM, 40-100% inhibition [16]) [16] Cu2+ <3> (<3> 10 mM, 40-100% inhibition [16]) [16] EDTA <3, 4> (<3> above 10 mM [16]; <4> completely inactivates the enzyme by complexation of Mg2+ [13]) [13, 16] Mg2+ <3> (<3> 10 mM, 40-100% inhibition [16]) [16] Mn2+ <3> (<3> 10 mM, 40-100% inhibition [16]) [16] N-(7-Dimethyl-amino-4-methyl coumarinyl) maleimide <10> [14] NEM <7> [9] Ni2+ <3> (<3> 10 mM, 40-100% inhibition [16]) [16] PCMB <7> [9] Zn2+ <3> (<3> 10 mM, 40-100% inhibition [16]) [16] monoiodoacetate <7> [9] Additional information <4, 12> (<4> l-monapterin does not inhibit activity [3]; <12> NaCl 100 mM and KCl 100 mM have no effect on activity [12]) [3, 12] Cofactors/prosthetic groups NADPH <4> (<4> required [3]) [3] Additional information <3> (<3> no cofactor required [16]) [16] Activating compounds EDTA <3> (<3> 1-5 mM, 110-120% activation [16]) [16] dithioerythritol <4, 10, 12> (<4, 10, 12> increased catalytic activity with 10 mM [12,13,14]) [12, 13, 14] Additional information <4> (<4> NADPH is not necessary for activity [3,13]) [3, 13] Metals, ions Ba2+ <7> (<7> stimulatory effect, 5 mM greatly accelerates activity [9]) [9] Ca2+ <5, 7, 12> (<12> 65% of the activity with equal concentration of Mg2+ [12]; <7> stimulatory effect and additive effect at Mg2+ , 1 mM [9]; <5> less than 15% of activity with Mg2+ with 5 mM [2]) [2, 9, 12] Co2+ <5> (<5> free metal enzyme activity reaches 60% of activity of wild type with 0.50 mM and concentrations higher than 0.15 mM completely inactivates the enzyme. Less than 15% of activity with Mg2+ with 5 mM [2]) [2] Mg2+ <4, 5, 7, 11, 12> (<4,5> required [1, 3]; <4, 11> necessary for activity [3, 7]; <12> optimum concentration is 8 mM [12]; <7> stimulates, Km of 0.2 mM and 2 mM at pH 8.5 and 37ﬀ C [9]; <5> free metal enzyme activity is 15% of the wild type with 8 mM, it is not bound to the enzyme [2]) [1, 2, 3, 7, 9, 12] Mn2+ <5, 7> (<7> stimulatory effect but additional competitive or inhibitory effect [9]; <5> less than 15% of activity with Mg2+ with 5 mM [2]) [2, 9]

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Ni2+ <5, 7> (<7> no effect on enzyme activity [9]; <5> 71% of the activity with Mg2+ with NiCl2 , 5 mM [2]) [2, 9] Sr2+ <7> (<7> 5 mM greatly accelerates activity [9]) [9] Zn2+ <5, 7, 12> (<5> the zinc/enzyme-subunit ratio of the wild-type enzyme is 0.8 [2]; <7> no effect on enzyme activity [9]; <5> free metal enzyme activity reaches 85% of activity of wild type with 0.05 mM and 8 mM Mg2+ [2]; <5> bound to the enzyme [2]; <12> bound to the Ne -atoms of the three His residues HisA23, HisA48, and HisA50, one to each of the two active sites [8]; <5> concentrations higher than 0.15 mM completely inactivates the enzyme [2]) [1, 2, 8, 9] Additional information <3, 5, 7> (<3> no metal ion required [16]; <7> Cu2+ has no effect on activity [9]; <5> Cu2+ , Fe2+ , less than 15% of activity with Mg2+ with 5 mM of each ion [2]) [2, 9, 16] Turnover number (min–1) 0.01 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> mutant enzyme P87L [11]) [11] 0.012 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> mutant enzyme R16C [11]) [11] 0.02 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> mutant enzyme R25Q [11]) [11] 0.032 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> wild-type enzyme [11]) [11] Additional information <4, 5> (dihydroneopterin triphosphate, <5> 0.277 mU/min/mg [10]; <4> 0.080 mU/min/mg [10]) [10] Specific activity (U/mg) 0.00023 <4> (<4> SSCR activity [16]) [16] 0.00034 <1> (<1> SSCR activity [16]) [16] 0.00039 <9> (<9> PTPS activity [16]) [16] 0.000611 <4> (<4> during the third trimester, enzyme assays at 37 C and pH 7.4 [27]) [27] 0.000714 <4> (<4> during the first trimester, enzyme assays at 37 C and pH 7.4 [27]) [27] 0.000782 <4> (<4> during the second trimester, enzyme assays at 37 C and pH 7.4 [27]) [27] 0.00113 <3> (<3> PTPS activity [16]) [16] 0.00891 <1> (<1> PTPS activity [16]) [16] 0.01303 <4> (<4> PTPS activity [16]) [16] 0.0253 <4> (<4> pH 7.4, 37 C, no differences observed in the enzyme activity between pregnant and non-pregnant women [24]) [24] 0.044 <4> (<4> R16C mutant enzyme [17]) [17] 0.0574 <4> [1] 0.0632 <1> [15] 0.074 <4> (<4> R25Q mutant enzyme [17]) [17] 0.077 <4> [10] 0.09767 <9> (<9> SSCR activity [16]) [16] 0.09936 <3> (<3> SSCR activity [16]) [16]

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0.1 <4> [13] 0.12 <4> (<4> wild-type enzyme [17]) [11, 17] 0.129 <4> (<4> S19A mutant enzyme [17]) [17] 0.2766 <5> [10] 0.2883 <12> [12] 0.5167 <11> [7] 4.6 <10> [14] 40 <4> [3] 57.4 <4> [1] Additional information <2, 4, 5, 8> (<4> in cells treated with mixed inflammatory cytokines IFNg, TNFa and IL-1, PTPS messenger RNA abundance and enzyme activity are 10fold and 3fold higher than in untreated cells, respectively [21]; <8> 0.3 microU/mg protein [25]; <2> 1.4 mU/mg in brain determined with the ONPG-assay with the o-nitrophenyl-b-d-glactopyranoside substrate of a mouse heterozygous for the Pts-lacZ allele (encoding a fusion protein of N-terminal 35 amino acids of 6-pyruvoyl-tetrahydropterin synthase and b-galactosidase), 1 U is defined as 1 OD at 420 nm per minute at pH 7.5 and 37 C [25]; <4> 10fold more Flag-tag fused PTPS per total protein in cytoplasm compared to nucleus, no activity of Flag-tag fused PTPS in the nucleus [25]; <4> 18 microU/mg total protein [25]; <2> 2.2 mU/mg in liver determined with the ONPG-assay with the o-nitrophenyl-b-d-glactopyranoside substrate of a mouse heterozygous for the Pts-lacZ allele (encoding a fusion protein of N-terminal 35 amino acids of 6-pyruvoyl-tetrahydropterin synthase and b-galactosidase), 1 U is defined as 1 OD at 420 nm per minute at pH 7.5 and 37 C [25]; <2> 3.5 mU/mg in liver determined with the ONPGassay with the o-nitrophenyl-b-d-glactopyranoside substrate of a mouse homozygous for the Pts-lacZ allele (encoding a fusion protein of N-terminal 35 amino acids of 6-pyruvoyl-tetrahydropterin synthase and b-galactosidase), 1 U is defined as 1 OD at 420 nm per minute at pH 7.5 and 37 C [25]; <4> 5.3 pmol/min/10*10 platelets, PTPS activity does not change significantly after treatment with glucocorticoids in a dose equivalent to at least 100 mg prednisone for at least 7 days, enzyme assays at 37 C [26]) [10, 11, 13, 21, 25, 26] Km-Value (mM) 0.0005 <5> (7,8-dihydroneopterin triphosphate, <5> mutant E133Q pH 7.4, 37 C [2]) [2] 0.00177 <5> (7,8-dihydroneopterin triphosphate, <5> mutant H89N pH 7.4, 37 C [2]) [2] 0.0022 <10> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin) [14] 0.0022 <10> (7,8-dihydroneopterin triphosphate, <10> pH 7.4, 37 C [14]) [14] 0.0055 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> S19A mutant enzyme [17]) [17] 0.008 <5> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <5> wild-type enzyme [2,10]; <5> pH 7.4, 37 C [22]) [2, 10, 22] 0.008 <5> (7,8-dihydroneopterin triphosphate, <5> pH 7.4, 37 C [2,10]) [2, 10]

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0.0081 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> wild-type enzyme [17]) [17] 0.0085 <4> (7,8-dihydroneopterin triphosphate, <4> pH 7.4, 37 C [10]) [10] 0.0091 <12> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin) [12] 0.0091 <12> (7,8-dihydroneopterin triphosphate, <12> pH 7.4, 37 C [12]) [12] 0.01 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin) [3, 13] 0.01 <4> (7,8-dihydroneopterin triphosphate, <4> pH 7.5, 37 C [13]; <4> pH 7.4, 37 C, shows positive cooperativity [3]) [3, 13] 0.011 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> wild-type enzyme [11]) [11] 0.011 <4> (7,8-dihydroneopterin triphosphate, <4> pH 7.4, 37 C [11]) [11] 0.012 <11> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin) [7] 0.0124 <11> (7,8-dihydroneopterin triphosphate, <11> 90 KDa protein pH 7.4, 37 C [7]) [7] 0.0128 <7> (7,8-dihydroneopterin triphosphate, <7> pH 8.5, 37 C, 1 mM Mg2+ [9]) [9] 0.013 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> mutant enzyme P87L [11]) [11] 0.0169 <7> (7,8-dihydroneopterin triphosphate, <7> pH 8.5 37 C, 5 mM Mg2+ [9]) [9] 0.0305 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> mutant enzyme R25Q [11]) [11] 0.0308 <4> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <4> mutant enzyme R16C [11]) [11] 0.1 <1> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin) [15] 0.1 <1> (7,8-dihydroneopterin triphosphate, <1> pH 7.5, 37 C [15]) [15] 0.92 <3> (sepiapterin, <3> pH 7.5, 37 C [16]) [16] 1.8 <5> (6-(l-erythro-1,2-dihydroxypropyl 3-monophosphate)-7,8-dihydropterin, <5> pH 7.4, 37 C [22]) [22] 5 <5> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <5> mutant enzyme E133Q [2]) [2] 17.7 <5> (6-(l-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin, <5> mutant enzyme H89N [2]) [2] pH-Optimum 6.5-7 <3, 12> [12, 16] 7.4 <5, 6, 12> (<5,6,12> assay at [2,5,12]) [2, 5, 12] 7.5 <1, 4, 10> (<1> assay at [15]; <4,10> in Tris-HCl 100 mM [3,13,14]) [3, 13, 14, 15] 7.5-8 <11> [7] 8.5 <7> [9]

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Temperature optimum ( C) 37 <1, 4, 5, 12> (<1,4,5,12> assay at [2,3,4,12,13,15]) [2, 3, 4, 12, 13, 15] 60-80 <3> (<3> 3.2fold increase in activity compared to that at 37 C [16]) [16]

4 Enzyme Structure Molecular weight 68000 <4, 10> (<4,10> gel filtration [3,6,14]) [3, 6, 14] 72000 <7> (<7> native PAGE [9]) [9] 83000 <1, 4, 12> (<4> gel filtration [1]; <12> HPLC gel filtration [12]; <4> gel filtration, seems to have 4 identical subunits [13]; <1> gel filtration of active enzyme seems to have larger molecular mass forms [15]) [1, 4, 12, 13, 15] 85000-87000 <7> (<7> gel filtration [9]) [9] 90000 <11> (<11> gel filtration [7]) [7] Subunits ? <1, 4, 5, 10, 11, 12> (<10> x * 16000, SDS-PAGE [14]; <4> x * 19000, SDSPAGE [4,13]; <10,11,12> x * 17000, SDS-PAGE [7,12,14]; <1> x * 37500, SDSPAGE [15]; <4> x * 16500, SDS-PAGE [3]; <5> x * 15855, calculation from nucleotide sequence [2]; <4> x * 16199, mutant protein R16C, electrospray mass spectrometry [11]; <4> x * 16000, SDS-PAGE and electrospray ionization-mass spectometry [1,10]; <4> x * 16224, mutant protein R25Q, electrospray mass spectrometry [11]) [1, 2, 3, 4, 7, 10, 11, 12, 13, 14, 15] dimer <1, 4, 5> (<4> 2 * or 1 * 16255, wild-type enzyme occurs as monomeric or dimeric form, electrospray mass spectrometry [11]; <1> 2 * 37500, also evidence for higher multimeric forms, SDS-PAGE [15]) [11, 15, 22] hexamer <4, 5> (<4> 6 * 97000, SDS-PAGE [10]; <4> 6 * 16254, electronionization mass spectrometry [10]) [10, 22] monomer <4> (<4> 1 * or 2 * 16255, wild-type enzyme occurs as monomeric or dimeric form, electrospray mass spectrometry [11]) [11] pentamer <4> (<4> 5 * 16000, SDS-PAGE [1]; <4> 5 * 16257, electrospray ionization-mass spectrometry [1]) [1] tetramer <4, 7, 10, 11, 12> (<11,12> 4 * 17000, SDS-PAGE [7,12]; <7> 4 * 18500, SDS-PAGE [9]; <4> 4 * 16500, SDS-PAGE [3]; <4> 4 * 19000, SDSPAGE [4,13]; <10> 4 * 17208, electrospray ionization mass spectrometry [6]) [3, 4, 6, 7, 9, 12, 13] trimer <4, 5> (<4> 3 * 47000, SDS-PAGE [10]; <5> 3 * 15899, electron-ionization mass spectrometry [10]) [10] Additional information <5> (<5> each subunit consists of 144 residues with 2497 non-hydrogen atoms [22]) [22] Posttranslational modification glycoprotein <4> [4] no glycoprotein <10> (<10> no carbohydrates bound [14]) [14]

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Additional information <10, 12> (<12> mature protein lacks 4 N-terminal amino acids residues [12]; <10> free of mannose and glucose residues [14]) [12, 14]

5 Isolation/Preparation/Mutation/Application Source/tissue COS-1 cell <8> (<8> monkey kidney cells, CRL 1650 [25]) [25] MOLT-4 cell <11> (<11> acute lymphoblastic leukemia cell line, 65% homology with rat enzyme [7]) [7] SK-N-BE(2) cell <4> (<4> neuroblastoma cells, CRL 2271 [25]) [25] adrenal medulla <6> (<6> partial purification [5]) [5, 8] blood platelet <4> [26] brain <2, 5> (<2> in brain nuclear b-galactosidase expression in neurons located in prominent catecholaminergic cell groups such as substantia nigra and locus coeruleus, in the ventral part of the hypothalamus, in the hippocampus and in the amygdala. Nuclear and cytoplasmic b-galactosidase expression in germinal periventricular layer [25]) [25] chorion <4> (<4> tissue from normal pregnant women [27]) [27] erythrocyte <4> [24] fat body <7> (<7> larvae [9]) [9] fibroblast <4> (<4> cells from patients with BH4 deficiency [11]) [11] head <1> [3, 15] hypophysis <4> [3, 8] kidney <2> (<2> in kidney very weak b-galactosidase expression in adult and strong expression in newborn mice heterozygous for the Pts-lacZ allele (encoding a fusion protein of N-terminal 35 amino acids of 6-pyruvoyl-tetrahydropterin synthase and b-galactosidase). Localization in the cytoplasm but not in the nucleus in all cells lining the proximal convoluted tube in adult and newborn mice. No staining in the medulla of newborn mice. Nuclear localization in the thin limb of the Henle loops in adult mice [25]) [25] larva <7> (<7> fat bodies [9]) [8, 9] liver <4, 5, 10, 12> (<10> high homology with rat protein but different amine terminus [6]) [1, 3, 4, 6, 8, 10, 12, 13, 14] monocyte <4> [18] pituitary gland <4> (<4> heat instability, it is present in the anterior pituitary endothelial cells but not in posterior pituitary [3]) [3] umbilical vein endothelium <4> [21] Localization cytoplasm <2, 4, 5> (<4> immunofluorescence, Western blot analysis [25]; <5> weak staining in immunohistochemistry [25]) [25] nucleus <2, 4, 5> (<4> immunofluorescence, Western blot analysis [25]; <5> strong staining in immunohistochemistry [25]) [25]

244

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Purification <1> (ammonium sulfate precipitation, Affigel Blue chromatography, DEAEBio-gel chromatography, Affigel Blue chromatography, SDS-PAGE) [15] <3> (extraction, chromatography on a column of Ni-NTA gel) [16] <4> [8, 11] <4> (affinity chromatography, gel filtration , SDS-PAGE) [10] <4> (affinity chromatography, gel filtration, SDS-PAGE) [1] <4> (ammonium sulfate precipitation, DEAE-Sephadex A50 column) [3] <4> (ammonium sulfate precipitation, hydroxyapatite column, gel filtration, DEAE-Fractogel 650S column, SDS-PAGE) [4, 13] <4> (extraction, chromatography on an amylose resin column) [20] <5> (affinity chromatography, gel filtration, SDS-PAGE) [1, 2] <6> (SDS-PAGE) [5] <7> (ammonium sulfate precipitation, hydroxyapatite column, gel filtration) [9] <10> [6] <10> (ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration, ion exchange, hydroxyapatite chromatography) [14] <11> (affinity chromatography, gel filtration) [7] <12> [8] <12> (ammonium sulfate precipitation, hydroxyapatite column, butyl-toyopearl chromatography, gel filtration, HPLC ion exchange column) [12] Crystallization <4> (X-ray crystallography) [20] <5> (X-ray crystallography, sitting drop vapor diffusion) [22] <12> [8] Cloning <3> (expression in Escherichia coli) [16, 19] <4> (expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion protein) [10] <4> (expression in Cos-1 cells) [25] <4> (expression in Escherichia coli) [1, 20] <4> (expression in Escherichia coli and COS-1 cells, wild-type enzyme and 4 naturally occurring mutants) [11] <5> (cloned in Escherichia coli) [1, 2, 10] <5> (expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion protein) [10] <11> (cloned in Escherichia coli) [7] <12> (cloned) [12] Engineering C10A <4> (<4> 50% decrease in activity [25]) [25] C42A <5> (<5> no catalytic activity, complete loss of metal binding site and activity, it is the only Cys in the active site [2]) [2] D96N <4> (<4> mutant enzyme that causes PTPS deficiency [23]) [23]

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D119-145 <4> (<4> Flag-tag fused PTPS with C-terminal deletion, no detectable activity, no more nuclear staining [25]) [25] D143-145 <4> (<4> Flag-tag fused PTPS with C-terminal deletion, no detectable activity, no more nuclear staining [25]) [25] DV57 <4> (<4> mutant enzyme DV57 is incorrectly folded and thus unstable [11]) [11] d1-11 <4> (<4> Flag-tag fused PTPS with N-terminal deletion, 97% decrease in activity [25]) [25] E133Q <5> (<5> 1.3% of wild-type activity but similar affinity for the substrate [2]) [2] H23L <5> (<5> complete loss of metal binding site and activity [2]) [2] H48L <5> (<5> complete loss of metal binding site and activity [2]) [2] H50L <5> (<5> complete loss of metal binding site and activity [2]) [2] H89N <5> (<5> 4.3% of wild-type activity but similar affinity for the substrate [2]) [2] K143A <4> (<4> 35% decrease in activity [25]) [25] P87L <4> (<4> phosphorylated, 30% of activity of wild type enzyme [11]) [11] R16C <4> (<4> 37% of activity of wild type enzyme. Forms stable homomultimers and exhibit significant activity in vitro, but no activity in COS-1 cells [11]; <4> 89% decrease in activity [25]) [11, 25] R25G <4> (<4> mutant enzyme that causes PTPS deficiency [23]) [23] R25Q <4> (<4> 61% of activity of wild type enzyme. Phosphorylated, forms stable homomultimers and exhibit significant activity in vitro, but no activity in COS-1 cells [11]; <4> 96% decrease in activity [25]) [11, 25] R87L <4> (<4> has substantial activity but enhanced sensitivity to local unfolding [11]) [11] T106M <4> (<4> mutant enzyme that causes PTPS deficiency [23]) [23] V56M <4> (<4> mutant enzyme that causes PTPS deficiency [23]) [23] V70D <4> (<4> mutant enzyme that causes PTPS deficiency [23]) [23] Additional information <4> (<4> fusion protein of PTPS with a N-terminally fused Flag-tag set on 100% of activity, 55% decreased activity of fusion protein of PTPS with a C-terminally fused Flag-tag, 49% increase in activity of fusion protein consisting of PTPS and both N-terminally fused Flag-tag and a N-terminally fused nuclear localization signal [25]) [25] Application medicine <4, 7> (<7> the decrease of this enzyme is the most frequent cause of atypical phenylketonuria [9]; <4> lack of tetrahydrobiopterin leads to hyperphenylalaninemia and a deficiency of biogenic amine neurotransmitters such as dopamine and serotonin and severe progressive mental retardation [1]) [1, 9]

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6 Stability Temperature stability 80 <3, 4, 5, 10> (<10> 5 min, 25% loss of activity [14]; <4,5> stable [10]; <3> 24% retention of activity after 10 min [16]) [10, 14, 16] Additional information <1, 4, 10> (<1,4,10> the human pituitary gland enzyme is heat instable in contrast to the enzyme from human, rat and salmon liver, and Drosophila heads [3]) [3] General stability information <4>, stable at all steps of purification [3] Storage stability <1>, -20 C, PIPES 50 mM, stable for 1 year [15] <3>, -70 C, stable [16] <4>, -20 C and -70 C, Tris HCl, several months, stable [4, 13] <4>, -70 C, stable for several months [13] <5>, 4 C, Tris-HCl, 4 weeks, no degradation [2] <12>, -70 C, stable for at least 1 year [12]

References [1] Thçny, B.; Leimbacher, W.; Blau, N.; Heizmann, C.W.; Burgisser, D.: Human liver 6-pyruvoyl-tetrahydropterin synthase: expression of the cDNA, purification and preliminary characterization of the recombinant protein. Adv. Exp. Med. Biol., 338, 187-190 (1993) [2] Burgisser, D.M.; Thçny, B.; Redweik, U.; Hess, D.; Heizmann, C.W.; Huber, R.; Nar, H.: 6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif. Site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modeling of substrate binding. J. Mol. Biol., 253, 358-369 (1995) [3] Guzman, J.; Redweik, U.; Schoedon, G.; Hunziker, P.; Wiestler, O.D.; Heizmann, C.W.; Blau, N.: Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from human pituitary gland. Enzyme, 46, 287-298 (1992) [4] Takikawa, S.; Curtius, H.Ch.; Redweik, U.; Ghisla, S.: Purification of 6-pyruvoyl-tetrahydropterin synthase from human liver. Biochem. Biophys. Res. Commun., 134, 646-651 (1986) [5] Le van, Q.; Katzenmeier, G.; Schwarzkopf, B.; Schmid, C.; Bacher, A.: Biosynthesis of biopterin studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase. Biochem. Biophys. Res. Commun., 151, 512-517 (1988) [6] Hauer, C.R.; Leimbacher, W.; Hunziker, P.; Neuheiser, F.; Blau, N.; Heizmann, C.W.: 6-Pyruvoyl tetrahydropterin synthase from salmon liver amino acid sequence analysis by tandem mass spectrometry. Biochem. Biophys. Res. Commun., 182, 953-959 (1992)

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[7] Ashida, A.; Hatakeyama, K.; Kagamiyama, H.: cDNA cloning, expression in Escherichia coli and purification of human 6-pyruvoyl-tetrahydropterin synthase. Biochem. Biophys. Res. Commun., 195, 1386-1393 (1993) [8] Auerbach, G.; Nar, H.: The pathway from GTP to tetrahydropterin: threedimensional structures of GTP cyclohydrolase I and 6-pyruvoyl tetrahydropterin synthase. Biol. Chem., 378, 185-192 (1997) [9] Masada, M.: Enzymatic properties of 6-pyruvoyl tetrahydropterin synthase purified from fat bodies of silkworm larvae. Adv. Exp. Med. Biol., 338, 191194 (1993) [10] Burgisser, D.M.; Thçny, B.; Redweik, U.; Hunziker, P.; Heizmann, C.W.; Blau, N.: Expression and characterization of recombinant human and rat liver 6pyruvoyl tetrahydropterin synthase. Modified cysteine residues inhibit the enzyme activity. Eur. J. Biochem., 219, 497-502 (1994) [11] Oppliger, T.; Thçny, B.; Nar, H.; Burgisser, D.; Huber, R.; Heizmann, C.W.; Blau, N.: Structural and functional consequences of mutations in 6-pyruvoyltetrahydropterin synthase causing hyperphenylalaninemia in humans. J. Biol. Chem., 270, 29498-29506 (1995) [12] Inoue, Y.; Kawasaki, Y.; Harada, T.; Hatekeyama, K.; Kagamiyama, H.: Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase. J. Biol. Chem., 266, 20791-20796 (1991) [13] Takikawa, S.; Curtius, H.C.; Redweik, U.; Leimbacher, W.; Ghisla, S.: Biosynthesis of tetrahydropterin. Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from human liver. Eur. J. Biochem., 161, 295-302 (1986) [14] Hasler, T.; Curtius, H.C.: Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver. Eur. J. Biochem., 180, 205211 (1989) [15] Park, Y.S.; Kim, J.H.; Jacobson, K.B.; Yim, J.J.: Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from Drosophila melanogaster. Biochim. Biophys. Acta, 1038, 186-194 (1990) [16] Woo, H.J.; Hwang, Y.K.; Kim, Y.J.; Kang, J.Y.; Choi, Y.K.; Kim, C.G.; Park, Y.S.: Escherichia coli 6-pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8-dihydropterin. FEBS Lett., 523, 234-238 (2002) [17] Scherer-Oppliger, T.; Leimbacher, W.; Blau, N.; Thony, B.: Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II. J. Biol. Chem., 274, 31341-31348 (1999) [18] Leitner, K.L.; Meyer, M.; Leimbacher, W.; Peterbauer, A.; Hofer, S.; Heufler, C.; Mueller, A.; Heller, R.; Werner, E.R.; Thoeny, B.; Werner-Felmayer, G.: Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase. Biochem. J., 373, 681-688 (2003) [19] Woo, H.J.; Kang, J.Y.; Choi, Y.K.; Park, Y.S.: Production of sepiapterin in Escherichia coli by coexpression of cyanobacterial GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase. Appl. Environ. Microbiol., 68, 3138-3140 (2002)

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[20] Bracher, A.; Eisenreich, W.; Schramek, N.; Ritz, H.; Gotze, E.; Herrmann, A.; Gutlich, M.; Bacher, A.: Biosynthesis of pteridines. NMR studies on the reaction mechanisms of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase. J. Biol. Chem., 273, 28132-28141 (1998) [21] Linscheid, P.; Schaffner, A.; Blau, N.; Schoedon, G.: Regulation of 6-pyruvoyltetrahydropterin synthase activity and messenger RNA abundance in human vascular endothelial cells. Circulation, 98, 1703-1706 (1998) [22] Ploom, T.; Thony, B.; Yim, J.; Lee, S.; Nar, H.; Leimbacher, W.; Richardson, J.; Huber, R.; Auerbach, G.: Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl tetrahydropterin synthase. J. Mol. Biol., 286, 851-860 (1999) [23] Liu, T.-T.; Hsiao, K.-J.; Lu, S.-F.; Wu, S.-J.; Wu, K.-F.; Chiang, S.-H.; Liu, X.Q.; Chen, R.-G.; Yu, W.-M.: Mutation analysis of the 6-pyruvoyl-tetrahydropterin synthase gene in Chinese hyperphenylalaninemia caused by tetrahydrobiopterin synthesis deficiency. Hum. Mutat., 11, 76-83 (1998) [24] Tachibana, D.; Fukumasu, H.; Shintaku, H.; Fukumasu, Y.; Yamamasu, S.; Ishiko, O.; Yamano, T.; Ogita, S.: Decreased plasma tetrahydrobiopterin in pregnant women is caused by impaired 6-pyruvoyl tetrahydropterin synthase activity. Int. J. Mol. Med., 9, 49-52 (2002) [25] Elzaouk, L.; Laufs, S.; Heerklotz, D.; Leimbacher, W.; Blau, N.; Resibois, A.; Thoeny, B.: Nuclear localization of tetrahydrobiopterin biosynthetic enzymes. Biochim. Biophys. Acta, 1670, 56-68 (2004) [26] Franscini, N.; Bachli, E.B.; Blau, N.; Fischler, M.; Walter, R.B.; Schaffner, A.; Schoedon, G.: Functional tetrahydrobiopterin synthesis in human platelets. Circulation, 110, 186-192 (2004) [27] Iwanaga, N.; Yamamasu, S.-I.; Tachibana, D.; Nishio, J.; Nakai, Y.; Shintaku, H.; Ishiko, O.: Activity of synthetic enzymes of tetrahydrobiopterin in the human placenta. Int. J. Mol. Med., 13, 117-120 (2004)

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(+)-d-Cadinene synthase

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1 Nomenclature EC number 4.2.3.13 Systematic name 2-trans,6-trans-farnesyl-diphosphate diphosphate-lyase (cyclizing, (+)-d-cadinene-forming) Recommended name (+)-d-cadinene synthase Synonyms (+)-d-cadinene synthase <1> [12] 2-trans,6-trans-farnesyl-diphosphate diphosphate-lyase (cyclizing, (+)-a-cadinene-forming) d-cadinene synthase EC 4.6.1.11 (formerly) cyclase, d-cadinene d-cadinene cyclase d-cadinene synthase farnesyl diphosphate-d-cadinene cyclase CAS registry number 166800-09-5

2 Source Organism <1> <2> <3> <4> <5>

Gossypium hirsutum (no sequence specified) [1, 2, 7, 9, 10, 11, 12] Gossypium arboreum (no sequence specified) [3, 8, 9, 11] Gossypium barbadense (no sequence specified) [4, 5, 7, 8] Gossypium arboreum (UNIPROT accession number: Q39760) [6] Gossypium arboreum (UNIPROT accession number: Q39761) [6]

3 Reaction and Specificity Catalyzed reaction 2-trans,6-trans-farnesyl diphosphate = (+)-d-cadinene + diphosphate (<1,3> cyclization reaction [7])

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Reaction type cyclization (<1> elimination of diphosphate [12]) elimination of diphosphate Natural substrates and products S 2-trans,6-trans-farnesyl diphosphate <1, 2, 3, 4, 5> (<1> transcription of cad1-C is activated in stems treated with Verticillium dahliae [9]; <4, 5> the fungal-elicited production of a (+)-d-cadinene synthase is consistent with a role for this enzyme as the first committed step in the pathways leading to the related phytoalexins gossypol and lacinilene C in cotton [6]; <3> the first step enzyme in the conversion of farnesyl diphosphate to sesquiterpene phytoalexins. The reaction is induced by the infection of cotton with Verticillium dahliae [8]; <2> amount of enzyme does not increase significantly in stems treated with Verticillium dahliae due to a high basal level of enzyme in untreated stems [9]; <1> the sesquiterpenoid (+)-a-cadinene is an intermediate in phytoalexin biosynthesis [1]; <3> the product (+)-d-cadinene is metabolically converted to deoxyhemigossypol, deoxyhemigossypol 6-methyl ether, hemigossypol and hemigossypol 6-methyl ether [5]; <1,2> the enzyme is encoded by a complex gene family that based on homology can be divided into two subfamilies, cad1A and cad1-C [9]) (Reversibility: ?) [1, 5, 6, 7, 8, 9] P (+)-d-cadinene + diphosphate <1, 2, 3> [1, 5, 7, 8, 9] Substrates and products S 2-trans,6-trans-farnesyl diphosphate <1, 2, 3, 4, 5> (<3> nerolidyl diphosphate is an intermediate [5]; <1> transcription of cad1-C is activated in stems treated with Verticillium dahliae [9]; <4,5> the fungal-elicited production of a (+)-d-cadinene synthase is consistent with a role for this enzyme as the first committed step in the pathways leading to the related phytoalexins gossypol and lacinilene C in cotton [6]; <3> the first step enzyme in the conversion of farnesyl diphosphate to sesquiterpene phytoalexins. The reaction is induced by the infection of cotton with Verticillium dahliae [8]; <2> amount of enzyme does not increase significantly in stems treated with Verticillium dahliae due to a high basal level of enzyme in untreated stems [9]; <1> the sesquiterpenoid (+)-a-cadinene is an intermediate in phytoalexin biosynthesis [1]; <3> the product (+)-d-cadinene is metabolically converted to deoxyhemigossypol, deoxyhemigossypol 6-methyl ether, hemigossypol and hemigossypol 6-methyl ether [5]; <1,2> the enzyme is encoded by a complex gene family that based on homology can be divided into two subfamilies, cad1-A and cad1-C [9]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 12] P (+)-d-cadinene + diphosphate <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6, 7, 8, 9] S nerolidyl diphosphate <3> (Reversibility: ?) [5] P (+)-d-cadinene + diphosphate <3> [5] S nerolidyl diphosphate <2> (Reversibility: ?) [8] P (+)-d-cadinene + a-bisabolol + b-bisabolene + b-farnesene + ? <2> [8]

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Metals, ions Mg2+ <2, 4, 5> (<4,5> required [6]; <2> 2.5 mM MgCl2 required for optimal activity, enzyme form CAD1-A [3]; <2> 15 mM MgCl2 required for optimal activity, enzyme form CAD1-C [3]) [3, 6] Turnover number (min–1) 0.033 <4> (2-trans,6-trans-farnesyl diphosphate, <4> fusion protein composed of the pXC1-encoded protein and the histidine leader peptide derived from pET28, pH 7, 30 C [6]) [6] Specific activity (U/mg) 0.0000487 <1> (<1> [3 H] farnesyl diphosphate [12]) [12] 0.000405 <1> (<1> 27 days postanthesis [11]) [11] 0.0005 <1> (<1> 60 days postanthesis [11]) [11] 0.000746 <1> (<1> 35 days postanthesis [11]) [11] 0.000988 <1> (<1> 40 days postanthesis [11]) [11] 0.021 <2> (<2> CAD1-A isozyme, expressed in Escherichia coli [3]) [3] 0.03 <4> (<4> recombinant protein [6]) [6] 0.036 <2> (<2> CAD1-A isozyme, removal of 15 amino acids by thrombin cleavage [3]) [3] 0.433 <1> [1] Additional information <1, 2, 3> (<1,2> specific activity of the enzyme increases concomitantly with the cad1 transcripts in developing cottonseeds [11]; <1> enzyme activity appears between 26 and 33 days postanthesis, reaches a maximum at 38 days postanthesis and then sharply declines [10]; <1,3> in the early stages of infection with a defoliating isolate of the pathogen Verticillium dahliae, the enzyme activity in resistant plants increases more quickly than in susceptible plants [7]; <1,2> in roots the activity increases on day 7 post germination. In cotyledons and hypocotyls the activity decreases on day 7 post germination [9]) [7, 9, 10, 11] Km-Value (mM) 0.00065 <2> (nerolidyl diphosphate, <2> pH 7.5, 30 C, recombinant CDN1C1 enzyme [8]) [8] 0.0017 <4> (2-trans,6-trans-farnesyl diphosphate, <4> pH 7, 30 C, fusion protein composed of the pXC1-encoded protein and the histidine leader peptide derived from pET28 [6]) [6] 0.00605 <2> (2-trans,6-trans-farnesyl diphosphate, <2> pH 7.5, 30 C, recombinant CDN1-C1 enzyme [8]) [8] 0.007 <2> (2-trans,6-trans-farnesyl diphosphate, <2> isoenzyme CAD1-A, pH 8.7, 30 C [3]) [3] 0.0106 <1> (2-trans,6-trans-farnesyl diphosphate) [12] pH-Optimum 6.7-8 <4, 5> [6] 7-7.5 <2> (<2> enzyme form CAD1-C [3]) [3] 8.7 <2> (<2> enzyme form CAD1-A [3]) [3]

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pH-Range 6.8-8.7 <2> (<2> 62% of maximal activity at pH 6.8, enzyme form CAD1-A [3]) [3] 7-8.7 <2> (<2> 60% of maximal activity at pH 8.7, enzyme form CAD1-C [3]) [3]

4 Enzyme Structure Molecular weight 45000 <1> (<1> gel filtration [2]) [2] 64000 <1> (<1> SDS-PAGE [12]) [12] 64060 <2> (<2> calculated from DNA sequence, CAD1-A isozyme [3]) [3] 64100 <4> (<4> calculated from DNA sequence [6]) [6] 64120 <5> (<5> calculated from DNA sequence [6]) [6] Subunits monomer <1> (<1> 1 * 64000-65000, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture <4> (<4> time-dependent 10fold increase in mRNA in response to a challenge by preparation from Verticillium dahliae [6]) [6] cotyledon <1, 2> (<1> stimulated to produce sesquiterpenoid phytoalexins by inoculation with Xanthomonas campestris pv. malvacearum, or by injection of oligogalacturonide elicitor [1]) [1, 9] flower <1, 2> [9] leaf <1, 2> [9] pericarp <1, 2> [9] petal <1, 2> [9] root <1, 2> [9] seed <1> [10, 11] stele <3> (<3> infected with Verticillium dahliae. Verticillium dahliae initiates a signal in the stele tissue that results in an increased steady-state level of d-cadinene synthase mRNA and an increased activity of d-cadinene synthase [5]) [4, 5] stem <1, 2> [9] Purification <1> (N-terminal fusion of 10x His-tag, purification by affinity chromatography under native and denaturing conditions) [12] <1> (slurry homogenization and centrifugation) [7] <1> (to homogeneity, several chromatographic steps, including hydroxyapatite, phenyl-agarose and anion-exchange chromatography) [2] <2> (CAD1-A isoenzyme) [3]

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<2> (affinity chromatography, chromatography on glutathione-Sepharose-4B column and anion-exchange column) [8] <4> (purification of a recombinant enzyme using histidine affinity chromatography) [6] <5> (purification of a recombinant enzyme using histidine affinity chromatography) [6] Crystallization <2> (modeled from amino acid sequence of CDN1-C1, in SWISS-MODEL automated homology modeling server) [8] Cloning <1> (expressed in Escherichia coli) [12] <2> (expression in Escherichia coli) [11] <2> (expression in Escherichia coli of CDN1-C1) [8] <2> (expression in Escherichia coli of the CAD1-A isoenzyme, 80% of homology with CAD1-C isoenzyme) [3] <4> (expression in Escherichia coli of a cDNA isolated and amplified from a cell culture infected with Verticillium dahliae) [6] <5> (expression in Escherichia coli of a cDNA isolated and amplified from a cell culture infected with Verticillium dahliae) [6]

6 Stability General stability information <1>, loss of activity during freezing and thawing is gradually restored during incubation at 0 C, complete recovery after 4-5 h [1] Storage stability <1>, -20 C, stable for at least 1 year [1] <4,5>, -20 C, stable for at least 4 weeks [6]

References [1] Davis, G.D.; Essenberg, M.: (+)-d-Cadinene is a product of sesquiterpene cyclase activity in cotton. Phytochemistry, 39, 553-567 (1995) [2] Davis, E.M.; Tsuji, J.; Davis, G.D.; Pierce, M.L.; Essenberg, M.: Purification of (+)-d-cadinene synthase, a sesquiterpene cyclase from bacteria-inoculated cotton foliar tissue. Phytochemistry, 41, 1047-1055 (1996) [3] Chen, X.Y.; Wang, M.; Chen, Y.; Davisson, V.J.; Heinstein, P.: Cloning and heterologous expression of a second (+)-d-cadinene synthase from Gossypium arboreum. J. Nat. Prod., 59, 944-951 (1996) [4] Benedict, C.R.; Alchanati, I.; Harvey, P.J.; Liu, J.; Stipanovic, R.D.; Bell, A.A.: The enzymatic formation of d-cadinene from farnesyl diphosphate in extracts of cotton. Phytochemistry, 39, 327-331 (1995)

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4.2.3.13

(+)-d-Cadinene synthase

[5] Alchanati, I.; Acreman Patel, J.A.; Liu, J.; Benedict, C.R.; Stipanovic, R.D.; Bell, A.A.; Cui, Y.; Magill, C.W.: The enzymatic cyclization of nerolidyl diphosphate by d-cadinene synthase from cotton stele tissue infected with Verticillium dahlia. Phytochemistry, 47, 961-967 (1998) [6] Chen, X.Y.; Chen, Y.; Heinstein, P.; Davisson, V.J.: Cloning, expression, and characterization of (+)-d-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis. Arch. Biochem. Biophys., 324, 255-266 (1995) [7] Bianchini, G.M.; Stipanovic, R.D.; Bell, A.A.: Induction of d-cadinene synthase and sesquiterpenoid phytoalexins in cotton by Verticillium dahliae. J. Agric. Food Chem., 47, 4403-4406 (1999) [8] Benedict, C.R.; Lu, J.L.; Pettigrew, D.W.; Liu, J.; Stipanovic, R.D.; Williams, H.J.: The cyclization of farnesyl diphosphate and nerolidyl diphosphate by a purified recombinant d-cadinene synthase. Plant Physiol., 125, 1754-1765 (2001) [9] Tan, X.P.; Liang, W.Q.; Liu, C.J.; Luo, P.; Heinstein, P.; Chen, X.Y.: Expression pattern of (+)-d-cadinene synthase genes and biosynthesis of sesquiterpene aldehydes in plants of Gossypium arboreum L. Planta, 210, 644-651 (2000) [10] Martin, G.S.; Liu, J.; Benedict, C.R.; Stipanovic, R.D.; Magill, C.W.: Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-d-cadinene synthase. Phytochemistry, 62, 31-38 (2003) [11] Meng, Y.L.; Jia, J.W.; Liu, C.J.; Liang, W.Q.; Heinstein, P.; Chen, X.Y.: Coordinated accumulation of (+)-d-cadinene synthase mRNAs and gossypol in developing seeds of Gossypium hirsutum and a new member of the cad1 family from G. arboreum. J. Nat. Prod., 62, 248-252 (1999) [12] Townsend, B.J.; Poole, A.; Blake, C.J.; Llewellyn, D.J.: Antisense suppression of a (+)-d-cadinene synthase gene in cotton prevents the induction of this defense response gene during bacterial blight infection but not its constitutive expression. Plant Physiol., 138, 516-528 (2005)

255

Pinene synthase

4.2.3.14

1 Nomenclature EC number 4.2.3.14 Systematic name geranyl-diphosphate diphosphate-lyase (cyclizing, pinene-forming) Recommended name pinene synthase Synonyms (-)-(1S,5S)-pinene synthase (-)-(4S)-limonene/(-)-(1S,5S)-a-pinene synthase <1> [12] (-)-b-pinene synthase <3> [14] (-)-limonene/(-)-a-pinene synthase <1> [12] (-)-pinene cyclase (II) (-)-pinene synthase <1> [11, 13, 15] b-geraniolene synthase b-pinene synthase <3> [14] monoterpene cyclase II pinene cyclase Additional information [3, 4, 5, 6] CAS registry number 110637-20-2

2 Source Organism <1> Abies grandis (no sequence specified) [1, 2, 8, 9, 11, 12, 13, 15] <2> Salvia officinalis (no sequence specified) [3, 4, 5, 6, 7, 10] <3> Artemisia annua (no sequence specified) [14]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate = pinene + diphosphate Reaction type cyclization, C-O bond cleavage

256

4.2.3.14

Pinene synthase

Natural substrates and products S geranyl diphosphate <1, 3> (Reversibility: ?) [13, 14] P pinene + diphosphate <1, 3> [13, 14] S geranyl diphosphate <1, 2> (<1> defense against bark beetle attack, induced by stem wounding [1, 2, 8, 9]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10] P (-)-a-pinene + (-)-b-pinene + diphosphate <1, 2> (<2> camphene as side product [3,5,6,10]; <1> ratio a and b form 42:58% [1]; <1> small amounts of other monoterpenes as side products [8]; <1> ratio 32:64%, small amount of myrcene as side product [2]; <2> camphene and small amounts of other monoterpenes as side products [4]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10] Substrates and products S (2S,3R)-methanogeranyl diphosphate <1> (<1> racemic [11]) (Reversibility: ?) [11, 13] P (E)-ocimene + diphosphate <1> [11, 13] S (3R)-linalyl diphosphate <2> (Reversibility: ?) [5] P (-)-a-pinene + (-)-b-pinene + diphosphate S (3S)-linalyl diphosphate <2> (Reversibility: ?) [5, 6] P (-)-a-pinene + (-)-b-pinene + diphosphate <2> [5, 6] S 6,7-dihydrogeranyl diphosphate <1> (Reversibility: ?) [11, 13] P 7-methyl-3-methylene-1-octene + diphosphate <1> [11, 13] S geranyl diphosphate <1, 3> (Reversibility: ?) [11, 13, 14] P pinene + diphosphate <1, 3> [11, 13, 14] S geranyl diphosphate <1> (Reversibility: ?) [12, 15] P (-)-a-pinene + (-)-b-pinene S geranyl diphosphate <3> (Reversibility: ?) [14] P b-pinene + a-pinene + diphosphate <3> (<3> major product b-pinene, minor product a-pinene, ratio 94:6 [14]) [14] S geranyl diphosphate <1, 2> (<1> defense against bark beetle attack, induced by stem wounding [1, 2, 8, 9]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10] P (-)-a-pinene + (-)-b-pinene + diphosphate <1, 2> (<2> camphene as side product [3,5,6,10]; <1> ratio a and b form 42:58% [1]; <1> small amounts of other monoterpenes as side products [8]; <1> ratio 32:64%, small amount of myrcene as side product [2]; <2> camphene and small amounts of other monoterpenes as side products [4]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10] S neryl diphosphate <2> (Reversibility: ?) [5] P (-)-a-pinene + (-)-b-pinene + diphosphate Inhibitors (+)-a-pinene <1> [8] (-)-a-Pinene <1> [8] Diethyldicarbonate <1> (<1> IC50: 0.64 mM [8]) [8] Diphosphate <1> (<1> IC50: 0.17 mM [8]) [8] Geranyl diphosphate <1> (<1> inhibitory above 0.033 mM, complete inhibition above 0.066 mM [8]) [8]

257

Pinene synthase

4.2.3.14

phosphate <1> (<1> IC50: 51 mM [8]) [8] SDS <1> (<1> 0.1% for 1 h 4 C, complete loss of activity, can partially be restored by adding Tween 20 or Triton X-100 [8]) [8] Zwittergent 3-12 <1> [8] p-hydroxymercuribenzoate <1> (<1> IC50: 0.002 mM [8]) [8] Activating compounds Triton X-100 <1> (<1> 0.1-1% [8]) [8] Tween 20 <1> (<1> 0.1-1% [8]) [8] Metals, ions K+ <1> (<1> required, optimum concentration 0.5 M [1]) [1] Mg2+ <1> (<1> ineffective [1]; <1> poor substitute for Mn2+ [8]) [1, 8] Mn2+ <1> (<1> required [1]; <1> required, maximum activity at 0.1 mM, higher concentrations inhibitory [8]) [1, 8] Turnover number (min–1) 0.000167 <1> (Geranyl diphosphate) [8] Specific activity (U/mg) 0.0135 <1> (<1> crude extract [2]) [2] 0.142 <1> (<1> partially purified enzyme [2]) [2] Km-Value (mM) 0.002 <2> (neryl diphosphate) [5, 6] 0.003 <2> (geranyl diphosphate) [5, 6] 0.0035 <2> ((3R)-linalyl diphosphate) [5, 6] 0.0035 <2> ((3S)-linalyl diphosphate) [5, 6] 0.006 <1> (geranyl diphosphate) [8] pH-Optimum 7.8 <1> [8] pH-Range 7-8.3 <1> (<1> 80% of maximum activity [8]) [8] Temperature optimum ( C) 42 <1> [8]

4 Enzyme Structure Molecular weight 55000 <2> (<2> gel filtration [7]) [7] 62000 <1> (<1> SDS-PAGE [2,8,9]) [2, 8, 9] 63000 <1> (<1> gel filtration [2,8]) [2, 8] 64000 <1> (<1> calculated molecular weight of recombinant protein without transit peptide [1]) [1] 67000 <3> (<3> calculated from cDNA [14]) [14] 715000 <1> (<1> calculated from theoretical amino acid sequence of recombinant pre-protein from E. coli [1]) [1]

258

4.2.3.14

Pinene synthase

Subunits monomer <1> (<1> 1 * 62000, SDS-PAGE, gel filtration [8]) [8]

5 Isolation/Preparation/Mutation/Application Source/tissue inflorescence <3> [14] leaf <2, 3> [3, 4, 10, 14] stem <1, 3> [2, 8, 9, 14] Additional information <3> (<3> not detected in roots [14]) [14] Purification <1> [8, 9] <1> (partial) [2] <1> (recombinant enzyme) [13] <2> [10] <2> (partial) [3, 4, 5, 7] Renaturation <1> (renaturation after SDS-PAGE with 1% Tween 20, recovery 5-10%) [8] Cloning <1> [11] <1> (expressed in Escherichia coli) [13] <1> (in Escherichia coli) [1] <3> (cDNA clone QH6 expressed in Escherichia coli BL21(DE3)) [14] Engineering C372S <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C372S/C480S <1> (<1> compared to wild-type enzyme the proportion of apinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C372S/C480S/F597W <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C372S/C480S/S485C <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C372S/C480S/S485C/F597W <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C372S/F597W <1> (<1> compared to wild-type enzyme the proportion of apinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C372S/M398I/Y450C/I451F/C480S/G481A/I484P/S485C/A594S/A596V/ F597W/Y599H <1> (<1> altered product spectrum compared to wild-type

259

Pinene synthase

4.2.3.14

enzyme, relative product distribution for wild-type enzyme in parenthesis: tricyclene 2.1 (0), a-pinene 30.7 (29.5), camphene 30.1 (0.1), b-pinene 1.9 (63.4), mycrene 1.0 (1.8), limonene 3.6 (3.6), camphene hydrate 22.3 (0), activity of the mutant enzyme is 38% of wild-type activity [15]) [15] C372S/S485C <1> (<1> compared to wild-type enzyme the proportion of apinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C372S/S485C/F597W <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C372S/Y450C/I451F/C480S/G481A/I484P/S485C <1> (<1> altered product spectrum compared to wild-type enzyme, relative product distribution for wild-type enzyme in parenthesis: tricyclene 0.7 (0), a-pinene 57.3 (29.5), camphene 9.3 (0.1), b-pinene 4.4 (63.4), mycrene 1.0 (1.8), limonene 4.8 (3.6), camphene hydrate 14.0 (0), activity of the mutant enzyme is 77% of wild-type activity [15]) [15] C372S/Y450C/I451F/C480S/G481A/I484P/S485C/A594S/A596V/F597W/Y599H <1> (<1> altered product spectrum compared to wild-type enzyme, relative product distribution for wild-type enzyme in parenthesis: tricyclene 2.2 (0), a-pinene 32.0 (29.5), camphene 30.1 (0.1), b-pinene 1.7 (63.4), mycrene 1.0 (1.8), limonene 3.7 (3.6), camphene hydrate 21.3 (0), activity of the mutant enzyme is 36% of wild-type activity [15]) [15] C372S/Y450C/I451F/C480S/G481A/I484P/S485C/F597W <1> (<1> altered product spectrum compared to wild-type enzyme, relative product distribution for wild-type enzyme in parenthesis: tricyclene 1.8 (0), a-pinene 40.6 (29.5), camphene 26.2 (0.1), b-pinene 2.2 (63.4), mycrene 0.9 (1.8), limonene 3.3 (3.6), camphene hydrate 20.8 (0), activity of the mutant enzyme is 65% of wild-type activity [15]) [15] C480S <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C480S/F597W <1> (<1> compared to wild-type enzyme the proportion of apinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C480S/S485C <1> (<1> compared to wild-type enzyme the proportion of apinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] C480S/S485C/F597W <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] F597W <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] S485C <1> (<1> compared to wild-type enzyme the proportion of a-pinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15]

260

4.2.3.14

Pinene synthase

S485C/F597W <1> (<1> compared to wild-type enzyme the proportion of apinene increases at the expense of b-pinene, and the proportion of camphene and camphene hydrate increase [15]) [15] Y450C/I451F/C480S/G481A/I484P/S485C/A594S/A596V/F597W/Y599H <1> (<1> altered product spectrum compared to wild-type enzyme, relative product distribution for wild-type enzyme in parenthesis: tricyclene 1.8 (0), apinene 43.8 (29.5), camphene 23.9 (0.1), b-pinene 2.6 (63.4), mycrene 0.8 (1.8), limonene 4.5 (3.6), camphene hydrate 13.3 (0), activity of the mutant enzyme is 41% of wild-type activity [15]) [15] Y450C/I451F/C480S/G481A/I484P/S485C/F597W <1> (<1> altered product spectrum compared to wild-type enzyme, relative product distribution for wild-type enzyme in parenthesis: tricyclene 1.7 (0), a-pinene 51.6 (29.5), camphene 22.2 (0.1), b-pinene 3.4 (63.4), mycrene 0.7 (1.8), limonene 4.2 (3.6), camphene hydrate 9.6 (0), activity of the mutant enzyme is 27% of wild-type activity [15]) [15] Additional information <1> (<1> (-)-(4 S)-limonene synthase (LS) and (-)-(4 S)-limonene/(-)-(1S,5S)-a-pinene synthase (LPS) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis is carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gives rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) alters the product distribution to an intermediate between LS and LPS, and results in a 5fold increase in relative velocity [12]; <1> structural modeling followed by directed mutagenesis in (-)-pinene synthase is used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produces an enzyme in which more than half of the product is channeled through an alternative pathway. It is also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions are necessary before catalysis is significantly altered [15]) [12, 15]

6 Stability General stability information <1>, extremely labile towards coextracted phenolic compounds, stable after DEAE-cellulose treatment, unstable in absence of dithiothreitol, 60% loss of activity within 1 h, 4 C [8] Storage stability <1>, 4 C, stable for 3 weeks, -20 C stable for several month [8]

261

Pinene synthase

4.2.3.14

References [1] Bohlmann, J.; Steele C.L.; Croteau, R.: Monoterpene synthases from grand fir (Abies grandis): cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)pinene synthase. J. Biol. Chem., 272, 21784-21792 (1997) [2] Gijzen, M.; Lewinsohn, E.; Croteau, R: Characterization of the constitutive and wound-inducible monoterpene cyclases of grand fir (Abies grandis). Arch. Biochem. Biophys., 289, 267-273 (1991) [3] Wagschal, K.C.; Pyun H.J.; Coates, R.M.; Croteau, R.: Monoterpene biosynthesis: Isotope effects associates with bicyclic olefin formation catalyzed by pinene synthases from sage (Salvia officinalis). Arch. Biochem. Biophys., 308, 477-487 (1994) [4] Croteau, R.; Wheeler, C.J.: Isotopically sensitive branching in the formation of cyclic monoterpenes: Proof that (-)-a-pinene and (-)-b-pinene are synthesized by the same monoterpene cyclase via deprotonation of a common intermediate. Biochemistry, 26, 5383-5389 (1987) [5] Croteau, R.; Satterwhite, D.M.; Cane, D.E.; Chang, C.C.: Biosynthesis of monoterpenes: Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophospahte to (+)- and (-) pinene and (+)- and (-)-camphene. J. Biol. Chem., 263, 10063-10071 (1988) [6] Croteau, R.; Satterwhite, D.M.: Biosynthesis of monoterpenes: Stereochemical implications of acyclic and monocyclic olefin formation by (+)- and (-)-pinene cyclases from sage. J. Biol. Chem., 264, 15309-15315 (1989) [7] Croteau, R.; Satterwhite, D.M.; Wheeler, C.J.; Felton, N.M.: Biosynthesis of monoterpenes: Stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (+)-a- and (-)b-pinene. J. Biol. Chem., 264, 2075-2080 (1989) [8] Lewinsohn, E.; Gijzen, M.; Croteau, R.: Wound-inducible pinene cyclase from grand fir: Purification, characterization, and renaturation after SDSPAGE. Arch. Biochem. Biophys., 293, 167-173 (1992) [9] Gijzen, M.; Lewinsohn, E.; Croteau, R.: Antigenic cross-reactivity among monoterpene cyclases from grand fir and induction of these enzymes upon stem wounding. Arch. Biochem. Biophys., 294, 670-674 (1992) [10] Pyun, H.J.; Wagschal, K.C.; Jung, D.; Coates, R.M.; Croteau, R.: Stereochemistry of the proton elimination in the formation of (+)- and (-)-a-pinene by monoterpene cyclases from sage (Salvia officinalis). Arch. Biochem. Biophys., 308, 488-496 (1994) [11] Schwab, W.; Williams, D.C.; Davis, E.M.; Croteau, R.: Mechanism of monoterpene cyclization: Stereochemical aspects of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase. Arch. Biochem. Biophys., 392, 123-136 (2001) [12] Katoh S, Hyatt D, Croteau R.: Altering product outcome in Abies grandis (-)-limonene synthase and (-)-limonene/(-)-a-pinene synthase by domain

262

4.2.3.14

Pinene synthase

swapping and directed mutagenesis. Arch. Biochem. Biophys., 425, 65-76 (2004) [13] Schwab, W.; Williams, D.C.; Croteau, R.: Mechanism of monoterpene cyclization: stereochemistry of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase. J. Mol. Catal. B, 19-20, 415-421 (2002) [14] Lu, S.; Xu, R.; Jia, J.-W.; Pang, J.; Matsuda, S.P.T.; Chen, X-Y.: Cloning and functional characterization of a b-pinene synthase from Artemisia annua that shows a circadian pattern of expression. Plant Physiol., 130, 477-486 (2002) [15] Hyat, D.C.; Croteau, R.: Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis. Arch. Biochem. Biophys., 439, 222-233 (2005)

263

Myrcene synthase

4.2.3.15

1 Nomenclature EC number 4.2.3.15 Systematic name geranyl-diphosphate diphosphate-lyase (myrcene-forming) Recommended name myrcene synthase Synonyms AtTPS10 <4> [2] myrcene/(E)-b-ocimene synthase <4> [2] CAS registry number 197462-59-2

2 Source Organism <1> <2> <3> <4>

Abies grandis (no sequence specified) [1] Perilla frutescens (no sequence specified) [4] Ochtodes secundiramea (no sequence specified) [3] Arabidopsis thaliana (UNIPROT accession number: Q9FVI8) [2]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate = myrcene + diphosphate Reaction type elimination, C-O bond cleavage Natural substrates and products S geranyl diphosphate <1, 3, 4> (<1> induced by stem wounding, enhances resistance to insects [1]) (Reversibility: ?) [1, 2, 3] P myrcene + diphosphate <1, 3, 4> [1, 2, 3] Substrates and products S geranyl diphosphate <4> (Reversibility: ?) [2] P (+)-limonene <4> (<4> less than 5% of total hydrocarbon product [2]) [2]

264

4.2.3.15

S P S P S P S P S P S P S P S P

Myrcene synthase

geranyl diphosphate <4> (Reversibility: ?) [2] (-)-limonene <4> (<4> less than 5% of total hydrocarbon product [2]) [2] geranyl diphosphate <4> (Reversibility: ?) [2] (E)-b-ocimene <4> (<4> 20% of total hydrocarbon product [2]) [2] geranyl diphosphate <4> (Reversibility: ?) [2] 2-carene <4> (<4> less than 5% of total hydrocarbon product [2]) [2] geranyl diphosphate <4> (Reversibility: ?) [2] tricyclene <4> (<4> less than 5% of total hydrocarbon product [2]) [2] geranyl diphosphate <2> (Reversibility: ?) [4] myrcene + sabinene + linalool + limonene + diphosphate (<2> formation of 53.8% myrcene, 20.9% sabinene, 19.8% linalool and 5.5% limonene [4]) geranyl diphosphate <1, 3, 4> (<1> induced by stem wounding, enhances resistance to insects [1]) (Reversibility: ?) [1, 2, 3] myrcene + diphosphate <1, 3, 4> (<4> 56% of total hydrocarbon product [2]) [1, 2, 3] linalyl diphosphate <3> (Reversibility: ?) [3] acyclic and cyclic monoterpenes <3> [3] neryl diphosphate <3> (Reversibility: ?) [3] limonene <3> [3]

Metals, ions K+ <1> (<1> required, optimum concentration of 0.5 M [1]) [1] Mg2+ <1, 3> (<3> required for activity [3]; <1> ineffective [1]) [1, 3] Mn2+ <1> (<1> required [1]) [1] pH-Optimum 7.2 <3> [3]

4 Enzyme Structure Molecular weight 69000 <3> [3] 72500 <1> (<1> calculated MW of recombinant protein expressed in Escherichia coli [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <2> (<2> young [4]) [4] stem <1> [1] suspension culture <3> [3] Cloning <1> (expressed in Escherichia coli) [1] <2> (expression in Escherichia coli) [4] <4> (expression in Escherichia coli) [2]

265

Myrcene synthase

4.2.3.15

References [1] Bohlmann, J.; Steele C.L.; Croteau, R.: Monoterpene synthases from grand fir (Abies grandis): cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase. J. Biol. Chem., 272, 21784-21792 (1997) [2] Bohlmann, J.; Martin, D.; Oldham, N.J.; Gershenzon, J.: Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-b-ocimene synthase. Arch. Biochem. Biophys., 375, 261-269 (2000) [3] Wise, M.L.; Rorrer, G.L.; Polzin, J.J.; Croteau, R.: Biosynthesis of marine natural products: isolation and characterization of a myrcene synthase from cultured tissues of the marine red alga Ochtodes secundiramea. Arch. Biochem. Biophys., 400, 125-132 (2002) [4] Hosoi, M.; Ito, M.; Yagura, T.; Adams, R.P.; Honda, G.: cDNA isolation and functional expression of myrcene synthase from Perilla frutescens. Biol. Pharm. Bull., 27, 1979-1985 (2004)

266

(4S)-Limonene synthase

4.2.3.16

1 Nomenclature EC number 4.2.3.16 Systematic name geranyl-diphosphate diphosphate-lyase [cyclizing, (4S)-limonene-forming] Recommended name (4S)-limonene synthase Synonyms (-)-limonene synthase <7> [15] CAS registry number 110639-20-8

2 Source Organism <1> <2> <3> <4> <5> <6> <7>

Abies grandis (no sequence specified) [2, 12] Mentha sp. (no sequence specified) [1, 3, 5, 6, 7, 9, 10] Perilla frutescens (no sequence specified) [4] Ricciocarpos natans (no sequence specified) [8] Mentha spicata (no sequence specified) [11, 14] Agastache rugosa (no sequence specified) [13] Picea abies (UNIPROT accession number: Q675L1) [15]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate = (-)-(4S)-limonene + diphosphate Reaction type C-O bond cleavage Natural substrates and products S geranyl diphosphate <1, 2, 3, 4> (<1> induced by stem wounding, enhances resistance to insects [2]; <2> essential for menthol production [9]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10] P (-)-(4S)-limonene + diphosphate <1, 2, 3, 4> (<2> small amounts of (-)-a-pinene, (-)-b-pinene and myrcene as side products [3,5,6]; <1> small amounts of (-)-a-pinene, (-)-b-pinene and b-phellandrene as side products [2]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]

267

(4S)-Limonene synthase

4.2.3.16

Substrates and products S (3R)-linalyl diphosphate <2> (Reversibility: ?) [6] P ? S (3S)-linalyl diphosphate <2> (Reversibility: ?) [6] P ? S 6,7-dihydrogeranyl diphosphate <5> (<5> reaction yields only achiral, olefin products from 6,7-dehydrogeranyl diphosphate, no significant amounts of terpenols or homoterpenols are formed [11]) (Reversibility: ?) [11, 14] P ? <5> [11, 14] S geranyl diphosphate <1, 2, 3, 4, 5, 6> (<2> (3S)-linalyl diphosphate as intermediate [10]; <5> cyclization proceeds via preliminary isomerization to the bound tertiary intermediate 3S-linalyl diphosphate [14]; <1> induced by stem wounding, enhances resistance to insects [2]; <2> essential for menthol production [9]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14] P (-)-(4S)-limonene + diphosphate <1, 2, 3, 4, 5, 6> (<2> small amounts of (-)-a-pinene, (-)-b-pinene and myrcene as side products [3,5,6]; <1> small amounts of (-)-a-pinene, (-)-b-pinene and b-phellandrene as side products [2]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14] S neryl diphosphate <2> (Reversibility: ?) [6] P ? Inhibitors 6-cyclopropylidene-(3E)-methyl-hex-2-en-1-yl diphosphate <2> (<2> Ki : 0.0003 mM, irreversible covalent modification [1]) [1] diethyl dicarbonate <2> (<2> reversible with hydroxylamine, pretreatment with geranyl diphosphate and MnCl2 prevents inhibition [10]) [1, 6, 10] N-ethylmaleimide <2> (<2> reversible by cysteine treatment [6]) [6] sodium diphosphate <2> (<2> Ki : 1 mM [6]) [6] sodium phosphate <2> (<2> Ki : 30 mM [6]) [6] benzyl bromide <2> [6] linalyl sulfonium ion <2> [6, 10] p-hydroxymercuribenzoate <2> (<2> pretreatment with geranyl diphosphate and MnCl2 prevents inhibition [6]) [1, 6, 10] Activating compounds sodium phosphate <2> (<2> stimulating at low concentrations [6]) [6] Metals, ions K+ <1> (<1> required, optimum concentration of 0.5 M [2]) [2] Mg2+ <1, 2, 4> (<2> cofactor [3]; <1> ineffective [2]; <2> Mn2+ preferred [6]; <4> can substitute for Mn2+ [8]) [2, 3, 6, 8] Mn2+ <1, 2, 4> (<1,2,4> required [2,8,10]; <2> required, optimum activity with 2 mM, inhibitory at higher concentrations [6]) [2, 6, 8, 10] Additional information <2> (<2> Ca2+ , Cd2+ , Co2+ , Cu2+ , Fe2+ , Ni2+ , Zn2+ activated less than 5% [6]) [6]

268

4.2.3.16

(4S)-Limonene synthase

Km-Value (mM) 0.001 <2> (neryl diphosphate) [6] 0.00125 <4> (geranyl diphosphate) [8] 0.0018 <2> (geranyl diphosphate) [6] 0.0019 <2> ((3R)-linalyl diphosphate) [6] 0.0023 <2> ((3S)-linalyl diphosphate) [6] Ki-Value (mM) 0.0003 <2> (6-cyclopropylidene-(3E)-methyl-hex-2-en-1-yl diphosphate, <2> irreversible covalent modification [1]) [1] 1 <2> (sodium diphosphate) [6] 30 <2> (sodium phosphate) [6] pH-Optimum 6.5 <4> [8] 6.7 <2> [6] pH-Range 6.1-7.2 <2> (<2> half maximum activity at both pH [6]) [6]

4 Enzyme Structure Molecular weight 51000 <4> (<4> gel filtration [8]) [8] 56000 <2> (<2> SDS-PAGE [7, 9]; <2> SDS-PAGE, gel filtration [3, 5]) [3, 5, 7, 9] 59800 <2> (<2> calculated from cDNA sequence [3]) [3] 70000 <6> (<6> SDS-PAGE [13]) [13] 73500 <1> (<1> calculated MW of recombinant protein expressed in Escherichia coli [2]) [2] Subunits monomer <2> (<2> 1 * 56000, gel filtration, SDS-PAGE [5]) [5]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <2, 3> (<2> glandular trichome secretory cells [1, 3, 5, 9]) [1, 3, 4, 5, 6, 7, 9, 10] stem <1> [2] Localization leucoplast <2> [9] Purification <2> [3, 5, 7] <2> (partial) [6, 9] <4> (partial) [8] <5> [14]

269

(4S)-Limonene synthase

4.2.3.16

Cloning <1> [12] <1> (expressed in Escherichia coli) [2] <2> (expressed in Escherichia coli) [3] <3> (expressed in Escherichia coli) [4] <5> (expressed in Escherichia coli) [14] <6> [13] <7> (expression in Escherichia coli) [15] Engineering Additional information <1> (<1> exchange of the predicted helix D through F region gives rise to an (-)-limonene/(-)-a-pinene synthase product outcome [12]) [12]

6 Stability pH-Stability 7 <4> (<4> most stable [8]) [8] General stability information <2>, unstable in crude secretory cell extract, stable after DEAE-cellulose chromatography [6] Storage stability <2>, 4 C, stable for 1 week without loss of activity after DEAE-cellulose chromatography [6]

References [1] Croteau, R.; Alonso, W.R.; Koepp, A.E.; Shim, J.H.; Cane, D.E.: Irreversible inactivation of monoterpene cyclases by a mechanism-based inhibitor. Arch. Biochem. Biophys., 307, 397-404 (1993) [2] Bohlmann, J.; Steele C.L.; Croteau, R.: Monoterpene synthases from grand fir (Abies grandis): cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)pinene synthase. J. Biol. Chem., 272, 21784-21792 (1997) [3] Colby, S.M.; Alonso, W.R.; Katahira, E.J.; McGarvey, D.J.; Croteau, R.: 4Slimonene synthase from the oil glands of spearmint (Mentha spicata): cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase. J. Biol. Chem., 268, 23016-23024 (1993) [4] Yuba, A.; Yazaki, K.; Tabata, M.; Honda, G.; Croteau, R.: cDNA cloning, characterization, ana functional expression of 4S-(-)-limonene synthase from Perilla frutescens. Arch. Biochem. Biophys., 332, 280-287 (1996) [5] Alonso, W.R.; Rajaonarivony, J.I.M.; Gershenzon; J.; Croteau, R.: Purification of 4S-limonene synthase, a monoterpene cyclase from the glandular

270

4.2.3.16

[6]

[7] [8] [9]

[10] [11]

[12]

[13] [14]

[15]

(4S)-Limonene synthase

trichomes pf peppermint (Mentha x piperita) and spearmint (Mentha spicata). J. Biol. Chem., 267, 7582-7587 (1992) Rajaonarivony, J.I.M.; Gershenzon; J.; Croteau, R.: Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita). Arch. Biochem. Biophys., 296, 49-57 (1992) Alonso, W.R.; Crock, J.E.; Cotreau, R.: Production and characterization of polyclonal antibodies in rabbits to 4S-limonene synthase from spearmint (Mentha spicata). Arch. Biochem. Biophys., 301, 58-63 (1993) Adam, K.P.; Crock, J.; Croteau, R.: Partial purification and characterization of a monoterpene cyclase, limonene synthase, from the liverwort Ricciocarpos natans. Arch. Biochem. Biophys., 332, 352-356 (1996) Turner, G.; Gershenzon, J.; Nielson, E.E.; Froehlich, J.E.; Croteau, R.: Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of the oil gland secretory cells. Plant Physiol., 120, 879-886 (1999) Rajaonarivony, J.I.M.; Gershenzon, J.; Miyazaki, J.; Croteau, R.: Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases. Arch. Biochem. Biophys., 299, 77-82 (1992) Schwab, W.; Williams, D.C.; Davis, E.M.; Croteau, R.: Mechanism of monoterpene cyclization: Stereochemical aspects of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase. Arch. Biochem. Biophys., 392, 123-136 (2001) Katoh S, Hyatt D, Croteau R.: Altering product outcome in Abies grandis (-)-limonene synthase and (-)-limonene/(-)-a-pinene synthase by domain swapping and directed mutagenesis. Arch. Biochem. Biophys., 425, 65-76 (2004) Maruyama, T.; Ito, M.; Kiuchi, F.; Honda, G.: Molecular cloning, functional expression and characterization of d-limonene synthase from Schizonepeta tenuifolia. Biol. Pharm. Bull., 24, 373-377. (2001) Schwab W, Williams DC, Croteau R.: Mechanism of monoterpene cyclization: stereochemistry of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase. J. Mol. Catal. B, 19, 415-421 (2002) Martin, D.M.; Faeldt, J.; Bohlmann, J.: Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol., 135, 1908-1927 (2004)

271

Taxadiene synthase

4.2.3.17

1 Nomenclature EC number 4.2.3.17 Systematic name geranylgeranyl-diphosphate diphosphate-lyase (cyclizing, taxa-4,11-dieneforming) Recommended name taxadiene synthase Synonyms geranylgeranyl pyrophosphate cyclase taxa-4(5),11(12)-diene synthase CAS registry number 169277-52-5

2 Source Organism <1> Taxus brevifolia (no sequence specified) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] <2> Taxus canadensis (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction geranylgeranyl diphosphate = taxa-4,11-diene + diphosphate Reaction type cyclization Natural substrates and products S (E,E,E)-geranylgeranyl diphosphate <1> (<1> first committed step of taxol biosynthesis [10]) (Reversibility: ?) [10] P taxa-4(5),11(12)-diene + diphosphate S geranylgeranyl diphosphate <1, 2> (<1,2> catalyzes the first step in taxol biosynthesis [1,2,3,4,5,6,7]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] P taxa-4,11-diene + diphosphate <1, 2> [1, 2, 3, 4, 5, 6, 7, 8] Substrates and products S (E,E,E)-geranylgeranyl diphosphate <1> (<1> first committed step of taxol biosynthesis [10]) (Reversibility: ?) [10] P taxa-4(5),11(12)-diene + diphosphate

272

4.2.3.17

Taxadiene synthase

S (E,E,E)-geranylgeranyl diphosphate <1> (<1> mechanism of cyclization. Incubation of (R)-[4-2H1]geranylgeranyl diphosphate gives a 10:10:80 mixture of [5b-2H1]taxa-3(4),11(12)-diens, [5b-2H1]taxa-4(20),11(12)diene and unlabeled taxa-4(5),11(12)-diene [10]) (Reversibility: ?) [10] P taxa-4,11-diene + diphosphate S (10,11R)-dihydrogeranylgeranyl diphosphate <1> (Reversibility: ?) [11] P (1S,12R)-11,12-dihydroisocembrene + diphosphate S (10,11S)-dihydrogeranylgeranyl diphosphate <1> (Reversibility: ?) [11] P (1S,12S)-11,12-dihydroisocembrene + diphosphate S 11-desmethylgeranylgeranyl diphosphate <1> (Reversibility: ?) [11] P (1S)-12-desmethylisocembrene + diphosphate S (2E,6E,10Z)-geranylgeranyl diphosphate <1> (Reversibility: ?) [11] P (1S,11,12Z)-isocembrene + diphosphate S 7-fluorogeranylgerannyl diphosphate <1> (Reversibility: ?) [9] P (1R,3E,7Z,11R)-7-fluoro-4,8,12,15,15-pentamethyl-bicyclo[9.3.1]pentadeca-3,7,12-triene (<1> i.e. endo-7-fluoroverticillene, 25% yield [9]) S 7-fluorogeranylgerannyl diphosphate <1> (Reversibility: ?) [9] P (4Z,11R)-5-fluoro-4,14,15,15-tetramethyl-8-methylene-bicyclo[9.3.1]pentadeca-4,14-diene (<1> i.e. 4(20)-methylene-7-fluoroverticillene, 26% yield [9]) S 7-fluorogeranylgeranyl diphosphate <1> (<1> verticille-12-yl carbocation intermediate with an 11R stereocenter, the reaction gives three main products and two further isomers [9]) (Reversibility: ?) [9] P (1S,3E,7Z,11R)-7-fluoro-4,8,15,15-tetramethyl-12-methylene-bicyclo[9.3.1]pentadeca-3,7-diene (<1> i.e. exo-7-fluoroverticillene, 37% yield [9]) S cyclopropylidene <1> (Reversibility: ?) [6] P ? S geranylgeranyl diphosphate <1, 2> (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] P taxa-4(5),11(12)-diene + diphosphate <1, 2> [1, 2, 3, 4, 5, 6, 7, 8] S geranylgeranyl diphosphate <1> (<1> taxa-4(20),11(12)-diene is formed as a small amount [6]) (Reversibility: ?) [6] P taxa-4(20),11(12)-diene + diphosphate <1> [6] S geranylgeranyl diphosphate <1, 2> (<1, 2> catalyzes the first step in taxol biosynthesis [1, 2, 3, 4, 5, 6, 7]; <1> isocembrenyl cation is possibly an intermediate in the cyclization of geranylgeranyl diphosphate to taxadiene [11]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 11] P taxa-4,11-diene + diphosphate <1, 2> [1, 2, 3, 4, 5, 6, 7, 8] Metals, ions Mg2+ <2> [1] Turnover number (min–1) 0.00004 <1> (geranylgeranyl diphosphate, <1> M1 preprotein [6]) [6] 0.0000417 <1> (geranylgeranyl diphosphate, <1> thioredoxin fusion [6]) [6] 0.000133 <1> (geranylgeranyl diphosphate, <1> M93 [6]) [6] 0.00017 <1> (geranylgeranyl diphosphate, <1> M79 [6]) [6] 0.000177 <1> (geranylgeranyl diphosphate, <1> native enzyme [6]) [6]

273

Taxadiene synthase

4.2.3.17

Specific activity (U/mg) 0.0003 <2> [1] Km-Value (mM) 0.0026 <2> (geranylgeranyl diphosphate) [1] 0.003 <1> (geranylgeranyl diphosphate, <1> native enzyme [6]) [6] 0.0031 <1> (geranylgeranyl diphosphate, <1> thioredoxin fusion [6]) [6] 0.0105 <1> (geranylgeranyl diphosphate, <1> M1 preprotein [6]) [6] 0.014 <1> (geranylgeranyl diphosphate, <1> M93 [6]) [6] 0.0159 <1> (geranylgeranyl diphosphate, <1> M79 [6]) [6] 0.025 <2> (Mg2+ ) [1] pH-Optimum 8.5 <2> [1]

4 Enzyme Structure Molecular weight 98300 <1> (<1> calculation from amino acid sequence [2]) [2] Subunits ? <2> (<2> 79000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue stem <1> [3] Purification <1> [2, 4, 6] <2> [1] Cloning <1> (expression in Escherichia coli) [2] <1> (overexpression in Escherichia coli) [5, 6, 7] Engineering H579R <1> (<1> accepts farnesyl diphosphate as substrate [8]) [8] Application medicine <1> (<1> chemotherapeutic agent against a range of cancers, including ovarian and breast cancer [1, 2, 3, 4, 5, 6, 7]) [1, 2, 3, 4, 5, 6, 7]

274

4.2.3.17

Taxadiene synthase

References [1] Hezari, M.; Ketchum, R.E.B.; Gibson, D.M.; Croteau, R.: Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures. Arch. Biochem. Biophys., 337, 185-190 (1997) [2] Wildung, M.R.; Croteau, R.: A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis. J. Biol. Chem., 271, 9201-9204 (1996) [3] Koepp, A.E.; Hezari, M.; Zajicek, J.; Stofer Vogel, B.; LaFever, R.; Lewis, N.G.; Croteau, R.: Cyclization of geranylgeranyl diphosphateto taxa-4(5),11(12)-diene is the comitted step of taxol biosynthesis in pacific yew. J. Biol. Chem., 270, 8686-8690 (1995) [4] Lin, X.; Hezari, M.; Koepp, A.E.; Floss, H.G.; Croteau, R.: Mechanism of taxadiene synthase, a diterpene cyclase that catalyzes the first step of taxol biosynthesis in pacific yew. Biochemistry, 35, 2968-2977 (1996) [5] Williams, D.C.; Carroll, B.J.; Jin, Q.; Rithner, C.D.; Lenger, S.R.; Floss, H.G.; Coates, R.M.; Williams, R.M.; Croteau, R.: Intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to the taxadiene precursor of taxol catalyzed by recombinant taxadiene synthase. Chem. Biol., 7, 969-977 (2000) [6] Williams, D.C.; Wildung, M.R.; Jin, A.Q.; Dalal, D.; Oliver, J.S.; Coates, R.M.; Croteau, R.: Heterologous xpression and characterization of a “Pseudomature“ form of taxadiene synthase involved in paclitaxel (taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction. Arch. Biochem. Biophys., 379, 137-146 (2000) [7] Huang, Q.; Roessner, C.A.; Croteau, R.; Scott, A.I.: Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol. Bioorg. Med. Chem., 9, 2237-2242 (2001) [8] Huang, Q.; Williams, H.J.; Roessner, C.A.; Scott, A.I.: Sesquiterpenes produced by truncated taxadiene synthase. Tetrahedron Lett., 41, 9701-9704 (2000) [9] Jin, Y.; Williams, D.C.; Croteau, R.; Coates, R.M.: Taxadiene synthase-catalyzed cyclization of 6-fluorogeranylgeranyl diphosphate to 7-fluoroverticillenes. J. Am. Chem. Soc., 127, 7834-7842 (2005) [10] Jin, Q.; Williams, D.C.; Hezari, M.; Croteau, R.; Coates, R.M.: Stereochemistry of the macrocyclization and elimination steps in taxadiene biosynthesis through deuterium labeling. J. Org. Chem., 70, 4667-4675 (2005) [11] Chow, S.Y.; Williams, H.J.; Huang, Q.; Nanda, S.; Scott, A.I.: Studies on taxadiene synthase: interception of the cyclization cascade at the isocembrene stage with GGPP analogues. J. Org. Chem., 70, 9997-10003 (2005)

275

Abietadiene synthase

4.2.3.18

1 Nomenclature EC number 4.2.3.18 Systematic name (+)-copalyl-diphosphate diphosphate-lyase (cyclizing, (-)-abietadiene-forming) Recommended name abietadiene synthase Synonyms (-)-abietadiene synthase PtTPS-LAS <1> [9] abietadiene cyclase abietadiene/levopimaradiene synthase <1> [9] cyclase, abietadiene CAS registry number 157972-08-2

2 Source Organism <1> Pinus taeda (no sequence specified) [9] <2> Abies grandis (no sequence specified) [1, 2, 3, 4, 5, 6, 7, 8]

3 Reaction and Specificity Catalyzed reaction (+)-copalyl diphosphate = (-)-abietadiene + diphosphate Reaction type cyclization elimination of diphosphate Natural substrates and products S (+)-copalyl-diphosphate <2> (Reversibility: ?) [1, 2, 3, 4, 5, 7, 8] P (-)-abietadiene + diphosphate Substrates and products S (+)-copalyl-diphosphate <2> (Reversibility: ?) [1, 2, 3, 4, 5, 7, 8] P (-)-abietadiene + diphosphate

276

4.2.3.18

Abietadiene synthase

S 8a-hydroxy-17-nor copalyl diphosphate <2> (Reversibility: ?) [1] P 17-normanoyl oxide + diphosphate Inhibitors 14,15-dihydro-15-aza-geranylgeranyl diphosphate <2> [4] 14,15-dihydro-15-azageranylgeranyl diphosphate <2> [3] Geranylgeranyl diphosphate <2> (<2> substrate inhibition [2,3]) [2, 3, 4] norpimarenylamine <2> [5] Metals, ions Mg2+ <2> (<2> 10 mM selectively enables the conversion of copalyl diphosphate [4]) [4] Turnover number (min–1) 0.05 <2> (copalyl diphosphate, <2> mutant E589A [5]) [5] 0.2 <2> (copalyl diphosphate, <2> mutant E778A [5]) [5] 0.4 <2> (copalyl diphosphate, <2> mutant T617A [5]) [5] 0.75 <2> (copalyl diphosphate) [2] 2 <2> (copalyl diphosphate, <2> mutants D361A, D402A, D402E, D404N, D404A, D404E, D404N, D405A, D405E, D405N, D621A, E499A, R365A, R411A, R454A, W358A, Y520A [4]) [4] Km-Value (mM) 0.0002 <2> (copalyl diphosphate, <2> mutants D404N, R411A [4]) [4] 0.0003 <2> (copalyl diphosphate, <2> mutants E773A, E778A [5]; <2> mutants D402A, D402N, D404E [4]) [4, 5] 0.00035 <2> (copalyl diphosphate) [2, 3] 0.0004 <2> (copalyl diphosphate, <2> D404A [3]; <2> wild-type, mutant D766A [5]; <2> mutants W358A, D361A, D404A, D405E [4]) [3, 4, 5] 0.0005 <2> (copalyl diphosphate, <2> mutant N765A [5]; <2> mutants D402E, D405E, R454A, E499A Y520A [4]; <2> mutants rAS:K86A/R87A [7]) [4, 5, 7] 0.0007 <2> (copalyl diphosphate, <2> mutants R762A, E699A, D621A [5]; <2> mutants ASD:107-868 [7]; <2> mutants D405N, R365A [4]) [4, 5, 7] 0.0008 <2> (copalyl diphosphate, <2> mutants T848A, Y841F [5]) [5] 0.0009 <2> (copalyl diphosphate, <2> mutant rAS:D96A [7]) [7] 0.001 <2> (copalyl diphosphate, <2> mutants E589A, R584A [5]) [5] 0.002 <2> (copalyl diphosphate, <2> mutants R586A, T769A [5]) [5] 0.003 <2> (copalyl diphosphate, <2> mutant S721A [5]) [5] 132 <2> (copalyl diphosphate) [3, 4] 138 <2> (copalyl diphosphate, <2> mutant D404A [3]) [3] pH-Optimum 8.7 <2> (<2> with copalyl diphosphate as substrate [4]) [4]

277

Abietadiene synthase

4.2.3.18

4 Enzyme Structure Molecular weight 80000 <2> (<2> gel filtration [2]) [2] 90000 <2> (<2> gel filtration [3]) [3] Subunits monomer <2> (<2> 1 * 80000, SDS-PAGE [2]) [2, 3]

5 Isolation/Preparation/Mutation/Application Localization plastid <1> [9] soluble <2> [2] Purification <2> [2, 8] Cloning <1> (expression of PtTPS-LAS in pET100 vector) [9] <2> (expression in Escherichia coli) [1, 2, 8] Engineering D361A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D402A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D402E <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D402N <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D404A <2> (<2> unreactive with geranylgeranyl diphosphate [3]) [3, 4] D404E <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D404N <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D405A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D405E <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D405N <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] D621A <2> (<2> unreactive with (+)-copalyl diphosphate [3]) [3, 5] D625A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] D766A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5]

278

4.2.3.18

Abietadiene synthase

D845A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] D:107-868 <2> (<2> lower turnover with copalyl diphosphate than wild-type [7]) [7] D:85-849 <2> (<2> no turnover with copalyl diphosphate [7]) [7] E499A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] E589A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] E699A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] E773A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] E778A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] N765A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] R365A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] R411A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] R454A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] R584A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] R586A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] R762A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] S721A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] T617A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] T769A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] T848A <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] W358A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] Y520A <2> (<2> lower turnover with geranylgeranyl diphosphate than wildtype [4]) [4] Y841F <2> (<2> no effect on geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [5]) [5] rAS:D96A <2> (<2> nearly the same turnover with copalyl diphosphate like wild-type [7]) [7] rAS:K86A/R87A <2> (<2> lower turnover with copalyl diphosphate than wild-type [7]) [7]

279

Abietadiene synthase

4.2.3.18

References [1] Ravn, M.M.; Coates, R.M.; Flory, J.E.; Peters, R.J.; Croteau, R.: Stereochemistry of the cyclization-rearrangement of (+)-copalyl diphosphate to (-)-abietadiene catalyzed by recombinant abietadiene synthase from Abies grandis. Org. Lett., 2, 573-576 (2000) [2] Peters, R.J.; Flory, J.E.; Jetter, R.; Ravn, M.M.; Lee, H.J.; Coates, R.M.; Croteau, R.B.: Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the “pseudomature“ recombinant enzyme. Biochemistry, 39, 15592-15602 (2000) [3] Peters, R.J.; Ravn, M.M.; Coates, R.M.; Croteau, R.B.: Bifunctional abietadiene synthase: Free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites. J. Am. Chem. Soc., 123, 8974-8978 (2001) [4] Peters, R.J.; Croteau, R.B.: Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. Biochemistry, 41, 1836-1842 (2002) [5] Peters, R.J.; Croteau, R.B.: Abietadiene synthase catalysis: mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement. Proc. Natl. Acad. Sci. USA, 99, 580-584 (2002) [6] Ravn, M.M.; Peters, R.J.; Coates, R.M.; Croteau, R.: Mechanism of abietadiene synthase catalysis: stereochemistry and stabilization of the cryptic pimarenyl carbocation intermediates. J. Am. Chem. Soc., 124, 6998-7006 (2002) [7] Peters, R.J.; Carter, O.A.; Zhang, Y.; Matthews, B.W.; Croteau, R.B.: Bifunctional abietadiene synthase: mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations. Biochemistry, 42, 2700-2707 (2003) [8] Vogel, B.S.; Wildung, M.R.; Vogel, G.; Croteau, R.: Abietadiene synthase from grand fir (Abies grandis). cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase involved in resin acid biosynthesis. J. Biol. Chem., 271, 23262-23268 (1996) [9] Ro, D.K.; Bohlmann, J.: Diterpene resin acid biosynthesis in loblolly pine (Pinus taeda): Functional characterization of abietadiene/levopimaradiene synthase (PtTPS-LAS) cDNA and subcellular targeting of PtTPS-LAS and abietadienol/abietadienal oxidase (PtAO, CYP720B1). Phytochemistry, 67, 1572-1578 (2006)

280

ent-Kaurene synthase

4.2.3.19

1 Nomenclature EC number 4.2.3.19 Systematic name ent-copalyl-diphosphate diphosphate-lyase (cyclizing; ent-kaurene-forming) Recommended name ent-kaurene synthase Synonyms KS <3, 5> [14, 15] ent-kaurene synthase B ent-kaurene synthetase B Additional information <1, 2, 4, 6, 7, 8, 9, 10, 11, 12> (<1, 2, 4, 6, 7, 8, 9, 10, 11, 12> part of bifunctional enzyme catalyzing the two step reaction from trans-geranylgeranyl-diphosphate to ent-kaurene, first step is catalyzed by EC 5.5.1.13 (ent-kaurene synthase A) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] CAS registry number 9055-64-5

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11> <12>

Triticum aestivum (no sequence specified) [9, 12] Pisum sativum (no sequence specified) [9, 12] Arabidopsis thaliana (no sequence specified) [15] Helianthus annuus (no sequence specified) [7, 8] Oryza sativa (no sequence specified) [14] Cucurbita maxima (no sequence specified) [2,9,10,11] Fusarium moniliforme (no sequence specified) [1] Gibberella fujikuroi (no sequence specified) [11] Marah macrocarpus (no sequence specified) [5,6,7,8] Phaeosphaeria sp. (UNIPROT accession number: O13284) [3] Gibberella fujikuroi (UNIPROT accession number: Q9ZWQ2) [4] Phaeosphaeria sp. (no sequence specified) [13]

281

ent-Kaurene synthase

4.2.3.19

3 Reaction and Specificity Catalyzed reaction ent-copalyl diphosphate = ent-kaurene + diphosphate Reaction type cyclization diphosphate lysis Natural substrates and products S ent-copalyl diphosphate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12> (<1, 2, 4, 6, 7, 8, 9, 10, 11, 12> involved in biosynthesis of gibberellins [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13]; <3> second step in plant hormone gibberellin biosynthesis, regulation of the gibberellin biosynthesis, the KS is not rate-determining, overview [15]; <5> the enzyme is involved in gibberellin metabolism and biosynthesis of diterpene phytoalexins, overview [14]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15] P ent-kaurene + diphosphate <1, 2, 4, 6, 7, 8, 9, 10, 11, 12> (<2> additionally an unidentified acid labile compound is formed that is not found in products from pea and pumpkin [9]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] Substrates and products S ent-copalyl diphosphate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12> (<1, 2, 4, 6, 7, 8, 9, 10, 11, 12> involved in biosynthesis of gibberellins [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13]; <3> second step in plant hormone gibberellin biosynthesis, regulation of the gibberellin biosynthesis, the KS is not rate-determining, overview [15]; <5> the enzyme is involved in gibberellin metabolism and biosynthesis of diterpene phytoalexins, overview [14]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15] P ent-kaurene + diphosphate <1, 2, 4, 6, 7, 8, 9, 10, 11, 12> (<2> additionally an unidentified acid labile compound is formed that is not found in products from pea and pumpkin [9]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] Inhibitors 2’-isopropyl-4’(trimethylammonium chloride)-5’-methylphenyl piperidine-1carboxylate <9, 10> (<9> strong inhibition of EC 5.5.1.13 activity, very weak inhibition of EC 4.2.3.19 activity [5]; <9,10> Amo-1618 [3,5]) [3, 5] 2-(N,N-dimethyl-N-octylammonium bromide)-p-methan-1-ol <7, 9> (<7> Q-64 [1]; <9> strong inhibition of EC 5.5.1.13 activity, very weak inhibition of EC 4.2.3.19 activity [5]) [1, 5] Ba2+ <6> [10] Ca2+ <6> [10] Cu2+ <6, 7> [1, 10] EDTA <7, 9, 10> [1, 3, 5] Hg2+ <7> [1] N,N-dimethylaminoethyl 2,2-diphenylphenylether <7, 9> (<9> strong inhibition of EC 5.5.1.13 activity, very weak inhibition of EC 4.2.3.19 activity [5]; <7> SKF 3301A [1]) [1, 5]

282

4.2.3.19

ent-Kaurene synthase

N-ethylmaleimide <7, 9> [1, 5] phosphone D <7, 9> (<9> strong inhibition of EC 5.5.1.13 activity, very weak inhibition of EC 4.2.3.19 activity [5]) [1, 5] phosphone S <7, 9> (<9> strong inhibition of EC 5.5.1.13 activity, very weak inhibition of EC 4.2.3.19 activity [5]) [1, 5] sulfhydryl groups <7> [1] copalyl diphosphate <7, 9> (<7> slight substrate inhibition at concentrations above 0.0007 mM [1]; <9> substrate inhibition above 0.0002 mM, might be an effect of contaminants in substrate preparations [5]) [1, 5] deoxycholate <7> [1] iodacetamide <7> [1] p-hydroxymercuribenzoate <7, 9> [1, 5] Additional information <8> (<8> wide variety of quaternary ammonium compounds with structural similarities to geranylgeranyl diphosphate tested for inhibition, substances shows significant effects on AB activity, but low effects on B activity [11]) [11] Activating compounds 2-mercaptoethanol <7> [1] cysteine <7> [1] dithiothreitol <7, 9> (<7> stimulates activity in presence of EDTA [1]; <9> 0.001-10 mM, eliminated in presence of 0.1 mM EDTA [5]) [1, 5] Additional information <5> (<5> expression of KS4, KS7, and KS8 is induced by elicitor treatment [14]) [14] Metals, ions Ca2+ <7, 9> (<7,9> no effect [1,5]) [1, 5] Co2+ <6, 7, 9> (<6,7,9> can partially replace Mg2+ [1,5,10]) [1, 5, 10] Mg2+ <6, 7, 9, 10> (<6,10> required [3,10]; <7> required, optimum concentration 5 mM [1]) [1, 3, 5, 10] Mn2+ <6, 7, 9> (<6,7,9> can partially replace Mg2+ [1,5,10]) [1, 5, 10] Ni2+ <7, 9> (<7> can partially replace Mg2+ [1]; <9> very low activity [5]) [1, 5] Specific activity (U/mg) 0.005 <9> (<9> partially purified enzyme [5]) [5] 0.01 <6> (<6> purified enzyme [10]) [10] 0.123 <7> (<7> purified enzyme [1]) [1] Additional information <3> (<3> activity in wild-type and transgenic KSoverexpressing plants [15]) [15] Km-Value (mM) 0.0004 <6> (copalyl diphosphate) [10] 0.0006 <9> (copalyl diphosphate) [5] 0.001 <7> (copalyl diphosphate) [1] pH-Optimum 6.2-7.2 <9> (<9> TES buffer [5]) [5] 6.8-7.5 <6> [10]

283

ent-Kaurene synthase

4.2.3.19

6.9 <7, 9> (<9> imidazole buffer [5]; <7> TES or HEPES buffer [1]) [1, 5] 7.2-8.2 <9> (<9> Tris buffer [5]) [5] 7.5 <7> (<7> phosphate buffer [1]) [1]

4 Enzyme Structure Molecular weight 45000 <9> (<9> gel filtration, molecular weight found to be less than 45000 Da [5]) [5] 81000 <6, 9> (<9> sucrose density gradient centrifugation [6]; <6> SDSPAGE, purified enzyme [10]; <6> SDS-PAGE, native enzyme [2]) [2, 6, 10] 83000 <9> (<9> gel filtration [6]) [6] 89000 <6> (<6> molecular weight of native enzyme predicted from DNA sequence [2]) [2] 90000 <6> (<6> gel filtration [10]) [10] 107000 <11> (<11> predicted from cDNA sequence [4]) [4] 130000 <6> (<6> SDS-PAGE, cloned fusion protein [2]) [2] 131000 <6> (<6> molecular weight of cloned fusion protein predicted from DNA sequence [2]) [2] 132000 <10> (<10> SDS-PAGE, cloned fusion protein [3]) [3] 430000 <7> (<7> sucrose density gradient centrifugation, crude enzyme [1]) [1] 450000 <7> (<7> sucrose density gradient centrifugation, purified enzyme [1]) [1] 490000 <7> (<7> gel filtration [1]) [1] Subunits tetramer <11> (<11> 4 * 107000 [4]) [4]

5 Isolation/Preparation/Mutation/Application Source/tissue node <1> [12] root <1, 4, 9> (<1,4,9> perennial: very low activity [8,12]) [8, 12] scutellum <1> (<1> highest activity 3 days after imbibition [12]) [12] seed <6, 9> [2, 5, 6, 8, 10] seedling <1, 4, 5> (<4> no activity in fresh crude extracts from seedlings for ent-kaurene formation from geranyl-geranyl diphosphate, activity detected after some days of storage in liquid nitrogen, little effect of liquid nitrogen storage on EC 4.2.3.19 activity [7]; <1> shoots of seedlings [9]; <4> cotyledons contain over 90% of activity [8]) [7, 8, 9, 14] shoot base <1> (<1> highest activity 4 days after imbibition [12]) [12] Additional information <1> (<1> no activity in internodes and leaves [12]) [12]

284

4.2.3.19

ent-Kaurene synthase

Localization chloroplast <2> (<2> low activity [12]) [12] plastid <1, 2, 5, 6> (<1> stroma [12]; <1> probably in stroma, no activity in leaf chloroplasts [9]) [2, 9, 12, 14] Purification <6> (cloned enzyme from Escherichia coli and native enzyme from endosperm) [2] <6> (separation of activities A and B achieved by hydrophobic interaction chromatography) [10] <7> (partial, copurified with ent-kaurene synthase A (EC 5.5.1.13) activity) [1] <9> (partial) [5, 6] <12> (from Escherichia coli) [13] Cloning <3> (overexpression of CPS and KS in transgenic Arabidopsis thaliana plants, expression in Escherichia coli) [15] <5> (DNA and amino acid sequence determination of genes KS1-9, chromosomal localizations, gene KS9 is a pseudogene, phylogenetic analysis, overview) [14] <6> (expressed in Escherichia coli as fusion protein with maltose-binding protein, enzyme exclusively shows EC 4.2.3.19 activity) [2] <10> (expressed in Escherichia coli JM109 as glutathione-S-transferase fusion protein, enzyme shows EC 4.2.3.19 and EC 5.5.1.13 activity) [3] <11> (expressed in Escherichia coli JM109, enzyme shows EC 4.2.3.19 and EC 5.5.1.13 activity) [4] <12> (in Escherichia coli as glutathione-S-transferase fusion protein) [13] Engineering D132A <12> (<12> low activity [13]) [13] D320A <12> (<12> low activity [13]) [13] D656A <12> (<12> no activity [13]) [13] Additional information <3, 5, 12> (<12> several N- and C-terminal truncated enzymes produced, all found to be inactive [13]; <3> construction of transgenic Arabidopsis thaliana plants, using the Agrobacterium tumefaciens infection system, overexpressing KS leads to increased ent-kaurene production but not to an increase in bioactive gibberellins, no altered morphology or phenotype compared to wild-type plants [15]; <5> construction of transgenic rice mutants expressing mutated KS-like genes, the mutant plants show reduced enzyme activity, phenotype analysis and effects on gibberellin metabolism, overview [14]) [13, 14, 15]

6 Stability General stability information <4>, no activity in fresh crude extracts from seedlings for ent-kaurene formation from geranyl-geranyl diphosphate, activity detected after some days

285

ent-Kaurene synthase

4.2.3.19

of storage in liquid nitrogen, little effect of liquid nitrogen storage on EC 4.2.3.19 activity [7] <7>, sensitive on freezing and thawing [1] Storage stability <4>, -180 C, no activity in fresh crude extracts from seedlings for ent-kaurene formation from geranyl-geranyl diphosphate, activity detected after some days of storage in liquid nitrogen, little effect of liquid nitrogen storage on EC 4.2.3.19 activity [7] <7>, -20 C, 25% glycerol, several months, no loss of activity for purified enzyme [1]

References [1] Fall, R.R.; West, C.A.: Purification and properties of kaurene synthetase from Fusarium moniliforme. J. Biol. Chem., 246, 6913-6928 (1971) [2] Yamaguchi, S.; Saito, T.; Abe, H.; Yamane, H.; Murofushi, N.; Kamiya, Y.: Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.). Plant J., 10, 203-213 (1996) [3] Kawaide, H.; Imai, R.; Sassa, T.; Kamiya, Y.: ent-Kaurene synthetase from the fungus Phaeosphaeria sp. L487. cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase in fungal gibberellin biosynthesis. J. Biol. Chem., 272, 21706-21712 (1997) [4] Toyomasu, T.; Kawaide, H.; Ishizaki, A.; Shinoda, S.; Otsuka, M.; Mitsuhashi, W.; Sassa, T.: Cloning of a full-length cDNA encoding ent-kaurene synthase from Gibberella fujikuroi: functional analysis of a bifunctional diterpene cyclase. Biosci. Biotechnol. Biochem., 64, 660-664 (2000) [5] Frost, R.G.; West, C.A.: Properties of kaurene synthetase from Marah macrocarpus. Plant Physiol., 59, 22-29 (1977) [6] Duncan, J.D.; West, C.A.: Properties of kaurene synthetase from Marah macrocarpus endosperm: evidence for the participation of separate but interacting enzymes. Plant Physiol., 68, 1128-1134 (1981) [7] Shen-Miller, J.; West, C.A.: Kaurene synthetase activity in Helianthus annuus. Increases in enzyme activity after storage of seedlings in liquid nitrogen. Plant Physiol., 74, 439-441 (1984) [8] Shen-Miller, J.; West, C.A.: Distribution and ent-kaurene synthetase in Helianthus annuus and Marah macrocarpus. Phytochemistry, 24, 461-464 (1985) [9] Aach, H.; Boese, G.; Graebe, J.E.: ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localization of ent-kaurene synthetase in plastids of three species. Planta, 197, 333-342 (1995) [10] Saito, T.; Abe, H.; Yamane, H.; Sakurai, A.; Murofushi, N.; Takio, K.; Takahashi, N.; Kamiya, Y.: Purification and properties of ent-kaurene synthase B from immature seeds of pumpkin. Plant Physiol., 109, 1239-1245 (1995)

286

4.2.3.19

ent-Kaurene synthase

[11] Saito, T.; Yamane, H.; Sakurai, A.; Murofushi, N.; Takahashi, N.; Kamiya, Y.: Inhibition of ent-kaurene synthase by quaternary ammonium growth retardants. Biosci. Biotechnol. Biochem., 60, 1040-1042 (1996) [12] Aach, H.; Bode, H.; Robinson, D.G.; Graebe, J.E.: ent-Kaurene synthase is located in proplastids of meristematic shoot tissues. Planta, 202, 211-219 (1997) [13] Kawaide, H.; Sassa, T.; Kamiya, Y.: Functional analysis of the two interacting cyclase domains in ent-kaurene synthase from the fungus Phaeosphaeria sp. L487 and a comparison with cyclases from higher plants. J. Biol. Chem., 275, 2276-2280 (2000) [14] Sakamoto, T.; Miura, K.; Itoh, H.; Tatsumi, T.; Ueguchi-Tanaka, M.; Ishiyama, K.; Kobayashi, M.; Agrawal, G.K.; Takeda, S.; Abe, K.; Miyao, A.; Hirochika, H.; Kitano, H.; Ashikari, M.; Matsuoka, M.: An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiol., 134, 1642-1653 (2004) [15] Fleet, C.M.; Yamaguchi, S.; Hanada, A.; Kawaide, H.; David, C.J.; Kamiya, Y.; Sun, T.P.: Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins. Plant Physiol., 132, 830-839 (2003)

287

(R)-Limonene synthase

4.2.3.20

1 Nomenclature EC number 4.2.3.20 Systematic name geranyldiphosphate diphosphate lyase [(+)-(R)-limonene-forming] Recommended name (R)-limonene synthase Synonyms (+)-limonene synthase CitMTSE1 <1> [7] Cl(+)LIMS1 <2> [5] Cl(+)LIMS2 d-limonene synthase <1> [7] dLMS CAS registry number 155807-65-1

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8>

Citrus unshiu (no sequence specified) [7] Citrus limon (no sequence specified) [5] Perilla frutescens (no sequence specified) [6] Carum carvi (no sequence specified) [1] Citrus limon (UNIPROT accession number: Q8L5K3) [2] Schizonepeta tenuifolia (UNIPROT accession number: Q9FUW5) [3] Citrus limon (UNIPROT accession number: Q8L5K1) [2] Agastache rugosa (UNIPROT accession number: Q940E7) [4]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate = (+)-(4R)-limonene + diphosphate (<5> requires a divalent metal ion (preferably Mn2+ ) for catalysis [2]; <4> forms the first step of carvone biosynthesis in caraway [1])

288

4.2.3.20

(R)-Limonene synthase

Reaction type cyclization Natural substrates and products S geranyl diphosphate <4, 5, 6> (Reversibility: ?) [1, 2, 3] P (+)-(R)-limonene + diphosphate Substrates and products S geranyl diphosphate <4, 5, 6, 7> (Reversibility: ?) [1, 2, 3] P (+)-(R)-limonene + diphosphate <4, 5, 7> (<5,7> product is exclusively R-(+)-limonene [2]; <4> high stereoselectivity, 98.4% of (+)-product [1]) [1, 2] S geranyl diphosphate <1> (Reversibility: ?) [7] P (4R)-limonene + diphosphate Inhibitors geranyl diphosphate <5, 7> (<5,7> substrate inhibition above 0.01 mM [2]) [2] K+ <5, 7> (<5,7> maximum inhibition above 100 mM [2]) [2] Mn2+ <5, 7> (<5,7> above 0.6 mM, activation below [2]) [2] Activating compounds Mn2+ <5, 7> (<5,7> inhibition above 0.6 mM, activation below [2]) [2] Metals, ions Mg2+ <1> (<1> Mn2+ or Mg2+ required [7]) [7] Mn2+ <1> (<1> Mn2+ or Mg2+ required [7]) [7] Additional information <4, 5, 6, 7, 8> (<4,5,7> divalent metal ion required, probably Mn2+ [1,2]; <6,8> DDxxD motif, binding site for substrate-divalent metal cation complex [3,4]) [1, 2, 3, 4] Km-Value (mM) 0.0007 <5, 7> (geranyl diphosphate) [2] pH-Optimum 7 <5, 7> [2] 7.5 <4> [1] 7.6 <6> (<6> assay at [3]) [3] Temperature optimum ( C) 31 <6> (<6> assay at [3]) [3]

4 Enzyme Structure Subunits ? <1, 6, 8> (<8> x * 60000, SDS-PAGE [4]; <6> x * 62000, SDS-PAGE, x * 64000, deduced from gene sequence [3]; <1> 1 * 70000, recombinant GST fusion protein, SDS-PAGE [7]) [3, 4, 7]

289

(R)-Limonene synthase

4.2.3.20

5 Isolation/Preparation/Mutation/Application Source/tissue flower <1> (<1> powerfully expressed at anthesis [7]) [7] fruit <5> (<5> peel of young developing fruit [2]) [2] Localization plastid <3> [6] soluble <4> [1] Cloning <1> (functional expression of four cDNA clones in Escherichia coli) [7] <2> (limonene cyclase enzyme Cl(+)LIMS2 produced exclusively (+)-limonene as the major product of the enzymatic catalysis. In a chimeric enzyme, substitution of ClgTS domain III and domain IV by their counterparts from Cl(+)LIMS2 changed product specificity to that of Cl(+)LIMS2, (+)-limonene being the major product formed) [5] <3> (introduction of limonene synthase cDNA with three different sorting signals (to localize either in cytosol or the endoplasmic reticulum) in Nicotiana tabacum. Full-length and modified enzyme are subcloned in a binary vector under the El2 promoter, which is a strong constitutive promoter, to yield pBin-FullLC1, pBin-DEltaLC1 and pBin.ERLC1 to plastidial, cytosolic and endoplasmic localization, respectively. These plasmids are introduced via Agrobacterium tumefaciens into tobacco. More than 10 transgenic tobacco plants for each construct, i.e. 15 plastid localization, 17 cytosol localization, 11 endoplasmic reticulum localization are established) [6] <5> [2] <6> [3]

References [1] Bouwmeester, H.J.; Gershenzon, J.; Konings, M.C.J.M.; Croteau, R.: Biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway. I. Demonstration of enzyme activities and their changes with development. Plant Physiol., 117, 901-912 (1998) [2] Lucker, J.; El Tamer, M.K.; Schwab, W.; Verstappen, F.W.; van der Plas, L.H.; Bouwmeester, H.J.; Verhoeven, H.A.: Monoterpene biosynthesis in lemon (Citrus limon). cDNA isolation and functional analysis of four monoterpene synthases. Eur. J. Biochem., 269, 3160-3171 (2000) [3] Maruyama, T.; Ito, M.; Kiuchi, F.; Honda, G.: Molecular cloning, functional expression and characterization of d-limonene synthase from Schizonepeta tenuifolia. Biol. Pharm. Bull., 24, 373-377 (2001) [4] Maruyama, T.; Saeki, D.; Ito, M.; Honda, G.: Molecular cloning, functional expression and characterization of d-limonene synthase from Agastache rugosa. Biol. Pharm. Bull., 25, 661-665 (2002)

290

4.2.3.20

(R)-Limonene synthase

[5] El Tamer, M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; van Tunen, A.J.; Voragen, A.G.J.; de Maagd, R.A.; Bouwmeester, H.J.: Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specificity. Arch. Biochem. Biophys., 411, 196-203 (2003) [6] Ohara, K.; Ujihara, T.; Endo, T.; Sato, F.; Yazaki, K.: Limonene production in tobacco with Perilla limonene synthase cDNA. J. Exp. Bot., 54, 2635-2642 (2003) [7] Shimada, T.; Endo, T.; Fujii, H.; Hara, M.; Ueda, T.; Kita, M.; Omura, M.: Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu Marc. Plant Sci., 166, 49-58 (2004)

291

Vetispiradiene synthase

4.2.3.21

1 Nomenclature EC number 4.2.3.21 Systematic name trans,trans-farnesyl-diphosphate diphosphate-lyase (cyclizing, vetispiradiene-forming) Recommended name vetispiradiene synthase Synonyms HVS pemnaspirodiene synthase synthase, vetispiradiene vetispiradiene cyclase vetispiradiene-forming farnesyl pyrophosphate cyclase CAS registry number 192465-18-2

2 Source Organism <1> Nicotiana tabacum (no sequence specified) [2] <2> Hyoscyamus muticus (no sequence specified) [1, 3, 5] <3> Solanum tuberosum (UNIPROT accession number: Q9XIZ0) [4]

3 Reaction and Specificity Catalyzed reaction trans,trans-farnesyl diphosphate = vetispiradiene + diphosphate Reaction type internal cyclization Natural substrates and products S trans,trans-farnesyl diphosphate <3> (<3> infection of Phytophthora infestans with potato tubers causes transient increase in the transcript level of vetispiradiene synthase. Wound-induced expression of the squalene

292

4.2.3.21

Vetispiradiene synthase

synthase is suppressed in favor of the expression of vetispiradiene synthase [4]) (Reversibility: ?) [4] P vetispiradiene + diphosphate Substrates and products S trans,trans-farnesyl diphosphate <1, 2, 3> (<2> mechanism involves an initial rapid equilibrium of enzyme with substrate to form an enzymesubstrate complex, followed by a slower conversion of farnesyl diphosphate to an enzyme-bound hydrocarbon, and a subsequent rate-limiting step [3]; <3> infection of Phytophthora infestans with potato tubers causes transient increase in the transcript level of vetispiradiene synthase. Wound-induced expression of the squalene synthase is suppressed in favor of the expression of vetispiradiene synthase [4]) (Reversibility: ?) [1, 2, 3, 4] P vetispiradiene + diphosphate Turnover number (min–1) 0.04 <2> (trans,trans-farnesyl diphosphate, <2> recombinant enzyme [3]) [3] Km-Value (mM) 0.002-0.005 <2> (trans,trans-farnesyl diphosphate, <2> native enzyme [3]) [3] 0.0035 <2> (trans,trans-farnesyl diphosphate, <2> recombinant enzyme [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [2] tuber <3> (<3> infected with Phytophtora infestans [4]) [4] Purification <2> (recombinant vetispiradiene synthase, a chimeric 5-epi-aristolochene synthase and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity) [3] Cloning <2> (expression in Escherichia coli. Highest level of sesquiterpene production occurs when the enzymes are expressed in strain DH5a from the trc promoter of the high-copy plasmid pTrc99A in M9 medium supplemented with 0.2% v/v glycerol at 30 C) [5] <2> (expression of 3 cDNA clones in Escherichia coli) [1] <2> (hexahistidyl-tagged enzyme expressed in Escherichia coli, genes for vetispiradiene synthase, a chimeric 5-epi-aristolochene synthase and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity) [3]

293

Vetispiradiene synthase

4.2.3.21

References [1] Back, K.; Chappell, J.: Cloning and bacterial expression of a sesquiterpene cyclase from Hyoscyamus muticus and its molecular comparison to related terpene cyclases. J. Biol. Chem., 270, 7375-7381 (1995) [2] Keller, H.; Czernic, P.; Ponchet, M.; Ducrot, P.H.; Back, K.; Chappell, J.; Ricci, P.; Marco, Y.: Sesquiterpene cyclase is not a determining factor for elicitorand pathogen-induced capsidiol accumulation in tobacco. Planta, 205, 467476 (1998) [3] Mathis, J.R.; Back, K.; Starks, C.; Noel, J.; Poulter, C.D.; Chappell, J.: Pre-steady-state study of recombinant sesquiterpene cyclases. Biochemistry, 36, 8340-8348 (1997) [4] Yoshioka, H.; Yamada, N.; Doke, N.: cDNA cloning of sesquiterpene cyclase and squalene synthase, and expression of the genes in potato tuber infected with Phytophthora infestans. Plant Cell Physiol., 40, 993-998 (1999) [5] Martin, V.J.; Yoshikuni, Y.; Keasling, J.D.: The in vivo synthesis of plant sesquiterpenes by Escherichia coli. Biotechnol. Bioeng., 75, 497-503 (2001)

294

Germacradienol synthase

4.2.3.22

1 Nomenclature EC number 4.2.3.22 Systematic name 2-trans,6-trans-farnesyl-diphosphate diphosphate-lyase [(1E,4S,5E,7R)-germacra-1(10),5-dien-11-ol-forming] Recommended name germacradienol synthase Synonyms (+)-germacrene D synthase <4, 6> [1, 2] (-)-germacrene D synthase <3, 4, 5, 7> [1, 2, 4, 5] GerD <7> [4] TPS <7> [4] TPS1 <3> [5] germacradienol/germacrene D synthase <2> [3] sesquiterpene synthase <1, 2> [6, 7] sesquiterpene synthase 1 <3> [5] terpene cyclase <2> [7] terpenoid cyclase <7> [4] CAS registry number 211049-88-6

2 Source Organism <1> <2> <3> <4> <5> <6> <7>

Streptomyces coelicolor (no sequence specified) [6] Streptomyces coelicolor A3(2) (no sequence specified) [3, 7] Populus trichocarpa x Populus deltoides (no sequence specified) [5] Solidago canadensis (no sequence specified) [2] Solidago canadensis (UNIPROT accession number: Q70EZ7) [1] Solidago canadensis (UNIPROT accession number: Q70EZ6) [1] Vitis vinifera (UNIPROT accession number: Q6Q3H3) [4]

295

Germacradienol synthase

4.2.3.22

3 Reaction and Specificity Catalyzed reaction 2-trans,6-trans-farnesyl diphosphate + H2 O = (1E,4S,5E,7R)-germacra1(10),5-dien-11-ol + diphosphate (<2> reaction mechanism [7]; <2> catalytic mechanism via intermediate is (7R)-germacra-1(10),4-diene-11-ol [3]) 2-trans,6-trans-farnesyl diphosphate = (-)-(7S)-germacrene D + diphosphate (<4> enantioselectivity and catalytic mechanisms of (-)-germacrene D synthase and (+)-germacrene D synthase [2]; <5,6> enantioselectivity and catalytic mechanisms of (-)-germacrene D synthase and (+)-germacrene D synthase and molecular modeling [1]) Natural substrates and products S 2-trans,6-trans-farnesyl diphosphate <2, 3, 4, 5, 7> (<3> (-)-germacrene D is synthesized by leaves after attack of forest tent caterpillars, Malacosoma disstria, as a volatile phytoalexin, together with others substances, reaction is a step in terpenoid biosynthesis [5]; <4> strictly enantio-specific reaction of (-)-germacrene D synthase [2]) (Reversibility: ?) [1, 2, 3, 4, 5] P (-)-(7S)-germacrene D + diphosphate (<2> GC-MS product analysis [3]) S 2-trans,6-trans-farnesyl diphosphate <4, 6> (<4> strictly enantio-specific reaction of (+)-germacrene D synthase [2]) (Reversibility: ?) [1, 2] P (+)-(7S)-germacrene D + diphosphate S 2-trans,6-trans-farnesyl diphosphate + H2 O <1, 2> (<1> enzyme is essential for geosmin biosynthesis [6]; <2> enzyme is involved in geosmin biosynthesis [7]) (Reversibility: ?) [6, 7] P (1E,4S,5E,7R)-germacra-1(10),5-dien-11-ol + diphosphate S 2-trans,6-trans-farnesyl diphosphate + H2 O <2> (<2> key step in biosynthesis of geosmin [3]) (Reversibility: ?) [3] P (4S,7R)-germacra-1(10)E,5E-dien-11-ol + diphosphate S Additional information <7> (<7> cell terpenoid profile determination [4]) (Reversibility: ?) [4] P ? Substrates and products S 2-trans,6-trans-farnesyl diphosphate <2, 3, 4, 5, 7> (<3> (-)-germacrene D is synthesized by leaves after attack of forest tent caterpillars, Malacosoma disstria, as a volatile phytoalexin, together with others substances, reaction is a step in terpenoid biosynthesis [5]; <4, 5, 7> strictly enantiospecific reaction of (-)-germacrene D synthase [1, 2, 4]; <3> enantio-specific [5]; <2, 4> strictly enantio-specific reaction of (-)-germacrene D synthase, mechanism [2,3]) (Reversibility: ?) [1, 2, 3, 4, 5] P (-)-(7S)-germacrene D + diphosphate (<2, 3, 4, 7> GC-MS product analysis [2,3,4,5]) S 2-trans,6-trans-farnesyl diphosphate <4, 6> (<4,6> strictly enantio-specific reaction of (+)-germacrene D synthase [1,2]; <4> strictly enantio-spe-

296

4.2.3.22

P S

P S

P S

P

Germacradienol synthase

cific reaction of (+)-germacrene D synthase, mechanism [2]) (Reversibility: ?) [1, 2] (+)-(7S)-germacrene D + diphosphate (<4> GC-MS product analysis [2]) 2-trans,6-trans-farnesyl diphosphate + H2 O <1, 2> (<1> enzyme is essential for geosmin biosynthesis [6]; <2> enzyme is involved in geosmin biosynthesis [7]; <2> cyclization reaction, mechanism of full length enzyme and N-terminal catalytic domain [7]; <1> reaction is catalyzed by one of two sesquiterpene domains of the enzyme [6]) (Reversibility: ?) [6, 7] (1E,4S,5E,7R)-germacra-1(10),5-dien-11-ol + diphosphate 2-trans,6-trans-farnesyl diphosphate + H2 O <2> (<2> key step in biosynthesis of geosmin [3]; <2> cyclization reaction, detailed mechanism, an intermediate is (7R)-germacra-1(10),4-diene-11-ol [3]) (Reversibility: ?) [3] (4S,7R)-germacra-1(10)E,5E-dien-11-ol + diphosphate (<2> GC-MS and NMR product configuration analysis [3]) Additional information <2, 7> (<7> cell terpenoid profile determination [4]; <2> no activity with geranylgeranyl diphosphate [7]; <2> stereospecificity, the 1,3-hydride shift of the (-)-germacrene D synthase of Streptomyces coelicolor A3(2) is opposite to the enzyme from Solidago canadensis [3]) (Reversibility: ?) [3, 4, 7] ?

Inhibitors EDTA <2> [7] Activating compounds Additional information <3> (<3> mechanical wounding specifically activates enzyme expression in a diurnal rhythm, spatial expression pattern analysis, overview [5]) [5] Metals, ions Co2+ <2> (<2> can substitute for Mg2+ [7]) [7] Cu2+ <2> (<2> can substitute for Mg2+ [7]) [7] Fe2+ <2> (<2> can substitute for Mg2+ [7]) [7] Fe3+ <2> (<2> can substitute for Mg2+ [7]) [7] Mg2+ <2, 4, 5, 6, 7> (<4> anhydrous MgSO4 is used in the assay [2]; <2> required, preferred divalent cation [7]) [1, 2, 4, 7] Mn2+ <2> (<2> can substitute for Mg2+ [7]) [7] Ni2+ <2> (<2> can substitute for Mg2+ [7]) [7] Zn2+ <2> (<2> can substitute for Mg2+ [7]) [7] Additional information <2> (<2> enzyme activity is dependent on divalent cations [7]) [7] Turnover number (min–1) 0.02 <5> (2-trans,6-trans-farnesyl diphosphate, <5> 25 C, recombinant (-)-germacrene D synthase [1]) [1] 0.03 <6> (2-trans,6-trans-farnesyl diphosphate, <6> 25 C, recombinant (+)-germacrene D synthase [1]) [1]

297

Germacradienol synthase

4.2.3.22

3.2 <2> (2-trans,6-trans-farnesyl diphosphate, <2> pH 8.2, 30 C, recombinant N-terminal domain [7]) [7] 6.2 <2> (2-trans,6-trans-farnesyl diphosphate, <2> pH 8.2, 30 C, recombinant enzyme [7]) [7] Km-Value (mM) 0.000062 <2> (2-trans,6-trans-farnesyl diphosphate, <2> pH 8.2, 30 C, recombinant enzyme [7]) [7] 0.000115 <2> (2-trans,6-trans-farnesyl diphosphate, <2> pH 8.2, 30 C, recombinant N-terminal domain [7]) [7] 0.0029 <6> (2-trans,6-trans-farnesyl diphosphate, <6> 25 C, recombinant (+)-germacrene D synthase [1]) [1] 0.003 <5> (2-trans,6-trans-farnesyl diphosphate, <5> 25 C, recombinant (-)-germacrene D synthase [1]) [1] Additional information <5, 6> (<6> kinetic analysis, recombinant (+)-germacrene D synthase, i.e. clone Sc19 [1]; <5> kinetic analysis, recombinant (-)-germacrene D synthase, i.e.clone Sc11 [1]) [1] pH-Optimum 7 <7> (<7> assay at [4]) [4] 8.2 <2> (<2> assay at [7]) [7] pH-Range 5.5-9.5 <2> [7] Temperature optimum ( C) 25 <5, 6> (<5,6> assay at [1]) [1] 30 <2, 4, 7> (<2,4,7> assay at [2,4,7]) [2, 4, 7]

4 Enzyme Structure Subunits ? <2, 3> (<2> x * 40000, recombinant enzyme, SDS-PAGE [7]; <3> x * 65000, about, recombinant enzyme, SDS-PAGE [5]) [5, 7] Additional information <5, 6> (<6> enantiospecific (+)-germacrene D synthases reveals a functionally active variant of the universal isoprenoidbiosynthesis aspartate-rich motif involving residues H406, G444, N448, and E520, molecular modeling [1]; <5> enantiospecific (-)-germacrene D synthases reveals a functionally active variant of the universal isoprenoidbiosynthesis aspartate-rich motif involving residues Y406, S444, D448, and I520, molecular modeling [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue flower <7> [4] flower bud <7> [4]

298

4.2.3.22

Germacradienol synthase

fruit <7> (<7> young [4]) [4] leaf <3, 4, 5, 6> (<5,6> young [1]) [1, 2, 5] Additional information <3> (<3> acropetal, but not basipedal, expression after induction by wounding [5]) [5] Purification <2> (recombinant enzyme from Escherichia coli strain BL21(DE3), soluble enzyme and enzyme solubilized and refolded from inclusion bodies, by ion exchange chromatography and gel filtration) [7] <4> (partially from leaves by ion exchange chromatography) [2] <5> (recombinant N-terminally His-tagged, thioredoxin-fusion (-)-germacrene D synthase from Escherichia coli strain AD494(DE3)) [1] <6> (recombinant N-terminally His-tagged, thioredoxin-fusion (+)-germacrene D synthase from Escherichia coli strain AD494(DE3)) [1] Renaturation <2> (solubilization of recombinant enzyme from inclusion bodies by 0.02% Triton X-100 and 100 mM NaOH, and refolding) [7] Cloning <1> (construction of an ordered library of Supercos-1 clones, DNA sequence determination and analysis, subcloning in Escherichia coli) [6] <2> (expression in Escherichia coli strain BL21(DE3)) [7] <3> (DNA and amino acid sequence determination and analysis, phylogenetic tree, functional expression in Escherichia coli) [5] <5> (genetic library construction, DNA sequence determination and analysis, expression of N-terminally His-tagged, thioredoxin-fusion (-)-germacrene D synthase in Escherichia coli strain AD494(DE3)) [1] <6> (genetic library construction, DNA sequence determination and analysis, expression of N-terminally His-tagged, thioredoxin-fusion (+)-germacrene D synthase in Escherichia coli strain AD494(DE3)) [1] <7> (DNA and amino acid sequence determination and analysis of Vitis vinifera cv. Gewrztraminer, phylogenetic tree, expression in Escherichia coli strain BL21(DE3)-RIL) [4] Engineering Additional information <1, 2> (<1> construction of a cyc2 disruption mutant inactive in geosmin biosynthesis [6]; <2> expression of isolated N-terminal and C-terminal domains reveals that the N-terminal domain is responsible for the catalytic activity, while the C-terminal domain is barely active [7]) [6, 7]

References [1] Prosser, I.; Altug, I.G.; Phillips, A.L.; Koenig, W.A.; Bouwmeester, H.J.; Beale, M.H.: Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-

299

Germacradienol synthase

4.2.3.22

biosynthesis aspartate-rich motif. Arch. Biochem. Biophys., 432, 136-144 (2004) [2] Schmidt, C.O.; Bouwmeester, H.J.; Franke, S.; Konig, W.A.: Mechanisms of the biosynthesis of sesquiterpene enantiomers (+)- and (-)-germacrene D in Solidago canadensis. Chirality, 11, 353-362 (1999) [3] He, X.; Cane, D.E.: Mechanism and stereochemistry of the germacradienol/ germacrene D synthase of Streptomyces coelicolor A3(2). J. Am. Chem. Soc., 126, 2678-2679 (2004) [4] Luecker, J.; Bowen, P.; Bohlmann, J.: Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of monoand sesquiterpene synthases in grapevine flowers and berries. Phytochemistry, 65, 2649-2659 (2004) [5] Arimura, G.-i.; Huber, D.P.W.; Bohlmann, J.: Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid volatiles in hybrid poplar (Populus trichocarpa x deltoides): cDNA cloning, functional characterization, and patterns of gene expression of (-)-germacrene D synthase, PtdTPS1. Plant J., 37, 603-616 (2004) [6] Gust, B.; Challis, G.L.; Fowler, K.; Kieser, T.; Chater, K.F.: PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. USA, 100, 1541-1546 (2003) [7] Cane, D.E.; Watt, R.M.: Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis. Proc. Natl. Acad. Sci. USA, 100, 1547-1551 (2003)

300

Germacrene-A synthase

4.2.3.23

1 Nomenclature EC number 4.2.3.23 Systematic name 2-trans,6-trans-farnesyl-diphosphate diphosphate-lyase [(+)-(R)-germacreneA-forming] Recommended name germacrene-A synthase Synonyms (+)-(10R)-germacrene A synthase <6> [4] (+)-germacrene A synthase <1, 4, 5> [2, 3, 5] GAS <4, 5> [2, 3] GASlo <2> [6] GASsh <3> [6] Sc1 <6> [4] germacrene A synthase <2, 3> [6] guaiadiene synthase <5> [2] sesquiterpene cyclase <1> [5] sesquiterpene synthase <6> [4] CAS registry number 213763-55-4

2 Source Organism <1> <2> <3> <4> <5> <6> <7>

Cichorium intybus (no sequence specified) [5] Cichorium intybus (UNIPROT accession number: Q8LSC3) [6] Cichorium intybus (UNIPROT accession number: Q8LSC2) [6] Ixeris dentata (no sequence specified) [3] Ixeris dentata (UNIPROT accession number: Q6YN71) [2] Solidago canadensis (UNIPROT accession number: Q9AR67) [4] Artemisia annua (UNIPROT accession number: Q1PDD2) [1]

301

Germacrene-A synthase

4.2.3.23

3 Reaction and Specificity Catalyzed reaction 2-trans,6-trans-farnesyl diphosphate = (+)-(10R)-germacrene A + diphosphate (<2, 3> structure modeling and reaction mechanism, overview [6]; <4> the catalytic site is located at the N-terminal 44 amino acid residues [3]) Natural substrates and products S 2-trans,6-trans-farnesyl diphosphate <1, 2, 3, 4, 5, 6, 7> (<1> the product germacrene A is released after reaction to be further processing by oxidations and/or glucosylations and cyclization by a germacrene cyclase [5]) (Reversibility: ?) [1, 2, 3, 4, 5, 6] P (+)-(10R)-germacrene A + diphosphate (<6> 98% of total product is (+)-(10R)-germacrene A, 2% is a-humulene in feeding experiments in vivo [4]) S Additional information <1, 2, 3, 4, 6, 7> (<7> (+)-germacrene A synthesis is a step in isoprenoid biosynthesis, overview [1]; <1,2,3,4> (+)-germacrene A synthesis is the committed step in the biosynthesis of bitter sesquiterpene lactones in chicory [3,5,6]; <6> sesquiterpene hydrocarbon profile of leaf extracts, overview [4]) (Reversibility: ?) [1, 3, 4, 5, 6] P ? Substrates and products S 2-trans,6-trans-farnesyl diphosphate <1, 2, 3, 4, 5, 6, 7> (<1> the product germacerene A is released after reaction to be further processing by oxidations and/or glucosylations and cyclization by a germacrane cyclase [5]) (Reversibility: ?) [1, 2, 3, 4, 5, 6] P (+)-(10R)-germacrene A + diphosphate (<6> 98% of total product is (+)-(10R)-germacrene A, 2% is a-humulene in feeding experiments in vivo [4]; <5,7> GC-MS product analysis [1,2]; <6> chiral GC-MS product analysis [4]; <4> reaction product analysis by GC-MS and NMR [3]; <1> reaction product solubilization and analysis in pentane, determination of germacrene A configuration [5]) S Additional information <1, 2, 3, 4, 6, 7> (<7> (+)-germacrene A synthesis is a step in isoprenoid biosynthesis, overview [1]; <1,2,3,4> (+)-germacrene A synthesis is the committed step in the biosynthesis of bitter sesquiterpene lactones in chicory [3,5,6]; <6> sesquiterpene hydrocarbon profile of leaf extracts, overview [4]; <4> no activity with geranyl diphosphate [3]) (Reversibility: ?) [1, 3, 4, 5, 6] P ? Inhibitors EDTA <5> [2] Activating compounds Tween 20 <1> (<1> at 0.1%, activates the enzyme by 5fold [5]) [5]

302

4.2.3.23

Germacrene-A synthase

Metals, ions Mg2+ <1, 5, 6, 7> (<1> required [5]) [1, 2, 4, 5] Turnover number (min–1) 0.00035 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant mutant T433A [2]) [2] 0.00037 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant mutant T433A/A435T [2]) [2] 0.016 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant wild-type enzyme [2]) [2] 0.017 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant mutant S434A [2]) [2] 0.044 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant mutant A435T [2]) [2] Specific activity (U/mg) 6.24 <1> (<1> purified enzyme [5]) [5] Km-Value (mM) 0.0025 <6> (2-trans,6-trans-farnesyl diphosphate, <6> pH 7.0, 25 C, recombinant enzyme [4]) [4] 0.0032 <3> (2-trans,6-trans-farnesyl diphosphate, <3> pH 7.0, 30 C [6]) [6] 0.0047 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant mutant T433A [2]) [2] 0.0066 <1> (2-trans-6-trans-farnesyl diphosphate, <1> pH 7.0, 30 C [5]) [5] 0.0069 <2> (2-trans-6-trans-farnesyl diphosphate, <2> pH 7.0, 30 C [6]) [6] 0.0076 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant mutant T433A/A435T [2]) [2] 0.0106 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant mutant S434A [2]) [2] 0.011 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant wild-type enzyme [2]) [2] 0.0251 <5> (2-trans,6-trans-farnesyl diphosphate, <5> pH 7.0, 25 C, recombinant mutant A435T [2]) [2] 11 <4> (2-trans,6-trans-farnesyl diphosphate, <4> pH 7.5-8.0, 25 C [3]) [3] 14.9 <4> (2-trans,6-trans-farnesyl diphosphate, <4> pH 7.5-8.0, 37 C [3]) [3] Additional information <2, 3> [6] pH-Optimum 6.7 <1> (<1> about, broad optimum [5]) [5] 6.8 <2> [6] 7 <3, 5, 6, 7> (<5,6,7> assay at [1,2,4]) [1, 2, 4, 6] 7.5-8 <4> (<4> recombinant enzyme [3]) [3] pH-Range 5.1-7.3 <1> (<1> half maximal activities at pH 5.1 and pH 7.3 [5]) [5]

303

Germacrene-A synthase

4.2.3.23

Temperature optimum ( C) 25 <5, 6> (<5,6> assay at [2,4]) [2, 4] 30 <1, 7> (<1,7> assay at [1,5]) [1, 5] 37 <4> (<4> recombinant enzyme [3]) [3]

4 Enzyme Structure Molecular weight 54000 <1> (<1> gel filtration [5]) [5] Subunits ? <4, 6> (<6> x * 64700, about, DNA sequence calculation [4]; <4> x * 67100, DNA sequence calculation [3]) [3, 4] monomer <1> (<1> 1 * 56000, about, SDS-PAGE [5]) [5] Additional information <2, 3, 6> (<2,3> isozyme primary sequence and three-dimensional structure modeling [6]; <6> molecular modeling, structure analysis, enzyme contains the conserved motif DDXXD/E [4]) [4, 6]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1, 2, 4, 6, 7> (<4> expression levels decline near the onset of flowering [3]; <2> head leaves, very low expression in green leaves [6]) [1, 3, 4, 5, 6] root <1, 4> (<1> high enzyme activity [5]) [3, 5] seedling <2, 3> (<2,3> normal and etiolated [6]) [6] shoot <2> (<2> head core tissue [6]) [6] taproot <2, 3> (<2> especially in outer tissue, highest expression level of the long isozyme [6]; <3> highest expression level of the short isozyme in outer and inner tissues [6]) [6] trichome <7> [1] Additional information <2, 3> (<2> both isozymes are expressed in all tissues but in varying levels, overview [6]; <3> nearly no expression of the short isozyme in head leaves and in green leaves, both isozymes are expressed in all tissues but in varying levels, overview [6]) [6] Localization cytosol <1> [5] Purification <1> (201fold from roots by anion exchange and dye-ligand affinity chromatography) [5] <2> (recombinant long isozyme from Escherichia coli, native isozyme partially from chicory heads by anion exchange chromatography) [6] <3> (recombinant short isozyme from Escherichia coli, native isozyme partially from chicory heads by anion exchange chromatography) [6] <4> (partially, recombinant enzyme) [3]

304

4.2.3.23

Germacrene-A synthase

<5> (recombinant wild-type and mutant enzymes from bacteria) [2] <6> (recombinant thioredoxin fusion enzyme from Escherichia coli by nickel chelate affinity chromatography) [4] Cloning <2> (DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in Escherichia coli) [6] <3> (DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in Escherichia coli) [6] <4> (DNA sequence determination and analysis) [3] <5> (expression of wild-type and mutant enzymes in bacteria) [2] <6> (DNA library construction, phage display, DNA and amino acid sequence determination and analysis, functional expression in Escherichia coli as N-terminal thioredoxin fusion protein) [4] <7> (cloning from a constructed glandular trichome cDNA library, DNA and amino acid sequence determination and analysis, phylogenetic analysis, functional expression in Escherichia coli strain BL21-DE3-RIL) [1] Engineering A435T <5> (<5> site-directed mutagenesis, altered kinetics but similar activity compared to the wild-type enzyme [2]) [2] S434A <5> (<5> site-directed mutagenesis, activity and kinetics similar to the wild-type enzyme [2]) [2] S434T/A435T <5> (<5> site-directed mutagenesis, inactive mutant [2]) [2] T433A <5> (<5> site-directed mutagenesis, altered kinetics and reduced activity compared to the wild-type enzyme [2]) [2] T433A/A435T <5> (<5> site-directed mutagenesis, altered kinetics and reduced activity compared to the wild-type enzyme [2]) [2] T433A/S434A <5> (<5> site-directed mutagenesis, inactive mutant [2]) [2] T433A/S434A/A435T <5> (<5> site-directed mutagenesis, inactive mutant [2]) [2] T433A/S434T <5> (<5> site-directed mutagenesis, inactive mutant [2]) [2] Additional information <4> (<4> deletion of the N-terminal 44 amino acid residues leads to loss of 90% of catalytic activity [3]) [3]

6 Stability General stability information <6>, glycerol stabilizes the enzyme [4] Storage stability <1>, -80 C, partially purified root enzyme, in 15 mM MOPS, pH 7.0, 10 mM MgCl2 , 2 mM DTT, 30% v/v glycerol, stable for several months [5]

305

Germacrene-A synthase

4.2.3.23

References [1] Bertea, C.M.; Voster, A.; Verstappen, F.W.; Maffei, M.; Beekwilder, J.; Bouwmeester, H.J.: Isoprenoid biosynthesis in Artemisia annua: cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library. Arch. Biochem. Biophys., 448, 3-12 (2006) [2] Chang, Y.J.; Jin, J.; Nam, H.Y.; Kim, S.U.: Point mutation of (+)-germacrene A synthase from Ixeris dentata. Biotechnol. Lett., 27, 285-288 (2005) [3] Kim, M.-Y.; Chang, Y.-J.; Bang, M.-H.; Baek, N.-I.; Jin, J.; Lee, C.-H.; Kim, S.U.: cDNA isolation and characterization of (+)-germacrene A synthase from Ixeris dentata form. albiflora Hara. J. Plant Biol., 48, 178-186 (2005) [4] Prosser, I.; Phillips, A.L.; Gittings, S.; Lewis, M.J.; Hooper, A.M.; Pickett, J.A.; Beale, M.H.: (+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis. Phytochemistry, 60, 691-702 (2002) [5] de Kraker, J.W.; Franssen, M.C.; de Groot, A.; Konig, W.A.; Bouwmeester, H.J.: (+)-Germacrene A biosynthesis. The committed step in the biosynthesis of bitter sesquiterpene lactones in chicory. Plant Physiol., 117, 1381-1392 (1998) [6] Bouwmeester, H.J.; Kodde, J.; Verstappen, F.W.; Altug, I.G.; de Kraker, J.W.; Wallaart, T.E.: Isolation and characterization of two germacrene A synthase cDNA clones from chicory. Plant Physiol., 129, 134-144 (2002)

306

Amorpha-4,11-diene synthase

4.2.3.24

1 Nomenclature EC number 4.2.3.24 Systematic name 2-trans,6-trans-farnesyl-diphosphate diphosphate-lyase (amorpha-4,11-dieneforming) Recommended name amorpha-4,11-diene synthase Synonyms ADS <1> [5] CAS registry number 259213-60-0

2 Source Organism <1> Artemisia annua (no sequence specified) (<1> BCA2 [2, 4, 6]) [1, 2, 3, 4, 5, 6, 7] <2> Artemisia annua (UNIPROT accession number: Q9AR04) [8]

3 Reaction and Specificity Catalyzed reaction 2-trans,6-trans-farnesyl diphosphate = amorpha-4,11-diene + diphosphate (<1> enzyme first catalyzes a 1,6 ring closure with a subsequent 1,10 closure [1]; <1> mechanism includes a bisabolyl carbocation and 1,3-hydride shift, detailed analysis [5]; <1> mechanism involves isomerzation of farnesyl diphosphate to (R)-nerolidyl diphosphate, ionization, and C-1,C-6-ring closure to generate a bisabolyl cation, followed by a 1,3-hydride shift, 1,10-ring closure, and deprotonation at either C-12 or C-13 [4]) Natural substrates and products S Additional information <2> (<2> key enzyme in the biosynthetic pathway of the antimalarial drug artemisinin [8]) (Reversibility: ?) [8] P ?

307

Amorpha-4,11-diene synthase

4.2.3.24

Substrates and products S farnesyl diphosphate <1, 2> (Reversibility: ?) [1, 2, 4, 7, 8] P amorpha-4,7-diene + diphosphate (<1> configuration is (1S,6R,7R,10R)amorpha-4,11-diene [4]; <2> major product, minor product is beat-sesquiphellandrene [8]; <1> more than 90% of product, minor products are amorpha-4,7(11)-diene, g-humulene, amorpha-4-en-7-ol and others [1]) S farnesyl diphosphate + H2 O <1> (Reversibility: ?) [3] P amorpha-4,11-diene + diphosphate (<1> additionally production of 15 different sesquiterpenoids, detailed reaction scheme [3]) S geranyl diphosphate + H2 O <1> (<1> substrate in the presence of Mn2+ but not with Mg2+ or Co2+ [3]) (Reversibility: ?) [3] P amorpha-4,11-diene + diphosphate S Additional information <1, 2> (<2> key enzyme in the biosynthetic pathway of the antimalarial drug artemisinin [8]; <1> H-1si-proton of substrate is transferred during the cyclization reaction to carbon 10 of amorphadiene, as a result of a 1,3-hydride-shift follwing initial 1,6 ring closure, while the H-1re-proton of substrate is retained on C-6 of the product [4]; <1> no substrate: geranyl diphosphate [1]; <1> significant increased product selectivity in the presence of Mn2+ or Co2+ [3]) (Reversibility: ?) [1, 3, 4, 8] P ? Inhibitors Co2+ <1> (<1> inhibitory above 0.5 mM [3]) [3] Cu2+ <1> (<1> complete inhibition [3]) [3] Mn2+ <1> (<1> inhibitory above 0.5 mM [3]) [3] Ni2+ <1> (<1> inhibition [3]) [3] Zn2+ <1> (<1> complete inhibition [3]) [3] Metals, ions Co2+ <1> (<1> increase in product selectivity at pH 6.5, inhibitory above 0.5 mM [3]) [3] Mg2+ <1> (<1> Km -value 0.07 mM [1]) [1, 3] Mn2+ <1> (<1> increase in product selectivity at pH 6.5, inhibitory above 0.5 mM [3]; <1> Km -value 0.013 mM [1]) [1, 3] Specific activity (U/mg) 3.53 <1> (<1> pH 7.5, 35 C, recombinant enzyme [2]) [2] Km-Value (mM) 0.0006 <1> (farnesyl diphosphate, <1> pH 7.0 [7]) [7] 0.0009 <1> (<1> pH 7.5 [1]) [1] 0.6 <1> (farnesyl diphosphate, <1> presence of Co2+, pH 7.5, 30 C [3]) [3] 0.7 <1> (farnesyl diphosphate, <1> presence of Co2+, pH 6.5, 30 C [3]) [3] 1.6 <1> (farnesyl diphosphate, <1> presence of Mg2+ , pH 9.5, 30 C [3]) [3] 2 <1> (farnesyl diphosphate, <1> presence of Mg2+ , pH 7.5, 30 C [3]) [3] 3.3 <1> (farnesyl diphosphate, <1> presence of Mg2+ , pH 6.5, 30 C [3]) [3] 4.2 <1> (farnesyl diphosphate, <1> presence of Mn2+ , pH 7.5, 30 C [3]) [3]

308

4.2.3.24

Amorpha-4,11-diene synthase

8 <1> (farnesyl diphosphate, <1> presence of Mn2+ , pH 6.5, 30 C [3]) [3] 16.9 <1> (geranyl diphosphate, <1> presence of Mn2+ , pH 6.5, 30 C [3]) [3] 28.2 <1> (geranyl diphosphate, <1> presence of Mn2+ , pH 7.5, 30 C [3]) [3] pH-Optimum 6.5 <1> [3] 6.5-7 <1> (<1> broad [7]) [7] 7.5-9 <1> (<1> broad [1]) [1] pH-Range 7.5 <1> (<1> pH-minimum, increasing enzyme activity with increasing pH above 7.5 [3]) [3]

4 Enzyme Structure Molecular weight 56000 <1> (<1> gel filtration [7]) [7] Subunits ? <1, 2> (<1> x * 61000, SDS-PAGE of recombinant protein with His6-tag [5]; <1,2> x * 63900, calculated [1,8]; <1> x * 63900, calculated, x * 63500, SDSPAGE [2]; <1> x * 65000, SDS-PAGE, recombinant enzyme with His-tag [3]) [1, 2, 3, 5, 8] Posttranslational modification Additional information <1> (<1> sequence contains no plastidial targeting sequence [2]) [2]

5 Isolation/Preparation/Mutation/Application Purification <1> (partial) [7] <1> (recombinant enzyme with his-tag) [3] Cloning <1> [1, 2, 3] Application synthesis <1, 2> (<1> expression of enzyme plus mevalonate isoprenoid pathway genes from Saccharomyces cerevisiae in Escherichia coli results in synthesis of amorpha-4,11-diene up to 0.024 mg caryophyllene per ml [6]; <2> production of amorpha-4,11-diene, expression in Nicotiana tabacum gives 0.2 to 1.7 ng per g fresh weight [8]) [6, 8]

309

Amorpha-4,11-diene synthase

4.2.3.24

References [1] Mercke, P.; Bengtsson, M.; Bouwmeester, H.J.; Posthumus, M.A.; Brodelius, P.E.: Molecular cloning, expression, and characterization of amorpha-4,11diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L.. Arch. Biochem. Biophys., 381, 173-180 (2000) [2] Chang, Y.-J.; Song, S.-H.; Park, S.-H.; Kim, S.-U.: Amorpha-4,11-diene synthase of Artemisia annua: cDNA isolation and bacterial expression of a terpene synthase involved in artemisinin biosynthesis. Arch. Biochem. Biophys., 383, 178-184 (2000) [3] Picaud, S.; Olofsson, L.; Brodelius, M.; Brodelius, P.E.: Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L. Arch. Biochem. Biophys., 436, 215-226 (2005) [4] Picaud, S.; Mercke, P.; He, X.; Sterner, O.; Brodelius, M.; Cane, D.E.; Brodelius, P.E.: Amorpha-4,11-diene synthase: mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate. Arch. Biochem. Biophys., 448, 150-155 (2006) [5] Kim, S.H.; Heo, K.; Chang, Y.J.; Park, S.H.; Rhee, S.K.; Kim, S.U.: Cyclization mechanism of amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis. J. Nat. Prod., 69, 758-762 (2006) [6] Martin, V.J.; Pitera, D.J.; Withers, S.T.; Newman, J.D.; Keasling, J.D.: Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol., 21, 796-802 (2003) [7] Bouwmeester, H.J.; Wallaart, T.E.; Janssen, M.H.A.; Van Loo, B.; Jansen, B.J.M.; Posthumus, M.A.; Schmidt, C.O.; De Kraker, J.-W.; Konig, W.A.; Franssen, M.C.R.: Amorpha-4,11-diene synthase catalyzes the first probable step in artemisinin biosynthesis. Phytochemistry, 52, 843-854 (1999) [8] Wallaart, T.E.; Bouwmeester, H.J.; Hille, J.; Poppinga, L.; Maijers, N.C.A.: Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta, 212, 460-465 (2001)

310

S-Linalool synthase

4.2.3.25

1 Nomenclature EC number 4.2.3.25 Systematic name geranyl-diphosphate diphosphate-lyase [(3S)-linalool-forming] Recommended name S-linalool synthase Synonyms LIS <1, 2, 4, 6> [1, 2, 3, 4, 5, 6, 8] LIS1 <2, 5> [2] LIS2 <2> [2] S-LIS <1> [8] linalool synthase <2> [5, 6] Additional information <2, 5, 6> (<2,5,6> the enzyme belongs to the family of isoprenoid synthases [2]) [2] CAS registry number 160477-81-6

2 Source Organism <1> <2> <3> <4> <5> <6> <7>

Vitis vinifera (no sequence specified) [8] Clarkia breweri (no sequence specified) [1, 2, 4, 5, 6] Cereus peruvianus (no sequence specified) [7] Clarkia breweri (UNIPROT accession number: Q96376) [3] Clarkia concinna (no sequence specified) [2] Oenothera arizonica (no sequence specified) [2] no activity in Petunia hybrida [4]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate + H2 O = (3S)-linalool + diphosphate (<2> reaction mechanism [1])

311

S-Linalool synthase

4.2.3.25

Natural substrates and products S geranyl diphosphate + H2 O <1, 2, 3, 4, 6> (<3> enantio-specific reaction, the enzyme plays a pivotal role in production of flavor and aroma of ripe cactus fruits [7]; <1> enantio-specific reaction, the enzyme plays a pivotal role in production of flavor of ripe grape berries, production of free and bound S-linalool [8]; <2> recombinant enzyme in transgenic Petunia hybrida plants [4]; <2> wild-type enzyme in Clarkia breweri and recombinant enzyme in transgenic Lycopersicon esculentum plants, the enzyme is involved in the terpenoid pathway, overview [6]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] P (3S)-linalool + diphosphate (<1,2,3> product identification [4,7,8]) S Additional information <5> (<5> Clarkia concinna is a non-scented species but does express linalool synthase at a low level [2]) (Reversibility: ?) [2] P ? Substrates and products S (S)-linalyl diphosphate + H2 O <2> (<2> low activity, 15% of the maximal activity with geranyl diphosphate [1]) (Reversibility: ?) [1] P (3S)-linalool + diphosphate S geranyl diphosphate + H2 O <1, 2, 3, 4, 6> (<3> enantio-specific reaction, the enzyme plays a pivotal role in production of flavor and aroma of ripe cactus fruits [7]; <1> enantio-specific reaction, the enzyme plays a pivotal role in production of flavor of ripe grape berries, production of free and bound S-linalool [8]; <2> recombinant enzyme in transgenic Petunia hybrida plants [4]; <2> wild-type enzyme in Clarkia breweri and recombinant enzyme in transgenic Lycopersicon esculentum plants, the enzyme is involved in the terpenoid pathway, overview [6]; <1,2> enantio-specific reaction [6,8]; <3> enantio-specific reaction, 85% optical puritiy of S-linalool [7]; <2> enantiospecific reaction [1]; <2> recombinant enzyme from transgenic Petunia hybrida plants [4]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] P (3S)-linalool + diphosphate (<1,2,3> product identification [1,4,7,8]) S Additional information <2, 3, 5> (<5> Clarkia concinna is a non-scented species but does express linalool synthase at a low level [2]; <2> no activity with (R)-linalyl diphosphate [1]; <3> spontaneous enzyme-independent formation of racemic linalool from geranyl diphosphate in presence of Fe2+ [7]) (Reversibility: ?) [1, 2, 7] P ? Inhibitors SDS <2> (<2> leads to complete loss of activity at about 0.1% [1]) [1] Additional information <2> (<2> no effects by 0.1% w/v Triton X-100 [1]) [1] Activating compounds DTT <2, 3> (<2> required for stability [1]) [1, 4, 7]

312

4.2.3.25

S-Linalool synthase

Metals, ions Mg2+ <2, 3> (<2> half as effective as Mn2+ [1]) [1, 4, 5, 7] Mn2+ <2, 3> (<3> preferred cation [7]; <2> absolutely required for activity, preferred divalent cation [1]) [1, 4, 5, 7] Additional information <2, 3> (<3> divalent cation is required for activity, no activity with Cu2+ , Co2+ , and Ca2+ , in presence of Fe2+ spontaneous enzyme-independent formation of racemic linalool from geranyl diphosphate occurs [7]; <2> no activity with Cu2+ , Zn2+ , Ca2+ , Co2+ [1]) [1, 7] Specific activity (U/mg) 0.0004 <2> (<2> purified enzyme [1]) [1] Additional information <2> (<2> microgramm emitted linalool per g fresh weight [5]) [5] Km-Value (mM) 0.0009 <2> (geranyl diphosphate, <2> pH 7.4, 20 C [1]) [1] 0.0056 <3> (Mn2+ , <3> pH 6.6, 30 C [7]) [7] 0.018 <3> (geranyl diphosphate, <3> pH 6.6, 30 C [7]) [7] 0.045 <2> (Mn2+ , <2> pH 7.4, 20 C [1]) [1] 0.06 <3> (Mg2+ , <3> pH 6.6, 30 C [7]) [7] 0.33 <2> (Mg2+ , <2> pH 7.4, 20 C [1]) [1] pH-Optimum 6.6 <3> (<3> 6.9 <3> (<3> 7.2 <2> (<2> 7.4 <2> (<2> 7.8 <2> (<2>

small scale assay at [7]) [7] large scale assay at [7]) [7] assay at, recombinant enzyme [4]) [4] broad optimum [1]) [1] assay at [5]) [5]

pH-Range 6.4-8.4 <2> (<2> 80% of maximal activity within this range [1]) [1] Temperature optimum ( C) 20 <2> (<2> assay at [1]) [1] 30 <2, 3> (<2> assay at, recombinant enzyme [4]; <3> small scale assay at [7]) [4, 7] 34 <3> (<3> large scale assay at [7]) [7]

4 Enzyme Structure Molecular weight 53000 <3> (<3> gel filtration [7]) [7] 73000 <2> (<2> gel filtration [1]) [1] Subunits monomer <2> (<2> 1 * 79000, SDS-PAGE [1]) [1] Additional information <4, 5> (<4,5> the enzyme contains the conserved DDXXD motif [2,3]) [2, 3]

313

S-Linalool synthase

4.2.3.25

5 Isolation/Preparation/Mutation/Application Source/tissue anther <4> (<4> expression in flower buds, not in open flowers [3]) [3] exocarp <1> (<1> activity occurs during ripening [8]) [8] filament <4> (<4> expression in days 1-3 of open flowers [3]) [3] flower <2, 4> (<2> monoterpenes are produced in unopened buds and in open flowers with a peak at 2-3 days of anthesis [5]) [3, 5] flower bud <4> (<4> expression mainly in epidermis [3]) [3] fruit <1, 3> (<1> exocarp, activity occurs during ripening [8]; <3> ripening, very low levels in green immature fruits, enzyme levels increase during fruit ripening [7]) [7, 8] leaf <1> (<1> adult [8]) [8] petal <2, 4> (<2> high enzyme activity, linalool is emitted [5]; <2> highest expression level of LIS2 [2]; <4> of flower buds and open flowers, expression mainly in epidermis [3]) [2, 3, 5] pistil <2> (<2> high enzyme activity, linalool is mostly converted to linalool oxide [5]) [5] sepal <2> (<2> very low expression level of LIS1 [2]) [2] stamen <2, 4> (<2> high enzyme activity, linalool is mostly converted to linalool oxide [5]) [2, 3, 5] stigma <2, 4, 5> (<5> low enzyme expression [2]; <2> highest expression level of LIS1 [2]; <4> of open flowers, high expression level in the secretory zone, between the papillate epidermis and parenchyma cells, converging into the style as a central region of transmitting tissue [3]) [1, 2, 3] style <2, 4> (<4> high expression level, in the transmitting tissue but not in the vascular system or the stylar epidermis [3]) [2, 3] Additional information <1, 2, 4> (<2> floral tissue-specific expression levels of LIS1 and LIS2, overview, no expression in vegetative tissues [2]; <1> no activity determinable in cultured cell [8]; <2> the monoterpene is emitted soon after its synthesis [5]; <4> tissue specific expression dependent on developmental stage of flowers, no activity in leaves, no expression in sepals [3]) [2, 3, 5, 8] Purification <2> (native enzyme 26fold from stigmata of freshly opened flowers by anion exchange and hydroxyapatite chromatography to over 95% purity) [1] <3> (partially from fruits by anion exchange chromatography) [7] Cloning <2> (cloning in a binary vector for introduction into Agrobacterium tumefaciens strain LBA4404) [6] <2> (functional expression in transgenic Petunia hybrida plants) [4] <2> (gene LIS2, partial genomic library construction, DNA sequence determination and analysis, promoter sequence analysis, gene structure) [2] <4> (partial protein sequencing and cDNA library construction, DNA and amino acid determination and analysis) [3]

314

4.2.3.25

S-Linalool synthase

<5> (partial genomic library construction, DNA sequence determination and analysis, promoter sequence analysis, gene structure) [2] <6> (partial genomic library construction, DNA sequence determination and analysis, gene structure) [2] Engineering Additional information <2> (<2> construction of transgenic Petunia hybrida plants from wild-type W115 expressing Clarkia breweri LIS in all tissues with enantio-specific formation of (S)-linalool, which is converted to non-volatile b-d-glucopyranoside by an endogenous glucosyltransferase, in leaves, sepals, corolla, stem, and ovary, but not in nectaris, roots, pollen, and style, plants show also increased a-terpineol levels [4]; <2> construction of transgenic tomato plants using the Agrobacterium tumefaciens transfection system, enzyme expression under control of a tomato-late-ripening-specific E8 promoter leads to accumulation of S-linalool and 8-hydroxylinalool in ripening fruits, volatile profiles of wild-type Lycopersicon esculentum plants and transgenic plants expressing LIS [6]) [4, 6] Application biotechnology <2> (<2> the enzyme can be used to modify the flavor/nuritional value of vegetables, e.g. tomato fruits, by enzyme expression in transgenic plants [6]) [6] nutrition <2> (<2> the enzyme can be used to modify the flavor/nuritional value of vegetables, e.g. tomato fruits, by enzyme expression in transgenic plants [6]) [6]

6 Stability General stability information <2>, DTT is required for enzyme stability, in absence of DTT the purified enzyme loses 95% of its activity within 2 h at 4 C [1] <2>, hexane and pentane stabilize the enzyme [1]

References [1] Pichersky, E.; Lewinsohn, E.; Croteau, R.: Purification and characterization of S-linalool synthase, an enzyme involved in the production of floral scent in Clarkia breweri. Arch. Biochem. Biophys., 316, 803-807 (1995) [2] Cseke, L.; Dudareva, N.; Pichersky, E.: Structure and evolution of linalool synthase. Mol. Biol. Evol., 15, 1491-1498 (1998) [3] Dudareva, N.; Cseke, L.; Blanc, V.M.; Pichersky, E.: Evolution of floral scent in Clarkia: novel patterns of S-linalool synthase gene expression in the C. breweri flower. Plant Cell, 8, 1137-1148 (1996) [4] Luecker, J.; Bouwmeester, H.J.; Schwab, W.; Blaas, J.; van der Plas, L.H.; Verhoeven, H.A.: Expression of Clarkia S-linalool synthase in transgenic petunia

315

S-Linalool synthase

4.2.3.25

plants results in the accumulation of S-linalyl-b-d-glucopyranoside. Plant J., 27, 315-324 (2001) [5] Pichersky, E.; Raguso, R.A.; Lewinsohn, E.; Croteau, R.: Floral scent production in Clarkia (Onagraceae). I. Localization and developmental modulation of monoterpene emission and linalool synthase activity.55. Plant Physiol., 106, 1533-1540 (1994) [6] Lewinsohn, E.; Schalechet, F.; Wilkinson, J.; Matsui, K.; Tadmor, Y.; Nam, K.H.; Amar, O.; Lastochkin, E.; Larkov, O.; Ravid, U.; Hiatt, W.; Gepstein, S.; Pichersky, E.: Enhanced levels of the aroma and flavor compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol., 127, 1256-1265 (2001) [7] Sitrit, Y.; Ninio, R.; Bar, E.; Golan, E.; Larkov, O.; Ravid, U.; Lewinsohn, E.: SLinalool synthase activity in developing fruit of the columnar cactus koubo [Cereus peruvianus (L.) Miller]. Plant Sci., 167, 1257-1262 (2004) [8] de Billerbeck, G.M.; Cozzolino, F.; Ambid, C.: Evidence of the presence of (S)-linalool and of (S)-linalool synthase activity in Vitis vinifera L., cv. Muscat de Frontignan. Spec. Publ.-R. Soc. Chem., 300, 271-277 (2005)

316

R-Linalool synthase

4.2.3.26

1 Nomenclature EC number 4.2.3.26 Systematic name geranyl-diphosphate diphosphate-lyase [(3R)-linalool-forming] Recommended name R-linalool synthase Synonyms QH1 <1> [1] QH5 <1> [1] CAS registry number 254993-26-5

2 Source Organism <1> Artemisia annua (no sequence specified) [1] <2> Mentha citrata (UNIPROT accession number: Q8H2B4) (<2> BCA2 [2]) [2]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate + H2 O = (3R)-linalool + diphosphate Substrates and products S geranyl diphosphate + H2 O <1, 2> (Reversibility: ?) [1, 2] P (3R)-linalool + diphosphate Inhibitors EDTA <1> (<1> up to 95% loss of activity, isoenzyme QH1, full reactivation by 10 mM Mg2+ [1]) [1] Metals, ions Mg2+ <1, 2> (<2> required [2]; <1> Km -value 4.6 mM, isoenzyme QH1 [1]) [1, 2] Mn2+ <2> (<2> may substitute for Mg2+ [2]) [2] Additional information <1> (<1> Mn2+ may not substitute for Mg2+ [1]) [1]

317

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4.2.3.26

Turnover number (min–1) 0.24 <2> (geranyl diphosphate, <2> pH 6.5 [2]) [2] Km-Value (mM) 0.025 <2> (geranyl diphosphate, <2> pH 6.5 [2]) [2] 0.064 <1> (geranyl diphosphate, <1> isoenzyme QH1, pH 7.0, 30 C [1]) [1] pH-Optimum 6.5 <2> [2] pH-Range 7.5 <2> (<2> half-maximal velocity [2]) [2]

4 Enzyme Structure Subunits ? <1, 2> (<1> x * 65700, isoenzyme QH1, x * 67400, isoenzyme QH5, calculated [1]; <2> x * 70500, calculated [2]) [1, 2] Posttranslational modification Additional information <2> (<2> sequence contains a plastidial transit peptide [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue epidermis <1> (<1> of stem, isoenzyme QH5, induction by wounding [1]) [1] inflorescence <1> (<1> both isoenzyme QH1 and QH5 [1]) [1] leaf <1> (<1> both isoenzyme QH1 and QH5, induction by wounding [1]) [1] stem <1> (<1> epidermis of, isoenzyme QH5, induction by wounding [1]) [1] Additional information <1> (<1> not: stem stele, root [1]) [1] Purification <1> (both isoenzyme QH1 and QH5, partial) [1] <2> (recombinant protein) [2] Renaturation <1> (EDTA-inactivated isoenzyme QH1, full reactivation by 10 mM Mg2+ ) [1]

6 Stability General stability information <2>, addition of bovine serum albumine, cytochrome c, or carbonic anhydrase stabilizes with concommittant increase in both Km - and kcat -value [2]

318

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R-Linalool synthase

References [1] Jia, J.W.; Crock, J.; Lu, S.; Croteau, R.; Chen, X.Y.: (3R)-Linalool synthase from Artemisia annua L.: cDNA isolation, characterization, and wound induction. Arch. Biochem. Biophys., 372, 143-149 (1999) [2] Crowell, A.L.; Williams, D.C.; Davis, E.M.; Wildung, M.R.; Croteau, R.: Molecular cloning and characterization of a new linalool synthase. Arch. Biochem. Biophys., 405, 112-121 (2002)

319

Isoprene synthase

4.2.3.27

1 Nomenclature EC number 4.2.3.27 Systematic name dimethylallyl-diphosphate diphosphate-lyase (isoprene-forming) Recommended name isoprene synthase Synonyms ISPS <6, 7, 8> [3, 6, 13] CAS registry number 139172-14-8

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10>

Bacillus subtilis (no sequence specified) [1] Quercus robur (no sequence specified) [5] Mucuna pruriens (no sequence specified) [11] Salix discolor (no sequence specified) [8, 9] Populus alba + Populus tremula (UNIPROT accession number: Q9AR86) [12] Populus canescens (no sequence specified) [13] Populus alba (UNIPROT accession number: Q50L36) [3] Populus tremoloides (no sequence specified) [4,6,10] Quercus petraea (no sequence specified) [2] Mucuna sp. (no sequence specified) [7]

3 Reaction and Specificity Catalyzed reaction dimethylallyl diphosphate = isoprene + diphosphate Natural substrates and products S dimethylally diphosphate <6> (<6> substrate limitations can be responsinsible for great variations of enzyme activity from leaf to leaf [13]) (Reversibility: ir) [13]

320

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Isoprene synthase

P isoprene + diphosphate (<6> isoprene is a volatile product emmitted to the atmosphere [13]) S dimethylallyl diphosphate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10> (<1> in presence of a divalent cation [1]; <4> light- and age-dependent regulation of the thylakoid membrane-bound enzyme, the relation of soluble and membranebound isozymes is not affected by light and leaf age, overview [9]; <4> light-dependent regulation of the thylakoid membrane-bound enzyme, overview [8]; <6> substrate limitations can be responsible for great variations of enzyme activity from leaf to leaf [13]; <2> the enzyme is positively regulated by light [5]) (Reversibility: ir) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] P isoprene + diphosphate (<4,5,6> isoprene is a volatile product emmitted to the atmosphere [8,9,12,13]; <2,8> isoprene is emitted into the atmosphere following a diurnal cycle in vivo [5,6]; <3,10> isoprene is emmitted to the atmosphere [7,11]) S Additional information <1, 8> (<8> the acid-catalyzed non-enzymatic conversion of dimethylallyl diphosphate is negligible at physiologic pH [10]; <1> the enzyme activity parallels fluctuations of isoprene release during growth of Bacillus subtilis and is highly regulated, overview [1]) (Reversibility: ?) [1, 1] P ? Substrates and products S dimethylally diphosphate <6> (<6> substrate limitations can be responsible for great variations of enzyme activity from leaf to leaf [13]) (Reversibility: ir) [13] P isoprene + diphosphate (<6> isoprene is a volatile product emmitted to the atmosphere [13]; <6> i.e. 2-methyl-1,3-butadiene [13]) S dimethylallyl diphosphate <1, 2, 3, 4, 5, 6, 7, 8, 9, 10> (<1> in presence of a divalent cation [1]; <4> light- and age-dependent regulation of the thylakoid membrane-bound enzyme, the relation of soluble and membranebound isozymes is not affected by light and leaf age, overview [9]; <4> light-dependent regulation of the thylakoid membrane-bound enzyme, overview [8]; <6> substrate limitations can be responsible for great variations of enzyme activity from leaf to leaf [13]; <2> the enzyme is positively regulated by light [5]; <7> the enzyme is absolutely specific for dimethylally diphosphate [3]) (Reversibility: ir) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] P isoprene + diphosphate (<7> product identification [3]; <4,5,6> isoprene is a volatile product emmitted to the atmosphere [8,9,12,13]; <2,8> isoprene is emitted into the atmosphere following a diurnal cycle in vivo [5, 6]; <3, 10> isoprene is emmitted to the atmosphere [7,11]; <4,5,6,10> i.e. 2-methyl-1,3-butadiene [7, 8, 9, 12, 13]; <2, 3> i.e. 2-methyl-1,3-butadiene, product determination by gas chromatography [5, 11]; <8> i.e. 2methyl-1,3-butadiene, volatile product, product determination by gas chromatography [10]; <8> product stoichiometry determination, isoprene is volatile 2-methyl-1,3-butadiene emitted by the plant into the atmo-

321

Isoprene synthase

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sphere, where it plays an important role, the enzyme is positively regulated by light [4]) S Additional information <1, 7, 8> (<8> the acid-catalyzed non-enzymatic conversion of dimethylallyl diphosphate is negligible at physiologic pH [10]; <1> the enzyme activity parallels fluctuations of isoprene release during growth of Bacillus subtilis and is highly regulated, overview [1]; <7> substrate specifcity, no enzymatic activity with isoprenyl diphosphate or geranyl diphosphate as substrates, overview [3]) (Reversibility: ?) [1, 3, 10] P ? Inhibitors 1,10-phenanthroline <1> (<1> weak, 20% inhibition at 10 mM [1]) [1] Ca2+ <1, 10> (<1> inhibitory at higher concentrations [1]; <10> stimulates at up to 2 mM, inhibits at 2-30 mM [7]) [1, 7] Co2+ <1> (<1> inhibits at 1 mM [1]) [1] diethyldicarbonate <4> (<4> 34-59% inhibition at 1.1 mM, dimethylally diphosphate and Mg2+ protect the soluble enzyme, but not the solubilized thylakoid membrane isozyme [9]) [9] dimethylallyl diphosphate <8> (<8> substrate inhibition above 10 mM [4]) [4] diphosphate <4> (<4> 69-100% inhibition [9]) [9] EDTA <1> (<1> 40% inhibition at 1 mM, 70% inhibition at 10 mM [1]) [1] Fe2+ <1> (<1> inhibits at 1 mM [1]) [1] Mg2+ <1> (<1> activates at low concentration below 0.1 mM, inhibits at higher concentrations [1]) [1] Mn2+ <1> (<1> activates at low concentration below 0.1 mM, inhibits at higher concentrations [1]) [1] NEM <4> (<4> complete inactivation at 1.1 mM, dimethylally diphosphate and Mg2+ protect the isozyme partially [9]) [9] phenylglyoxal <4> (<4> 50 mM, complete inactivation, dimethylally diphosphate and Mg2+ protect the isozyme partially [9]) [9] Zn2+ <1> (<1> inhibits at 1 mM [1]) [1] Additional information <1, 4, 6, 7, 8> (<6> no inhibition by PMSF [13]; <4> no inhibition by phosphate [9]; <1> no inhibition by EGTA [1]; <8> enzyme expression and protein levels are highly reduced under elevated O3 but not under elevated CO2 in field-grown aspen trees [6]; <7> enzyme expression is highly reduced in the dark [3]; <6> no inhbition by PMSF [13]) [1, 3, 6, 9, 13] Activating compounds Additional information <2, 3, 4, 7, 8, 10> (<2,3> enzyme activity is induced by light, positive regulation [5,11]; <4> light-dependent regulation of the thylakoid membrane-bound enzyme, overview [8]; <7> the enzyme is induced by heat stress and continuous light irradiation [3]; <8,10> the enzyme is positively regulated by light [4,7]) [3, 4, 5, 7, 8, 11]

322

4.2.3.27

Isoprene synthase

Metals, ions Ca2+ <10> (<10> stimulates at up to 2 mM, inhibits at 2-30 mM [7]) [7] Co2+ <7> (<7> activates, 23% of the activity with Mg2+ [3]) [3] Fe2+ <7> (<7> 2% of the activity with Mg2+ [3]) [3] Mg2+ <1, 2, 3, 4, 5, 6, 7, 8, 9, 10> (<2,8> dependent on [4,5,10]; <7> activates at 20 mM, best divalent cation [3]; <1> activates at low concentration below 0.1 mM, inhibits at higher concentrations [1]; <10> dependent on, best at 15 mM, 20fold higher activity compared to Mn2+ [7]; <4> dependent on, isozyme activity profiles, optimal at 10-20 mM [9]; <4> dependent on, optimal at 10-15 mM, activity profile at 0-25 mM MgCl2 [8]; <6> dependent on, optimal at 20 mM [13]; <5> dependent on, the enzyme contains the Mg2+ -binding DDXXD motif, typical for monoterpene synthases [12]; <9> the enzyme is strictly dependent on divalent cations, Mg2+ is the most effective [2]) [1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13] Mn2+ <1, 4, 5, 7, 8, 10> (<4,5,8> can substitute for Mg2+ [4,8,12]; <1> activates at low concentration below 0.1 mM, inhibits at higher concentrations [1]; <7> activates, 34% of the activity with Mg2+ [3]; <10> can partially substitute for Mg2+ , 20fold lower activity compared to Mg2+ [7]) [1, 3, 4, 7, 8, 12] Ni2+ <7> (<7> 2% of the activity with Mg2+ [3]) [3] Zn2+ <7> (<7> 3% of the activity with Mg2+ [3]) [3] Additional information <1, 4, 7, 8> (<4> no effect by Ca2+ [8]; <8> the enzyme is strictly dependent on divalent cations, no activity with Zn2+ and Ca2+ [4]; <7> the enzyme requires divalent cations, no activity with Cu2+ and Ca2+ [3]; <1> the enzyme requires low levels of divalent cations [1]) [1, 3, 4, 8] Turnover number (min–1) 1.7 <8> (dimethylallyl diphosphate) [4] Specific activity (U/mg) 0.00039 <7> (<7> leaf cell culture [3]) [3] 0.001 <4> (<4> purified thylakoid membrane-bound isozyme [9]) [9] 0.011 <4> (<4> purified soluble isozyme [9]) [9] 0.012 <5> (<5> purified recombinant enzyme with or without leader sequence [12]) [12] 0.333 <2> (<2> partially purified enzyme [5]) [5] 0.521 <8> (<8> purified enzyme [4]) [4] Additional information <2, 4, 10> (<10> activity and isoprene emission during leaf development, overview [7]; <4> activity in subcellular fractions during partial purification, overview [8]; <2> Quercus robur is a strong emitter of isoprene, measurements of three trees over three days [5]) [5, 7, 8] Km-Value (mM) 1.2 <6> (dimethylallyl diphosphate, <6> pH 7.8, 40 C, recombinant C-terminally His-tagged enzyme [13]) [13] 1.2 <6> (dimethylallyl diphosphate, <6> pH 7.8, 40 C, recombinant C-terminally His-tagged enzyme [13]) [13] 2.45 <6> (dimethylallyl diphosphate, <6> pH 7.8, 40 C, native enzyme [13]) [13]

323

Isoprene synthase

4.2.3.27

2.45 <6> (dimethylallyl diphosphate, <6> pH 7.8, 40 C, native enzyme [13]) [13] 3.1 <6> (dimethylallyl diphosphate, <6> pH 7.8, 40 C, recombinant N-terminally His-tagged enzyme [13]) [13] 3.1 <6> (dimethylallyl diphosphate, <6> pH 7.8, 40 C, recombinant N-terminally His-tagged enzyme [13]) [13] 3.68 <6> (dimethylallyl diphosphate, <6> pH 7.8, 40 C, recombinant untagged enzyme [13]) [13] 3.68 <6> (dimethylallyl diphosphate, <6> pH 7.8, 40 C, recombinant untagged enzyme [13]) [13] 8 <4, 8> (dimethylallyl diphosphate, <4> pH 7.8 [8]) [4, 8] Additional information <4> (<4> soluble and membrane-bound isozymes [9]) [9] pH-Optimum 6.2 <1> [1] 7-9 <8> [10] 7.3 <2> (<2> first optimum [5]) [5] 7.7 <2> (<2> second optimum [5]) [5] 7.8-8.5 <6, 10> (<6> recombinant C-terminally His-tagged enzyme, recombinant untagged enzyme, and native enzyme [13]) [7, 13] 8 <4, 7, 8> (<7> recombinant enzyme [3]; <8> assay at [4]; <4> soluble isozyme [9]) [3, 4, 9] 8.5 <9> [2] 9 <6> (<6> recombinant N-terminally His-tagged enzyme [13]) [13] 10 <4> (<4> thylakoid membrane-bound isozyme [9]) [8, 9] pH-Range 5.4-11.2 <10> [7] 5.5-10 <8> [10] 6.3-9.2 <2> (<2> sharp decrease in activity below pH 6.3 [5]) [5] 6.5-9.5 <7> (<7> activity range [3]) [3] Additional information <1> (<1> pH profile [1]) [1] Temperature optimum ( C) 28 <8> (<8> assay at [10]) [10] 30 <10> (<10> assay at [7]) [7] 32 <8> (<8> assay at [4]) [4] 35 <4, 9> (<4> assay at [9]) [2, 9] 37 <1> (<1> assay at [1]) [1] 40 <5, 6, 7> (<5> assay at [12]; <7> recombinant enzyme [3]; <6> recombinant N-terminally His-tagged enzyme, recombinant untagged enzyme, and native enzyme [13]) [3, 12, 13] 40-45 <3> (<3> optimal temperature for isoprene emission in vivo in warm and cooler regions, respectively [11]) [11] 40-50 <6> (<6> recombinant C-terminally His-tagged enzyme [13]) [13] 50 <2> [5]

324

4.2.3.27

Isoprene synthase

Temperature range ( C) 15-50 <2> (<2> exponential increase of activity from 15 C to 40 C, optimum activity at 50 C, inactivation at 60 C [5]) [5] 25-50 <7> (<7> about half-maximal activity at 25 C and 50 C, temperature profile [3]) [3] 26-45 <3> (<3> no activity in vivo below 20 C and above 45 C, temperature dependence of activity, it is not closely related to electron flux or CO2 assimilation arte, overview [11]) [11] 40-55 <6> (<6> recombinant N-terminally His-tagged enzyme, recombinant untagged enzyme, and native enzyme [13]) [13] 40-60 <6> (<6> recombinant C-terminally His-tagged enzyme [13]) [13]

4 Enzyme Structure Molecular weight 52000 <6> (<6> native enzyme, gel filtration [13]) [13] 53000 <6> (<6> recombinant untagged enzyme, gel filtration [13]) [13] 62000 <6> (<6> recombinant untagged enzyme, native PAGE [13]) [13] 90000-100000 <9> (<9> gel filtration [2]) [2] 98000-137000 <8> (<8> gel filtration [4]) [4] Subunits dimer <8> (<8> 1 * 58000 + 1 * 63000, SDS-PAGE [4]) [4] monomer <6> (<6> 1 * 68000, C-terminally His-tagged enzyme, SDS-PAGE, 1 * 78000, N-terminally His-tagged enzyme, SDS-PAGE [13]) [13] Additional information <5, 7> (<7> the enzyme contains the aspartate-rich motif DDxxD [3]; <5> the enzyme contains the Mg2+ -binding DDXXD motif, typical for monoterpene synthases [12]) [3, 12] Posttranslational modification proteolytic modification <5, 7> (<5> the enzyme contains a plastidial leader sequence [12]; <7> the enzyme contains a plastidial targeting sequence [3]) [3, 12]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <7> (<7> from leaves [3]) [3] leaf <2, 3, 4, 6, 7, 8, 9, 10> (<10> activity during leaf development, highest activity an isoprene emission in about 14-days-old leaves, overview [7]) [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13] Localization chloroplast <4, 5, 6> (<4> soluble and membrane-bound isozymes [9]; <5> the enzyme contains a plastidial leader sequence [12]; <6> the enzyme is localized in the stroma and is also attached to the stromal side of the thylakoid membrane [13]) [8, 9, 12, 13]

325

Isoprene synthase

4.2.3.27

chloroplast stroma <4> (<4> soluble isozyme [9]) [9] plastid <7> (<7> the enzyme contains a plastidial targeting sequence [3]) [3] soluble <4> (<4> soluble enzyme form [8,9]) [8, 9] thylakoid membrane <4> (<4> tightly membrane-bound enzyme form with a stromal facing, accessible to trypsin treatment [8]; <4> tightly membranebound isozyme with a stromal facing, membrane association via a membrane tail, accessible to trypsin treatment [9]) [8, 9] Additional information <4, 6> (<4> study of subcellular localization, overview [8]; <6> subcellular localization study, overview [13]) [8, 13] Purification <1> (native enzyme partially by anion exchange chromatography) [1] <2> (native enzyme partially from leaves by ammonium sulfate fractionation) [5] <4> (separation of soluble and membrane-bound isozymes, by ammonium sulfate fractionation, chloroplast preparation, PEG precipitation, hydrophobic interaction and ion exchange chromatography, and gel filtration, the thylakoid membrane isozyme is irreversibly solubilized by pH 10.0 treatment, overview) [9] <5> (recombinant His-tagged enzyme from Escherichia coli strain TG1) [12] <6> (native enzyme partially from leaves, recombinant His-tagged enzyme without plastidial targeting sequence from Escherichia coli strain TG1 by nickel affinity chromatography) [13] <7> (recombinant enzyme from Escherichia coli) [3] <8> (native enzyme 4000fold from leaves to homogeneity, ammonium sulfate fractionation of cell-free supernatant, dialysis, ion exchange chromatography, ultrafiltration, and gel filtration) [4] <8> (native enzyme partially from leaves by ammonium sulfate fractionation) [10] <8> (recombinant His-tagged ISPS from Escherichia coli strain BL21(DE3) by nickel affinity chromatography) [6] <9> (native enzyme from leaves) [2] <10> (native enzyme partially by ammonium sulfate fractionation and anion exchange chromatography) [7] Cloning <5> (gene ispS, DNA and amino acid sequence determination and analysis, sequence comparisons, expression of His-tagged enzyme with or without leader sequence in Escherichia coli strain TG1) [12] <6> (gene ispS, expression of N-terminally or C-terminally His-tagged enzyme without plastidial targeting sequence in Escherichia coli strain TG1) [13] <7> (gene ispS, DNA and amino acid sequence determination and analysis, expression in Escherichia coli, expression as GFP-fusion protein in onion peels and tobacco leaves, transformations by particle bombardement) [3] <8> (expression analysis under different atmospheric conditions, overview, expression of the His-tagged ISPS in Escherichia coli strain BL21(DE3)) [6]

326

4.2.3.27

Isoprene synthase

6 Stability General stability information <1>, the enzyme activity is very labile and only reproducibly detected when suitable protectants are used in the extraction buffer, especially PMSF is highly stabilizing [1] <8>, glycerol stabilizes the enzyme in the early stages of purification, PEG in the late stages [4] Storage stability <2>, -80 C, 30-60 C ammonium sulfate fraction, resuspended in 50 mM TrisHCl, pH 8.5, 20 mM MgCl2 , 5% glycerol, 2 mM DTT, stable [5] <8>, -70 C, partially purified enzyme, 40-55% ammonium sulfate fraction, in phosphate buffered saline, pH 7.4, 1mM DTT, stable for at least 1 month [10]

References [1] Sivy, T.L.; Shirk, M.C.; Fall, R.: Isoprene synthase activity parallels fluctuations of isoprene release during growth of Bacillus subtilis. Biochem. Biophys. Res. Commun., 294, 71-75 (2002) [2] Schnitzler, J.P.; Arenz, R.; Steinbrecher, R.; Lehning, A.: Characterization of an isoprene synthase from leaves of Quercus petraea. Bot. Acta, 109, 216221 (1996) [3] Sasaki, K.; Ohara, K.; Yazaki, K.: Gene expression and characterization of isoprene synthase from Populus alba. FEBS Lett., 579, 2514-2518 (2005) [4] Silver, G.M.; Fall, R.: Characterization of aspen isoprene synthase, an enzyme responsible for leaf isoprene emission to the atmosphere. J. Biol. Chem., 270, 13010-13016 (1995) [5] Lehning, A.; Zimmer, I.; Steinbrecher, R.; Bruggemann, N.; Schnitzler, J.P.: Isoprene synthase activity and its relation to isoprene emission in Quercus robur L. leaves. Plant Cell Environ., 22, 495-504 (1999) [6] Calfapietra, C.; Wiberley, A.E.; Falbel, T.G.; Linskey, A.R.; Mugnozza, G.S.; Karnosky, D.F.; Loreto, F.; Sharkey, T.D.: Isoprene synthase expression and protein levels are reduced under elevated O3 but not under elevated CO2 (FACE) in field-grown aspen trees. Plant Cell Environ., 30, 654-661 (2007) [7] Kuzma, J.; Fall, R.: Leaf isoprene emission rate is dependent on leaf development and the level of isoprene synthase. Plant Physiol., 101, 435-440 (1993) [8] Wildermuth, M.C.; Fall, R.: Light-dependent isoprene emission (characterization of a thylakoid-bound isoprene synthase in Salix discolor chloroplasts). Plant Physiol., 112, 171-182 (1996) [9] Wildermuth, M.C.; Fall, R.: Biochemical characterization of stromal and thylakoid-bound isoforms of isoprene synthase in willow leaves. Plant Physiol., 116, 1111-1123 (1998) [10] Silver, G.M.; Fall, R.: Enzymatic synthesis of isoprene from dimethylallyl diphosphate in aspen leaf extracts. Plant Physiol., 97, 1588-1591 (1991)

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Isoprene synthase

4.2.3.27

[11] Monson, R.K.; Jaeger, C.H.; Adams, W.W.; Driggers, E.M.; Silver, G.M.; Fall, R.: Relationships among isoprene emission rate, photosynthesis, and isoprene synthase activity as influenced by temperature. Plant Physiol., 98, 1175-1180 (1992) [12] Miller, B.; Oschinski, C.; Zimmer, W.: First isolation of an isoprene synthase gene from poplar and successful expression of the gene in Escherichia coli. Planta, 213, 483-487 (2001) [13] Schnitzler, J.P.; Zimmer, I.; Bachl, A.; Arend, M.; Fromm, J.; Fischbach, R.J.: Biochemical properties of isoprene synthase in poplar (Populus x canescens). Planta, 222, 777-786 (2005)

328

2-Hydroxypropyl-CoM lyase

4.2.99.19

1 Nomenclature EC number 4.2.99.19 (transferred to EC 4.4.1.23, 2-hydroxypropyl-CoM lyase. The enzyme was incorrectly classified as acting on a C-O bond rather than a C-S bond) Recommended name 2-hydroxypropyl-CoM lyase

329

threo-3-Hydroxyaspartate ammonia-lyase

4.3.1.16

1 Nomenclature EC number 4.3.1.16 Systematic name threo-3-hydroxy-l-aspartate ammonia-lyase (oxaloacetate-forming) Recommended name threo-3-hydroxyaspartate ammonia-lyase Synonyms l-threo-3-hydroxyaspartate dehydratase threo-3-hydroxyaspartate ammonia-lyase threo-3-hydroxyaspartate dehydratase CAS registry number 248270-70-4

2 Source Organism <1> Pseudomonas sp. (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction threo-3-hydroxy-l-aspartate = oxaloacetate + NH3 Reaction type deamination Substrates and products S l-threo-3-hydroxyaspartate <1> (<1> no substrates are d-threo or d,lerythro-3-hydroxyaspartate [1]) (Reversibility: ?) [1] P oxaloacetate + NH3 <1> [1] Inhibitors Cu2+ <1> [1] EDTA <1> [1] hydroxylamine <1> [1] Zn2+ <1> [1]

330

4.3.1.16

threo-3-Hydroxyaspartate ammonia-lyase

Cofactors/prosthetic groups pyridoxal 5’-phosphate <1> [1] Metals, ions Ca2+ <1> (<1> activates [1]) [1] Co2+ <1> (<1> activates [1]) [1] Fe2+ <1> (<1> activates [1]) [1] Mg2+ <1> (<1> activates [1]) [1] Mn2+ <1> (<1> activates [1]) [1] Specific activity (U/mg) 8 <1> [1] Km-Value (mM) 0.74 <1> (l-threo-3-hydroxyaspartate) [1]

4 Enzyme Structure Molecular weight 59000 <1> (<1> gel filtration [1]) [1] Subunits Additional information <1> (<1> SDS-PAGE analysis shows 39000 subunit, either monomer or dimer [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> [1]

References [1] Wada, M.; Matsumoto, T.; Nakamori, S.; Sakamoto, M.; Kataoka, M.; Liu, J.Q.; Itoh, N.; Yamada, H.; Shimizu, S.: Purification and characterization of a novel enzyme, l-threo-3-hydroxyaspartate dehydratase, from Pseudomonas sp. T62. FEMS Microbiol. Lett., 179, 147-151 (1999)

331

L-Serine

ammonia-lyase

4.3.1.17

1 Nomenclature EC number 4.3.1.17 Systematic name l-serine ammonia-lyase Recommended name l-serine ammonia-lyase Synonyms dehydratase, l-serine EC 4.2.1.13 l-Hydroxy amino acid dehydratase l-SD l-SD1 l-SD2 l-serine deaminase l-serine dehydratase <4> [28] SD SDH <4, 5> [28, 29, 30, 34, 35] SdaA Name <19> (<19> commentary [32]) [32] serine deaminase serine dehydratase <5> [30, 34] TdcG <3> [42] Additional information <4> (<4> l-serine dehydratase and cystathionine bsynthase are the same enzyme [1]) [1] CAS registry number 9014-27-1

2 Source Organism <1> Gallus gallus (no sequence specified) [7] <2> Mus musculus (no sequence specified) [36, 37, 38, 39] <3> Escherichia coli (no sequence specified) (<3> a-subunit of Fdh3 [3, 9, 22, 23]) [3, 9, 17, 22, 23, 42, 44] <4> Homo sapiens (no sequence specified) [1, 28, 40, 43, 46] <5> Rattus norvegicus (no sequence specified) (<5> isozyme PPA1, chloroplast precursor [24]; <5> NAT2 [4]) [4, 7, 10, 11, 12, 14, 24, 26, 27, 29, 30, 33, 34, 35, 41, 43, 45]

332

4.3.1.17

<6> <7> <8> <9> <10> <11> <12> <13> <14> <15> <16> <17> <18> <19> <20>

L-serine

ammonia-lyase

Saccharomyces cerevisiae (no sequence specified) [25] Ovis aries (no sequence specified) [33] Klebsiella aerogenes (no sequence specified) [17] Corynebacterium sp. (no sequence specified) [8] Clostridium acidi-urici (no sequence specified) [13, 17] Arthrobacter globiformis (no sequence specified) [5, 6, 17] Clostridium sticklandii (no sequence specified) [18] Lactobacillus fermentum (no sequence specified) [16, 17] Pseudomonas cepacia (no sequence specified) [17] Lactobacillus murinus (no sequence specified) [2] Clostridium propionicum (no sequence specified) [19] Corynebacterium glycinophilum (no sequence specified) [15] Peptostreptococcus asaccharolyticus (no sequence specified) [17, 20, 21] Campylobacter jejuni (no sequence specified) [32] Paracoccus seriniphilus (no sequence specified) [31]

3 Reaction and Specificity Catalyzed reaction l-serine = pyruvate + NH3 Reaction type elimination Natural substrates and products S l-Ser <3, 15> (<3> the metabolic role may be related to serine toxicity [9]; <15> constitutive enzyme, repressed by glucose [2]) (Reversibility: ?) [2, 9] P ? S l-serine <4, 5, 7, 19, 20> (Reversibility: ir) [28, 29, 30, 31, 32, 33, 34, 35] P pyruvate + NH3 <4, 5, 7, 19, 20> [28, 29, 30, 31, 32, 33, 34, 35] Substrates and products S d-serine <2> (<2> a,b-elimination reaction [38]; <2> elimination reaction [36]) (Reversibility: ?) [36, 37, 38] P pyruvate + NH3 S d-serine <2> (<2> racemization reaction [36,37,38]) (Reversibility: r) [36, 37, 38] P l-serine S l-Leu <17> (<17> 3% of the activity with l-Ser [15]) (Reversibility: ?) [15] P 3-Hydroxy-2-oxopropionate + NH3 S l-Ser <3, 15> (<3> the metabolic role may be related to serine toxicity [9]; <15> constitutive enzyme, repressed by glucose [2]) (Reversibility: ?) [2, 9] P ?

333

L-serine

ammonia-lyase

4.3.1.17

S l-Ser <1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18> (<9, 11, 15, 17, 18> specific for l-Ser [2, 5, 8, 15, 21]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27] P pyruvate + NH3 <1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18> [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27] S l-Thr <5, 17> (<17> 1% of the activity with l-Ser [15]) (Reversibility: ?) [12, 15] P 3-hydroxy-2-oxobutyrate + NH3 S l-Trp <17> (<17> 17% of the activity with l-Ser [15]) (Reversibility: ?) [15] P ? S l-allo-threonine <3> (<3> at 4.6% of the rate with l-serine [44]) (Reversibility: ?) [44] P 2-oxobutyrate + NH3 S l-serine <2, 3, 4, 5, 7, 19, 20> (<2> a,b-elimination reaction [38]; <2,4> elimination reaction [36,39,40]) (Reversibility: ir) [28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45] P pyruvate + NH3 <4, 5, 7, 19, 20> [28, 29, 30, 31, 32, 33, 34, 35] S l-serine <2, 4> (<2> racemization reaction [36,37,38]; <4> reacemization reaction [40]; <2> recemization reaction [39]) (Reversibility: r) [36, 37, 38, 39, 40] P d-serine S l-serine O-sulfate <2> (<2> elimination reaction [36]) (Reversibility: ?) [36] P O-sulfopyruvate + NH3 S l-serine-O-sulfate <2> (<2> elimination reaction [39]) (Reversibility: ?) [39] P ? + NH3 S l-serine-O-sulfate <4> (<4> elimination reaction [40]) (Reversibility: ?) [40] P O-sulfopyruvate + NH3 S l-threo-3-hydroxyaspartate <2> (Reversibility: ?) [36] P oxaloacetate + NH3 S l-threonine <5> (Reversibility: ?) [34] P 2-oxo-butanoate + NH3 <5> [34] S l-threonine <2> (<2> a,b-elimination reaction [38]) (Reversibility: ?) [38] P 3-hydroxy-2-butenoic acid + NH3 S l-threonine <2, 3, 4, 5> (<3> at 2.7% of the rate with l-serine [44]) (Reversibility: ?) [36, 43, 44] P 2-oxobutyrate + NH3 S b-chloro-l-alanine <2> (Reversibility: ?) [36] P 3-chloropropenoic acid + NH3 S Additional information <2, 3> (<3> no substrate: l-threonine [42]; <2> racemization and elimination activities reside at the same active site of enzyme. Racemization activity is specific to serine, elimination activity

334

4.3.1.17

L-serine

ammonia-lyase

has a broader specificity for l-amino acids with a suitable leaving group at the b-carbon [36]; <2> ration of elimination reaction/racemization reaction for substrate l-serine is 3.7 [38]; <3> no substrate: d-serine. Extremely poor substrate: l-cysteine [44]) (Reversibility: ?) [36, 38, 42, 44] P ? Inhibitors 1,10-phenanthroline <3, 12, 18> (<18> no effect [21]) [3, 18, 21] ATP <2> (<2> inhibitory to l-serine O-sulfate dehydration reaction, activating for racemization reraction [39]) [39] AlK(SO4 )2 <3> [3] Co2+ <2> (<2> inhibition of both activities [37]) [37] Cu2+ <2> (<2> inhibition of both activities [37]) [37] CuCl2 <3> (<3> 0.7 mM, 50% inhibition [3]) [3] d-His <3> [23] d-Ser <3, 9, 11, 12, 16> (<11,12> competitive [5,18]) [3, 5, 8, 18, 19] d-serine <3, 19> (<3> competitive [44]; <19> 100 mM, 40% inhibition [32]) [32, 44] EDTA <2, 12, 18> (<18> no effect [21]; <2> inhibition of both activities [37]) [18, 21, 37] ethanolamine <3, 18> [3, 21] Fe2+ <2> (<2> slight inhibition of both activities [37]) [37] Gly <17, 18> [15, 21] glycylglycine <18> [21] HgCl2 <11> [5] hydroxylamine <4> [1] l-Ala <5, 9> [4, 8] l-Cys <9, 11, 12, 16, 17, 18> (<11,18> competitive [5,21]) [5, 8, 15, 18, 19, 21] l-cysteine <3> (<3> competitive [44]) [44] l-His <3> [23] l-Thr <15> (<15> competitive [2]) [2] l-threonine <19> (<19> 100 mM, 56% inhibition [32]) [32] l-Trp <9, 17> [8, 15] l-Val <17> [15] l-cysteine <19> (<19> 50 mM, 95% inhibition [32]) [32] MnCl2 <17> [15] Ni2+ <2> (<2> slight inhibition of both activities [37]) [37] PCMB <5, 18> (<5> NH+4 protects [12]) [12, 21] pyruvate <15> (<15> competitive [2]) [2] Zn2+ <2> (<2> inhibition of both activities [37]) [37] ZnSO4 <3> [3] homocysteine <5> (<5> noncompetitive with respect to substrate, competitive with regard to pyridoxal 5’-phosphate [14]) [14] imidazole <3> [23] Additional information <3, 12> (<12> NaBH4 has no effect [18]; <3> not inhibitory: l-alanine [44]) [18, 44]

335

L-serine

ammonia-lyase

4.3.1.17

Cofactors/prosthetic groups pyridoxal 5’-phosphate <4, 5, 7, 8, 12, 13, 14, 15, 16, 18> (<13> prosthetic group [16]; <5> coenzyme [10,12]; <15> required, maximal activity at 0.34 mM [2]; <5> K+ and NH+4 decrease the dissociation constant of pyridoxal 5’-phosphate. Tris and the substrate compete for pyridoxal 5’-phosphate with the enzyme because of Schiffs base formation. Influence of substrate concentration on the Km -value of pyridoxal 5’-phosphate [10]; <5> one mol of enzyme contains two mol of pyridoxal phosphate [7]; <5> Km : 0.0003 mM [7]; <8, 12, 13, 14, 16, 18> pyridoxal-5’-phosphate-independent enzyme [16, 17, 18, 19, 21]; <4> pyridoxal 5’-phosphate is sandwiched between F40 and A222 [46]) [2, 7, 10, 12, 16, 17, 18, 19, 21, 28, 29, 30, 33, 34, 35, 46] Additional information <19, 20> (<20> not dependent on pyridoxal 5’-phosphate [31]; <19> pyridoxal 5’-phosphate independent [32]) [31, 32] Activating compounds ATP <2> [38] Sulfhydryl reagents <10> (<10> required [13]) [13] Additional information <3> (<3> the enzyme is inactive in crude extract and can be activated with iron and dithiothreitol. The activation requires oxygen, and is inhibited by free radical scavengers and by diethylenentriamine pentaacetic acid, which prevents Fe cycling [23]) [23] Metals, ions Ca2+ <2> (<2> may partially replace Mg2+ [39]) [37, 39] Fe2+ <3, 10, 13, 17, 19, 20> (<10,13> required [13,16,17]; <10> Km : 0.1 mM [13]; <19> iron-sulfur enzyme [32]; <3> requires activation [17]; <17> slight activation by FeCl2 [15]; <13> Km : 0.55 mM [16]; <20> required for activity, Fe-S cluster [31]) [13, 15, 16, 17, 31, 32] Iron <3, 16, 18> (<18> enzyme contains one [4Fe-4S]cluster per heterodimer [17]; <18> enzyme probably contains a diamagnetic [4Fe-4S]2+cluster which is converted by oxidation and loss of iron ion to a paramegnetic [3Fe-4S]+ cluster resulting in inactivation of the enzyme [20]; <16> iron-sulfur-dependent enzyme [19]; <18> the enzyme contains 3.8 mol Fe and 5.6 mol inorganic sulfur per mol of heterodimer, indicating the presence of an [Fe-S]center [21]; <3> 7.7 mol iron per mol dimer, two (4Fe-4S)2+ clusters per dimer in anaerobically isolated enzyme, exposure to air results in loss of clusters and concomitant loss of enzyme activity [42]; <3> presence of (4Fe-4S)2+ and of (2Fe-2S)2+ clusters involved in catalytic turnover [44]) [17, 19, 20, 21, 42, 44] K+ <4, 5> (<5> slight stimulation [12]; <4> stimulating [46]) [12, 46] KCl <11> (<11> nonspecific requirement for a monovalent or bivalent cation. Half-maximal activity is produced with 22.5 mM KCl [5]) [5] Mg2+ <2> (<2> required by both isoforms A and B [39]; <2> required, both activities [37]) [37, 39] MgCl2 <9, 11> (<9> activates [8]; <11> nonspecific requirement for a monovalent or bivalent cation. Half-maximal activity with 1.0 mM MgCl2 [5]) [5, 8] Mn2+ <2, 15> (<15> increases activity [2]; <2> may partially replace Mg2+ [39]) [2, 37, 39] NH+4 <5> (<5> slight stimulation [12]) [12] 336

4.3.1.17

L-serine

ammonia-lyase

Turnover number (min–1) 0.0033 <2> (d-serine, <2> wild-type, pH 7.4, 37 C [38]) [38] 0.012 <2> (d-serine, <2> mutant Q155D, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 0.028 <2> (d-threonine, <2> mutant Q155D, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 0.033 <2> (l-serine, <2> mutant Q155D, presence of ATP, pH 7.4, 37 C [38]; <2> wild-type, a,b-elimination, pH 7.4, 37 C [38]) [38] 0.042 <2> (d-serine, <2> wild-type, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 0.166 <2> (l-serine, <2> wild-type, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 0.3 <2> (d-threonine, <2> wild-type, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 0.49 <2> (l-serine O-sulfate, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 2.6 <2> (b-chloro-l-alanine, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 3.2 <2> (d-serine, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 3.8 <2> (l-serine, <2> pH 8.0, 37 C, presence of 1 mM ATP, racemization reaction [36]) [36] 4 <2> (l-serine, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 10.5 <2> (l-threonine, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 14.5 <2> (d-serine, <2> pH 8.0, 37 C, presence of 1 mM ATP, racemization reaction [36]) [36] 31 <2> (l-threo-3-hydroxyaspartate, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 436 <3> (l-serine, <3> pH 8.0, 37 C [44]) [44] 544 <3> (l-serine, <3> anaerobically isolated enzyme, pH 8.0 [42]) [42] Specific activity (U/mg) 0.262 <17> [15] 0.649 <13> [16] 96 <4> (<4> substrate l-threonine, pH 8.3, 37 C [43]) [43] 110 <9> [8] 115 <19> (<19> recombinant SdaA [32]) [32] 116 <5> (<5> substrate l-threonine, pH 8.3, 37 C [43]) [43] 137 <4> (<4> substrate l-serine, pH 8.3, 37 C [43]) [43] 174.2 <5> (<5> isoenzyme II [4]) [4] 200 <5> (<5> substrate l-serine, pH 8.3, 37 C [43]) [43] 226.9 <5> (<5> isoenzyme I [4]) [4] 278.3 <5> [11] 307 <3> (<3> anaerobically isolated enzyme, pH 8.0 [42]) [42] 660 <11> [5]

337

L-serine

ammonia-lyase

4.3.1.17

Additional information <4, 11, 12, 16, 18> (<11> activity stain in polyacrylamide gels [6]) [1, 6, 18, 19, 21] Km-Value (mM) 0.49 <2> (l-serine O-sulfate, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 0.8 <18> (l-Ser) [21] 2.67 <3> (l-Serine, <3> pH 8.0, 37 C [44]) [44] 3.2 <2> (d-Serine, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 3.8 <2> (l-Serine, <2> pH 8.0, 37 C, presence of 1 mM ATP, racemization reaction [36]) [36] 4 <2> (l-Serine, <2> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [36]) [36] 4.8 <3> (l-Serine, <3> anaerobically isolated enzyme, pH 8.0 [42]) [42] 5 <16> (l-Ser) [19] 5.1 <20> (l-Serine, <20> 51 C, pH 7.6 [31]) [31] 7 <11> (l-Ser) [5] 7.8 <10> (l-Ser) [13] 8.33 <15> (l-Ser) [2] 8.5 <2> (d-Serine, <2> wild-type, a,b-elimination, pH 7.4, 37 C [38]) [38] 9 <2> (d-Serine, <2> wild-type, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 9 <2> (l-Serine, <2> wild-type, a,b-elimination, pH 7.4, 37 C [38]) [38] 10 <2> (l-Serine, <2> wild-type, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 11 <2> (l-Serine, <2> mutant Q155D, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 12 <2> (d-Serine, <2> mutant Q155D, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 14.5 <2> (d-Serine, <2> pH 8.0, 37 C, presence of 1 mM ATP, racemization reaction [36]) [36] 14.6 <19> (l-Serine) [32] 30 <2> (l-Serine, <2> pH 8.0, 37 C, racemization reaction [37]) [37] 45 <5> (l-Serine, <5> pH 8.3, 37 C [43]) [43] 49 <2> (d-Serine, <2> pH 8.0, 37 C, racemization reaction [37]) [37] 50 <4> (l-Serine, <4> pH 8.3, 37 C [43]) [43] 50 <5> (l-Threonine, <5> 37 C, pH 8.3, recombinant SDH [34]) [34] 55 <2> (d-Threonine, <2> wild-type, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 57 <4, 5> (l-Threonine, <4,5> pH 8.3, 37 C [43]) [43] 60 <2> (d-Threonine, <2> mutant Q155D, a,b-elimination, presence of ATP, pH 7.4, 37 C [38]) [38] 65 <13> (l-Ser) [16] 67 <5> (l-Serine, <5> 37 C, pH 8.3, recombinant SDH [34]) [34] 70 <5> (l-Ser) [7] 75 <2> (d-Serine, <2> pH 8.0, 37 C, elimination reaction [37]) [37]

338

4.3.1.17

L-serine

ammonia-lyase

75 <5> (l-Ser, <5> in presence of 20 mM NH4 Cl [12]) [12] 75 <2> (l-Serine, <2> pH 8.0, 37 C, elimination reaction [37]) [37] 100 <5> (l-Ser, <5> without NH+4 [12]) [12] 130 <5> (l-Thr, <5> in presence and in absence of 20 mM NH4 Cl [12]) [12] 177 <5> (l-Serine, <5> pH 7.4, 25 C [45]) [45] 182 <5> (l-Serine, <5> pH 7.4, 25 C, animals treated with thioacetamide for 97 days [45]) [45] 420 <3> (l-Ser) [3] Additional information <5> [4] Ki-Value (mM) 0.7 <3> (CuCl2 ) [3] 0.9 <3> (l-Alanine, <3> pH 8.0, 37 C [44]) [44] 1.41 <3> (d-Serine, <3> pH 8.0, 37 C [44]) [44] pH-Optimum 6 <15> [2] 7.5-8 <11> [5] 7.6 <20> [31] 8 <13> [16] 8-9.5 <2> [37] 8.4 <18> [21] 8.6 <5> [7] 8.8 <17> [15] 9 <9> [8] pH-Range 6.5 <2> (<2> negligible activity below [37]) [37] 6.5-9.5 <9> (<9> pH 6.5: about 40% of maximal activity, pH 9.5: about 90% of maximal activity [8]) [8] 7-9.5 <17> (<17> pH 7: about 35% of maximal activity, pH 9.5: about 35% of maximal activity [15]) [15] Temperature optimum ( C) 40 <17> [15] 45 <13> [16] 51 <20> [31] Temperature range ( C) 18-50 <17> (<17> 18 C: about 50% of maximal activity, 50 C: about 25% of maximal activity [15]) [15]

4 Enzyme Structure Molecular weight 30500 <7> (<7> gel filtration [33]) [33] 35000-40000 <5> (<5> gel filtration [33]) [33] 53000 <20> (<20> gel filtration [31]) [31]

339

L-serine

ammonia-lyase

4.3.1.17

55000 <2> (<2> isoforms A and B, gel filtration [39]) [39] 57000 <16> (<16> gel filtration in presence of 10 mM Fe2+ , dimeric enzyme form [19]) [19] 60000 <4, 5> (<4,5> gel filtration [11,43]) [11, 43] 63500 <5> [7] 64000 <5> (<5> ultracentrifugation [33]) [33] 66000 <5> (<5> gel filtration [27]; <5> gel filtration, recombinant SDH [34]) [27, 34] 66810 <5> (<5> laseer light scattering [33]) [33] 68000 <5> (<5> sucrose density gradient centrifugation [11]) [11] 72000 <10> (<10> gel filtration [13]) [13] 78000 <2> (<2> gel filtration [37]) [37] 101700 <3> (<3> dynamic light scattering [42]) [42] 107000 <3> (<3> gel filtration [44]) [44] 123000 <19> (<19> gel filtration [32]) [32] 130000 <17> (<17> gel filtration [15]) [15] 150000 <13> (<13> gel filtration [16]) [16] 180000 <18> (<18> gradient PAGE [21]) [21] 200000 <18> (<18> gel filtration [21]) [21] 230000 <16> (<16> gel filtration in absence of Fe2+ , octameric enzyme form [19]) [19] 250000 <12> (<12> gel filtration [18]) [18] Subunits ? <2> (<2> x * 36121, MALDI- MS, x * 36123, calculated [36]) [36] dimer <2, 3, 4, 5, 16, 18, 19, 20> (<5> 2 * 35000, SDS-PAGE [11]; <5> 2 * 34000, SDS-PAGE [27,33]; <2> 2 * 37000, SDS-PAGE [37]; <18> 1 * 25000 + 1 * 30000, the enzyme is composed of two different subunits in a 1:1 stoichiometry, forming heterodimers to heterooctamers [17]; <5> 2 * 34000 [7]; <16> 1 * 26000 + 1 * 10000, enzyme either exists as a heterooctamer or a heterodimer, SDS-PAGE [19]; <20> 1 * 14500 + 1 * 40000, SDS-PAGE [31]; <5> 2 * 34000, SDS-PAGE, recombinant SDH [34]; <19> 2 * 51758, electrospray mass spectrometry [32]; <4> 2 * 33000, SDS-PAGE, 2 * 34702, calculated [43]; <3> 2 * 51000, His-tagged protein, SDS-PAGE and calculated [44]; <3> 2 * 52910, calculated for His-tagged protein, 2 * 48460, calculated for native protein, 2 * 46000, SDS-PAGE of His-tagged protein [42]) [7, 11, 17, 19, 27, 31, 32, 33, 34, 37, 42, 43, 44] hexamer <18> (<18> 3 * 25000 + 3 * 30000, the enzyme is composed of two different subunits in a 1:1 stoichiometry, forming heterodimers to heterooctamers [17]) [17] monomer <7> (<7> 1 *30500, SDS-PAGE, immunoblot [33]) [33] octamer <12, 16, 18> (<18> 4 * 25000 + 4 * 30000, the enzyme is composed of two different subunits in a 1:1 stoichiometry, forming heterodimers to heterooctamers [17]; <16> 4 * 26000 + 4 * 30000, enzyme either exists as a heterooctamer or a heterodimer, SDS-PAGE [19]; <12> 4 * 27000 + 4 * 30000, SDS-PAGE [18]) [17, 18, 19]

340

4.3.1.17

L-serine

ammonia-lyase

tetramer <13, 18> (<13> 4 * 40000, SDS-PAGE [16]; <18> 2 * 25000 + 2 * 30000, the enzyme is composed of two different subunits in a 1:1 stoichiometry, forming heterodimers to heterooctamers [17]) [16, 17]

5 Isolation/Preparation/Mutation/Application Source/tissue brain <2, 4> [39, 40] kidney <1> [7] liver <1, 4, 5> (<4> fetal [1]; <5> confined to periportal region [43]; <5> enzyme activity increases with age of animals and also in response to the amount of surplus amino acids from dietary protein. Induction is mainly controlled at the level of transcription and seems not to be related to gluconeogenesis [41]; <4> predominantly localized in perivenous region [43]; <5> suffering chronic injury caused by ingestion of thioacetamide. After 97 days of intake, enzyme activity is about 60% lower than in controls. No significant differences in Km -value are found, while enzyme protein level is reduced. Enzyme is localized to periportal zone of the hepatic acinus [45]) [1, 4, 7, 10, 11, 12, 14, 24, 29, 41, 43, 45, 46] Localization cytosol <5> [24] soluble <15> [2] Purification <2> (both isoforms A and B) [39] <2> (recombinant enzyme expressed in insect cells) [37] <3> (anaerobic purification of His-tagged enzyme, purified protein is brown in colour) [42] <3> (recombinant protein, purification under anaerobic conditions) [44] <3> (use of gene fusion of the structural gene sdaA to purify l-serine deaminase 1) [22] <4> [1] <4> (expression in Escherichia coli with N-terminal His-tag, purification protocol from inclusion bodies) [40] <4> (recombinant SDH) [28] <4> (recombinant protein expressed in Escherichia coli) [43] <5> [11, 12, 33] <5> (recombinant SDH) [34] <5> (recombinant protein expressed in Escherichia coli) [43] <7> (partially purified) [33] <9> [8] <10> [13] <11> [5] <12> (partial) [18] <13> [16] <16> (partial) [19]

341

L-serine

ammonia-lyase

<17> <18> <19> <20>

4.3.1.17

(strain AJ-3170) [15] [21] (recombinant His-tagged SdaA) [32] [31]

Crystallization <4> (crystal structure obtained by molecular replacement shows a homodimer and a fold typical for b-family pyridoxal 5’-phosphate-dependent enzymes. Each monomer serves as an active unit) [46] <4> (hanging-drop vapour diffusion, 0.002 ml protein solution containing 20-30 mg/ml SDH in 20 mM Tris-HCl, pH 7.6 and 150 mM NaCl is mixed with 0.002 ml reservoir solution containing 800 mM ammonium sulfate in 100 mM Tris-HCl, pH 7.0-8.0, crystals diffract to 2.5 A) [28] <5> (crystal structures of apo-SDH and holo-SDH, crystallized with Omethylserine, at 2.8 A and 2.6 A resolution, respectively) [29] Cloning <4> (expression in Escherichia coli) [28] <5> (expression in Escherichia coli) [27, 34] <19> (expression in Escherichia coli) [32] Engineering H152S <2> (<2> ratio of elimination reaction to racemization is 1.4 pared to 3.7 in wild-type [38]) [38] N154F <2> (<2> ratio of elimination reaction to racemization is 0.33 pared to 3.7 in wild-type [38]) [38] P153S <2> (<2> ratio of elimination reaction to racemization is 0.24 pared to 3.7 in wild-type [38]) [38] Q155D <2> (<2> ratio of elimination reaction to racemization is 0.25 pared to 3.7 in wild-type [38]) [38]

comcomcomcom-

Application analysis <20> (<20> online-determination of l-serine concentation in bioreactor [31]) [31]

6 Stability pH-Stability 5 <9> (<9> 37 C, 30 min, about 50% loss of activity [8]) [8] 5.5 <9> (<9> 37 C, 30 min, about 40% loss of activity [8]) [8] 6-7 <9> (<9> 37 C, 30 min, about 25% loss of activity [8]) [8] 8 <9> (<9> 37 C, 30 min, about 45% loss of activity [8]) [8] 8.5 <9> (<9> 37 C, 30 min, 50% loss of activity [8]) [8] Temperature stability 0 <3> (<3> 30 min, 45% loss of activity [3]) [3] 30 <9> (<9> pH 6.2, 30 min, stable up to [8]) [8] 37 <3> (<3> complete loss of activity within 30 min. Fe2+ , l-Ser d-Ser or ethanolamine decrease the loss of activity at 37 C [3]) [3]

342

4.3.1.17

L-serine

ammonia-lyase

40 <9> (<9> pH 6.2, 30 min, about 50% loss of activity [8]) [8] 45 <4, 15> (<4> 10 min, stable up to [1]; <15> 10 min, 70% loss of activity [2]) [1, 2] 50 <4, 9> (<4> 10 min, about 10% loss of activity [1]; <9> about 90% loss of activity [8]) [1, 8] 55 <4, 5, 15> (<15> 10 min, complete loss of activity [2]; <4> 10 min, about 50% loss of activity [1]; <5> complete loss of activity after 4 min. NH4 Cl, KCl or (NH4 )2 SO4 stabilize [12]) [1, 2, 12] 60 <4> (<4> 10 min, about 75% loss of activity [1]) [1] Oxidation stability <19>, purified SdaA is inactivated with a half-life of approx. 1.5 h upon exposure to air, inactivated SdaA can be reactivated to approx. 60% of its activity under strict anaerobic conditiones with Fe2+ and dithiothreitol [32] <20>, complete loss of activity in crude extracts after exposure to air for 1 h [31] <12, 18>, inactivated upon exposure to air, reactivated by Fe2+ under aerobic conditions [17, 18] <16, 18>, rapid loss of activity upon exposure to air, reactivation by Fe2+ [19, 21] General stability information <3>, it is absolutely critical that dilutions of the purified enzyme be made in buffer containing 50% glycerol, otherwise the enzyme rapidly loses activity [44] <3>, unstable in all attempts at purification [3] <5>, suszeptible to proteases e.g. trypsin [34] <12>, l-Cys and d-Ser stabilize enzyme activity [18] <16>, extreme instability does not permit purification to homogeneity [19] Storage stability <3>, -10 C, 40% glycerol, loss of activity within 1 week [3] <3>, 0 C, 45% loss of activity after 30 min [3] <3>, unstable within the cell in the presence of its inducers, Gly and Leu, but not in their absence [9] <4>, -80 C, stable for more than 2 weeks [43] <4>, 0 C, stable for at least 2 weeks [43] <5>, -80 C, at least 1 month, no loss of activity [34] <11>, -20 C, pH 7.0-8.0, stable over many months, enzyme in crude extract [5] <11>, -70 C, 0.6 mg/ml protein, 30% loss of activity after 1 month, purified enzyme [5] <17>, -20 C, 40% loss of activity after 24 h, 0.76 mg/ml protein concentration, partially purified enzyme [15] <17>, 4 C, 90% loss of activity after 24 h, 0.76 mg/ml protein concentration, partially purified enzyme [15]

343

L-serine

ammonia-lyase

4.3.1.17

References [1] Porter, P.N.; Grishaver, M.S.; Jones, O.W.: Characterization of human cystathionine b-synthase. Evidence for the identity of human l-serine dehydratase and cystathionine b-synthase. Biochim. Biophys. Acta, 364, 128-139 (1974) [2] Farias, M.E.; Strasser de Saad, A.M.; Pesce de Ruiz Holgado, A.A.; Oliver, G.: Evidence for the presence of l-serine dehydratase in Lactobacillus murinus. J. Gen. Appl. Microbiol., 31, 563-567 (1985) [3] Newman, E.B.; Kapoor, V.: In vitro studies on l-serine deaminase activity of Escherichia coli K12. Can. J. Biochem., 58, 1292-1297 (1980) [4] Yeung, Y.G.; Yeung, D.: Comparative studies on threonine and serine dehydratases in rat liver. Int. J. Biochem., 11, 161-164 (1980) [5] Gannon, F.; Bridgeland, E.S.; Jones, K.M.: l-Serine dehydratase from Arthrobacter globiformis. Biochem. J., 161, 345-355 (1977) [6] Gannon, F.; Jones, K.M.: An activity stain for l-serine dehydratase in polyacrylamide gels. Anal. Biochem., 79, 594-596 (1977) [7] Grillo, M.A.: Serine dehydratase in animal tissues. Acta Vitaminol. Enzymol., 27, 51-61 (1973) [8] Morikawa, Y.; Nakamura, N.; Kimura, K.: Purification and some properties of l-serine dehydratase of Corynebacterium sp.. Agric. Biol. Chem., 38, 531-537 (1974) [9] Isenberg, S.; Newman, E.B.: Studies on l-serine deaminase in Escherichia coli K-12. J. Bacteriol., 118, 53-58 (1974) [10] Simon, D.; Kroeger, H.: Interactions of l-serine dehydratase from rat liver with its coenzyme and substrates. Biochim. Biophys. Acta, 334, 208-217 (1974) [11] Simon, D.; Hoshino, J.; Kroeger, H.: l-Serine dehydratase from rat liver. Purification and some properties. Biochim. Biophys. Acta, 321, 361-368 (1973) [12] Hoshino, J.; Simon, D.; Kroeger, H.: Influence of monovalent cations on the activity of l-serine (L-threonine) dehydratase from rat liver. Eur. J. Biochem., 27, 388-394 (1972) [13] Sagers, R.D.; Carter, J.E.: l-Serine dehydratase (Clostridium acidiurici). Methods Enzymol., 17B, 351-356 (1971) [14] Pestana, A.; Sandoval, I.V.; Sols, A.: Inhibition by homocysteine of serine dehydratase and other pyridoxal 5’-phosphate enzymes of the rat through cofactor blockage. Arch. Biochem. Biophys., 146, 373-379 (1971) [15] Kubota, K.; Yokozeki, K.; Ozaki, H.: Effect of l-serine dehydratase activity onL-serine production by Corynebacterium glycinophilum and an examination of the properties of the enzyme. J. Ferment. Bioeng., 67, 391-394 (1989) [16] Farias, M.E.; Strasser de Saad, A.M.; Pesce de Ruiz Holgado, A.A.; Oliver, G.: Purification and properties of l-serine dehydratase from Lactobacillus fermentum ATCC 14931. Curr. Microbiol., 22, 205-211 (1991)

344

4.3.1.17

L-serine

ammonia-lyase

[17] Grabowski, R.; Hofmeister, A.E.M.; Buckel, W.: Bacterial l-serine dehydratases: a new family of enzymes containing iron-sulfur clusters. Trends Biochem. Sci., 18, 297-300 (1993) [18] Zinecker, H.; Andreesen, J.R.; Pich, A.: Partial purification of an iron-dependent l-serine dehydratase from Clostridium sticklandii. J. Basic Microbiol., 38, 147-155 (1998) [19] Hofmeister, A.E.M.; Grabowski, R.; Linder, D.; Buckel, W.: l-Serine and lthreonine dehydratase from Clostridium propionicum. Two enzymes with different prosthetic groups. Eur. J. Biochem., 215, 341-349 (1993) [20] Hofmeister, A.E.M.; Albracht, S.P.J.; Buckel, W.: Iron-sulfur cluster-containing l-serine dehydratase from Peptostreptococcus asaccharolyticus: correlation of the cluster type with enzymatic activity. FEBS Lett., 351, 416-418 (1994) [21] Grabowski, R.; Buckel, W.: Purification and properties of an iron-sulfur containing and pyridoxal-phosphate-independent l-serine dehydratase from Peptostreptococcus asaccharolyticus. Eur. J. Biochem., 199, 89-94 (1991) [22] Su, H.; Moniakis, J.; Newman, E.B.: Use of gene fusion of the structural gene sdaA to purify l-serine deaminase 1 from Escherichia coli K-12. Eur. J. Biochem., 211, 521-527 (1993) [23] Newman, E.B.; Walker, C.; Ziegler-Skylakis, K.: A possible mechanism for the in vitro activation of l-serine deaminase activity in Escherichia coli K12. Biochem. Cell Biol., 68, 723-728 (1990) [24] Hopgood, M.F.; Ballard, F.J.: The relative stability of liver cytosol enzymes incubated in vitro. Biochem. J., 144, 371-376 (1974) [25] Ramos, F.; Wiame, J.M.: Occurrence of a catabolic l-serine (l-threonine) deaminase in Saccharomyces cerevisiae. Eur. J. Biochem., 123, 571-576 (1982) [26] Suda, M.; Nakagawa, H.: l-Serine dehydratase (rat liver). Methods Enzymol., 17B, 346-351 (1971) [27] Ogawa, H.; Takasugawa, F.; Wakaki, K.; Kishi, H.; Eskandarian, M.R.; Kobayashi, M.; Date, T.; Huh, N.H.; Pitot, H.C.: Rat liver serine dehydratase. J. Biol. Chem., 274, 12855-12869 (1999) [28] Sun, L.; Li, X.; Dong, Y.; Yang, M.; Liu, Y.; Han, X.; Zhang, X.; Pang, H.; Rao, Z.: Crystallization and preliminary crystallographic analysis of human serine dehydratase. Acta Crystallogr. Sect. D, 59, 2297-2299 (2003) [29] Yamada, T.; Komoto, J.; Takata, Y.; Ogawa, H.; Pitot, H.C.; Takusagawa, F.: Crystal structure of serine dehydratase from rat liver. Biochemistry, 42, 12854-12865 (2003) [30] Imai, S.; Kanamoto, R.; Yagi, I.; Kotaru, M.; Saeki, T.; Iwami, K.: Response of the induction of rat liver serine dehydratase to changes in the dietary protein requirement. Biosci. Biotechnol. Biochem., 67, 383-387 (2003) [31] Laroche, M.; Pukall, R.; Ulber, R.: Recovery and characterization of a lserine dehydratase from the marine bacterium Paracoccus seriniphilus for the construction of bioanalytical systems. Chem.-Ing.-Tech., 75, 146-149 (2003)

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L-serine

ammonia-lyase

4.3.1.17

[32] Velayudhan, J.; Jones, M.A.; Barrow, P.A.; Kelly, D.J.: l-serine catabolism via an oxygen-labile l-serine dehydratase is essential for colonization of the avian gut by Campylobacter jejuni. Infect. Immun., 72, 260-268 (2004) [33] Ogawa, H.; Gomi, T.; Takusagawa, F.; Masuda, T.; Goto, T.; Kan, T.; Huh, N.H.: Evidence for a dimeric structure of rat liver serine dehydratase. Int. J. Biochem. Cell Biol., 34, 533-543 (2002) [34] Ogawa, H.; Takusagawa, F.; Wakaki, K.; Kishi, H.; Eskandarian, M.R.; Kobayashi, M.; Date, T.; Huh, N.-H.; Pitot, H.C.: Rat liver serine dehydratase. Bacterial expression and two folding domains as revealed by limited proteolysis. J. Biol. Chem., 274, 12855-12860 (1999) [35] Xue, H.H.; Fujie, M.; Sakaguchi, T.; Oda, T.; Ogawa, H.; Kneer, N.M.; Lardy, H.A.; Ichiyama, A.: Flux of the l-serine metabolism in rat liver. The predominant contribution of serine dehydratase. J. Biol. Chem., 274, 16020-16027 (1999) [36] Strisovsky, K.; Jiraskova, J.; Mikulova, A.; Rulisek, L.; Konvalinka, J.: Dual substrate and reaction specificity in mouse serine racemase: identification of high-affinity dicarboxylate substrate and inhibitors and analysis of the b-eliminase activity. Biochemistry, 44, 13091-13100 (2005) [37] Strisovsky, K.; Jiraskova, J.; Barinka, C.; Majer, P.; Rojas, C.; Slusher, B.S.; Konvalinka, J.: Mouse brain serine racemase catalyzes specific elimination of l-serine to pyruvate. FEBS Lett., 535, 44-48 (2003) [38] Foltyn, V.N.; Bendikov, I.; De Miranda, J.; Panizzutti, R.; Dumin, E.; Shleper, M.; Li, P.; Toney, M.D.; Kartvelishvily, E.; Wolosker, H.: Serine racemase modulates intracellular d-serine levels through an a,b-elimination activity. J. Biol. Chem., 280, 1754-1763 (2005) [39] Neidle, A.; Dunlop, D.S.: Allosteric regulation of mouse brain serine racemase. Neurochem. Res., 27, 1719-1724 (2002) [40] Nagayoshi, C.; Ishibashi, M.; Kita, Y.; Matsuoka, M.; Nishimoto, I.; Tokunaga, M.: Expression, refolding and characterization of human brain serine racemase in Escherichia coli with N-terminal His-tag. Protein Pept. Lett., 12, 487-490 (2005) [41] Kanamoto, R.; Fujita, K.; Kumasaki, M.; Imai, S.; Kotaru, M.; Saeki, T.; Iwami, K.: Inverse correlation between the nitrogen balance and induction of rat liver serine dehydratase (SDH) by dietary protein. Biosci. Biotechnol. Biochem., 68, 888-893 (2004) [42] Burman, J.D.; Harris, R.L.; Hauton, K.A.; Lawson, D.M.; Sawers, R.G.: The iron-sulfur cluster in the l-serine dehydratase TdcG from Escherichia coli is required for enzyme activity. FEBS Lett., 576, 442-444 (2004) [43] Kashii, T.; Gomi, T.; Oya, T.; Ishii, Y.; Oda, H.; Maruyama, M.; Kobayashi, M.; Masuda, T.; Yamazaki, M.; Nagata, T.; Tsukada, K.; Nakajima, A.; Tatsu, K.; Mori, H.; Takusagawa, F.; Ogawa, H.; Pitot, H.C.: Some biochemical and histochemical properties of human liver serine dehydratase. Int. J. Biochem. Cell Biol., 37, 574-589 (2005) [44] Cicchillo, R.M.; Baker, M.A.; Schnitzer, E.J.; Newman, E.B.; Krebs, C.; Booker, S.J.: Escherichia coli l-serine deaminase requires a [4Fe-4S] cluster in catalysis. J. Biol. Chem., 279, 32418-32425 (2004)

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L-serine

ammonia-lyase

[45] Lopez-Flores, I.; Barroso, J.B.; Valderrama, R.; Esteban, F.J.; Martinez-Lara, E.; Luque, F.; Peinado, M.A.; Ogawa, H.; Lupianez, J.A.; Peragon, J.: Serine dehydratase expression decreases in rat livers injured by chronic thioacetamide ingestion. Mol. Cell. Biochem., 268, 33-43 (2005) [46] Sun, L.; Bartlam, M.; Liu, Y.; Pang, H.; Rao, Z.: Crystal structure of the pyridoxal-5’-phosphate-dependent serine dehydratase from human liver. Protein Sci., 14, 791-798 (2005)

347

D-Serine

ammonia-lyase

4.3.1.18

1 Nomenclature EC number 4.3.1.18 Systematic name d-serine ammonia-lyase (pyruvate-forming) Recommended name d-serine ammonia-lyase Synonyms d-hydroxy amino acid dehydratase d-serine deaminase <2, 5> [21, 23] d-serine dehydrase d-serine hydrolase (deaminating) d-serine ammonia lyase <5> [22] d-serine dehydratase <2, 5> [18, 20] DSD <2, 5> [18, 20] dehydratase, d-serine DsdA <2> [19, 21] Dsdase EC 4.2.1.14 CAS registry number 9015-88-7

2 Source Organism <1> Gallus gallus (no sequence specified) [1] <2> Escherichia coli (no sequence specified) (<2> gene leg-1 [11]) [3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21] <3> Klebsiella pneumoniae (no sequence specified) [8] <4> Escherichia intermedia (no sequence specified) [2] <5> Escherichia coli (UNIPROT accession number: P00926) (<5> a-subunit of Fdh3 [20]) [20, 22, 23]

348

4.3.1.18

D-serine

ammonia-lyase

3 Reaction and Specificity Catalyzed reaction d-Serine = pyruvate + NH3 (<2> reaction mechanism, the phosphate group of the substrate-pyridoxal-phosphate complex serves as the main anchoring point during catalysis, intermediate is the Schiff base of a-aminoacrylate and pyridoxal 5’-phosphate [7]; <2> transimination of the enzyme-linked cofactor pyridoxal 5’-phosphate is at least a two-step process, the first step of which is probably formation of a noncovalent complex between the enzyme and the amino acid [4]) Reaction type elimination Natural substrates and products S d-Ser <2, 4> (<4> adaptive enzyme, which enables cells to grow with dSer as the sole source of carbon and nitrogen [2]; <2> the enzyme enables cells to grow with d-Ser as the sole source of nitrogen [17]) (Reversibility: ?) [2, 17] P ? S d-serine <2, 5> (Reversibility: ?) [18, 19, 20, 21, 22] P pyruvate + NH3 <2> [18, 19] S l-serine <5> (Reversibility: ?) [23] P pyruvate + NH3 Substrates and products S d-allothreonine <2> (Reversibility: ?) [3] P ? S d-Ser <1, 2, 3, 4> (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17] P pyruvate + NH3 S d-Ser <2, 4> (<4> adaptive enzyme, which enables cells to grow with dSer as the sole source of carbon and nitrogen [2]; <2> the enzyme enables cells to grow with d-Ser as the sole source of nitrogen [17]) (Reversibility: ?) [2, 17] P ? S d-Thr <2, 3> (Reversibility: ?) [3, 8, 10, 11] P 3-hydroxy-2-oxobutyrate + NH3 S d-serine <2, 5> (<5> 25 C [20]) (Reversibility: ?) [18, 19, 20, 21, 22] P pyruvate + NH3 <2> [18, 19] S l-Ser <2, 3> (<2> very low activity [3]) (Reversibility: ?) [3, 8] P pyruvate + NH3 S l-serine <5> (Reversibility: ?) [23] P pyruvate + NH3 Inhibitors ATP <1> (<1> activation at low concentrations, inhibition at high concentrations [1]) [1] Ala <2> [6]

349

D-serine

ammonia-lyase

4.3.1.18

Alkylamines <2> (<2> inactivation via a transimination of pyridoxal 5’phosphate [6]) [6] dl-2,3-Diaminopropionic acid <2> [18] dl-O-methyl serine <2> [18] Gly <2> [6] Glycine <2> [18] Isoserine <2, 5> [13, 18, 20] l-2,3-diaminopropionic acid <2> [18] l-Alanine <2> [18] l-Serine <2> [18] O-methylserine <2> (<2> competitive [11]) [11] Tris <2> (<2> inactivation is prevented by the presence of sufficient K+ or NH+4 and less effectively by Na+ or pyridoxal 5’-phosphate [11]) [11] Cofactors/prosthetic groups AMP <1> (<1> activates [1]) [1] ATP <1> (<1> activation at low concentrations, inhibition at high concentrations [1]) [1] CMP <1> (<1> activates [1]) [1] IMP <1> (<1> activates [1]) [1] pyridoxal 5’-phosphate <2, 3, 5> (<2> 1 mol of pyridoxal 5’-phosphate is bound per mol of enzyme to one e-amino group [10]; <2> transimination of the enzyme-linked cofactor by d-Ser [4]; <3> Km : 0.0025 mM [8]; <2, 3> enzyme linked cofactor [6, 8, 11]; <2> 1 pyridoxal 5’-phosphate per d-serine dehydratase molecule [18]) [4, 6, 8, 10, 11, 18, 20] UMP <1> (<1> activates [1]) [1] Activating compounds pyridoxal 5’-deoxymethylenephosphonate <2> (<2> can substitute for pyridoxal 5’-phosphate, the enzyme exhibits 35-40% of the activity of the native enzyme [12]) [12] Turnover number (min–1) 0.112 <2> (d-allothreonine, <2> pH 5.7, 25 C [3]) [3] 0.12 <2> (l-Ser, <2> pH 5.8, 25 C [3]) [3] 0.14 <2> (l-Ser, <2> pH 8.9, 25 C [3]) [3] 0.153 <2> (l-Ser, <2> pH 7.8, 25 C [3]) [3] 0.92 <2> (d-allothreonine, <2> pH 8.9, 25 C [3]) [3] 0.95 <2> (d-Thr, <2> pH 5.8, 25 C [3]) [3] 1.8 <2> (l-allothreonine, <2> pH 7.8, 25 C [3]) [3] 9.4 <2> (d-Ser, <2> pH 5.7, 25 C [3]) [3] 15.7 <2> (d-serine) [21] 17.2 <2> (l-Thr, <2> pH 8.9, 25 C [3]) [3] 19.6 <2> (l-Thr, <2> pH 7.8, 25 C [3]) [3] 30 <2> (d-Ser, <2> pH 7.8, 5 C [3]) [3] 48 <2> (d-Ser, <2> pH 8.9, 25 C [3]) [3] 118 <2> (d-Ser, <2> pH 7.8, 25 C [3]) [3]

350

4.3.1.18

D-serine

ammonia-lyase

Specific activity (U/mg) 0.29 <3> (<3> l-Ser [8]) [8] 12 <3> (<3> d-Ser [8]) [8] 100 <2> [18] Km-Value (mM) 0.083 <2> (d-Thr, <2> pH 5.7, 25 C [3]) [3] 0.086 <2> (d-Ser, <2> pH 5.7, 25 C [3]) [3] 0.1 <2> (d-serine) [21] 0.24 <2> (d-Ser, <2> pH 7.8, 5 C [3]) [3] 0.35 <2> (d-Ser, <2> pH 7.8, 25 C [3]) [3] 0.35 <2> (d-serine, <2> 25 C, pH 7.8 [18]) [18] 0.68 <2> (d-Thr, <2> pH 7.8, 25 C [3]) [3] 0.7 <2> (d-Ser) [10] 0.92 <2> (d-Thr) [10] 1.1 <2> (d-allothreonine, <2> pH 5.7, 25 C [3]) [3] 1.3 <2> (d-Ser) [11] 1.68 <2> (d-Ser, <2> pH 8.9, 25 C [3]) [3] 2.4 <2> (d-allothreonine, <2> pH 7.8, 25 C [3]) [3] 2.8 <3> (d-Ser) [8] 3.2 <2> (d-Thr) [11] 3.6 <3> (d-Thr) [8] 4 <2> (l-Ser, <2> pH 7.8, 25 C [3]) [3] 4.9 <2> (d-allothreonine, <2> pH 8.9, 25 C [3]) [3] 11 <2> (l-Ser, <2> pH 5.8, 25 C [3]) [3] 12.3 <2> (l-Ser, <2> pH 8.9, 25 C [3]) [3] 16 <2> (d-Thr, <2> pH 8.9, 25 C [3]) [3] 20 <3> (l-Ser) [8] Ki-Value (mM) 0.035 <2> (dl-2,3-diaminopropionic acid, <2> 25 C, pH 7.8 [18]) [18] 0.09 <2> (l-2,3-diaminopropionic acid, <2> 25 C, pH 7.8 [18]) [18] 0.33 <2> (isoserine, <2> 25 C, pH 7.8 [18]) [18] 0.46 <5> (isoserine) [20] 3 <2> (l-serine, <2> 25 C, pH 7.8 [18]) [18] 4.9 <2> (glycine, <2> 25 C, pH 7.8 [18]) [18] 17 <2> (l-alanine, <2> 25 C, pH 7.8 [18]) [18] 70 <2> (dl-O-methyl serine, <2> 25 C, pH 7.8 [18]) [18] pH-Optimum 6.5-7.5 <3> [8] 7.8-8 <2> [11] 8 <2> [10] pH-Range 6-8.5 <3> (<3> pH 6.0: about 30% of maximal activity, pH 8.5: about 60% of maximal activity [8]) [8] 6.5-9 <2> (<2> pH 6.5: about 30% of maximal activity, pH 9.0: about 60% of maximal activity [11]) [11]

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D-serine

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4.3.1.18

4 Enzyme Structure Molecular weight 37300 <2> (<2> analytical ultracentrifugation [10]) [10] 40000 <2> (<2> sucrose density gradient centrifugation [11]) [11] 46000 <3> (<3> gel filtration [8]) [8] Subunits ? <2> (<2> x * 48790, sequence analysis of peptides [5]) [5] monomer <2, 3, 5> (<3> 1 * 40000, SDS-PAGE [8]; <2> 1 * 47920 [18]; <5> 1 * 47920, calculated from amino acid sequence [20]) [8, 18, 20]

5 Isolation/Preparation/Mutation/Application Source/tissue kidney <1> [1] Localization soluble <1> [1] Purification <1> [1] <2> [10, 11] <2> (purified from k12 mutant C6) [18] <2> (to homogeneity) [21] <3> [8] Crystallization <2> [10, 11] <2> (enzyme complexed with 3-amino-2-hydroxypropionate) [16] Cloning <2> (expression in Escherichia coli) [21] <5> (expression in Saccharomyces cerevisiae) [23] Engineering G279D <2> (<2> mutant G279D and G281D, loss of activity, the mutant enzymes form a Schiff base linkage with pyridoxal 5’-phosphate but do not hold the cofactor in a catalytically competent orientation. G279D has 225fold reduced cofactor affinity, the ability to retain a cofactor:glycine complex is decreased 765fold. Mutant G281D has 50fold decreased cofactor affinity, the ability to retain a cofactor:glycine complex is decreased 1970fold [14]) [14] G281D <2> (<2> mutant G279D and G281D, loss of activity, the mutant enzymes form a Schiff base linkage with pyridoxal 5’-phosphate but do not hold the cofactor in a catalytically competent orientation. G279D has 225fold reduced cofactor affinity, the ability to retain a cofactor:glycine complex is decreased 765fold. Mutant G281D has 50fold decreased cofactor affinity, the ability to retain a cofactor:glycine complex is decreased 1970fold [14]) [14]

352

4.3.1.18

D-serine

ammonia-lyase

Application degradation <2> (<2> the enzyme is applied to remove endogenous d-serine from organotypic hippocampal slices. Complete removal of d-serine virtually abolishes NMDA-elicited neurotoxicity [21]) [21] molecular biology <5> (<5> the dsdA gene is used as a selectable marker for transformation of Arabidopsis [22]) [22] pharmacology <2> (<2> the d-serine dehydratase gene is an excellent marker, especially in the construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed [17]) [17]

6 Stability Temperature stability 22 <2> (<2> 6 h, 10% loss of activity [10]) [10] 37 <2> (<2> stable at and below, enzyme from mutant strain EM1610 [9]) [9] 50 <2> (<2> enzyme from wild type strain EM1609 is stable, enzyme from mutant strain EM1610 is rapidly inactivated [9]) [9] 54 <2> (<2> 1 min, dilute solution, complete loss of activity [10]) [10] 55 <2> (<2> enzyme from wild type strain is rapidly inactivated above [9]) [9] General stability information <2>, repeated freezing and thawing over a period of 1 or 2 months causes a 20-30% loss of activity [10] <2>, resolved completely by dialysis against l-Cys or d-Cys, reactivated by addition of pyridoxal 5’-phosphate [11] Storage stability <2>, 0 C, stable almost indefinitely [10]

References [1] Grillo, M.A.: Serine dehydratase in animal tissues. Acta Vitaminol. Enzymol., 27, 51-61 (1973) [2] Faleev, N.G.; Vikha, Yu.K.; Martinkova, N.S.; Belikov, V.M.: Enzymes accompanying tyrosine phenol lyase and the problem of its substrate specificity. Curr. Microbiol., 9, 235-240 (1983) [3] Federiuk, C.S.; Bayer, R.; Shafer, J.A.: Characterization of the catalytic pathway for d-serine dehydratase. J. Biol. Chem., 258, 5379-5385 (1983) [4] Federiuk, C.S.; Shafer, J.A.: A reaction pathway for transimination of the pyridoxal 5’-phosphate in d-serine dehydratase by amino acids. J. Biol. Chem., 258, 5372-5378 (1983) [5] Schiltz, E.; Schmitt, W.: Sequence of Escherichia coli d-serine dehydratase. FEBS Lett., 134, 57-62 (1981)

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ammonia-lyase

4.3.1.18

[6] Federiuk, C.S.; Shafer, J.A.: Inactivation of d-serine dehydratase by alkylamines via a transimination of enzyme-linked cofactor. J. Biol. Chem., 256, 7416-7423 (1981) [7] Schnackerz, K.D.; Ehrlich, J.H.; Giesemann, W.; Reed, T.A.: Mechanism of action of d-serine dehydratase. Identification of a transient intermediate. Biochemistry, 18, 3557-3563 (1979) [8] Kikuchi, S.; Ishimoto, M.: A d-serine dehydratase acting also on l-serine from Klebsiella pneumoniae. J. Biochem., 84, 1133-1138 (1978) [9] McFall, E.: Escherichia coli K-12 mutant forming a temperature-sensitive dserine deaminase. J. Bacteriol., 121, 1074-1077 (1975) [10] Robinson, W.G.; Labow, R.: d-Serine dehydratase (Escherichia coli). Methods Enzymol., 17B, 356-360 (1971) [11] Dupourque, D.; Newton, W.A.; Snell, E.E.: Purification and properties of dserine dehydrase from Escherichia coli. J. Biol. Chem., 241, 1233-1238 (1966) [12] Bloom, F.R.: Isolation and characterization of catabolite-resistant mutants in the d-serine deaminase system of Escherichia coli K-12. J. Bacteriol., 121, 1085-1091 (1975) [13] Schackerz, K.D.; Feldmann, K.: Pyridoxal-5’-deoxymethylenephosphonate reconstituted d-serine dehydratase: a phosphorus-31 nuclear magnetic resonance study. Biochem. Biophys. Res. Commun., 95, 1832-1838 (1980) [14] Marceau, M.; Lewis, S.D.; Shafer, J.A.: The glycine-rich region of Escherichia coli d-serine dehydratase. Altered interactions with pyridoxal 5’-phosphate produced by substitutions of aspartic acid for glycine. J. Biol. Chem., 263, 16934-16941 (1988) [15] Pavlasova, E.; Stejskalova, E.; Sikyta, B.: Stability of hyperproduction of dserine deaminase and tryptophanase in Escherichia coli. Biotechnol. Lett., 9, 761-764 (1987) [16] Obmolova, G.; Tepliakov, A.; Harutyunyan, E.; Wahler, G.; Schnackerz, K.D.: Crystallization and preliminary X-ray studies of d-serine dehydratase from Escherichia coli. J. Mol. Biol., 214, 641-642 (1990) [17] Maas, W.K.; Maas, R.; McFall, E.: d-Serine deaminase is a stringent selective marker in genetic crosses. J. Bacteriol., 177, 459-461 (1995) [18] Schnackerz, K.D.; Tai, C.-H.; Potsch, R.K.W.; Cook, P.F.: Substitution of pyridoxal 5’-phosphate in d-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis. J. Biol. Chem., 274, 36935-36943 (1999) [19] Roesch, P.L.; Redford, P.; Batchelet, S.; Moritz, R.L.; Pellett, S.; Haugen, B.J.; Blattner, F.R.; Welch, R.A.: Uropathogenic Escherichia coli use d-serine deaminase to modulate infection of the murine urinary tract. Mol. Microbiol., 49, 55-67 (2003) [20] Schnackerz, K.D.; Keller, J.; Phillips, R.S.; Toney, M.D.: Ionization state of pyridoxal 5’-phosphate in d-serine dehydratase, dialkylglycine decarboxylase and tyrosine phenol-lyase and the influence of monovalent cations as inferred by (31)P NMR spectroscopy. Biochim. Biophys. Acta, 1764, 230238 (2006)

354

4.3.1.18

D-serine

ammonia-lyase

[21] Shleper, M.; Kartvelishvily, E.; Wolosker, H.: d-Serine is the dominant endogenous coagonist for NMDA receptor neurotoxicity in organotypic hippocampal slices. J. Neurosci., 25, 9413-9417 (2005) [22] Erikson, O.; Hertzberg, M.; Naesholm, T.: The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformation. Plant Mol. Biol., 57, 425-433 (2005) [23] Vorachek-Warren, M.K.; McCusker, J.H.: DsdA (D-serine deaminase): A new heterologous MX cassette for gene disruption and selection in Saccharomyces cerevisiae. Yeast, 21, 163-171 (2004)

355

Threonine ammonia-lyase

4.3.1.19

1 Nomenclature EC number 4.3.1.19 Systematic name l-threonine ammonia-lyase (2-oxobutanoate-forming) Recommended name threonine ammonia-lyase Synonyms EC 4.2.1.16 <4, 34> (<4,34> formerly [54]) [54] l-TDH <47> [52] l-threonine deaminase l-threonine dehydratase TD <3, 11> [50, 51, 53, 56] TDH TH <4, 34> [54] threonine deaminase <3, 11, 21> [51, 53, 55] ilvA <3> [53] tdcB <3> [49] threonine dehydrase threonine dehydratase <4, 34> [54] threonine dehydratase/deaminase <11> [56] CAS registry number 9024-34-4

2 Source Organism <1> Salmonella typhimurium (no sequence specified) (<1> gene ispS [7]) [7, 21, 34] <2> Candida utilis (no sequence specified) [4] <3> Escherichia coli (no sequence specified) [7, 16, 17, 19, 20, 24, 25, 26, 31, 38, 39, 40, 44, 45, 49, 53] <4> Rattus norvegicus (no sequence specified) [9, 32, 33, 35, 41, 43, 54] <5> Saccharomyces cerevisiae (no sequence specified) [4] <6> Euglena gracilis (no sequence specified) [6] <7> Geobacillus stearothermophilus (no sequence specified) [18] <8> Rhodopseudomonas sphaeroides (no sequence specified) [28]

356

4.3.1.19

<9> <10> <11> <12> <13> <14> <15> <16> <17> <18> <19> <20> <21> <22> <23> <24> <25> <26> <27> <28> <29> <30> <31> <32> <33> <34> <35> <36> <37> <38> <39> <40> <41> <42> <43> <44> <45> <46> <47>

Threonine ammonia-lyase

Ovis aries (no sequence specified) [22] Spinacia oleracea (no sequence specified) [13] Arabidopsis thaliana (no sequence specified) [50, 51, 56] Pseudomonas aeruginosa (no sequence specified) [12] Pseudomonas putida (no sequence specified) [23] Serratia marcescens (no sequence specified) [10, 42, 43] Corynebacterium sp. (no sequence specified) [15] Hansenula polymorpha (no sequence specified) [4] Sporobolomyces salmonicolor (no sequence specified) [4] Corynebacterium glutamicum (no sequence specified) [36, 39, 47, 48] Lycopersicon esculentum (no sequence specified) [37] Capra hircus (no sequence specified) [29] Salmonella enterica (no sequence specified) [55] Clostridium tetanomorphum (no sequence specified) [17] Thermus sp. (no sequence specified) [27] Proteus morganii (no sequence specified) [8, 14] Porphyridium cruentum (no sequence specified) [30] Yarrowia lipolytica (no sequence specified) [4] Hansenula henricii (no sequence specified) [4] Hansenula anomala (no sequence specified) [4] Pichia guilliermondii (no sequence specified) [4] Rhodosporidium toruloides (no sequence specified) [4] Candida maltosa (no sequence specified) [4, 5] Clostridium propionicum (no sequence specified) [1] Chloroflexus aurantiacus (no sequence specified) [2, 3] Coturnix coturnix japonica (no sequence specified) [54] Cryptococcus terreus (no sequence specified) [4] Brettanomyces anomalus (no sequence specified) [4] Pichia pinus (no sequence specified) [4] Trigonopsis variabilis (no sequence specified) [4] Thiobacillus acidophilus (no sequence specified) [11] Synechococcus PCC7002 PR-6 (no sequence specified) [30] Anacystis marina (no sequence specified) [30] Chroomonas salina (no sequence specified) [30] Hemiselmis virescens (no sequence specified) [30] Tetraselmis maculata (no sequence specified) [30] Cyclotella nana (no sequence specified) [30] Crataegus laevigata (no sequence specified) [46] Rattus norvegicus (UNIPROT accession number: NP446414) [52]

3 Reaction and Specificity Catalyzed reaction l-threonine = 2-oxobutanoate + NH3

357

Threonine ammonia-lyase

4.3.1.19

Reaction type Deamination elimination Natural substrates and products S l-Thr <1, 3, 4, 9, 15, 18, 19, 33> (<9> inducible enzyme [22]; <15,33> constitutive enzyme [3,15]; <3> first reaction of the Ile pathway [39]; <19> key enzyme in biosynthesis of Ile [37]; <18> the enzyme is an important element in the flux control of Ile biosynthesis [47]; <33> isoleucine-insensitive enzyme is subject to glucose-mediated catabolite repression [2]; <4> increase of activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh [9]; <1> the enzyme is formed under anaerobic conditions, the enzyme is induced by l-Ser and l-Thr, cAMP is required for the synthesis of the enzyme [21]) (Reversibility: ?) [2, 3, 9, 15, 21, 22, 37, 39, 47] P ? S l-serine <47> (Reversibility: ?) [52] P pyruvate + NH3 <47> [52] S l-threonine <3, 4, 11, 21, 34, 47> (Reversibility: ?) [49, 50, 51, 52, 53, 54, 55] P 2-oxobutanoate + NH3 <3, 11, 47> [49, 50, 51, 52, 53] Substrates and products S dl-allo-cystathionine <46> (<46> at 20% of the activity with l-Thr [46]) (Reversibility: ?) [46] P ? S l-Cys <24> (Reversibility: ?) [8] P ? S l-Ser <4, 6, 9, 10, 12, 13, 15, 23, 24, 31, 33, 46> (<20> no activity [29]; <6> dehydratase I: 26% of the activity with l-Thr, dehydratase II: 16% of the activity with l-Thr [6]; <15> 6% of the activity with l-Thr [15]; <12> 20% of the activity with l-Ser [12]; <9> rapid loss of activity during Ser deamination [22]; <46> 45% of the activity with l-Thr [46]; <33> isoleucine-insensitive enzyme [3]) (Reversibility: ?) [2, 3, 5, 6, 8, 12, 13, 14, 15, 22, 23, 27, 35, 46] P pyruvate + NH3 + H2 O S l-Thr <1, 3, 4, 9, 15, 18, 19, 33> (<9> inducible enzyme [22]; <15,33> constitutive enzyme [3,15]; <3> first reaction of the Ile pathway [39]; <19> key enzyme in biosynthesis of Ile [37]; <18> the enzyme is an important element in the flux control of Ile biosynthesis [47]; <33> isoleucine-insensitive enzyme is subject to glucose-mediated catabolite repression [2]; <4> increase of activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh [9]; <1> the enzyme is formed under anaerobic conditions, the enzyme is induced by l-Ser and l-Thr, cAMP is required for the synthesis of the enzyme [21]) (Reversibility: ?) [2, 3, 9, 15, 21, 22, 37, 39, 47] P ?

358

4.3.1.19

Threonine ammonia-lyase

S l-Thr + H2 O <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45> (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 48] P 2-oxobutanoate + NH3 + H2 O <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45> [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 48] S l-allothreonine <15> (<15> 5% of the activity l-Thr [15]) (Reversibility: ?) [15] P ? S l-homoserine <31> (Reversibility: ?) [5] P 2-oxobutanoate + NH3 + H2 O S l-serine <47> (Reversibility: ?) [52] P pyruvate + NH3 <47> [52] S l-threonine <3, 4, 11, 21, 34, 47> (Reversibility: ?) [49, 50, 51, 52, 53, 54, 55] P 2-oxobutanoate + NH3 <3, 11, 47> [49, 50, 51, 52, 53] S b-chloro-l-Ala <24> (Reversibility: ?) [8, 14] P ? S Additional information <4> (<4> when pyridoxamine 5’-phosphate is incubated with the apoenzyme in the presence of small quantities of keto acids, e.g. pyruvate or 2-oxobutanoate, small amounts of l-Ala or l-aminobutanoate are formed, the reaction is not reversible [32]) (Reversibility: ?) [32] P ? Inhibitors 2,2’-dithiodipyridine <25, 40, 41, 42, 43, 44, 45> [30] 2-oxobutanoate <14> [42] 2-oxoglutarate <14> [42] biliverdin <4> [33] Ca2+ <11> [50] carbamate <4> [33] citrate <14> [42] Cys <4, 20> (<4> l-Cys and d-Cys [43]) [29, 43] d-Thr <33> [2] dithiothreitol <42, 43> [30] formaldehyde <4> [33] formate <4> [33] glyoxylate <14> [42] Hg2+ <31> [5] hydrazine <25, 40, 41, 42, 43, 44, 45> [30] hydroxylamine <13, 20, 25, 32, 40, 41, 42, 43, 44, 45> (<45> reversed by pyridoxal 5’-phosphate [30]) [1, 23, 29, 30]

359

Threonine ammonia-lyase

4.3.1.19

Ile <3, 6, 7, 8, 10, 13, 15, 18, 19, 20, 23, 24, 31, 33, 39, 45, 46> (<20> no inhibition [29]; <10,23> feed-back inhibition [13,27]; <3> negative allosteric effector [38]; <33> 2 enzyme forms: an isoleucine-sensitive enzyme form and and isoleucine-insensitive form, pH-dependence of inhibition [2]; <10> HgCl2 treated enzyme is less sensitive [13]; <3> biodegradative enzyme is feedback-resistant [39]; <18> native enzyme is totally inhibited by 15 mM Ile, the heterologous catabolic enzyme from E. coli retains 60% of its original activity even in presence of 200 mM Ile [39]; <39> reversed by low concentrations of l-Val [11]; <24> Hg2+ protects [8]; <10> l-Val partially reverses inhibition [13]; <19> 2 different forms: one enzyme form is sensitive to inhibition by Ile, the other form is insensitive to inhibition by Ile [37]; <6> dehydratase I, 50% inhibition at 0.35 mM [6]; <3> competitive allosteric inhibitor, the enzyme exists in two distinct catalytically active species: a tetramer sensitive to l-Ile inhibition and a dimer insensitive to l-Ile inhibition [24]; <31> 50% inhibition at 0.14 mM Ile, 98% inhibition at 1 mM Ile [5]; <15> 0.06 mM, 50% inhibition [15]; <7> more sensitive at 40 C than at 65 C [18]; <3> 0.5 mM, wild type enzyme is completely inhibited at both pH 8.0 and pH 6.5, the mutant enzyme is sensitive only at pH 6.5. In contrast to the wild type enzyme 1 mM Val does not reverse l-Ile inhibition of the mutant enzyme [25]; <46> reversed by Vla [46]) [2, 5, 6, 8, 11, 13, 15, 18, 23, 24, 25, 27, 28, 29, 30, 37, 38, 39, 45, 46, 47] iodoacetamide <40, 41, 44, 45> (<44,45> weak [30]) [30] iodoacetate <20> [29] isoleucine <3, 11> (<3> feedback inhibition [53]; <11> activation below 0.01 mM, strong inhibition above, 50% inhibition at 0.064 mM, inhibition can be reversed by valine [50]; <11> allosteric effector inducing dimerization, inhibition is reversed by high concentrations of valine [51]) [50, 51, 53] isoniazid <25, 40, 41, 44, 45> [30] l-Arg <14> [42] l-isoleucine <11, 21> (<21> allosteric inhibition [55]; <11> at 1 mM the transgenic lines containing omr1-1, omr1-5, and omr1-8 have 85% activity, while the transgenic line containing omr1-7 has 70% activity, the wild-type has 20% activity [56]) [55, 56] l-Val <7, 20, 39> (<39> competitive [11]; <7> slight inhibition [18]) [11, 18, 29] Leu <6, 20> (<6> dehydratase I, 6 mM, 50% inhibition [6]) [6, 29] Mg2+ <11> [50] NEM <25, 40, 41, 42, 43, 44, 45> [30] NSD-1055 <25, 40, 41, 42, 43, 44, 45> [30] Na-chenodeoxycholate <4> [33] Na-cholate <4> [33] Na-cholatemethyl ester <4> [33] Na-deoxycholate <4> [33] Na-lithocholate <4> [33] NaBH4 <13, 32, 33> [1, 2, 23] nitrate <46> [46] PCMB <20, 31> [5, 29]

360

4.3.1.19

Threonine ammonia-lyase

Phosphoenolpyruvate <14> [42] Pyruvate <1, 3, 14> (<3> uncompetitive inhibition and substrate inhibition with respect to l-Thr, noncompetitive inhibition with respect to AMP [7]; <1> noncompetitive inhibition with respect to l-Thr, mixed type inhibition with respect to AMP [7]) [7, 31, 42] Semicarbazide <13, 40, 41, 44, 45> [23, 30] Ser <20, 33> (<33> d-Ser [2]; <20> l-Ser, competitive with respect to l-Thr [29]) [2, 29] Urea <20> [29] bicarbonate <4> [33] cysteamine <4> [43] homoserine <14> [42] methoxylamine <25, 40, 41, 42, 43, 44, 45> [30] p-Chloromercuriphenyl sulfonate <25, 40, 41, 42, 43, 44, 45> [30] phenylhydrazine <13, 32, 45> (<32,45> reversed by pyridoxal 5’-phosphate [1,30]) [1, 23, 30] Cofactors/prosthetic groups ADP <1, 3, 22, 24> (<22, 24> activates [14, 17]; <1> stimulation [7]; <3> no stimulation by ADP [7]) [7, 14, 17] AMP <3, 24> (<3,24> activates [14,17]) [14, 17] Pyridoxal 5’-phosphate <1, 3, 4, 10, 11, 12, 22, 23, 24, 31, 32, 33, 47> (<3, 12, 23, 32> required [1, 12, 27, 45]; <3, 22, 24> contains 4 mol of pyridoxal 5’phosphate per mol of enzyme [8, 14, 17]; <4> activates [35]; <10> tightly bound [13]; <1, 3, 10, 22, 24, 31, 33> cofactor [2, 5, 7, 8, 13, 17, 24, 38]; <1, 3, 24> contains 2 mol of pyridoxal 5’-phosphate per mol of enzyme [7,8,19]; <3> enzyme contains 1 mol of pyridoxal 5’-phosphate per 56000 Da subunit [38]; <4> Km : 0.000682 mM [32]; <4> reactivates after dissociation of the coenzyme [32, 35]; <31> Km : 0.00028 mM [5]; <11> 1 mol per monomer [50]; <11> 1 molecule per monomer [51]) [1, 2, 5, 7, 8, 12, 13, 14, 17, 19, 24, 27, 32, 35, 38, 45, 50, 51, 52] Pyridoxamine 5’-phosphate <4> (<4> reactivates after dissociation of the coenzyme [32,35]) [32, 35] Activating compounds Isoleucine <11> (<11> activation below 0.01 mM, strong inhibition above, 50% inhibition at 0.064 mM [50]) [50] l-Val <3, 10, 15, 31> (<3, 10, 15, 31> activates [5, 13, 15, 38, 45]) [5, 13, 15, 38, 45] NH+4 <11> (<11> 2.1fold increase in activity [50]) [50] Phosphate <2, 5, 16, 17, 26, 27, 28, 29, 30, 31, 35, 36, 37, 38> (<2, 5, 16, 17, 26, 27, 28, 29, 30, 31, 35, 36, 37, 38> increases activity [4,5]; <31> maximal stimulation at 200 mM [5]) [4, 5] Additional information <6, 24> (<6> dehydratase I and II: not affected by adenylates [6]; <24> no activation by AMP, ADP and ATP [8]) [6, 8]

361

Threonine ammonia-lyase

4.3.1.19

Metals, ions K+ <10, 11, 20, 46> (<10,20> stimulates [13,29]; <46> high requirement [46]; <11> 3fold increase in activity, half-maximal activation at 3.1 mM [50]) [13, 29, 46, 50] Li+ <10, 11> (<10> stimulates [13]; <11> 2.8fold increase in activity [50]) [13, 50] NH+4 <10, 20> (<10,20> stimulates [13,29]) [13, 29] Na+ <11> (<11> 1.7fold increase in activity [50]) [50] Rb+ <46> (<46> can partially replace K+ in activation [46]) [46] Additional information <31> (<31> no requirement for divalent cations [5]) [5] Turnover number (min–1) 243 <4> (l-Ser) [32] 287 <4> (l-Thr) [32] Specific activity (U/mg) 0.52 <20> [29] 1 <10> (<10> l-Thr [13]) [13] 2.1 <13> [23] 7.3 <10> (<10> l-Thr [13]) [13] 36.5 <3> [20] 48.3 <8> [28] 82.6 <24> [14] 210 <3> [24, 38] 230 <3> [19, 26] 318 <24> [8] 400 <11> (<11> recombinant threonine deaminase [50]) [50] 683 <1> [34] 989 <4> [41] Additional information <4, 6, 15, 23, 31, 32, 33, 34, 39> (<4> activity is much higher in rat liver than in quail liver, regardless of the nutritional state. The specific activity in the normal rat liver is 15times higher than that of the control quail group. The specific activity in rat liver after fasting increases by a factor of 2.3 over that of normal fed state [54]; <34> activity is much lower in quail liver than in rat liver, regardless of the nutritional state. The specific activity in the normal rat liver is 15times higher than that of the control quail group. Activities in liver of the fastened, the 1% threonine-enriched and the 5% threonine-enriched group are about 2, 1.3 and 1.5times higher, respectively, than that of the control group of quails [54]) [1, 2, 5, 6, 11, 15, 27, 54] Km-Value (mM) 0.25 <19> (l-Ser, <19> Ile-insensitive enzyme form [37]) [37] 0.25 <19, 39> (l-Thr, <19> Ile-sensitive enzyme form and Ile-insensitive form [37]) [11, 37] 1.3 <33> (l-Thr, <33> Ile-sensitive enzyme [2]) [2] 1.5 <24> (l-Thr, <24> in presence of 1 mM AMP [14]) [14]

362

4.3.1.19

Threonine ammonia-lyase

1.6 <33> (l-Thr, <33> Ile-sensitive enzyme [3]) [3] 1.7 <19> (l-Ser, <19> Ile-sensitive form [37]) [37] 2.5 <13, 31> (l-Thr, <31> in absence of phosphate [4]) [4, 23] 3 <1, 31> (l-Thr, <31> in presence of 50 mM phosphate [4]; <1> with AMP [7]) [4, 5, 7] 3.2 <24> (l-Thr) [8] 4 <31> (l-Thr, <31> in presence of 250 mM phosphate [4]) [4] 4.5 <1> (l-Thr, <1> wild type enzyme [34]) [34] 5 <3> (l-Ser, <3> in presence of AMP [17]) [17] 5.7 <7> (l-Thr, <7> at 40 C [18]) [18] 7.1 <24> (l-Ser) [8] 7.7 <32> (l-Thr) [1] 8 <1> (l-Thr, <1> with AMP [7]) [7] 8.9 <24> (l-Ser, <24> in presence of AMP [14]) [14] 10 <33> (l-Ser, <33> Ile-insensitive enzyme [2,3]) [2, 3] 11 <3> (l-Thr, <3> in presence of AMP [17]) [17] 13 <6> (l-Thr, <6> dehydratase II [6]) [6] 13.9 <7> (l-Thr, <7> at 65 C [18]) [18] 14 <23> (l-Thr) [27] 16 <20> (l-Thr) [29] 19 <1> (l-Thr, <1> mutant strain DU-21 [34]) [34] 20 <33> (l-Thr, <33> Ile-insensitive enzyme [2,3]) [2, 3] 21.3 <33> (l-Thr, <33> Ile-sensitive enzyme [2]) [2] 24 <24> (l-Thr, <24> in absence of AMP [14]) [14] 30.3 <24> (l-Ser, <24> in absence of AMP [14]) [14] 34 <6> (l-Thr, <6> dehydratase I [6]) [6] 40 <3, 13> (l-Ser, <3> in absence of AMP [17]) [17, 23] 61 <4> (l-Ser) [35] 61.8 <4> (l-Ser) [32] 70 <3> (l-Thr, <3> without AMP [7]) [7] 80 <1> (l-Ser, <1> mutant strain DU-21 [34]) [34] 90 <1> (l-Ser, <1> wild type enzyme [34]) [34] 91 <3> (l-Thr, <3> in absence of AMP [17]) [17] 99.5 <4> (l-Thr) [32] 125 <1> (l-Thr, <1> without AMP [7]) [7] 129 <4> (l-Thr) [35] 380 <32> (l-Ser) [1] Ki-Value (mM) 0.06 <15> (Ile) [15] 0.14 <31> (Ile) [5] pH-Optimum 7.4-9.2 <3> (<3> in presence of AMP [17]) [17] 7.5 <13> [23] 8 <11, 23> [27, 50] 8-9 <15, 20> (<15> Tris/HCl buffer and diethanolamine buffer [15]) [15, 29] 8-9.5 <24> (<24> in presence of 1 mM AMP [14]) [14]

363

Threonine ammonia-lyase

4.3.1.19

8.5 <6, 40> (<6> dehydratase II [6]) [6, 30] 8.7 <44> [30] 8.7-9.1 <12> [12] 8.8 <31, 42, 45> [5, 30] 8.9 <41, 43> [30] 9 <10, 24> (<24> in absence of AMP [14]) [13, 14] 9-9.8 <3> (<3> in absence of AMP [17]) [17] 9-10 <39> [11] 9.2-9.6 <7, 46> (<7> at 40 C and 65 C [18]) [18, 46] 9.4-9.6 <3> [20] 9.5 <25> [30] 9.5-11 <6> (<6> dehydratase I [6]) [6] 10 <24> (<24> with l-Thr and l-Ser as substrate [8]) [8] pH-Range 7-8 <13> (<13> 65% of maximal activity at pH 7 and at pH 8 [23]) [23] 7.5-9.5 <4, 20> (<20> pH 7.5: about 55% of maximal activity, pH 9.5: about 85% of maximal activity [29]; <4> activity increases from pH 7.5 to pH 9.5 [35]) [29, 35] 8.1-9.6 <31> (<31> 50% of maximal activity at pH 8.1 and 9.6 [5]) [5] 8.5-11 <24> (<24> pH 8.5: about 30% of maximal activity, pH 11.0: about 70% of maximal activity, with l-Thr as substrate [8]) [8] Temperature optimum ( C) 35 <31> (<31> in absence of phosphate [4]) [4] 37 <31> (<31> in presence of 50 mM phosphate [4]) [4] 44 <31> (<31> in presence of 250 mM phosphate [4]) [4] 65 <7> [18] 70 <20> [29] 85-90 <23> [27] Temperature range ( C) 45-80 <20> (<20> 45 C: about 25% of maximal activity, 80 C: about 35% of maximal activity [29]) [29] 60-98 <23> (<23> 60 C: about 30% of maximal activity, 98 C: about 15% of maximal activity [27]) [27]

4 Enzyme Structure Molecular weight 64000 <47> [52] 100000-115000 <23> (<23> gel filtration [27]) [27] 106000 <33> (<33> gel filtration [2]) [2] 118000 <14> (<14> gel filtration [42]) [42] 120000 <14, 24, 33> (<24> gel filtration [14]; <14> native gradient PAGE [42]) [3, 14, 42]

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140000 <11> (<11> native PAGE, in the presence of the allosteric effector isoleucine [51]) [51] 147000 <3> (<3> sedimentation equilibrium ultracentrifugation [16,17]) [16, 17] 160000 <32> (<32> gel filtration [1]) [1] 184000 <22> [17] 190000 <3, 31> (<31> gel filtration [5]; <3> density gradient centrifugation [26]) [5, 26] 200000 <7, 19> (<7> gel filtration [18]; <19> Ile-insensitive enzyme form, gel filtration [37]) [18, 37] 201000 <3> (<3> equilibrium sedimentation [25]) [25] 203800 <3> (<3> sedimentation equilibrium experiments [24]) [24] 210000 <3, 8, 18> (<8> gel filtration [28]; <3> meniscus depletion sedimentation equilibrium [26]) [26, 28, 47] 214000 <3> (<3> meniscus depletion equilibrium sedimentation, analytical ultracentrifugation [19]) [19] 228000 <24> (<24> calculation from sedimentation and diffusion data [8]) [8] 230000 <24> (<24> gel filtration [8]) [8] 250000 <6> (<6> dehydratase II, gel filtration [6]) [6] 268000 <11> (<11> native PAGE, in the absence of allosteric effector [51]) [51] 370000 <19> (<19> Ile-sensitive enzyme form, gel filtration [37]) [37] Subunits dimer <11, 33, 47> (<33> 2 * 55000, SDS-PAGE [2]; <11> 2 * 59567, mass spectrometry, addition of isoleucine induces dimerization, tetrmerization is restored by addition of high valine concentration [51]) [2, 51, 52] tetramer <1, 3, 7, 11, 14, 18, 24, 31, 32> (<3> 4 * 50000, SDS-PAGE [19,26]; <31> 4 * 46000, SDS-PAGE [5]; <24> 4 * 58000, SDS-PAGE [8]; <14> 4 * 30000, SDS-PAGE [42]; <3> x * 38000, SDS-PAGE [7,16]; <3> 4 * 57000, SDS-PAGE [20]; <1> x * 36000, SDS-PAGE [7]; <7> 4 * 49000, SDS-PAGE [18]; <24> 4 * 32000, SDS-PAGE [14]; <32> 4 * 39000, SDS-PAGE [1]; <3> 4 * 56000, SDS-PAGE [38]; <3> 4 * 53000, the enzyme exists in two distinct catalytically active species: a tetramer sensitive to l-Ile inhibition and a dimer insensitive to l-Ile inhibition, SDS-PAGE [24]; <3> 4 * 49800, equilibrium sedimentation of the enzyme dialyzed against 6 M guanidine hydrochloride [25]; <18> 4 * 46599, calculation from nucleotide sequence [47]; <11> 4 * 59567, mass spectrometry [51]; <11> 4 * 59800, SDS-PAGE [50]) [1, 5, 7, 8, 14, 16, 18, 19, 20, 24, 25, 26, 38, 42, 47, 50, 51]

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4.3.1.19

5 Isolation/Preparation/Mutation/Application Source/tissue hepatocyte <4> (<4> primary culture [9]) [9] leaf <10, 19> (<19> isoleucine-sensitive enzyme form occurs predominantly in younger leaves, isoleucine-insensitive enzyme form occurs predominantly in older leaves [37]) [13, 37] liver <4, 9, 20, 34, 47> [22, 29, 32, 33, 35, 41, 43, 52, 54] tissue culture <46> [46] Purification <1> (mutant strain DU-21 with an activator site-deficient enzyme form) [34] <3> [19, 20, 24, 25, 26, 38] <4> [35, 41] <6> (partial) [6] <7> [18] <8> [28] <9> [22] <10> [13] <11> (recombinant threonine deaminase) [50, 51] <13> [23] <14> [42] <15> (partial) [15] <20> [29] <23> (partial) [27] <24> [8, 14] <31> [5] <32> [1] <33> [2] <39> (partial) [11] Crystallization <3> (crystal structure at 2.8 A resolution) [44] <24> [14] Cloning <3> (expression in Brevibacterium flavum) [38, 39, 40] <3> (expression in Corynebacterium glutamicum) [39] <3> (expression of tdcB in Corynebacterium glutamicum ATCC 21799) [49] <3> (ilvA expression in Nicotiana tabaccum is effectively utilized as a selectable marker gene to identify tobacco transformants when coupled with l-Omethylthreonine as the selction agent) [53] <11> [56] <11> (expression in Escherichia coli) [50, 51] <18> [36]

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Engineering E520A <11> (<11> omr1-7 allele, tolerates high concentrations of l-Omethylthreonine [56]) [56] H542L <11> (<11> omr1-8 allele, tolerates high concentrations of l-Omethylthreonine [56]) [56] R499C <11> (<11> omr1-5 allele, tolerates high concentrations of l-Omethylthreonine [56]) [56] R499C/R544H <11> (<11> omr1-1 allele, tolerates high concentrations of lO-methylthreonine [56]) [56] Val323Ala <18> (<18> feedback inhibition by l-Ile is entirely abolished, so that the enzyme is always present in a relaxed high-activity state [36]) [36] Y449L <11> (<11> concentration of isoleucine needed to reach 50% inhibition increases by a factor 45, two different effector-binding sites are constituted in part by Y449 and Y543 [50]) [50] Y543L <11> (<11> concentration of isoleucine needed to reach 50% inhibition increases by a factor 38, two different effector-binding sites are constituted in part by Y449 and Y543 [50]) [50] Additional information <1, 3, 21> (<3> the enzyme from the regulatory mutant CU18 is indistinguishable from the wild type enzyme in molecular weight and subunit composition [25]; <1> mutant with an activator site-deficient enzyme form, the Km for l-Thr is increased 4fold as compared with the wild type enzyme [34]; <21> mutants lacking yjgF generate an isoleucineinsensitive protein [55]) [25, 34, 55] Application molecular biology <11> (<11> in contrast to the wild-type, all four transgenic TD lines are able to tolerate high concentrations of l-O-methylthreonine. This illustrates the potential use of these mutant omr genes as dominant selectable markers in plant transformation [56]) [56]

6 Stability Temperature stability 0 <10> (<10> 10 h, 81% loss of activity in presence of 0.05 mM Ile, stable in presence of 1 mM Ile [13]) [13] 27 <10> (<10> 10 h, 27% loss of activity in presence of 0.05 mM Ile, stable in presence of 1 mM Ile [13]) [13] 37 <6> (<6> 10 min, 16% loss of activity of dehydratase I and 36% loss of activity of dehydratase II, loss of activity of dehydratase I is prevented by 1 mM Ile, but that of dehydratase II is not [6]) [6] 45 <31> (<31> 15 min, complete loss of activity in absence of phosphate, 94% loss of activity in presence of 50 mM phosphate, 49% loss of activity in presence of 250 mM phosphate [4]) [4] 55 <12> (<12> 10 min, stable [12]) [12] 70 <23> (<23> 1 h, 15% loss of activity [27]) [27] 90 <23> (<23> rapid inactivation [27]) [27]

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General stability information <6>, loss of activity of dehydratase I at 37 C is prevented by 1 mM Ile, but that of dehydratase II is not [6] <8>, dithiothreitol, allothreonine and pyridoxal phosphate are all required to maintain a stable form of threonine dehydratase [28] <10>, l-Ile protects the enzyme against inactivation at low temperatures [13] Storage stability <3>, -20 C, 50% loss of activity after 3 weeks [19] <3>, -20 C, no loss of activity after several weeks [26] <7>, 4 C, pH 7.2 or 9.0, 24 h, complete inactivation after 7 days, stable in presence of egg albumin [18] <10>, 4 C, little loss of activity after 50 days [13] <20>, 0-4 C, stable for at least 2 months [29] <23>, -20 C, 0.05 M potassium phosphate, pH 7.2, enzyme concentration 0.06 mg/ml, half-life: 4 weeks [27] <39>, 4 C, loss of activity after storage of more than one day [11] <46>, 0 C, rapid loss of activity unless maintained in presence of Ile and potassium phosphate [46]

References [1] Hofmeister, A.E.M.; Grabowski, R.; Linder, D.; Buckel, W.: l-Serine and lthreonine dehydratase from Clostridium propionicum. Two enzymes with different prosthetic groups. Eur. J. Biochem., 215, 341-349 (1993) [2] Laakmann-Ditges, G.; Klemme, J.H.: Amino acid metabolism in the thermophilic phototroph, Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases. Arch. Microbiol., 149, 249-254 (1988) [3] Laakmann-Ditges, G.; Klemme, J.H.: Occurence of two l-threonine (L-serine) dehydratases in thermophile Chloroflexus aurantiacus. Arch. Microbiol., 144, 219-221 (1986) [4] Bode, R.; Birnbaum, D.: Threonine dehydratase activity from several yeast species is activated and affected by phosphate. FEMS Microbiol. Lett., 37, 189-192 (1986) [5] Bode, R.; Schult, I.; Birnbaum, D.: Purification and some properties of lthreonine dehydratase from Candida maltosa. J. Basic Microbiol., 26, 443451 (1986) [6] Oda, Y.; Nakano, Y.; Kitaoka, S.: Occurence and some properties of two threonine dehydratases in Euglena gracilis. J. Gen. Microbiol., 129, 57-61 (1983) [7] Kim, S.S.; Datta, P.: Chemical characterization of biodegradative threonine dehydratases from two enteric bacteria. Biochim. Biophys. Acta, 706, 27-35 (1982)

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[8] Yoshida, H.; Hanada, K.; Ohsawa, H.; Kumagai, H.; Yamada, H.: Biosynthetic threonine deaminase from Proteus morganii. Agric. Biol. Chem., 46, 1035-1042 (1982) [9] Mak, W.W.N.; Pitot, H.C.: Increase of l-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh. Biochem. J., 198, 499-504 (1981) [10] Crout, D.H.G.; Gregorio, M.V.M.; Miller, U.S.; Komatsubara, S.; Kisumi, M.; Chibata, I.: Stereochemistry of the conversions of l-threonine and d-threonine into 2-oxobutanoate by the l-threonine and d-threonine dehydratases of Serratia marcescens. Eur. J. Biochem., 106, 97-105 (1980) [11] Proteau, G.; Silver, M.: Effects of amino acids on Thiobacillus acidophilus. II. Threonine deaminase. Can. J. Microbiol., 26, 385-388 (1980) [12] Lam, V.M.S.; Yeung, Y.G.: Properties of threonine deaminase from Pseudomonas aeruginosa. FEMS Microbiol. Lett., 3, 219-221 (1978) [13] Sharma, R.K.; Mazumder, R.: Purification, properties, and feedback control of l-threonine dehydratase from spinach. J. Biol. Chem., 245, 3008-3014 (1970) [14] Kumagai, H.; Nishimura, T.; Yamada, H.: Biodegradative threonine deaminase from Proteus morganii. Agric. Biol. Chem., 42, 613-621 (1978) [15] Bell, S.C.; Turner, J.M.: Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine hydro-lyase (deaminating) in a species of Corynebacterium. Biochem. J., 164, 579-587 (1977) [16] Saeki, Y.; Ito, S.; Shizuta, Y.; Hayaishi, O.; Kagamiyama, H.; Wada, H.: Subunit structure of biodegradative threonine deaminase. J. Biol. Chem., 252, 2206-2208 (1977) [17] Shizuta, Y.; Hayaishi, O.: Regulation of biodegradative threonine deaminase. Curr. Top. Cell. Regul., 11, 99-146 (1976) [18] Muramatsu, N.; Nosoh, Y.: Some catalytic and molecular properties of threonine deaminase from Bacillus stearothermophilus. J. Biochem., 80, 485-490 (1996) [19] Koerner, K.; Rahimi-Laridjani, I.; Grimminger, H.: Purification of biosynthetic threonine deaminase from Escherichia coli. Biochim. Biophys. Acta, 397, 220-230 (1975) [20] Kagan, Z.S.; Dorozhko, A.I.; Kovaleva, S.V.; Yakovleva, L.I.: Studies of homogeneous biosynthetic l-threonine dehydratase from Escherichia coli K-12. Some kinetic properties and molecular multiplicity. Biochim. Biophys. Acta, 403, 208-220 (1975) [21] Luginbuhl, G.H.; Hofler, J.G.; Decedue, C.J.; Burns, R.O.: Biodegradative lthreonine deaminase of Salmonella typhimurium. J. Bacteriol., 120, 559-561 (1974) [22] Doonan, S.; Koerner, D.H.; Schmutzler, W.; Vernon, C.A.: Inducibility and some properties of the threonine dehydratase of sheep liver. Biochem. J., 144, 533-541 (1974) [23] Cohn, M.S.; Phillips, A.T.: Purification and characterization of a B6-independent threonine dehydratase from Pseudomonas putida. Biochemistry, 13, 1208-1214 (1974)

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[24] Calhoun, D.H.; Rimerman, R.A.; Hatfield, G.W.: Threonine deaminase from Escherichia coli. I. Purification and properties. J. Biol. Chem., 248, 35113516 (1973) [25] Calhoun, D.H.; Kuska, J.S.; Hatfield, G.W.: Threonine deaminase from Escherichia coli. II. Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression. J. Biol. Chem., 250, 127131 (1974) [26] Grimminger, H.; Rahimi-Laridjani, I.; Koerner, K.; Lingens, F.: Purification of threonine deaminase from Escherichia coli. FEBS Lett., 35, 273-275 (1973) [27] Higa, E.H.; Ramaley, R.F.: Purification and properties of threonine deaminase from the X-1 isolate of the genus Thermus. J. Bacteriol., 114, 556-562 (1973) [28] Barritt, G.J.; Morrison, J.F.: Purification and properties of threonine dehydratase from Rhodopseudomonas spheroides. Biochim. Biophys. Acta, 284, 508-520 (1972) [29] Nath, M.; Sanwal, G.G.: Threonine dehydratase from goat liver. Arch. Biochem. Biophys., 151, 420-426 (1972) [30] Desai, I.D.; Laub, D.; Anita, N.J.: Comparative characterization of l-threonine dehydratase in seven species of unicellular marine algae. Phytochemistry, 11, 277-287 (1972) [31] Park, L.S.; Datta, P.: Inhibition of Escherichia coli biodegradative threonine dehydratase by pyruvate. J. Bacteriol., 138, 1026-1028 (1979) [32] Leoncini, R.; Vannoni, D.; di Pietro, C.; Guerranti, R.; Rosi, F.; Pagani, R.; Marinello, E.: Restoration of rat liver l-threonine dehydratase activity by pyridoxamine 5 -phosphate: the half-transaminating activity of l-threonine dehydratase and its regulatory role. Biochim. Biophys. Acta, 1425, 411-418 (1998) [33] Pagani, R.; Leoncini, R.; Terzuoli, L.; Guerranti, R.; Marinello, E.: In vitro regulation of rat liver l-threonine deaminase by different effectors. Enzyme, 43, 122-128 (1990) [34] Burns, R.O.; Hofler, J.G.; Luginbuhl, G.H.: Threonine deaminase from Salmonella typhimurium. Substrate-specific patterns of inhibition in an activator site-deficient form of the enzyme. J. Biol. Chem., 254, 1074-1079 (1979) [35] Pagani, R.; Leoncini, R.; Pizzichini, M.; Vannoni, D.; Tabucchi, A.; Marinello: Properties of rat liver l-threonine deaminase. Enzyme Protein, 48, 90-97 (1994) [36] Sahm, H.; Eggeling, L.; Eikmanns, B.; Kraemer, R.: Construction of l-lysine-, l-threonine-, and l-isoleucine-overproducing strains of Corynebacterium glutamicum. Ann. N.Y. Acad. Sci., 782, 25-39 (1996) [37] Szamosi, I.; Shaner, D.L.; Singh, B.K.: Identification and characterization of a biodegradative form of threonine dehydratase in senescing tomato (Lycopersicon esculentum) leaf. Plant Physiol., 101, 999-1004 (1993) [38] Eisenstein, E.: Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli. J. Biol. Chem., 266, 5801-5807 (1991)

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[39] Guillouet, S.; Rodal, A.A.; An, G.H.; Lessard, P.A.; Sinskey, A.J.: Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production. Appl. Environ. Microbiol., 65, 3100-3107 (1999) [40] Hashiguchi, K.; Kojima, H.; Sato, K.; Sano, K.: Effects of an Escherichia coli ilvA mutant gene encoding feedback-resistant threonine deaminase on lisoleucine production by Brevibacterium flavum. Biosci. Biotechnol. Biochem., 61, 105-108 (1997) [41] Leoncini, R.; Guerranti, R.; Pagani, R.; Marinello, E.: An improved method for purification of l-threonine deaminase from rat liver. J. Biochem. Biophys. Methods, 20, 97-105 (1990) [42] Choi, B.B.; Kim, S.S.: Inhibition of the biodegradative threonine dehydratase from Serratia marcescens by a-keto acids and their derivatives. J. Biochem. Mol. Biol., 28, 118-123 (1995) [43] Leoncini, R.; Pagani, R.; Marinello, E.; Keleti, T.: Double inhibition of lthreonine dehydratase by aminothiols. Biochim. Biophys. Acta, 994, 52-58 (1989) [44] Gallagher, D.T.; Gilliland, G.L.; Xiao, G.; Zondlo, J.; Fisher, K.E.; Chinchilla, D.; Eisenstein; E.: Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. Structure, 6, 465-475 (1998) [45] Eisenstein, E.: Allosteric regulation of biosynthetic threonine deaminase from Escherichia coli: effects of isoleucine and valine on active-site ligand binding and catalysis. Arch. Biochem. Biophys., 316, 311-318 (1995) [46] Dougall, D.K.: Threonine deaminase from Paul’s Scarlet rose tissue cultures. Phytochemistry, 9, 959-964 (1979) [47] Moeckel, B.; Eggeling, L.; Sahm, H.: Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum. J. Bacteriol., 174, 8065-8072 (1992) [48] Morbach, S.; Kelle, R.; Winkels, S.; Sahm, H.; Eggeling, L.: Engineering the homoserine dehydrogenase and threonine dehydratase control points to analyse flux towards l-isoleucine in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol., 45, 612-620 (1996) [49] Guillouet, S.; Rodal, A.A.; An, G.H.; Gorret, N.; Lessard, P.A.; Sinskey, A.J.: Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum. Appl. Microbiol. Biotechnol., 57, 667-673 (2001) [50] Wessel, P.M.; Graciet, E.; Douce, R.; Dumas, R.: Evidence for two distinct effector-binding sites in threonine deaminase by site-directed mutagenesis, kinetic, and binding experiments. Biochemistry, 39, 15136-15143 (2000) [51] Halgand, F.; Wessel, P.M.; Laprevote, O.; Dumas, R.: Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine. Biochemistry, 41, 1376713773 (2002) [52] Scarselli, M.; Padula, M.G.; Bernini, A.; Spiga, O.; Ciutti, A.; Leoncini, R.; Vannoni, D.; Marinello, E.; Niccolai, N.: Structure and function correlations between the rat liver threonine deaminase and aminotransferases. Biochim. Biophys. Acta, 1645, 40-48 (2003)

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[53] Ebmeier, A.; Allison, L.; Cerutti, H.; Clemente, T.: Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation. Planta, 218, 751-758 (2004) [54] Akagi, S.; Sato, K.; Ohmori, S.: Threonine metabolism in Japanese quail liver. Amino Acids, 26, 235-242 (2004) [55] Schmitz, G.; Downs, D.M.: Reduced transaminase B (IlvE) activity caused by the lack of yjgF is dependent on the status of threonine deaminase (IlvA) in Salmonella enterica serovar typhimurium. J. Bacteriol., 186, 803-810 (2004) [56] Garcia, E.L.; Mourad, G.S.: A site-directed mutagenesis interrogation of the carboxy-terminal end of Arabidopsis thaliana threonine dehydratase/deaminase reveals a synergistic interaction between two effector-binding sites and contributes to the development of a novel selectable marker. Plant Mol. Biol., 55, 121-134 (2004)

372

erythro-3-Hydroxyaspartate ammonia-lyase

4.3.1.20

1 Nomenclature EC number 4.3.1.20 Systematic name erythro-3-hydroxy-lS -aspartate ammonia-lyase Recommended name erythro-3-hydroxyaspartate ammonia-lyase Synonyms 3-hydroxyaspartate dehydratase dehydratase, 3-hydroxyaspartate EC 4.2.1.38 erythro-3-hydroxyaspartate hydro-lyase (deaminating) erythro-b-hydroxyaspartate dehydratase hydroxyaspartate dehydratase CAS registry number 37290-74-7

2 Source Organism <1> Sus scrofa (no sequence specified) [3] <2> Micrococcus denitrificans (no sequence specified) [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction erythro-3-hydroxy-lS -aspartate = oxaloacetate + NH3 Reaction type elimination Natural substrates and products S erythro-3-hydroxyaspartate <2> (<2> b-hydroxyaspartate pathway [1]; <2> glycine-glycolate metabolism [2]) (Reversibility: ?) [1, 2] P ?

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erythro-3-Hydroxyaspartate ammonia-lyase

4.3.1.20

S Additional information <1> (<1> hydroxyaspartate dehydratase and aconitase activities reside on the same protein and at a common active site [3]) (Reversibility: ?) [3] P ? Substrates and products S erythro-3-hydroxyaspartate <2> (<2> b-hydroxyaspartate pathway [1]; <2> glycine-glycolate metabolism [2]) (Reversibility: ?) [1, 2] P ? S erythro-dl-b-hydroxyaspartate <2> (<2> irreversible, enzyme is highly substrate specific using only the l-isomer [1]) (Reversibility: ?) [1, 2] P oxaloacetate + NH3 <2> [1, 2] S Additional information <1, 2> (<2> enzyme does not use threo-dl-b-hydroxyaspartate, erythro and threo-dl-b-hydroxy-b methylaspartate, allo d-isomers of serine, threonine and allothreonine, d and l-aspartate, and isomers of tartrate [1]; <1> hydroxyaspartate dehydratase and aconitase activities reside on the same protein and at a common active site [3]) (Reversibility: ?) [1, 3] P ? Inhibitors 4-pyridinecarboxylic acid <2> [1] EDTA <2> (<2> 0.1 mM almost complete inhibition, activity restored by divalent cations [1]) [1, 2] hydroxylamine <2> [1, 2] maleate <2> (<2> competitive [1,2]) [1, 2] semicarbazide <2> [1] p-chloromercuribenzoate <2> (<2> GSH protects [1]) [1, 2] Cofactors/prosthetic groups Pyridoxal 5’-phosphate <2> (<2> activates [1]; <2> required for activity [2]) [1, 2] Metals, ions Ca2+ <2> (<2> restores activity after EDTA treatment [1]; <2> required for activity [2]) [1, 2] Mg2+ <2> (<2> restores activity after EDTA treatment [1]; <2> required for activity [2]) [1, 2] Mn2+ <2> (<2> restores activity after EDTA treatment [1]) [1] Specific activity (U/mg) 7.15 <2> [1, 2] Km-Value (mM) 0.43 <2> (erythro-l-b-hydroxyaspartate) [1, 2] Additional information <2> (<2> Km -value varied with pH, optimum at pH 7.8 [1]) [1] pH-Optimum 9.5 <2> [1, 2]

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pH-Range 5-9 <2> [1]

5 Isolation/Preparation/Mutation/Application Source/tissue heart <1> [3] Purification <2> (partial) [1, 2]

6 Stability pH-Stability 6-10 <2> (<2> rapid loss of activity below pH 6 and above pH 10 [1]) [1] 7.5-9.5 <2> (<2> most stable [1]) [1] Storage stability <2>, -15 C, with 0.01M Tris-HCl buffer, pH 8, 1 mM MgCl2 and 0.01 mM pyridoxal 5’-phosphate, 50% loss of activity in 60 days [1, 2] <2>, 2 C, with 0.01M Tris-HCl buffer, pH 8, 1 mM MgCl2 and 0.01 mM pyridoxal 5’-phosphate, 50% loss of activity in 10 days [1, 2]

References [1] Gibbs, R.G.; Morris, J.G.: Purification and properties of erythro-b-hydroxyaspartate dehydratase from Micrococcus denitrificans. Biochem. J., 97, 547-554 (1965) [2] Gibbs, R.G.; Morris, J.G.: Glycine-Glyoxylate metabolism: b-hydroxyaspartate pathway (Micrococcus denitrificans). Methods Enzymol., 17A, 981-992 (1970) [3] Porter, D.J.T.; Alston, T.A.; Bright, H.J.: CO2 adducts as reactive analogues of carboxylate substrates for aconitase and other enzymes of carbohydrate metabolism. J. Biol. Chem., 262, 6552-6563 (1987)

375

Aminodeoxygluconate ammonia-lyase

1 Nomenclature EC number 4.3.1.21 (deleted, identical to EC 4.3.1.9) Recommended name aminodeoxygluconate ammonia-lyase

376

4.3.1.21

3,4-Dihydroxyphenylalanine reductive deaminase

4.3.1.22

1 Nomenclature EC number 4.3.1.22 Systematic name 3,4-dihydroxy-l-phenylalanine ammonia-lyase (3,4-dihydroxyphenylpropanoate-forming) Recommended name 3,4-dihydroxyphenylalanine reductive deaminase Synonyms DOPA-reductive deaminase <1> [1] DOPARDA <1> [1] reductive deaminase <1> [1]

2 Source Organism <1> Rhodobacter sphaeroides (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction 3,4-dihydroxy-l-phenylalanine + 2 NADH = 3,4-dihydroxyphenylpropanoate + 2 NAD+ + NH3 Substrates and products S 3,4-dihydroxy-l-phenylalanine + NADH <1> (Reversibility: ?) [1] P 3,4-dihydroxyphenylpropanoate + NAD+ + NH3 S 3,4-dihydroxy-l-phenylalanine + NADPH <1> (<1> activity with NADPH is less than 40% of the activity with NADH [1]) (Reversibility: ?) [1] P 3,4-dihydroxyphenylpropanoate + NADP+ + NH3 Cofactors/prosthetic groups NADH <1> (<1> required [1]) [1] NADPH <1> (<1> less than 40% of the activity with NADH [1]) [1] Km-Value (mM) 0.21 <1> (3,4-dihydroxy-l-phenylalanine) [1]

377

3,4-Dihydroxyphenylalanine reductive deaminase

4.3.1.22

pH-Optimum 7 <1> [1] Temperature optimum ( C) 40 <1> [1]

4 Enzyme Structure Molecular weight 274000 <1> (<1> non-denaturing PAGE [1]) [1] Subunits tetramer <1> (<1> 1 * 110000 + 1 * 82000 + 1 * 43000 + 1 * 39000, SDSPAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> [1]

References [1] Ranjith, N.K.; Sasikala, C.; Ramana Ch, V.: Catabolism of l-phenylalanine and l-tyrosine by Rhodobacter sphaeroides OU5 occurs through 3,4-dihydroxyphenylalanine. Res. Microbiol., 158, 506-511 (2007)

378

Deacetylisoipecoside synthase

4.3.3.3

1 Nomenclature EC number 4.3.3.3 Systematic name deacetylisoipecoside dopamine-lyase (secologanin-forming) Recommended name deacetylisoipecoside synthase CAS registry number 192827-94-4

2 Source Organism <1> Alangium lamarckii (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction deacetylisoipecoside + H2 O = dopamine + secologanin Reaction type C-C bond formation C-N bond formation Natural substrates and products S dopamine + secologanin <1> (<1> involved in production of tetrahydroquinoline monoterpene alkaloide derivates [1]) (Reversibility: ?) [1] P (1S)-deacetylisoipecoside + H2 O <1> [1] Substrates and products S dopamine + secologanin <1> (<1> involved in production of tetrahydroquinoline monoterpene alkaloide derivates [1]) (Reversibility: ?) [1] P (1S)-deacetylisoipecoside + H2 O <1> [1] pH-Range 7.5 <1> [1]

379

Deacetylisoipecoside synthase

4.3.3.3

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [1] Purification <1> (partial) [1]

References [1] DeEknamkul, W.; Ounaroon, A.; Tanahashi, T.; Kutchan, T.; Zenk, M.H.: Enzymatic condensation of dopamine and secologanin by cell-free extracts of Alangium lamarckii. Phytochemistry, 45, 477-484 (1997)

380

Deacetylipecoside synthase

4.3.3.4

1 Nomenclature EC number 4.3.3.4 Systematic name deacetylipecoside dopamine-lyase (secologanin-forming) Recommended name deacetylipecoside synthase CAS registry number 192827-93-3

2 Source Organism <1> Alangium lamarckii (no sequence specified) [1, 2]

3 Reaction and Specificity Catalyzed reaction deacetylipecoside + H2 O = dopamine + secologanin Reaction type dopamine-secologanin condensation Natural substrates and products S dopamine + secologanin <1> (<1> involved in production of tetrahydroquinoline monoterpene alkaloide derivates [1]) (Reversibility: ?) [1, 2] P (1R)-deacetylipecoside + H2 O <1> [1] Substrates and products S dopamine + secologanin <1> (<1> highly specific for dopamine [2]; <1> involved in production of tetrahydroquinoline monoterpene alkaloide derivates [1]) (Reversibility: ?) [1, 2] P (1R)-deacetylipecoside + H2 O <1> [1] Inhibitors alangimarckine <1> (<1> Ki : 0.01 mM [2]) [2] dehydroalangimarckine <1> (<1> Ki : 0.01 mM [2]) [2] Additional information <1> (<1> not inhibited by dopamine, tyramine, tryptamine, cephaeline, emetine and tubulosine [2]) [2]

381

Deacetylipecoside synthase

4.3.3.4

Specific activity (U/mg) 0.078 <1> (<1> crude extract [2]) [2] 43.02 <1> (<1> purified enzyme [2]) [2] Km-Value (mM) 0.7 <1> (dopamine, <1> with fixed concentration of secologanin of 5 mM [2]) [2] 0.9 <1> (secologanin, <1> with fixed concentration of dopamine of 5 mM [2]) [2] pH-Optimum 7.5 <1> [2] pH-Range 7.5 <1> [1] Temperature optimum ( C) 45 <1> [2]

4 Enzyme Structure Molecular weight 30000 <1> (<1> gel filtration, SDS-PAGE [2]) [2] Subunits monomer <1> (<1> gel filtration, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [1, 2] Purification <1> [2] <1> (partial) [1]

6 Stability Storage stability <1>, -20 - 4 C, 50% loss of activity within 5 days, complete loss of activity within 7 days [2]

382

4.3.3.4

Deacetylipecoside synthase

References [1] DeEknamkul, W.; Ounaroon, A.; Tanahashi, T.; Kutchan, T.; Zenk, M.H.: Enzymatic condensation of dopamine and secologanin by cell-free extracts of Alangium lamarckii. Phytochemistry, 45, 477-484 (1997) [2] DeEknamkul, W.; Suttipanta, N.; Kutchan, T.: Purification and characterization of deacetylipecoside synthase from Alangium lamarckii Thw.. Phytochemistry, 55, 177-181 (2000)

383

Prenylcysteine lyase

1 Nomenclature EC number 4.4.1.18 (transferred to EC 1.8.3.5) Recommended name prenylcysteine lyase

384

4.4.1.18

Phosphosulfolactate synthase

4.4.1.19

1 Nomenclature EC number 4.4.1.19 Systematic name (2R)-O-phospho-3-sulfolactate hydrogen-sulfite-lyase (phosphoenolpyruvateforming) Recommended name phosphosulfolactate synthase Synonyms (2R)-phospho-3-sulfolactate synthase ComA CAS registry number 473575-53-0

2 Source Organism <1> Methanococcus jannaschii (no sequence specified) [1, 2]

3 Reaction and Specificity Catalyzed reaction (2R)-O-phospho-3-sulfolactate = hydrogen sulfite + phosphoenolpyruvate (<1> mechanism, enzyme is not a member of the enolase superfamily [2]; <1> catalyses the Michael addition of sulfite to phosphoenolpyruvate, requires Mg2+ , specifically requires phosphoenolpyruvate and its broad alkaline pH optimum suggests that it uses sulfite rather than bisulfite [1]) Reaction type addition Natural substrates and products S sulfite + phosphoenolpyruvate <1> (Reversibility: ir) [1] P (2R)-O-phospho-3-sulfolactate S Additional information <1> (<1> catalyzes the first step in coenzyme M biosynthesis [1]) (Reversibility: ?) [1] P ?

385

Phosphosulfolactate synthase

4.4.1.19

Substrates and products S sulfite + phosphoenolpyruvate <1> (Reversibility: ir) [1] P (2R)-O-phospho-3-sulfolactate <1> (<1> enantiomeric ratio of 84:16 Risomer:S-isomer [1]) [1] S Additional information <1> (<1> catalyzes the first step in coenzyme M biosynthesis [1]) (Reversibility: ?) [1] P ? Inhibitors EDTA <1> [1] LiCl <1> (<1> weak [1]) [1] MgCl2 <1> (<1> inhibition above 10 mM [1]) [1] phosphoenolpyruvate <1> (<1> substrate inhibition above 5 mM [1]) [1] sulfite <1> (<1> substrate inhibition above 3 mM [1]) [1] Additional information <1> (<1> further weak inhibitors, not inhibitory: K+ , Na+ , NH+4 [1]) [1] Metals, ions Mg2+ <1> (<1> required [1]) [1] Specific activity (U/mg) 2.4 <1> (<1> recombinant enzyme [1]) [1] Km-Value (mM) 9.8 <1> (phosphoenolpyruvate) [1] Ki-Value (mM) 1.9 <1> (phosphoenolpyruvate) [1] pH-Optimum 8.5 <1> [1] pH-Range 6-11 <1> [1]

4 Enzyme Structure Molecular weight 86700 <1> (<1> gel filtration [1]) [1] Subunits trimer <1> (<1> 3 * 32000, SDS-PAGE, 3 * 28000, deduced from gene sequence [1]) [1, 2]

5 Isolation/Preparation/Mutation/Application Renaturation <1> (5 mM MgCl2 restores activity of EDTA-treated enzyme) [1]

386

4.4.1.19

Phosphosulfolactate synthase

Crystallization <1> [2] Engineering K137N <1> (<1> no measurable enzymatic activity [1]) [1]

6 Stability Storage stability <1>, 4 C, 20 mM Tris-HCl, 1 mM MgCl2 , pH 8.0, 2 months, no loss of activity [1]

References [1] Graham, D.E.; Xu, H.; White, R.H.: Identification of coenzyme M biosynthetic phosphosulfolactate synthase: a new family of sulfonate-biosynthesizing enzymes. J. Biol. Chem., 277, 13421-13429 (2002) [2] Wise, E.L.; Graham, D.E.; White, R.H.; Rayment, I.: The structural determination of phosphosulfolactate synthase from Methanococcus jannaschii at 1.7 A resolution: an enolase that isn’t an enolase. J. Biol. Chem., 278, 4585845863 (2003)

387

Leukotriene-C4 synthase

4.4.1.20

1 Nomenclature EC number 4.4.1.20 Systematic name (7E,9E,11Z,14Z)-(5S,6R)-6-(glutathion-S-yl)-5-hydroxyicosa-7,9,11,14-tetraenoate glutathione-lyase (epoxide-forming) Recommended name leukotriene-C4 synthase Synonyms (7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7,9,11,14-tetraenoate:glutathione leukotriene-transferase (epoxide-ring-opening) EC 2.5.1.37 (formerly) LTC4 synthase <10, 11> [32, 35, 36, 37, 38] LTC4 synthetase LTC4S <4, 11> [28, 30, 31, 33, 38] LTCS <11> [29] leukotriene A4 :glutathione S-leukotrienyltransferase leukotriene C4 synthase <4, 10, 11> [28, 29, 30, 31, 32, 33, 34, 35, 37, 38] leukotriene C4 synthetase synthase, leukotriene C4 CAS registry number 90698-32-1

2 Source Organism <1> Cavia porcellus (no sequence specified) [3, 13, 16, 19, 23] <2> Mus musculus (no sequence specified) [3, 4, 7, 11, 12, 14, 16, 24, 25] <3> Homo sapiens (no sequence specified) [1, 2, 3, 5, 6, 8, 9, 10, 12, 14, 15, 16, 18, 19, 20, 21, 25] <4> Rattus norvegicus (no sequence specified) [10, 13, 16, 17, 18, 19, 22, 26, 27, 28, 30, 33] <5> Bos taurus (no sequence specified) [18] <6> Oryctolagus cuniculus (no sequence specified) [18] <7> Ovis aries (no sequence specified) [18] <8> Equus caballus (no sequence specified) [18] <9> no activity in Sus scrofa [18]

388

4.4.1.20

Leukotriene-C4 synthase

<10> Mus musculus (UNIPROT accession number: Q60860) [35] <11> Homo sapiens (UNIPROT accession number: Q16873) [29, 31, 32, 34, 36, 37, 38]

3 Reaction and Specificity Catalyzed reaction leukotriene C4 = leukotriene A4 + glutathione (<3> random rapid equilibrium mechanism [6]) Reaction type alkenyl group transfer Natural substrates and products S leukotriene C4 <4, 11> (Reversibility: r) [28, 29, 30, 32, 33, 34, 37, 38] P leukotriene A4 + glutathione Substrates and products S 14,15-leukotriene A4 + glutathione <1, 3> (Reversibility: ?) [1, 13] P ? S 14,15-leukotriene A4 methyl ester + glutathione <1> (Reversibility: ?) [13] P ? S leukotriene A4 + glutathione <1, 2, 3, 4, 5, 6, 7, 8> (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27] P leukotriene C4 + H2 O <1, 2, 3, 4, 5, 6, 7, 8> [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27] S leukotriene A4 + glutathione <4, 11> (<4> free acid, 4 C, pH 7.35 [28]; <4> pH 7.4, room temperature [30]) (Reversibility: r) [28, 29, 30, 34, 37, 38] P leukotriene C4 + H2 O S leukotriene A4 free acid + glutathione <4> (<4> pH 7.4, 37 C [33]) (Reversibility: r) [33] P leukotriene C4 + H2 O S leukotriene A4 methyl ester + glutathione <1, 2, 3, 4> (<3> 6.5% of the activity with leukotriene A4 [15]) (Reversibility: ?) [4, 13, 15, 19, 20, 23, 24] P leukotriene C4 methyl ester + H2 O S leukotriene A4 methyl ester + glutathione <4> (<4> 4ﬀ C, pH 7.35 [28]) (Reversibility: r) [28] P leukotriene C4 methyl ester S leukotriene A4 methylester + glutathione <4> (<4> pH 7.4, 37ﬀ C [33]) (Reversibility: r) [33] P leukotriene C4 methylester S leukotriene A5 + glutathione <3> (<3> 37% of the activity with leukotriene A4 [15]) (Reversibility: ?) [15] P ?

389

Leukotriene-C4 synthase

4.4.1.20

S leukotriene C4 <4, 11> (Reversibility: r) [28, 29, 30, 32, 33, 34, 37, 38] P leukotriene A4 + glutathione Inhibitors bovine serum albumin <4> (<4> 5 mg, 45% inhibition [17]) [17] Co2+ <3, 11> [21, 37] indomethacin <2> (<2> poor inhibitor [24]; <2> IC50: 1.0 mM [7]) [7, 24] KCl <3> (<3> 0.5 mM, about 25% inhibition [21]) [21] l-699,333 <3> (<3> i.e. 2,[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]-4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]ethoxy]butanoic acid, reversible, competitive against glutathione and non-competitive against leukotriene A4 [6]) [6] leukotriene A4 <3> (<3> substrate inhibition [6]) [6] leukotriene C4 <1, 3> (<3> end product inhibition [21]; <1> IC50: 0.0021 mM, inhibits reaction with leukotriene C4 methyl ester [23]) [21, 23] leukotriene D4 <3> [21] MK-0591 <4> [27] MK-886 <2, 3, 4, 11> (<2> IC50: 0.0031 mM [25]; <3> IC50: 0.0027 mM [25]) [1, 4, 25, 28, 37] N-ethylmaleimide <11> (<11> inhibits the recombinant enzyme [37]) [37] NEM <1, 3> (<3> IC50: 0.02 mM [10]; <1> IC50: 0.018 mM [13]) [10, 13, 16] NaCl <3> (<3> 0.5 mM, about 25% inhibition [21]) [21] Rose bengal <2> (<2> IC50: 0.05 mM [7,24]) [7, 24] sulfobromophthalein <4> (<4> IC50: 0.06 mM [26]) [26] triphenyltin chloride <2> (<2> poor inhibitor [24]) [24] cysteinyl-leukotriene <4> [28] diethylcarbamazine <3> (<3> IC50: 0.05 mM [10]) [10] estrone-3-sulfate <4> (<4> IC50: 1.9 mM [26]) [26] hexylglutathione <1, 4> (<1> IC50: 1.4 mM [13]; <4> S-hexylglutathione [17]) [13, 17] leukotriene C2 <3> (<3> IC50: 0.0011 mM [21]) [21] leukotriene E4 <3> [21] p-aminohippuric acid <4> (<4> IC50: 0.26 mM [26]) [26] probenecid <4> (<4> IC50: 17 mM [26]) [26] thymochinone <11> [34] Activating compounds 17-b-estradiol-3-sulfate <4> (<4> stimulates [26]) [26] 17-b-estradiol-3-sulfate-17 glucuronide <4> (<4> stimulates [26]) [26] A23187 <11> (<11> a calcium ionophore [32]) [32] actinomycin D <4> (<4> in addition to retinoic acid [33]) [33] estriol-3-sulfate <4> (<4> stimulates [26]) [26] Mg2+ <11> [37] retinoic acid <4> [33] daunorubicin <4> [33] lipopolysaccharide <4> (<4> from Escherichia coli serotype O26:B26, increases mRNA expression in heart, brain, liver and adrenal gland [30]) [30] mitomycin C <4> [33]

390

4.4.1.20

Leukotriene-C4 synthase

Metals, ions Ca2+ <3> (<3> stimulates [21]) [21] Co2+ <11> (<11> inhibits the enzyme [37]) [37] Mg2+ <2, 3, 11> (<2> enhances activity [16]; <3> 30 mM, 1.8fold stimulation of partially purified enzyme [21]; <11> augments the enzyme [37]) [16, 21, 37] Specific activity (U/mg) 0.000399 <4> (<4> differentiated cells [10]) [10] 0.00786 <1> [13] 0.0562 <4> (<4> with leukotriene A4 free acid as substrate [28]) [28] 0.0813 <4> (<4> with leukotriene A4 methyl ester as substrate [28]) [28] 0.729 <3> [12] 1.74 <3> [21] 4.135 <3> [9] 30.5 <2> [24] Km-Value (mM) 0.0004 <3> (glutathione, <3> pH 7.4, 25 C [6]) [6] 0.0036 <11> (leukotriene A4 , <11> human recombinant enzyme [37]) [37] 0.0056 <3> (leukotriene A4 , <3> pH 7.4, 25 C [10]) [10] 0.007 <3> (leukotriene A4 , <3> pH 6.5, 30 C [15]) [15] 0.0099 <3> (leukotriene A4 ) [3] 0.015 <1, 3> (leukotriene methyl ester, <3> pH 6.5, 30 C [15]; <1> 22 C, pH 7.6 [23]) [15, 23] 0.0166 <3> (leukotriene A4 ) [21] 0.0188 <4> (leukotriene A4 free acid, <4> 4 C, pH 7.35 [28]) [28] 0.0198 <4> (leukotriene A4 methyl ester, <4> 4 C, pH 7.35 [28]) [28] 0.02 <1> (leukotriene A4 methyl ester, <1> pH 8.0, 37 C [13]) [13] 0.036 <1> (leukotriene A4 , <1> pH 8.0, 37 C [13]) [13] 0.056 <4> (leukotriene A4 , <4> pH 7.4, 25 C [27]) [27] 0.07 <1> (14,15-leukotriene A4 methyl ester, <1> pH 8.0, 37 C [13]) [13] 0.13 <1> (14,15-leukotriene A4 , <1> pH 8.0, 37 C [13]) [13] 0.21 <4> (leukotriene A4 , <4> pH 6.5, 30 C [26]) [26] 0.36 <2> (glutathione, <2> pH 7.5, 37 C [11]) [11] 1.2 <3> (glutathione, <3> pH 7.4, 25 C [10]) [10] 1.6 <11> (glutathione, <11> human recombinant enzyme [37]) [37] Ki-Value (mM) 0.00036 <3> (leukotriene C2 ) [21] 0.0023 <3> (leukotriene A4 , <3> pH 7.4, 25 C [6]) [6] 0.01 <11> (thymochinone, <11> IC50 value [34]) [34] pH-Optimum 6.5 <3> [15] 7.6 <4> [17] 7.6-8.2 <1> [13] 7.8 <4> (<4> lung enzyme, reaction with leukotriene A4 methyl ester [19]) [19]

391

Leukotriene-C4 synthase

4.4.1.20

pH-Range 5-8.8 <3> (<3> pH 5.5: about 25% of maximal activity, pH 8.8: about 45% of maximal activity [15]) [15]

4 Enzyme Structure Molecular weight 16800 <4> (<4> calculated from amino acid sequence [28]) [28] 39200 <3> (<3> gel filtration [9]) [9] Subunits ? <3, 4, 5, 11> (<3,4,5> x * 18000, SDS-PAGE [3,18,20,30,33]; <11> x * 17000, SDS-PAGE [29]; <3> x * 16567, calculation from nucleotide sequence [3]) [3, 18, 20, 29, 30, 33] dimer <3, 11> (<3> 2 * 180000, SDS-PAGE [9]; <11> 2 * 18000, SDS-PAGE, gel filtration [37]) [9, 37] trimer <11> (<11> 3 * 18000, SDS-PAGE, electron crystallography [38]) [38] Additional information <3, 11> (<3> the major components migrate as 18000 Da, 37000 Da, 48000 Da and 60000 Da proteins in SDS-PAGE [12]; <11> forms homo-oligomers of LTCS fusion proteins with Renilla luciferase [29]) [12, 29]

5 Isolation/Preparation/Mutation/Application Source/tissue COS-7 cell <11> [36] HEK-293 cell <11> [29] HL-60 cell <3> (<3> monocytic leukemia cell line [9]) [9] HMC-1 cell <3> [5] KG-1 cell <3, 11> [3, 20, 37] RBL-1 cell <4> [33] RBL-2H3 cell <11> [36] THP-1 cell <3, 11> (<3> monocytic leukemia cell line [3,9,14]) [3, 9, 14, 37] U-937 cell <3> (<3> monocytic leukemia cell line [9,10,21]; <3> dimethyl sulfoxide-differentiated and undifferentiated [10]; <3> dimethylsulfoxide-differentiated U937 cells [21]) [9, 10, 21] adrenal gland <1, 4> (<1> low activity [13]) [13, 30] ascites <2> [11] basophilic leukemia cell line <4> [17, 22, 26] bed nucleus of stria terminalis <10> [35] brain <1, 2, 4, 10> (<1> low activity [13]) [13, 25, 30, 35] colon <1, 4> (<1> low activity [13]) [13, 30] eosinophil <3> [2, 3, 5] heart <1, 2, 3, 4> [13, 25, 30] ileum <4> [30]

392

4.4.1.20

Leukotriene-C4 synthase

kidney <1, 2, 3, 4> [2, 13, 25, 27] lateral habenular nucleus <10> [35] leukocyte <11> [34] liver <1, 4> (<1> low activity [13]; <4> enzyme activity is significantly altered during the development of nephrotic serum nephritis, alterations occur during the initial inflammatory phase of the disease [27]) [13, 19, 27, 30] lung <1, 2, 3, 4, 11> [2, 3, 9, 13, 19, 23, 25, 30, 37] mast cell <2, 3> (<2> Kirsten sarcoma-transfected mast cell [14]; <3> derived from cord blood, expression of two C4 leukotriene synthase isoenzymes [5]; <2> bone-marrow-derived [25]) [2, 3, 5, 14, 25] mastocytoma cell <2> [24] medial amygdaloid nucleus <10> [35] midbrain central gray <10> [35] monocyte <3> [9] nasal polyp <3> [19] neuron <10> [35] pancreas <3> [25] paraventricular nucleus of hypothalamus <10> [35] placenta <3> [25] platelet <3, 4, 5, 6, 7, 8> [1, 2, 3, 15, 18] polymorphonuclear leukocyte <11> [32] skeletal muscle <2, 3, 4> [25, 30] small intestine <1> (<1> low activity [13]) [13] spleen <1, 2, 4> (<2> slight [25]) [13, 25, 30] stomach <1> (<1> low activity [13]) [13] suprachiasmatic nucleus <10> [35] supraoptic nucleus <10> [35] ventral septal area <10> [35] Localization axon <10> (<10> emanating from the vasopressinergic magnocellular neurons of the hypothalamic paraventricular, supraoptic and suprachiasmatic nuclei as well as the retrochiasmatic area to the pars nervosa of the pituitary gland. It is also observed in the axons of the extrahypothalamic system [35]) [35] cytosol <1, 3, 4> (<4> 22% of the activity [17]; <1,3,4> the enzyme is chiefly membrane-bound, although the cytosol contains some activity [19]) [17, 19] endoplasmic reticulum <3, 11> (<3> the active site of leukotriene A4 synthase is localized in the lumen of the endoplasmic reticulum [8]) [8, 29] membrane <1, 3, 4, 11> (<3> associated with [15]; <3> bound to [6]; <1,3,4> the enzyme is chiefly membrane-bound, although the cytosol contains some activity [19]; <11> in close association with 5-lipoxygenase activating protein [36]) [2, 6, 8, 10, 14, 15, 19, 36, 37, 38] microsome <1, 2, 3, 4> (<4> 70% of the activity [17]) [3, 7, 8, 9, 13, 14, 17, 20, 22, 23, 24] nuclear envelope <3> (<3> the active site of leukotriene A4 synthase is localized in the lumen of the nucear envelope and endoplasmic reticulum [8]) [8]

393

Leukotriene-C4 synthase

4.4.1.20

nuclear membrane <11> [29] nuclear outer membrane <3> [8] particle-bound <4> [26] perinuclear space <3> [3] Purification <1> [23] <1> (partial) [13] <2> [14] <2> (partial) [24] <3> [2, 6, 9, 14, 20, 21] <3> (affinity chromatography purification based on specific interaction between leukotriene C4 synthase and microsomal glutathione S-transferase which ocurs in the presence of magnesium ion) [12] <4> [22] <4> (partial) [27] <5> [18] <11> (the recombinant enzyme from Schizosaccharomyces pombe, to apparent homogeneity) [38] <11> (to homogeneity) [37] Crystallization <11> (resolution of 4.5 A) [38] Cloning <2> (COS-7 cells transfected with either human or cloned mouse leukotriene C4 synthase cDNA by DEAE-dextran transfection) [25] <3> [2] <3> (COS-7 cells transfected with either human or cloned mouse leukotriene C4 synthase cDNA by DEAE-dextran transfection) [25] <4> [30] <4> (expression in Spodoptera frugiperda) [28] <8> [18] <11> [29, 31, 37] <11> (expressed in Schizosaccharomyces pombe) [38] Engineering A52S <3> (<3> mutation increases the Km -value for the recombinant enzyme for glutathione [3,16]) [3, 16] C56S <3, 11> (<3> mutant enzyme without altered function [16]; <11> not inhibited by N-ethylmaleimide [37]) [16, 37] C56S/C82 <3> (<3> mutant enzyme without altered function [16]) [16] C82V <3> (<3> mutant enzyme without altered function [16]) [16] E4K <11> (<11> causes allergic diseases in patients such as bronchial asthma or allergic dermatitis [31]) [31] LTCS(1-115) <11> (<11> C-terminally truncated protein, gives a bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29]

394

4.4.1.20

Leukotriene-C4 synthase

LTCS(1-24) <11> (<11> C-terminally truncated protein, gives no bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29] LTCS(1-58) <11> (<11> C-terminally truncated protein, gives no bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29] LTCS(1-88) <11> (<11> C-terminally truncated protein, gives a bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29] LTCS(114-150) <11> (<11> N-terminally truncated protein, gives a bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29] LTCS(23-115) <11> (<11> C- and N-terminally truncated protein, gives a bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29] LTCS(23-150) <11> (<11> N-terminally truncated protein, gives a bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29] LTCS(57-150) <11> (<11> N-terminally truncated protein, gives a bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29] LTCS(87-150) <11> (<11> N-terminally truncated protein, gives a bioluminescence resonance energy transfer signal when fused to Renilla luciferase [29]) [29] N55A <3> (<3> mutation increases the Km -value for the recombinant enzyme for glutathione [3,16]) [3, 16] R51H <3> (<3> fully active mutant enzyme [3,16]) [3, 16] R51I <3, 11> (<11> inactive [37]; <3> mutant enzyme without activity [16]) [16, 37] R51K <3> (<3> fully active mutant enzyme [3,16]) [3, 16] R51T <3> (<3> mutant enzyme without activity [16]) [16] V49F <3> (<3> mutation increases the Km -value for the recombinant enzyme for glutathione [3,16]) [3, 16] Y59F <3> (<3> mutation increases the Km -value for the recombinant enzyme for glutathione [3,16]) [3, 16] Y93F <3, 11> (<3> mutation increases the Km -value for the recombinant enzyme for glutathione [3]; <3> activity of the mutant enzyme is less than 1% of the wild-type enzyme, shift in pH optimum of the residual activity to that of spontaneous conjugation [3,16]; <11> reduces the enzyme activity to less than 1% of the wild-type enzyme, shift in the pH-optimum [37]) [3, 16, 37] Y97F <3> (<3> mutation increases the Km -value for the recombinant enzyme for glutathione [3]) [3] Application drug development <11> (<11> leukotrienes are an important therapeutic target in asthma and inflammatory diseases [34]) [34]

395

Leukotriene-C4 synthase

4.4.1.20

6 Stability Temperature stability 4 <3> (<3> t1=2 : 18 h [21]) [21] 25 <3> (<3> t1=2 : 18 h [21]) [21] 37 <1> (<1> 3 min, 50 min loss of activity, 10 mM glutathione completely protects against inactivation [13]) [13] 40 <4> (<4> 5 min, 90% loss of activity [17]) [17] General stability information <3>, partially purified enzyme requires substrate stabilization for long-term storage [21] <3>, reduced glutathione irreversibly inactivates the enzyme when present during freeze/thaw cycles and storage at concentrations above 5 mM [21] Storage stability <3>, -20 C, enzyme in the membrane fraction from human platelets can be stored without major loss of activity [15] <3>, -80 C, in presence of 2-4 mM glutathione, stable for up to 1 year [21]

References [1] Sala, A.; Garcia, M.; Zarini, S.; Rossi, J.C.; Folco, G.; Durand, T.: 14,15-dehydroleukotriene A4 : a specific substrate for leukotriene C4 synthase. Biochem. J., 328, 225-229 (1997) [2] Penrose, J.F.: LTC4 synthase: Enzymology, biochemistry, and molecular characterization. Clin. Rev. Allergy Immunol., 17, 133-152 (1999) [3] Penrose, J.F.; Austen, K.F.: The biochemical, molecular, and genomic aspects of leukotriene C4 synthase. Proc. Assoc. Am. Phys., 111, 537-546 (1999) [4] Hevko, J.M.; Murphy, R.C.: Formation of murine macrophage-derived 5oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) is catalyzed by leukotriene C4 synthase. J. Biol. Chem., 277, 7037-7043 (2002) [5] Sjostrom, M.; Jakobsson, P.-J.; Juremalm, M.; Ahmed, A.; Nilsson, G.; Macchia, L.; Haeggstrom, J.Z.: Human mast cells express two leukotriene C4 synthase isoenzymes and the CysLT1 receptor. Biochim. Biophys. Acta, 1583, 53-62 (2002) [6] Gupta, N.; Gresser, M.J.; Ford-Hutchinson, A.W.: Kinetic mechanism of glutathione conjugation to leukotriene A4 by leukotriene C4 synthase. Biochim. Biophys. Acta, 1391, 157-168 (1998) [7] Sçderstrçm, M.; Hammarstrçm, S.; Mannervik, B.: Leukotriene C synthase in mouse mastocytoma cells. An enzyme distinct from cytosolic and microsomal glutathione transferases. Biochem. J., 250, 713-718 (1988) [8] Christmas, P.; Weber, B.M.; McKee, M.; Brown, D.; Soberman, R.J.: Membrane localization and topology of leukotriene C4 synthase. J. Biol. Chem., 277, 28902-28908 (2002)

396

4.4.1.20

Leukotriene-C4 synthase

[9] Nicholson, D.W.; Ali, A.; Vaillancourt, J.P.; Calaycay, J.R.; Mumford, R.A.; Zamboni, R.J.; Ford-Hutchinson, A.W.: Purification to homogeneity and the N-terminal sequence of human leukotriene C4 synthase: a homodimeric glutathione S-transferase composed of 18-kDa subunits. Proc. Natl. Acad. Sci. USA, 90, 2015-2019 (1993) [10] Nicholson, D.W.; Ali, A.; Klemba, M.W.; Munday, N.A.; Zamboni, R.J.; FordHutchinson, A.W.: Human leukotriene C4 synthase expression in dimethyl sulfoxide-differentiated U937 cells. J. Biol. Chem., 267, 17849-17857 (1992) [11] Abe, M.; Hugli, T.E.: Characterization of leukotriene C4 synthetase in mouse peritoneal exudate cells. Biochim. Biophys. Acta, 959, 386-398 (1988) [12] Soederstroem, M.; Morgenstern, R.; Hammarstroem, S.: Protein-protein interaction affinity chromatography of leukotriene C4 synthase. Protein Expr. Purif., 6, 352-356 (1995) [13] Izumi, T.; Honda, Z.; Ohishi, N.; Kitamura, S.; Tsuchida, S.; Sato, K.; Shimizu, T.; Seyama, Y.: Solubilization and partial purification of leukotriene C4 synthase from guinea-pig lung: a microsomal enzyme with high specificity towards 5,6-epoxide leukotriene A4 . Biochim. Biophys. Acta, 959, 305-315 (1988) [14] Goppelt-Struebe, M.: Two step purification of human and murine leukotriene C4 synthase. Biochim. Biophys. Acta, 1256, 257-261 (1995) [15] Sçderstrçm, M.; Mannervik, B.; Garkov, V.; Hammarstrçm, S.: On the nature of leukotriene C4 synthase in human platelets. Arch. Biochem. Biophys., 294, 70-74 (1992) [16] Lam, B.K.; Frank Austen, K.: Leukotriene C4 synthase: a pivotal enzyme in cellular biosynthesis of the cysteinyl leukotrienes. Prostaglandins Other Lipid Mediat., 68-69, 511-520 (2002) [17] Yoshimoto, T.; Soberman, R.J.; Lewis, R.A.; Austen, K.F.: Isolation and characterization of leukotriene C4 synthetase of rat basophilic leukemia cells. Proc. Natl. Acad. Sci. USA, 82, 8399-8403 (1985) [18] Tornhamre, S.; Sjolinder, M.; Lindberg, A.; Ericsson, I.; Nasman-Glaser, B.; Griffiths, W.J.; Lindgren, J.A.: Demonstration of leukotriene-C4 synthase in platelets and species distribution of the enzyme activity. Eur. J. Biochem., 251, 227-235 (1998) [19] Wu, C.: Conversion of leukotrienes A4 to C4 in cell-free systems. Biochem. Biophys. Res. Commun., 134, 85-92 (1986) [20] Penrose, J.F.; Gagnon, L.; Goppelt-Struebe, M.; Myers, P.; Lam, B.K.; Jack, R.M.; Austen, K.F.; Soberman, R.J.: Purification of human leukotriene C4 synthase. Proc. Natl. Acad. Sci. USA, 89, 11603-11606 (1992) [21] Nicholson, D.W.; Klemba, M.W.; Rasper, D.M.; Metters, K.M.; Zamboni, R.J.: Purification of human leukotriene C4 synthase from dimethylsulfoxide-differentiated U937 cells. Eur. J. Biochem., 209, 725-734 (1992) [22] Soberman, R.J.; Yoshimoto, T.: Leukotriene C4 synthase from rat basophilic leukemia cell microsomes. Methods Enzymol., 163, 353-357 (1988) [23] Soberman, R.J.: Purification and properties of leukotriene C4 synthase from guinea pig lung microsomes. Methods Enzymol., 187, 335-337 (1990)

397

Leukotriene-C4 synthase

4.4.1.20

[24] Sçderstrçm, M.; Mannervik, B.; Mammarstrçm, S.: Leukotriene C4 synthase: characterization in mouse mastocytoma cells. Methods Enzymol., 187, 306-312 (1990) [25] Lam, B.K.; Penrose, J.F.; Rokach, J.; Xu, K.; Baldasaro, M.H.; Austen, K.F.: Molecular cloning, expression and characterization of mouse leukotriene C4 synthase. Eur. J. Biochem., 238, 606-612 (1996) [26] Bach, M.K.; Brashler, J.R.; Morton, D.R.: Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: a novel, particulate glutathione S-transferase. Arch. Biochem. Biophys., 230, 455465 (1984) [27] Petric, R.; Nicholson, D.W.; Ford-Hutchinson, A.W.: Renal leukotriene C4 synthase: characterization, partial purification and alterations in experimental glomerulonephritis. Biochim. Biophys. Acta, 1254, 207-215 (1995) [28] Schroder, O.; Sjostrom, M.; Qiu, H.; Stein, J.; Jakobsson, P.-J.; Haeggstrom, J.Z.: Molecular and catalytic properties of three rat leukotriene C4 synthase homologs. Biochem. Biophys. Res. Commun., 312, 271-276 (2003) [29] Svartz, J.; Blomgran, R.; Hammarstrom, S.; Soderstrom, M.: Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer. Biochim. Biophys. Acta, 1633, 90-95 (2003) [30] Schroder, O.; Sjostrom, M.; Qiu, H.; Jakobsson, P.J.; Haeggstrom, J.Z.: Microsomal glutathione S-transferases: selective up-regulation of leukotriene C4 synthase during lipopolysaccharide-induced pyresis. Cell. Mol. Life Sci., 62, 87-94 (2005) [31] Yoshikawa, K.; Matsui, E.; Kaneko, H.; Fukao, T.; Inoue, R.; Teramoto, T.; Shinoda, S.; Fukutomi, O.; Aoki, M.; Kasahara, K.; Kondo, N.: A novel single-nucleotide substitution, Glu 4 Lys, in the leukotriene C4 synthase gene associated with allergic diseases. Int. J. Mol. Med., 16, 827-831 (2005) [32] Zaitsu, M.; Hamasaki, Y.; Matsuo, M.; Ichimaru, T.; Fujita, I.; Ishii, E.: Leukotriene synthesis is increased by transcriptional up-regulation of 5-lipoxygenase, leukotriene A4 hydrolase, and leukotriene C4 synthase in asthmatic children. J. Asthma, 40, 147-154 (2003) [33] Abe, M.; Shibata, K.; Urata, H.; Sakata, N.; Katsuragi, T.: Induction of leukotriene C4 synthase after the differentiation of rat basophilic leukemia cells with retinoic acid and a low dose of actinomycin D and its suppression with methylprednisolone. J. Cell. Physiol., 196, 154-164 (2003) [34] Mansour, M.; Tornhamre, S.: Inhibition of 5-lipoxygenase and leukotriene C4 synthase in human blood cells by thymoquinone. J. Enzyme Inhib. Med. Chem., 19, 431-436 (2004) [35] Shimada, A.; Satoh, M.; Chiba, Y.; Saitoh, Y.; Kawamura, N.; Keino, H.; Hosokawa, M.; Shimizu, T.: Highly selective localization of leukotriene C4 synthase in hypothalamic and extrahypothalamic vasopressin systems of mouse brain. Neuroscience, 131, 683-689 (2005) [36] Mandal, A.K.; Skoch, J.; Bacskai, B.J.; Hyman, B.T.; Christmas, P.; Miller, D.; Yamin, T.-t.D.; Xu, S.; Wisniewski, D.; Evans, J.F.; Soberman, R.J.: The membrane organization of leukotriene synthesis. Proc. Natl. Acad. Sci. USA, 101, 6587-6592 (2004)

398

4.4.1.20

Leukotriene-C4 synthase

[37] Lam, B.K.: Leukotriene C4 synthase. Prostaglandins Leukot. Essent. Fatty Acids, 69, 111-116 (2003) [38] Schmidt-Krey, I.; Kanaoka, Y.; Mills, D.J.; Irikura, D.; Haase, W.; Lam, B.K.; Austen, K.F.; Kuehlbrandt, W.: Human leukotriene C4 synthase at 4.5.ANG. resolution in projection. Structure, 12, 2009-2014 (2004)

399

S-Ribosylhomocysteine lyase

4.4.1.21

1 Nomenclature EC number 4.4.1.21 Systematic name S-(5-deoxy-d-ribos-5-yl)-l-homocysteine-lyase 2,3-dione-forming]

[(4S)-4,5-dihydroxypentan-

Recommended name S-ribosylhomocysteine lyase Synonyms LuxS <4, 5, 8> [2, 7, 11, 12] LuxS protein <2> [3] S-ribosylhomocysteinase <5> [7] CAS registry number 37288-63-4 (not distinguished from EC 3.2.1.148, formerly 3.3.1.3)

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10>

Staphylococcus aureus (no sequence specified) [6, 13] Bacillus subtilis (no sequence specified) [1, 3, 5, 8, 9, 10] Escherichia coli (no sequence specified) [4, 6] Vibrio fischeri (no sequence specified) [12] Vibrio harveyi (no sequence specified) [2,7,9,10] Neisseria meningitidis (no sequence specified) [6] Streptococcus pneumoniae (no sequence specified) [14] Helicobacter pylori (no sequence specified) [11] no activity in Pseudomonas aeruginosa [6] Porphyromonas gingivalis (no sequence specified) [6]

3 Reaction and Specificity Catalyzed reaction S-(5-deoxy-d-ribos-5-yl)-l-homocysteine = l-homocysteine + (S)-4,5-dihydroxypentan-2,3-dione (<2> catalytic mechanism in which the metal ion catalyzes an intramolecular redox reaction, shifting the carbonyl group from the C-1 position to the C-3 position of the ribose. Subsequent b-elimination at the C-4 and C-5 position releases homocysteine as a free thiol [3])

400

4.4.1.21

S-Ribosylhomocysteine lyase

Reaction type C-S bond cleavage Natural substrates and products S S-ribosylhomocysteine <5> (Reversibility: ?) [7] P homocysteine + 4,5-dihydroxy-2,3-pentanedione (<5> the autoinducer Al-2, a five-carbon furanone results from the spontaneous cyclization of 4,5-dihydroxy-2,3-pentanedione [7]) S S-ribosylhomocysteine <1, 2, 3, 6, 10> (<3> enzyme is involved in synthesis of the autoinducer AI-2 that is an universal signal, which may be used by a variety of bacteria for communication among and between species and may be responsible for regulation of virulence genes in Escherichia coli O157:H7 [4]; <2> key step in biosynthesis pathway of type II autoinducer AI-2 [3]; <1,3,6,10> the enzyme is required for AI-2 synthesis, important metabolic function in recycling of S-adenosylhomocysteine [6]) (Reversibility: ?) [3, 4, 6] P l-homocysteine + 4,5-dihydroxy-2,3-pentanedione S Additional information <1, 5, 7, 8> (<5> the enzyme is involved in one of the the quorum sensing systems that function in parallel to control the density-dependent expression of the luciferase structural operon luxCDABE. Each system is composed of a sensor, sensor I or sensor 2, and its cognate autoinducer, AI-1 or AI-2. LuxS has a role in the enzymatic synthesis of AI-2 [2]; <8> growth phase regulation of flaA expression in Helicobacter pylori is luxS-dependent [11]; <7> LuxS is related on the one hand to down-regulation of competence, and on the other hand to attenuation of autolysis in cultures entering stationary phase. The impact of LuxS on competence, but not on autolysis, involves cel-cell communication [14]; <1> the LuxS/AI-2 (autoinducer 2) system does not appear to contribute to the overall fitness of Staphylococcus aureus RN6390B during intracellular growth in epithelial cells [13]) (Reversibility: ?) [2, 11, 13, 14] P ? Substrates and products S S-ribosylhomocysteine <5> (Reversibility: ?) [7] P homocysteine + 4,5-dihydroxy-2,3-pentanedione (<5> the autoinducer Al-2, a five-carbon furanone results from the spontaneous cyclization of 4,5-dihydroxy-2,3-pentanedione [7]) S S-ribosylhomocysteine <1, 2, 3, 5, 6, 10> (<2,5> mechanism [9]; <3> enzyme is involved in synthesis of the autoinducer AI-2 that is an universal signal, which may be used by a variety of bacteria for communication among and between species and may be responsible for regulation of virulence genes in Escherichia coli O157:H7 [4]; <2> key step in biosynthesis pathway of type II autoinducer AI-2 [3]; <1,3,6,10> the enzyme is required for AI-2 synthesis, important metabolic function in recycling of S-adenosylhomocysteine [6]) (Reversibility: ?) [3, 4, 5, 6, 9, 10] P l-homocysteine + 4,5-dihydroxy-2,3-pentanedione S Additional information <1, 4, 5, 7, 8> (<5> the enzyme is involved in one of the the quorum sensing systems that function in parallel to control the

401

S-Ribosylhomocysteine lyase

4.4.1.21

density-dependent expression of the luciferase structural operon luxCDABE. Each system is composed of a sensor, sensor I or sensor 2, and its cognate autoinducer, AI-1 or AI-2. LuxS has a role in the enzymatic synthesis of AI-2 [2]; <8> growth phase regulation of flaA expression in Helicobacter pylori is luxS-dependent [11]; <7> LuxS is related on the one hand to down-regulation of competence, and on the other hand to attenuation of autolysis in cultures entering stationary phase. The impact of LuxS on competence, but not on autolysis, involves cel-cell communication [14]; <1> the LuxS/AI-2 (autoinducer 2) system does not appear to contribute to the overall fitness of Staphylococcus aureus RN6390B during intracellular growth in epithelial cells [13]; <4> LuxS affects both luminescence regulation and colonization competence - however its quantitative contribution is small when compared to that of the AinS signal [12]) (Reversibility: ?) [2, 11, 12, 13, 14] P ? Metals, ions Fe2+ <2> (<2> contains [3]) [3] Zn2+ <2> (<2> zinc-dependent metalloenzyme, each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits [5]) [5] Zn2+ <2> (<2> the metal center is composed of a Zn2+ atom coordinated by two histidines, a cysteine, and a solvent molecule [8]) [8] Additional information <2> (<2> to gain insight into the catalytic mechanism of the unusual reaction and the function of the metal cofactor, an efficient expression and purification system is developed to produce LuxS enriched in either Fe2+ , Co2+ or Zn2+ [3]) [3] Turnover number (min–1) 0.03 <2> (S-ribosylhomocysteine) [10] 0.4 <5> (S-ribosylhomocysteine) [10] Additional information <2> (<2> turnover numbers of enzyme forms enriched in either Fe2+ , Co2+ or Zn2+ [3]) [3] Km-Value (mM) 0.0014 <2> (S-ribosylhomocysteine) [10] 0.0025 <2> (S-ribosylhomocysteine, <2> native enzyme [3]) [3] 0.039 <5> (S-ribosylhomocysteine) [10] 0.18 <2> (S-ribosylhomocysteine, <2> mutant enzyme E57D [3]) [3] Additional information <2> (<2> KM -values of enzyme forms enriched in either Fe2+ , Co2+ or Zn2+ [3]) [3]

4 Enzyme Structure Subunits dimer <2> [5, 8]

402

4.4.1.21

S-Ribosylhomocysteine lyase

5 Isolation/Preparation/Mutation/Application Purification <2> [1, 3, 10] <5> [10] Crystallization <2> (hanging drop vapor diffusion, inactive mutant C84A of Co2+ -substituted LuxS is cocrystallized with the 2-ketone intermediate and the structure is determined to 1.8 A resolution) [10] <2> (hanging-drop vapor diffusion method with ammonium sulfate as precipitant, structure at 1.6 A resolution) [8] <2> (hanging-drop vapour diffusion method with ammonium sulfate as the precipitant. The crystals belong to the enantiomorphic space groups P6(1)22 or P6(5)22 with approximate unit-cell parameters A = b = 63.6, c = 151.5 A. The crystals diffract X-rays to at least 1.55 A resolution on a synchrotronradiation source) [1] <2> (structure of LuxS is determined at 1.2 A resolution, together with the binary complexes of LuxS with S-ribosylhomocysteine and homocysteine to 2.2 A and 2.3 A resolution, hanging-drop vapour diffusion method) [5] Cloning <2> [3] <2> (expression in Escherichia coli) [1] <2> (expression in Escherichia coli BL21) [8] <5> (overexpression as glutathione-S-transferase fusion in Escherichia coli) [7] Engineering C84A <2> (<2> no catalytic activity [3]) [3] C84D <2> (<2> more than 220fold reduced activity [3]) [3] C84S <2> (<2> more than 220fold reduced activity [3]) [3] C85A <2> (<2> catalytically inactive mutant [10]) [10] E57A <2> (<2> no detectable activity [3]) [3] E57D <2> (<2> 220fold reduced activity [3]) [3] E57Q <2> (<2> no detectable activity [3]) [3]

6 Stability Storage stability <2>, -80 C, when stored in the frozen form, the LuxS proteins are stable for at least 6 months [3]

403

S-Ribosylhomocysteine lyase

4.4.1.21

References [1] Das, S.K.; Sedelnikova, S.E.; Baker, P.J.; Ruzheinikov, S.N.; Foster, S.; Hartley, A.; Horsburgh, M.J.; Rice, D.W.: Cloning, purification, crystallization and preliminary crystallographic analysis of Bacillus subtilis LuxS. Acta Crystallogr. Sect. D, 57, 1324-1325 (2001) [2] Miller, M.B.; Bassler, B.L.B.: Quorum sensing in bacteria. Annu. Rev. Microbiol., 55, 165-199 (2001) [3] Zhu, J.; Dizin, E.; Hu, X.; Wavreille, A.S.; Park, J.; Pei, D.: S-Ribosylhomocysteinase (LuxS) is a mononuclear iron protein. Biochemistry, 42, 47174726 (2003) [4] Anand, S.K.; Griffiths, M.W.: Quorum sensing and expression of virulence in Escherichia coli O157:H7. Int. J. Food Microbiol., 85, 1-9 (2003) [5] Ruzheinikov, S.N.; Das, S.K.; Sedelnikova, S.E.; Hartley, A.; Foster, S.J.; Horsburgh, M.J.; Cox, A.G.; McCleod, C.W.; Mekhalfia, A.; Blackburn, G.M.; Rice, D.W.; Baker, P.J.: The 1.2 A structure of a novel quorum-sensing protein, Bacillus subtilis LuxS. J. Mol. Biol., 313, 111-122 (2001) [6] Winzer, K.; Hardie, K.R.; Burgess, N.; Doherty, N.; Kirke, D.; Holden, M.T.; Linforth, R.; Cornell, K.A.; Taylor, A.J.; Hill, P.J.; Williams, P.: LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl3(2H)-furanone. Microbiology, 148, 909-922 (2002) [7] Schauder, S.; Shokat, K.; Surette, M.G.; Bassler, B.L.: The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol. Microbiol., 41, 463-476 (2001) [8] Hilgers, M.T.; Ludwig, M.L.: Crystal structure of the quorum-sensing protein LuxS reveals a catalytic metal site. Proc. Natl. Acad. Sci. USA, 98, 11169-11174 (2001) [9] Zhu, J.; Patel, R.; Pei, D.: Catalytic mechanism of S-ribosylhomocysteinase (LuxS): stereochemical course and kinetic isotope effect of proton transfer reactions. Biochemistry, 43, 10166-10172 (2004) [10] Rajan, R.; Zhu, J.; Hu, X.; Pei, D.; Bell, C.E.: Crystal structure of S-ribosylhomocysteinase (LuxS) in complex with a catalytic 2-ketone intermediate. Biochemistry, 44, 3745-3753 (2005) [11] Loh, J.T.; Forsyth, M.H.; Cover, T.L.: Growth phase regulation of flaA expression in Helicobacter pylori is luxS dependent. Infect. Immun., 72, 5506-5510 (2004) [12] Lupp, C.; Ruby, E.G.: Vibrio fischeri LuxS and AinS: comparative study of two signal synthases. J. Bacteriol., 186, 3873-3881 (2004) [13] Doherty, N.; Holden, M.T.; Qazi, S.N.; Williams, P.; Winzer, K.: Functional analysis of luxS in Staphylococcus aureus reveals a role in metabolism but not quorum sensing. J. Bacteriol., 188, 2885-2897 (2006) [14] Romao, S.; Memmi, G.; Oggioni, M.R.; Trombe, M.C.: LuxS impacts on LytA-dependent autolysis and on competence in Streptococcus pneumoniae. Microbiology, 152, 333-341 (2006)

404

S-(Hydroxymethyl)glutathione synthase

4.4.1.22

1 Nomenclature EC number 4.4.1.22 Systematic name S-(hydroxymethyl)glutathione formaldehyde-lyase (glutathione-forming) Recommended name S-(hydroxymethyl)glutathione synthase Synonyms EC 1.2.1.1 <1> (<1> formerly [1]) [1] Gfa <1> [1, 2] glutathione-dependent formaldehyde-activating enzyme <1> [1] CAS registry number 425642-27-9

2 Source Organism <1> Paracoccus denitrificans (no sequence specified) [1, 2]

3 Reaction and Specificity Catalyzed reaction S-(hydroxymethyl)glutathione = glutathione + formaldehyde (<1> the enzyme from Paracoccus denitrificans accelerates the spontaneous reaction in which the adduct of formaldehyde and glutathione is formed, i.e. the substrate for EC 1.1.1.284, S-(hydroxymethyl)glutathione dehydrogenase, in the formaldehyde-detoxification pathway [1]) Reaction type addition Natural substrates and products S S-(hydroxymethyl)glutathione <1> (<1> the enzyme accelerates the spontaneous reaction in which the adduct of formaldehyde and glutathione is formed, i.e. the substrate for EC 1.1.1.284, S-(hydroxymethyl)glutathione dehydrogenase, in the formaldehyde-detoxification pathway [1]) (Reversibility: ?) [1] P glutathione + formaldehyde

405

S-(Hydroxymethyl)glutathione synthase

4.4.1.22

Substrates and products S S-(hydroxymethyl)glutathione <1> (<1> the enzyme accelerates the spontaneous reaction in which the adduct of formaldehyde and glutathione is formed, i.e. the substrate for EC 1.1.1.284, S-(hydroxymethyl)glutathione dehydrogenase, in the formaldehyde-detoxification pathway [1]) (Reversibility: ?) [1] P glutathione + formaldehyde S glutathione + formaldehyde <1> (Reversibility: ?) [2] P S-(hydroxymethyl)glutathione Metals, ions Zn <1> (<1> the enzyme has a new fold with two zinc-sulfur centers, one that is structural (zinc tetracoordinated) and one catalytic (zinc apparently tricoordinated). In the complex of enzyme with glutathione, the catalytic zinc is displaced due to disulfide bond formation of glutathione with one of the zinc-coordinating cysteines [2]) [2] Specific activity (U/mg) 350 <1> [1]

4 Enzyme Structure Subunits ? <1> (<1> x * 21000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> [1, 2] Crystallization <1> (vapor diffusion method, crystallization of enzyme and enzyme in complex with glutathione) [2]

References [1] Gçnrich, M.; Bartoschek, S.; Hagemeier, C.H.; Griesinger, C.; Vorholt, J.A.: A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy. J. Biol. Chem., 277, 3069-3072 (2002) [2] Neculai, A.M.; Neculai, D.; Griesinger, C.; Vorholt, J.A.; Becker, S.: A dynamic zinc redox switch. J. Biol. Chem., 280, 2826-2830 (2005)

406

2-Hydroxypropyl-CoM lyase

4.4.1.23

1 Nomenclature EC number 4.4.1.23 Systematic name (R)-[or (S)-]2-hydroxypropyl-CoM:2-mercaptoethanesulfonate lyase (epoxyalkane-ring-forming) Recommended name 2-hydroxypropyl-CoM lyase Synonyms Ea-CoMT <3> [5] EaCoMT <2> [4] coenzyme M-epoxyalkane ligase epoxyalkane:2-mercaptoethanesulfonate transferase epoxyalkane:CoM transferase epoxyalkyl:CoM transferase epoxypropane:coenzyme M transferase epoxypropyl:CoM transferase transferase, epoxyalkyl:coenzyme M CAS registry number 244301-07-3

2 Source Organism <1> <2> <3> <4>

Rhodococcus rhodochrous (no sequence specified) [2] Mycobacterium sp. (no sequence specified) [4] Nocardioides sp. (no sequence specified) [5] Xanthobacter autotrophicus (no sequence specified) [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction (R)-[or (S)-]2-hydroxypropyl-CoM = (R)-[or (S)-]1,2-epoxypropane + HSCoM Reaction type C-S bond cleavage

407

2-Hydroxypropyl-CoM lyase

4.4.1.23

Natural substrates and products S epoxypropane + HS-CoM <1, 4> (<1,4> inducible enzyme [2]; <4> this enzyme forms component I of a four-component enzyme system, comprising EC 4.2.99.19 2-hydroxypropyl-CoM lyase, component I, EC 1.8.1.5 2-oxopropyl-CoM reductase (carboxylating), component II, EC 1.1.1.268 2-(R)-hydroxypropyl-CoM dehydrogenase, component II and EC 1.1.1.269 2-(S)-hydroxypropyl-CoM dehydrogenase, component IV that is involved in epoxyalkane carboxylation [1]; <1,4> enzyme is involved in propylene metabolism [2]) (Reversibility: ?) [1, 2] P 2-hydroxypropyl-CoM <1, 4> [1, 2] Substrates and products S 2-hydroxypropyl-CoM <3> (Reversibility: ?) [5] P 1,2-epoxypropane + HS-CoM S epoxyethane + HS-CoM <2> (Reversibility: ?) [4] P 2-hydroxyethyl-CoM <2> [4] S epoxypropane + 3-mercaptopropionate <4> (<4> 0.59% of the activity with HS-CoM [3]) (Reversibility: ?) [3] P 3-[(2-hydroxypropyl)thio]propanoic acid S epoxypropane + HS-CoA <4> (<4> 0.51% of the activity with HS-CoM [3]; <4> 0.11% of the activity with HS-CoM [3]) (Reversibility: ?) [3] P ? S epoxypropane + HS-CoM <1, 2, 4> (<4> catalyzes the reaction of CoM with R-epoxypropane at a rate approximately twice of that with S-epoxypropane [1]; <1,4> inducible enzyme [2]; <4> this enzyme forms component I of a four-component enzyme system, comprising EC 4.2.99.19 2hydroxypropyl-CoM lyase, component I, EC 1.8.1.5 2-oxopropyl-CoM reductase (carboxylating), component II, EC 1.1.1.268 2-(R)-hydroxypropylCoM dehydrogenase, component II and EC 1.1.1.269 2-(S)-hydroxypropylCoM dehydrogenase, component IV that is involved in epoxyalkane carboxylation [1]; <1,4> enzyme is involved in propylene metabolism [2]) (Reversibility: ?) [1, 2, 3, 4] P 2-hydroxypropyl-CoM <1, 2, 4> [1, 2, 3, 4] Inhibitors 2-mercaptoethanol <4> (<4> weak competitive [3]) [3] Metals, ions Co2+ <4> (<4> the inactive and Zn-deficient form of enzyme is activated by addition of ZnCl2 or CoCl2 [3]) [3] Zn <4> (<4> Zn plays a key role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate, removal of Zn results in loss of catalytic activity, the inactive and Zn-deficient form of enzyme is activated by addition of ZnCl2 or CoCl2 . Thermodynamic characterization of the interaction of CoM with an enzyme-bound Zn center [3]) [3] Zn2+ <4> (<4> the inactive and Zn-deficient form of enzyme is activated by addition of ZnCl2 or CoCl2 [3]) [3]

408

4.4.1.23

2-Hydroxypropyl-CoM lyase

Turnover number (min–1) 6.5 <4> (epoxypropane) [3] Specific activity (U/mg) 143 <2> (<2> epoxypropane as substrate [4]) [4] 615 <2> (<2> epoxyethane as substrate [4]) [4] Km-Value (mM) 0.0018 <4> (epoxypropane) [3] 0.034 <4> (CoM) [3] pH-Optimum 8-8.5 <4> [3] pH-Range 7-9.5 <4> (<4> pH 7.0: about 85% of maximal activity, pH 9.5: about 90% of maximal activity [3]) [3]

4 Enzyme Structure Subunits Additional information <4> (<4> this enzyme forms component I of a fourcomponent enzyme system, comprising EC 4.2.99.19 2-hydroxypropyl-CoM lyase, component I, EC 1.8.1.5 2-oxopropyl-CoM reductase (carboxylating), component II, EC 1.1.1.268 2-(R)-hydroxypropyl-CoM dehydrogenase, component II and EC 1.1.1.269 2-(S)-hydroxypropyl-CoM dehydrogenase, component IV that is involved in epoxyalkane carboxylation [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <4> [2, 3] Cloning <2> (expressed in Mycobacterium smegmatis) [4] <4> (expression in Escherichia coli) [2] Engineering C220A <4> (<4> largely catalytically inactive protein, 0.06% of wild-type activity [3]) [3]

References [1] Allen, J.R.; Clark, D.D.; Krum, J.G.; Ensign, S.A.: A role for coenzyme M (2mercaptoethanesulfonic acid) in a bacterial pathway of aliphatic epoxide carboxylation. Proc. Natl. Acad. Sci. USA, 96, 8432-8437 (1999)

409

2-Hydroxypropyl-CoM lyase

4.4.1.23

[2] Krum, J.G.; Ensign, S.A.: Heterologous expression of bacterial epoxyalkane:coenzyme M transferase and inducible coenzyme M biosynthesis in Xanthobacter strain Py2 and Rhodococcus rhodochrous B276. J. Bacteriol., 182, 2629-2634 (2000) [3] Krum, J.G.; Ellsworth, H.; Sargeant, R.R.; Rich, G.; Ensign, S.A.: Kinetic and microcalorimetric analysis of substrate and cofactor interactions in epoxyalkane:CoM transferase, a zinc-dependent epoxidase. Biochemistry, 41, 5005-5014. (2002) [4] Coleman, N.V.; Spain, J.C.: Epoxyalkane:coenzyme M transferase in the ethene and vinyl chloride biodegradation pathways of mycobacterium strain JS60. J. Bacteriol., 185, 5536-5545 (2003) [5] Mattes, T.E.; Coleman, N.V.; Spain, J.C.; Gossett, J.M.: Physiological and molecular genetic analyses of vinyl chloride and ethene biodegradation in Nocardioides sp. strain JS614. Arch. Microbiol., 183, 95-106 (2005)

410

Sulfolactate sulfo-lyase

4.4.1.24

1 Nomenclature EC number 4.4.1.24 Systematic name 3-sulfolactate bisulfite-lyase (pyruvate-forming) Recommended name sulfolactate sulfo-lyase Synonyms 3-sulfolactate sulfo-lyase <1> [1] Suy <1> [1] Additional information <1> (<1> the enzyme belongs to the altronate dehydratase family [1]) [1]

2 Source Organism <1> Paracoccus pantotrophus NKNCYSA (UNIPROT accession number: Q58Y44) [1]

3 Reaction and Specificity Catalyzed reaction 3-sulfolactate = pyruvate + bisulfite (<1> putative reaction mechanism [1]) Natural substrates and products S 3-sulfolactate <1> (<1> desulfonation step involved in cysteate dissimilation, metabolic pathway in the cell, overview [1]) (Reversibility: ?) [1] P pyruvate + bisulfite S Additional information <1> (<1> activity with 3-sulfopyruvate in vivo by cysteate-grown cells only after membrane permeabilization [1]) (Reversibility: ?) [1] P ? Substrates and products S 3-sulfolactate <1> (<1> desulfonation step involved in cysteate dissimilation, metabolic pathway in the cell, overview [1]) (Reversibility: ?) [1] P pyruvate + bisulfite

411

Sulfolactate sulfo-lyase

4.4.1.24

S Additional information <1> (<1> activity with 3-sulfopyruvate in vivo by cysteate-grown cells only after membrane permeabilization [1]) (Reversibility: ?) [1] P ? Inhibitors EDTA <1> (<1> 70% inhibition at 1 mM [1]) [1] Activating compounds Additional information <1> (<1> the enzyme is strongly inducible [1]) [1] Metals, ions Co2+ <1> (<1> can substitute for Fe2+ at 2mM [1]) [1] Fe2+ <1> (<1> required for activity [1]) [1] Mn2+ <1> (<1> can substitute for Fe2+ at 2mM [1]) [1] Specific activity (U/mg) 0.3 <1> (<1> purified enzyme [1]) [1]

4 Enzyme Structure Subunits ? <1> (<1> x * 8000, a-subunit, + x * 42000, b-subunit, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue culture condition:(R)-cysteate-grown cell <1> (<1> i.e. 2-amino-3-sulfopropionate, is the sole carbon and energy source of the organism with either nitrate or molecular oxygen as terminal electron acceptor [1]) [1] culture condition:sulfolactate-grown cell <1> [1] Purification <1> (from cysteate-grown cells by anion exchange chromatography and hydrophobic interaction chromatography, to near homogeneity) [1] Cloning <1> (gene suyAB, DNA and amino acid sequence determination and analysis) [1]

References [1] Rein, U.; Gueta, R.; Denger, K.; Ruff, J.; Hollemeyer, K.; Cook, A.M.: Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulfate exporter in Paracoccus pantotrophus NKNCYSA. Microbiology, 151, 737-747 (2005)

412

L-Cysteate

sulfo-lyase

4.4.1.25

1 Nomenclature EC number 4.4.1.25 Systematic name l-cysteate bisulfite-lyase (deaminating) (pyruvate-forming) Recommended name l-cysteate sulfo-lyase Synonyms CuyA <3> [1] l-cysteate sulpho-lyase (deaminating) <3> [1]

2 Source Organism <1> Desulfovibrio sp. (no sequence specified) [1] <2> Bilophila wadsworthia (no sequence specified) [1] <3> Silicibacter pomeroyi (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction l-cysteate + H2 O = pyruvate + bisulfite + NH3 Reaction type C-S bond cleavage Natural substrates and products S l-cysteate + H2 O <1, 2, 3> (<1,2,3> inducible enzyme [1]) (Reversibility: ?) [1] P pyruvate + bisulfite + NH3 Substrates and products S l-cysteate + H2 O <1, 2, 3> (<1,2,3> inducible enzyme [1]) (Reversibility: ?) [1] P pyruvate + bisulfite + NH3 S Additional information <3> (<3> enzyme also catalyzes the d-cysteine desulphhydrase reaction [1]) (Reversibility: ?) [1] P ?

413

L-Cysteate

sulfo-lyase

4.4.1.25

Cofactors/prosthetic groups pyridoxal 5’-phosphate <3> (<3> tightly bound to the enzyme [1]) [1] Km-Value (mM) 11.7 <3> (l-cysteate, <3> pH 9.0, 30 C [1]) [1] pH-Optimum 8.8-9 <3> (<3> enzyme in crude cell extract, 50 mM Tris buffer [1]) [1]

4 Enzyme Structure Molecular weight 100000 <3> (<3> gel filtration [1]) [1] Subunits ? <3> (<3> x * 39000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <3> [1]

6 Stability General stability information <3>, frozen samples lose about 90% of the activity on thawing [1] Storage stability <3>, 4 C, 2 months without significant loss of activity [1]

References [1] Denger, K.; Smits, T.H.; Cook, A.M.: l-Cysteate sulpho-lyase, a widespread pyridoxal 5’-phosphate-coupled desulphonative enzyme purified from Silicibacter pomeroyi DSS-3(T). Biochem. J., 394, 657-664 (2006)

414

2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase

4.6.1.12

1 Nomenclature EC number 4.6.1.12 Systematic name 2-phospho-4-(cytidine (cyclizing)

5’-diphospho)-2-C-methyl-d-erythritol

CMP-lyase

Recommended name 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase Synonyms 2C-methyl-d-erythrol-2,4-cyclodiphosphate synthase <8> [11] IspDF <3, 4> (<3, 4> bifunctional methylerythritol 4-phosphate cytidyltransferase methylerythritol 2,4-cyclodidiphosphate synthase. Complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-d-erythritol kinase is observed in solution [7]) [7] IspE <2> (<2> monofunctional enzyme [7]) [7] MEC synthase <2> [5] MECDP synthase <1, 5> [2, 3] MECDP-synthase <6> [1] MECP <2> [12] MECPS MECS <9> [13] YgbB protein <6> [1, 9] CAS registry number 287480-92-6

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9>

Thermus thermophilus (no sequence specified) [2] Escherichia coli (no sequence specified) [1, 5, 7, 8, 10, 12] Agrobacterium tumefaciens (no sequence specified) [7] Campylobacter jejuni (no sequence specified) [7] Shewanella oneidensis (no sequence specified) [3] Escherichia coli (UNIPROT accession number: P62617) [1,6,9] Plasmodium falciparum (UNIPROT accession number: P62368) [4] Haemophilus influenzae (UNIPROT accession number: P44815) [11] Ginkgo biloba (UNIPROT accession number: Q1EDG4) [13]

415

2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase

4.6.1.12

3 Reaction and Specificity Catalyzed reaction 2-phospho-4-(cytidine 5’-diphospho)-2-C-methyl-d-erythritol = 2-C-methyld-erythritol 2,4-cyclodiphosphate + CMP Reaction type P-O bond cleavage formation of diphosphate Natural substrates and products S 2-phospho-4-(cytidine 5’-diphospho)-2-C-methyl-d-erythritol <2> (<2> involved in mevalonate-independent biosynthesis of isoprenoids [8]) (Reversibility: ?) [8] P 2-C-methyl-d-erythritol 2,4-cyclodiphosphate + CMP S 2-phospho-4-(cytidine 5’-diphospho)-2C-methyl-d-erythritol <7> (<7> nonmevalonate pathway of isoprenoid biosynthesis [4]) (Reversibility: ?) [4] P 2C-methyl-d-erythritol-2,4-cyclodiphosphate + CMP S Additional information <2> (<2> depletion of MEC synthase has an early and significant impact on cell wall biosynthesis and leads ultimately to cell death [5]) (Reversibility: ?) [5] P ? Substrates and products S 2-phospho-4-(cytidine 5’-diphospho)-2-C-methyl-d-erythritol <2, 9> (<2> involved in mevalonate-independent biosynthesis of isoprenoids [8]) (Reversibility: ?) [1, 8, 12, 13] P 2-C-methyl-d-erythritol 2,4-cyclodiphosphate + CMP S 2-phospho-4-(cytidine 5’-diphospho)-2C-methyl-d-erythritol <7> (<7> at low rate [4]) (Reversibility: ?) [4] P 2-phospho-2C-methyl-d-erythritol 3,4-cyclophosphate + CMP S 2-phospho-4-(cytidine 5’-diphospho)-2C-methyl-d-erythritol <6, 7> (<7> nonmevalonate pathway of isoprenoid biosynthesis [4]) (Reversibility: ?) [1, 4, 6, 9] P 2C-methyl-d-erythritol-2,4-cyclodiphosphate + CMP S 4-diphosphocytidyl-2C-methyl-d-erythritol <7> (<7> at low rate [4]) (Reversibility: ?) [4] P 2C-methyl-d-erythritol 3,4-cyclophosphate + CMP S Additional information <2> (<2> depletion of MEC synthase has an early and significant impact on cell wall biosynthesis and leads ultimately to cell death [5]) (Reversibility: ?) [5] P ? Inhibitors EDTA <7> (<7> 5 mM. Activity can be restored by the addition of Mg2+ to a concentration of 10 mM [4]) [4]

416

4.6.1.12

2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase

Metals, ions Mg2+ <2, 6, 7> (<6> Mn2+ or Mg2+ required [9]; <2> dependent on [8]; <7> Mg2+ or Mn2+ are required [4]) [4, 8, 9] Mn2+ <2, 6, 7> (<6> Mn2+ or Mg2+ required [9]; <7> Mg2+ or Mn2+ are required [4]; <2> a Mn2+ with octahedral geometry, is positioned between the a and b phosphates acting in concert with the Zn2+ to align and polarize the substrate for catalysis [10]) [4, 9, 10, 12] Na+ <5> (<5> presence of tetrahedral Zn2+ in one of the metal-binding sites and an octahedral sodium ion in the second metal site in absence of substrate [3]) [3] Zinc <2> (<2> tightly binds one zinc ion per subunit of the trimer at the active site, which helps to position the substrate for direct attack of the 2phosphate group on the b-phosphate [8]) [8] Zn2+ <2, 5> (<2> active site contains a Zn2+ with tetrahedral coordination [10]; <5> presence of tetrahedral Zn2+ in one of the metal-binding sites and an octahedral sodium ion in the second metal site in absence of substrate [3]) [3, 10, 12] Additional information <6> (<6> the tetrahedrally arranged transition metal binding site, potentially occupied by Mn2+ , sits at the base of the active site cleft. A phosphate oxygen of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate and the side chains of Asp8, His10, and His42 occupy the metal side chains of Asp8, His10, and His42 occupy the metal side coordination sphere [6]) [6] Specific activity (U/mg) 0.75 <7> [4] Km-Value (mM) 0.252 <7> (4-diphosphocytidyl-2C-methyl-d-erythritol, <7> pH 7.0, 27 C [4]) [4] pH-Optimum 7 <7> [4]

4 Enzyme Structure Molecular weight 19300 <9> (<9> calculated from amino acid sequence after removal of 59 Nterminal amino acids comprising a chloroplast transit peptide [13]) [13] 26030 <9> (<9> calculated from amino acid sequence, including a N-terminal chloroplast transit peptide [13]) [13] Subunits ? <6, 7> (<6> x * 17000, SDS-PAGE [9]; <7> x * 21348, electrospray mass spectrometry [4]) [4, 9] hexamer <4> (<4> complex formation of IspDF with 4-diphosphocytidyl2C-methyl-d-erythritol kinase is observed in solution [7]) [7]

417

2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase

4.6.1.12

trimer <2, 5, 6> (<5> 3 * 17300 [3]; <6> homotrimeric quarternary structure built around a central hydrophobic cavity and three externally facing active sites [6]) [3, 6, 8, 10] Additional information <2, 3> (<3> complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-d-erythritol kinase is observed in solution [7]; <2> the monofunctional enzyme 2C-methyl-d-erythritol-4-phosphate cytidyltransferase and 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase shows physical association [7]) [7]

5 Isolation/Preparation/Mutation/Application Purification <1> [2] <2> [6, 9] <5> [3] <7> [4] Crystallization <1> (crystals grow in space group P4(1)2(1)2 from polyethylene glycol using the hanging-drop method) [2] <2> (3.1 A resolution crystal structure of the Met142/Leu144 mutant) [12] <4> (hanging drop vapor diffusion method, crystal structure of IspDF, a bifunctional methylerythritol 4-phosphate cytidyltransferase methylerythritol 2,4-cyclodidiphosphate synthase) [7] <5> (high-resolution structure, 16 A, of the enzyme in absence of substrate in the active site. Optimized crystals are obtained at a protein concentration of 35 mg/ml in a solution containing 4 M sodium formate and 5% glycerol. The crystals grow to avarage dimensions of 0.4 * 0.3 * 0.3 mM within a week) [3] <6> [8] <6> (hanging-drop vapour diffusion method, X-ray crystal structures refined to 2.8 A resolution. The first structure contains a bound Mn2+ cation and the second structure contains CMP, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, and Mn2+ ) [6] <6> (vapor-diffusion hanging drop method, crystal structure of the zinc enzyme in complex with cytidine 5’-diphosphate and Mn2+ is determined to 1.8 A resolution) [10] <8> (hanging drop vapor diffusion method) [11] Cloning <5> [3] <7> (expression in Escherichia coli) [4] <8> (cloned into pET17b for expression of the native polypeptide in Escherichia coli strain BL21) [11] <9> (expression in Escherichia coli strain NMW26 and Arabidopsis thaliana) [13]

418

4.6.1.12

2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase

Engineering Arg142Met, Glu144Leu <2> (<2> dual mutation with little influence on both the overall structure and the detail in the active site [12]) [12] Application medicine <7> (<7> the enzyme is a potential target for antimalarial drugs directed at the nonmevalonate pathway of isoprenoid biosynthesis [4]) [4]

References [1] Takagi, M.; Kuzuyama, T.; Kaneda, K.; Watanabe, H.; Dairi, T.; Seto, H.: Studies on the nonmevalonate pathway: Formation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate from 2-phospho-4-(cytidine 5’-diphospho)-2C-methyl-d-erythritol. Tetrahedron Lett., 41, 3395-3398 (2000) [2] Kishida, H.; Wada, T.; Unzai, S.; Kuzuyama, T.; Takagi, M.; Terada, T.; Shirouzu, M.; Yokoyama, S.; Tame, J.R.; Park, S.Y.: Structure and catalytic mechanism of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MECDP) synthase, an enzyme in the non-mevalonate pathway of isoprenoid synthesis. Acta Crystallogr. Sect. D, 59, 23-31 (2003) [3] Ni, S.; Robinson, H.; Marsing, G.C.; Bussiere, D.E.; Kennedy, M.A.: Structure of 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase from Shewanella oneidensis at 1.6 : identification of farnesyl pyrophosphate trapped in a hydrophobic cavity. Acta Crystallogr. Sect. D, D60, 1949-1957 (2004) [4] Rohdich, F.; Eisenreich, W.; Wungsintaweekul, J.; Hecht, S.; Schuhr, C.A.; Bacher, A.: Biosynthesis of terpenoids. 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (ispF) from Plasmodium falciparum. Eur. J. Biochem., 268, 3190-3197 (2001) [5] Campbell, T.L.; Brown, E.D.: Characterization of the depletion of 2-Cmethyl-d-erythritol-2,4-cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis. J. Bacteriol., 184, 5609-5618 (2002) [6] Richard, S.B.; Ferrer, J.L.; Bowman, M.E.; Lillo, A.M.; Tetzlaff, C.N.; Cane, D.E.; Noel, J.P.: Structure and mechanism of 2-C-methyl-d-erythritol 2,4cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway. J. Biol. Chem., 277, 8667-8672 (2002) [7] Gabrielsen, M.; Bond, C.S.; Hallyburton, I.; Hecht, S.; Bacher, A.; Eisenreich, W.; Rohdich, F.; Hunter, W.N.: Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis. J. Biol. Chem., 279, 52753-52761 (2004) [8] Steinbacher, S.; Kaiser, J.; Wungsintaweekul, J.; Hecht, S.; Eisenreich, W.; Gerhardt, S.; Bacher, A.; Rohdich, F.: Structure of 2C-methyl-d-erythritol2,4-cyclodiphosphate synthase involved in mevalonate-independent biosynthesis of isoprenoids. J. Mol. Biol., 316, 79-88 (2002) [9] Herz, S.; Wungsintaweekul, J.; Schuhr, C.A.; Hecht, S.; Lttgen, H.; Sagner, S.; Fellermeier, M.; Eisenreich, W.; Zenk, M.H.; Bacher, A.; Rohdich, F.: Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-

419

2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase

[10]

[11]

[12]

[13]

420

4.6.1.12

methyl-d-erythritol 2-phosphate to 2C-methyl-d-erythritol 2,4-cyclodiphosphate. Proc. Natl. Acad. Sci. USA, 97, 2486-2490 (2000) Kemp, L.E.; Bond, C.S.; Hunter, W.N.: Structure of 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase: an essential enzyme for isoprenoid biosynthesis and target for antimicrobial drug development. Proc. Natl. Acad. Sci. USA, 99, 6591-6596 (2002) Lehmann, C.; Lim, K.; Toedt, J.; Krajewski, W.; Howard, A.; Eisenstein, E.; Herzberg, O.: Structure of 2C-methyl-d-erythrol-2,4-cyclodiphosphate synthase from Haemophilus influenzae: Activation by conformational transition. Proteins, 49, 135-138 (2002) Sgraja, T.; Kemp, L.E.; Ramsden, N.; Hunter, W.N.: A double mutation of Escherichia coli 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase disrupts six hydrogen bonds with, yet fails to prevent binding of, an isoprenoid diphosphate. Acta Crystallogr. Sect. F, F61, 625-629 (2005) Kim, S.M.; Kuzuyama, T.: Cloning and characterization of 2-C-methyl-derythritol 2,4-cyclodiphosphate synthase (MECS) gene from Ginkgo biloba. Plant Cell Rep., 8, 829-835 (2006)

Phosphatidylinositol diacylglycerol-lyase

4.6.1.13

1 Nomenclature EC number 4.6.1.13 Systematic name 1-phosphatidyl-1d-myo-inositol 1,2-diacyl-sn-glycerol lyase (1d-myo-inositol-1,2-cyclic phosphate-forming) Recommended name phosphatidylinositol diacylglycerol-lyase Synonyms 1-phosphatidyl-d-myo-inositol inositolphosphohydrolase (cyclic-phosphateforming) 1-phosphatidylinositol phosphodiesterase EC 3.1.4.10 (formerly) Phosphatidylinositol diacylglycerol-lyase monophosphatidylinositol phosphodiesterase phosphatidylinositol phosphodiesterase phosphatidylinositol phospholipase C phosphatidylinositol-specific phospholipase C <1, 8, 14, 16, 20> [42, 43, 44, 45, 46] CAS registry number 37288-19-0 63551-76-8

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11>

Staphylococcus aureus (no sequence specified) [45] Mus musculus (no sequence specified) [6] Homo sapiens (no sequence specified) [3, 9, 16, 26] Rattus norvegicus (no sequence specified) [1, 6, 7, 8, 10, 11, 13, 20] Sus scrofa (no sequence specified) [4, 12, 22] Bos taurus (no sequence specified) [2,5,23] Oryctolagus cuniculus (no sequence specified) [15] Bacillus cereus (no sequence specified) [18,19,21,25,28,30,32,37,38,45] Bacillus sp. (no sequence specified) [31,33] Equus caballus (no sequence specified) [9] Apium graveolens (no sequence specified) [17]

421

Phosphatidylinositol diacylglycerol-lyase

<12> <13> <14> <15> <16> <17> <18> <19> <20>

4.6.1.13

Brassica oleracea (no sequence specified) [17] Allium cepa (no sequence specified) [17] Listeria monocytogenes (no sequence specified) [29, 36, 42, 45, 46] Streptomyces antibioticus (no sequence specified) [24] Bacillus thuringiensis (no sequence specified) [14, 21, 27, 34, 35, 39, 40, 41, 45] no activity in Solanum tuberosum [17] no activity in Pyrus malus [17] Narcissus sp. (no sequence specified) [17] Bacillus anthracis (no sequence specified) [43, 44]

3 Reaction and Specificity Catalyzed reaction 1-phosphatidyl-1d-myo-inositol = 1d-myo-inositol 1,2-cyclic phosphate + 1,2-diacyl-sn-glycerol (<8,9,16> mechanism [31,32,33,37,41]; <16> general acid/general base mechanism [34]) Reaction type P-O bond cleavage Natural substrates and products S 1-phosphatidyl-1d-myo-inositol <8, 14> (<8> natural substrate [37]; <14> natural aggregate substrate, PI-PLC is a virulence factor of the animal and human pathogen [36]) (Reversibility: ?) [36, 37] P 1d-myo-inositol 1,2-cyclic phosphate + diacylglycerol <8, 14> [36, 37] S Additional information <14, 16, 20> (<20> Bacillus anthracis enzyme down-modulates dendritic cell function und T cell responses, possibly by cleaving GPI-anchored proteins important for TLR-mediated dendritic cell activation [44]; <14> Listeria monocytogenes phosphatidylinositolspecific phospholipase C is an important determinant of Listeria monocytogenes pathogenesis by absence of the Vb b-strand, thus leading to greatly reduced activity on GPI-anchored proteins [46]; <14> the enzyme activates a host protein kinase C cascade which promotes escape of the bacterium from a macrophage-like cell phagosome [42]; <14> the enzyme contributes to listerial infection of epithelial cells and macrophages as a virulence factor cooperating with other factors such as listeriolysin O and phosphatidylcholine-preferring phospholipase C [45]; <16> the enzyme exhibits cytotoxicity against some cultivated cells [45]; <20> the enzyme may have a role in Bacillus anthracis pathogenesis [43]) (Reversibility: ?) [42, 43, 44, 45, 46] P ? Substrates and products S 1-phosphatidyl-1d-myo-inositol <14> (<14> natural aggregate substrate, two-site enzyme model with interfacial cooperativity between the active site and a lipid-binding subsite, presumably adjacent to the active site [36]) (Reversibility: ?) [36]

422

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Phosphatidylinositol diacylglycerol-lyase

P 1d-myo-inositol 1,2-cyclic phosphate + sn-1,2-diacylglycerol <14> [36] S 1-phosphatidyl-1d-myo-inositol <8, 9, 14, 16> (<8> natural substrate [37]; <8> a catalytic diad at the active site composed of Asp-274 and His-32 is involved in substrate-assisted catalysis, its function is to hydrogen-bond with the 2-OH of phosphatidylinositol to form a catalytic triad, catalytic mechanism [32]; <16> aggregated substrate is preferred over monomeric substrate [35]; <9> catalytic mechanism, role of Arg-69, the bidentate nature of Arg-69 is the origin of the large thio effects and stereoselectivity in PI-PLC, its function is to bring the phosphate group and the 2-OH group of inositol into proximity and to induce proper alignment for nucleophilic attack, and possibly to lower the pKa of the 2-OH [31]; <16> catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol, catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad, catalytic mechanism, active site structure [41]; <8> cleaves phosphatidylinositol in a rapid intramolecular transphosphorylation reaction forming the products, in a second reaction the cyclic phosphorylase activity of PI-PLC catalyzes the slow hydrolysis of 1d-myo-inositol 1,2-cyclic phosphate to d-myo-inositol 1-phosphate, utilizes His-32 and His-82 in a general acid catalysis mechanism [37]; <16> general acid/general base mechanism, enhanced activity when phosphatidylinositol is present in an interface compared to monomeric substrate [34]; <9> PLC accepts only nonphosphorylated phosphatidylinositol substrates and produces cyclic inositol phosphate as final product, which is hydrolyzed at a 1000fold lower rate, catalytic mechanism, uses a guanidinium group of Arg-69 during catalysis [33]; <16> the active site is located at the C-terminal side [40]; <16> Trp-47 and Trp-242 residues are important for enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces, micellar phosphatidylinositol is a better substrate than monomeric phosphatidylinositol [39]; <14> natural aggregate substrate, PI-PLC is a virulence factor of the animal and human pathogen [36]) (Reversibility: ?) [31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41] P 1d-myo-inositol 1,2-cyclic phosphate + diacylglycerol <8, 9, 14, 16> (<16> a cyclic phosphodiesterase activity of PI-PLC converts 1d-myo-inositol 1,2-cyclic phosphate to inositol 1-phosphate [35,39]; <8> PI-PLC catalyzes in a second step the slow hydrolysis of 1d-myo-inositol 1,2-cyclic phosphate to form myo-inositol 1-phosphate [32]; <16> PI-PLC catalyzes the hydrolysis of myo-inositol 1,2-cyclic phosphate to myo-inositol 1-phosphate [34]) [31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41] S butyl-fluorescein myo-inositol phosphate <8> (<8> two substrate molecules bind to enzyme, one at the active site and one at a subsite, causing an increase in activity, subsite interactions of PI-PLC [37]) (Reversibility: ?) [37] P d-myo-inositol 1,2-cyclic phosphate + butyl-fluorescein <8> [37] S lysophosphatidylinositol + H2 O <8, 16> (Reversibility: ?) [14, 18] P ? S methyl-fluorescein myo-inositol phosphate <8, 14> (<14> monomeric substrate, only the d-enantiomer is active [36]; <8> substrate binds only

423

Phosphatidylinositol diacylglycerol-lyase

P S

P

S

424

4.6.1.13

to the active site and not to the activator site [37]) (Reversibility: ?) [36, 37] d-myo-inositol 1,2-cyclic phosphate + methyl-fluorescein <8, 14> [36, 37] phosphatidylinositol + H2 O <1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 19> (<4,6,15> specific for [2,8,24]; <4> the low molecular weight enzyme form hydrolyzes both phosphatidylinositol and phosphatidylinositol 4,5diphosphate, the high molecular weight enzyme form shows much greater activity against phosphatidylinositol than phosphatidylinositol 4,5-diphosphate [1]; <4> substrate in three forms: 1. multilayer liposomes, 2. single bilayer vesicles of phosphatidylinositol, 3. phosphatidylinositol oriented as monolayer at the air-water interface [20]; <5> phosphatidylinositol monolayer at an air/water interface [12]; <4> hydrolysis of membrane-bound and extracted phosphatidylinositol [7]; <1,14> at first the enzyme catalyzes phosphate transfer within the molecule of phosphatidylinositol from glycerol OH to 2-OH of myo-inositol, resulting in diacylglycerol and myo-inositol 1,2-cyclic phosphate. Next myo-inositol 1,2-cyclic phosphate is hydrolyzed by the enzyme to inositol 1-phosphate. Since the reaction rate of the first step (phosphotransferase) is 1000 times as much as that of the second step (cyclic phosphodiesterase) myo-inositol 1,2-cyclic phosphate accumulates as one of the major products during enzyme action [45]; <16> degrades synthetic phosphatidylinositols in the following order dilauroyl > dimyristoly > dioleoyl > dipalmitoyl. At first the enzyme catalyzes phosphate transfer within the molecule of phosphatidylinositol from glycerol OH to 2-OH of myo-inositol, resulting in diacylglycerol and myo-inositol 1,2-cyclic phosphate. Next myo-inositol 1,2-cyclic phosphate is hydrolyzed by the enzyme to inositol 1-phosphate. Since the reaction rate of the first step (phosphotransferase) is 1000 times as much as that of the second step (cyclic phosphodiesterase) myo-inositol 1,2-cyclic phosphate accumulates as one of the major products during enzyme action [45]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45] diacylglycerol + myo-inositol 1,2-cyclic phosphate <3, 4, 5, 6, 7, 8, 10, 11, 15, 16> (<7> 1,2-cyclic phosphoinositol and phosphoinositol in the approximate propertion of 60% and 40% respectively plus diacylglycerol [15]; <4> the ratio between inositol 1,2-cyclic phosphate and inositol 1phosphate produced decreases with increasing pH [11]; <5,8> diacylglycerol and a mixture of myo-inositol 1-phosphate and myo-inositol 1,2cyclic phosphate [19,22]; <4> myo-inositol 1,2-cyclic phosphate, myo-inositol 1-phosphate and glycerophosphoinositol in the molar proportions approximately 2:1:1 [7]; <6> diacylglycerol + ? [2]; <3,10> myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate in almost equal amounts [9]; <11> 1,2-diacylglycerol and a mixture of 86% myo-inositol 1-phosphate and 14% myo-inositol 1,2-(cyclic)phosphate [17]; <16> myoinositol-1,2-cyclic phosphate appears as sole product [14]) [2, 7, 9, 11, 14, 15, 17, 19, 22, 24] phosphatidylinositol + H2 O <8> (<8> at first the enzyme catalyzes phosphate transfer within the molecule of phosphatidylinositol from glycerol

4.6.1.13

P S

P S P S

P

Phosphatidylinositol diacylglycerol-lyase

OH to 2-OH of myo-inositol, resulting in diacylglycerol and myo-inositol 1,2-cyclic phosphate. Next myo-inositol 1,2-cyclic phosphate is hydrolyzed by the enzyme to inositol 1-phosphate. Since the reaction rate of the first step (phosphotransferase) is 1000 times as much as that of the second step (cyclic phosphodiesterase) myo-inositol 1,2-cyclic phosphate accumulates as one of the major products during enzyme action [45]) (Reversibility: ?) [45] diacylglycerol + myo-inositol 1,2-cyclic phosphate + d-myo-inositol 1phosphate phosphatidylinositol 4,5-diphosphate + H2 O <4, 7> (<4> the low molecular weight enzyme form hydrolyzes both phosphatidylinositol and phosphatidylinositol 4,5-diphosphate, the high molecular weight enzyme form shows much greater activity against phosphatidylinositol than phosphatidylinositol 4,5-diphosphate [1]; <7> at 34.9% of the activity with phosphatidylinositol [15]) (Reversibility: ?) [1, 15] ? triphosphoinositide + H2 O <7> (<7> at 37% of the activity with phosphatidylinositol [15]) (Reversibility: ?) [15] ? Additional information <14, 16, 20> (<16> approximately 20% of the alkaline phosphodiesterase I activity is released from the apical surface of the pig LLC-PK1 cells by the action of the 1-phosphatidylinositol phosphodiesterase [27]; <16> not: phosphatidylcholine [35,39]; <20> Bacillus anthracis enzyme down-modulates dendritic cell function und T cell responses, possibly by cleaving GPI-anchored proteins important for TLRmediated dendritic cell activation [44]; <14> Listeria monocytogenes phosphatidylinositol-specific phospholipase C is an important determinant of Listeria monocytogenes pathogenesis by absence of the Vb bstrand, thus leading to greatly reduced activity on GPI-anchored proteins [46]; <14> the enzyme activates a host protein kinase C cascade which promotes escape of the bacterium from a macrophage-like cell phagosome [42]; <14> the enzyme contributes to listerial infection of epithelial cells and macrophages as a virulence factor cooperating with other factors such as listeriolysin O and phosphatidylcholine-preferring phospholipase C [45]; <16> the enzyme exhibits cytotoxicity against some cultivated cells [45]; <20> the enzyme may have a role in Bacillus anthracis pathogenesis [43]; <20> enzyme from Bacillus anthracis unlike the ortholog from Listeria monocytogenes shows high activity on glycosylphosphatidylinositol-anchored proteins [43]) (Reversibility: ?) [27, 35, 39, 42, 43, 44, 45, 46] ? <16> [35, 39]

Inhibitors AOT <8> (<8> bis(2-ethylhexyl)sulfosuccinate [28]) [28] ATP <4> (<4> Ca2+ -stimulated enzyme or Mg2+ -stimulated enzyme [8]) [8] Ba2+ <7> (<7> at 0.05-5 mM [15]) [15] blood plasma <4> [13]

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Phosphatidylinositol diacylglycerol-lyase

4.6.1.13

Ca2+ <3, 5, 8, 16> (<3> at high concentrations [16]; <16> above 1 mM [14]; <8> 2-5 mM, 50% inhibition [18]; <5> at pH 7.0, above 0.4 mM [22]) [14, 16, 18, 22] Cd2+ <9, 15> (<9> severe inhibition of wild-type PI-PLC [33]) [24, 33] chlorpromazine <5> (<5> at pH 7.0 [22]) [22] choline plasmalogen <3> [3] cinchocaine <5> (<5> at pH 7.0 [22]) [22] Co2+ <9> (<9> severe inhibition of wild-type PI-PLC [33]) [33] EDTA <3, 7, 15> (<3> 2 mM, 70% inhibition [16]) [15, 16, 24] EGTA <6, 7, 15> [5, 15, 24] Hg2+ <15> [24] HgCl2 <4> (<4> inhibits Ca2+ -stimulated enzyme [8]) [8] KCl <16> (<16> above 10 mM [14]) [14] La3+ <7> (<7> at 0.05-5 mM [15]) [15] lysophosphatidylcholine <3, 4> (<4> C12 , C14 and C16 lysophosphatidylcholines give progressive inhibition with increasing chain length [13]) [3, 13] Mg2+ <5, 7, 8, 16> (<16> above 1 mM [14]; <8> 2-5 mM, 50% inhibition [18]; <5> at pH 7.0, above 0.4 mM [22]) [14, 15, 18, 22] Mn2+ <7, 8> (<8> 2-5 mM, 50% inhibition [18]; <7> at 0.05-5 mM [15]) [15, 18] NaCl <8, 16> (<8> at high concentrations [18]; <16> above 1 mM [14]) [14, 18] Ni2+ <9> (<9> severe inhibition of wild-type PI-PLC [33]) [33] octadecylamine <5> (<5> decreases the film pressure of the phosphatidylinositol monolayer at an air/water interface at which cut-off occurs, as well as the rate of hydrolysis below this pressures [12]) [12] palmitoylcholine <5> (<5> decreases the film pressure of the phosphatidylinositol monolayer at an air/water interface at which cut-off occurs, as well as the rate of hydrolysis below this pressure [12]) [12] phosphatidylcholine <3, 5, 11> (<5> decreases the film pressure of the phosphatidylinositol monolayer at an air/water interface at which cut-off occurs, as well as the rate of hydrolysis below this pressure [12]; <11> 30% inhibition at concentrations equimolar with phosphatidylinositol [17]) [3, 12, 17] sphingomyelin <3> [3] Sr2+ <7> (<7> above 0.25 mM [15]) [15] tetradecyltrimethylammonium bromide <4> [8] Triton X-100 <4> [8] Zn2+ <8, 9, 15, 16> (<16> above 1 mM [14]; <8> 2-5 mM, 50% inhibition [18]; <9> severe inhibition of wild-type PI-PLC, complete inhibition of R69D mutant below 1 mM [33]) [14, 18, 24, 33] deoxycholate <3, 10> (<3,10> weak [9]) [9] hydrogenated phosphatidylcholine <3> [3] lipoproteins <4> (<4> from blood [13]) [13] lysocholine plasmalogen <3> [3] myo-inositol <16> (<16> weak [35]; <16> poor competitive inhibitor [39]) [35, 39] sphingosylphosphocholine <3> [3]

426

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Phosphatidylinositol diacylglycerol-lyase

vanadate <8> [28] Additional information <4> (<4> the enzyme is strongly inhibited when ovophosphatidylcholine and saturated phosphatidylcholines with acyl chain length of more than eight carbon atoms are mixed with its substrate [13]) [13] Activating compounds chlorpromazine <5> (<5> stimulates at pH 5.5 [22]) [22] cinchocaine <5> (<5> stimulates at pH 5.5 [22]) [22] diethyl ether <15> (<15> strongly activates [24]) [24] dimethylformamide <16> (<16> water-miscible, enhances phosphotransferase activity [35]) [35] dimethylsulfoxide <16> (<16> water-miscible, enhances phosphotransferase activity [35]) [35] isopropanol <16> (<16> 30%, activates [39]; <16> water-miscible, 30%, activates [40]; <16> water-miscible, maximum activation at 30%, activates regardless of the type of phosphatidylinositol substrate, enhances phosphotransferase activity [35]) [35, 39, 40] oleic acid <4, 5> (<4> marked stimulation with membrane-bound phosphatidylinositol as substrate, slight stimulation with isolated phosphatidylinositol as substrate [7]; <5> markedly increases rate of hydrolysis [12]) [7, 12] oleyl alcohol <5> (<5> increases the film pressure of the phosphatidylinositol monolayer at an air/water interface at which cut-off occurs, as well as increases the rate of hydrolysis at lower pressures [12]) [12] phosphatidic acid <4, 5, 11, 16> (<11> stimulates [17]; <4> activates [13]; <5> increases the film pressure of the phosphatidylinositol monolayer at an air/water interface at which cut-off occurs, as well as increases the rate of hydrolysis at lower pressure [12]; <16> binding to nonsubstrate anionic interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism [34]) [12, 13, 17, 34] phosphatidylcholine <16> (<16> activates [35,39]; <16> binding to nonsubstrate zwitterionic phosphatidylcholine interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism [34]; <16> PI-PLC is activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers, activation corresponds with partial insertion into the interface of Trp-47 and Trp-242 in the rim of the ab-barrel [40]) [34, 35, 39, 40] phosphatidylethanolamine <5> (<5> slightly increases rate of hydrolysis [12]) [12] phosphatidylglycerol <5, 16> (<5> increases the film pressure of the phosphatidylinositol monolayer at an air/water interface at which cut-off occurs, as well as increases the rate of hydrolysis at lower pressures [12]; <16> binding to nonsubstrate anionic interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism [34]) [12, 34] phosphatidylserine <16> (<16> binding to nonsubstrate anionic interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism [34]) [34]

427

Phosphatidylinositol diacylglycerol-lyase

4.6.1.13

SDS <15> (<15> strongly activates [24]) [24] Triton X-100 <8, 16> (<8,16> stimulates [14,18]) [14, 18] Tween 20 <16> (<16> slight stimulation [14]) [14] butyl-fluorescein myo-inositol phosphate <8> (<8> two molecules bind to enzyme, one at the active site and one at a subsite, causing an increase in activity, kinetics [37]) [37] decanoyllysophosphatidylcholine <4> (<4> activates [13]) [13] deoxycholate <4, 6, 15, 16> (<16> stimulates [14]; <4> required [8]; <6> stimulates activity of enzyme I, II and III at pH 7 [5]; <4> required for activity with membrane-bound phosphatidylinositol, slight stimulation with isolated phosphatidylinositol [7]) [5, 7, 8, 14, 24] diacylglycerol <3, 5, 8, 15> (<3,15> activates [3,24]; <5> 1 mM, 2fold increase in the activity at pH 5.5 and 4fold increase in activity at pH 7.0 [4]; <8> activates, increases the hydrolytic activity of PI-PLC on large unilamellar vesicles containing 5-40% phosphatidylinositol [38]) [3, 4, 24, 38] diheptanoyl phosphatidylcholine <16> (<16> activates [40]) [40] dihexanoyl phosphatidylcholine <14> (<14> activates, 4-5fold increase in catalytic efficiency, binds to a lipid-binding subsite, not to the active site, maximal activation at 0.4 mM [36]) [36] dihexanoylglycerophosphocholine <4> (<4> activates [13]) [13] dihexanoylphosphatidylcholine <8> (<8> non-substrate activator lipid, maximum PI-PLC activity at 0.7-1 mM [37]) [37] oleoylglycerophosphate <4> (<4> activates [13]) [13] palmitoylglycerophosphate <4> (<4> activates [13]) [13] phosphatidylmethanol <16> (<16> binding to nonsubstrate anionic interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism [34]) [34] Additional information <16> (<16> isopropanol and diheptanoylphosphatidylcholine activate the hydrolytic activity towards 1d-myo-inositol 1,2-cyclic phosphate, PI-PLC exhibits kinetic interfacial activation [35]; <16> PI-PLC exhibits several types of kinetic interfacial activation by interfaces, roles of Trp-47 and Trp-242 [39]) [35, 39] Metals, ions Ca2+ <2, 3, 4, 5, 6, 7, 9, 10, 11, 15> (<3, 4, 5, 6, 7, 10, 11, 15> required [2, 9, 10, 12, 13, 15, 17, 24]; <4,5> stimulates [7, 8, 22]; <4> optimal concentration: 1 mM [20]; <3> no requirement for the membrane enzyme solubilized with sodium cholate, activates the membrane enzyme after solubilization in octyl glucoside with maximal activity at 1 mM: the cytoplasmic enzyme has a total dependence on Ca2+ with optimum concentration between 0.5 mM and 1 mM [26]; <3> optimal concentrations are 0.3 mM and 8 mM using 0.2 mM and 0.4 mM phosphatidylinositol [16]; <6> requires 2-5 mM CaCl2 [5]; <2, 4> optimal concentration: 2.0-3.0 mM: the activity at pH 5.5 unlike that at pH 7.4 is not absolutely dependent on Ca2+ [6]; <7> 0.1 mM increases the activity 60%, 1 mM increases 6fold [15]; <9> 41fold activation of R69D mutant, slight activation of wild-type PI-PLC [33]) [2, 5, 6, 7, 8, 9, 10, 12, 13, 15, 16, 17, 20, 22, 24, 26, 33]

428

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Phosphatidylinositol diacylglycerol-lyase

Cd2+ <9> (<9> activates R69D mutant at low concentrations, no activation of wild-type PI-PLC [33]) [33] Cl- <16> (<16> 1 M, 2-3fold activation of wild-type PI-PLC [41]) [41] Co2+ <4, 9> (<4> facilitates enzyme penetration into substrate monolayers [20]; <9> activates R69D mutant at low concentrations, no activation of wild-type PI-PLC [33]) [20, 33] K+ <5> (<5> stimulates at pH 5.5 [22]) [22] Li+ <9> (<9> 5fold activation of R69D mutant, slight activation of wild-type PI-PLC [33]) [33] Mg2+ <4, 5, 9, 14> (<4> stimulates [8]; <5> MgCl2 , stimulates less effectively than CaCl2 [22]; <4> facilitates enzyme penetration into substrate monlayers [20]; <9> 35fold activation of R69D mutant, slight activation of wild-type PI-PLC [33]; <14> MgCl2 , activates [36]) [8, 20, 22, 33, 36] Mn2+ <3, 4, 9> (<4> stimulates [8]; <4> facilitates enzyme penetration into substrate monlayers [20]; <3> no requirement for the membrane enzyme solubilized with sodium cholate, activates the membrane enzyme after solubilization in octyl glucoside with maximal activity at 0.1 mM [26]; <9> activates R69D mutant at low concentrations, slight activation of wild-type PI-PLC [33]) [8, 20, 26, 33] Ni2+ <4> (<4> facilitates enzyme penetration into substrate monolayers [20]) [20] Sr2+ <9> (<9> 9fold activation of R69D mutant, slight activation of wild-type PI-PLC [33]) [33] Additional information <4, 5, 8, 9, 14, 16> (<8> no requirement for a divalent metal ion [18,19]; <5> activity is detected in absence of any additional divalent cation [4]; <4> divalent cations required [8]; <16> Ca2+ -independent [35]; <14> salts have an activity-enhancing effect [36]; <9> wild-type PI-PLC is calcium-independent [33]) [4, 8, 18, 19, 33, 35, 36] Turnover number (min–1) 66 <14> (methyl-fluorescein myo-inositol phosphate, <14> pH 7 [36]) [36] Additional information <8> [37] Specific activity (U/mg) 13.74 <8> [19] 16.1 <15> [24] 28.97 <4> [1] 30 <14> (<14> pH 7.2, phosphatidylinositol as substrate, in absence of MgCl2 [36]) [36] 401 <16> (<16> W47I mutant PI-PLC, phosphotransferase activity [39]) [39] 556 <16> (<16> wild-type PI-PLC, phosphotransferase activity [39]) [39] 558 <16> (<16> W47F mutant PI-PLC, phosphotransferase activity [39]) [39] 658 <16> (<16> W242F mutant PI-PLC, phosphotransferase activity [39]) [39] 684 <16> (<16> W242I mutant PI-PLC, phosphotransferase activity [39]) [39] 700 <14> (<14> pH 7.2, phosphatidylinositol as substrate, in presence of MgCl2 [36]) [36]

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Phosphatidylinositol diacylglycerol-lyase

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3560 <9> (<9> pH 7.5, 37 C, wild-type PI-PLC, in absence of Ca2+ [33]) [33] Additional information <8, 9, 16> (<16> phosphotransferase activity towards phosphatidylinositol in several aggregation states in the absence and presence of 30% isopropanol [35]; <16> specific activities of wild-type and mutant PI-PLCs towards 1D-myo-inositol 1,2-cyclic phosphate in the absence and presence of different activators [39]; <9> values for wild-type and several mutant PI-PLCs in presence of 0.1 mM or 1 mM Ca2+ and in absence of Ca2+ [33]) [14, 18, 31, 33, 34, 35, 39, 40, 41] Km-Value (mM) 0.0015 <3> (phosphatidylinositol, <3> cytoplasmic enzyme [26]) [26] 0.005 <3> (phosphatidylinositol, <3> membrane-bound enzyme [26]) [26] 0.027 <7> (phosphatidylinositol) [15] 0.061 <14> (methyl-fluorescein myo-inositol phosphate, <14> pH 7 [36]) [36] 0.1-0.15 <3, 10> (phosphatidylinositol) [9] 0.14 <8> (methyl-fluorescein myo-inositol phosphate, <8> pH 7, 25 C, in presence of dihexanoylphosphatidylcholine [37]) [37] 0.81 <8> (methyl-fluorescein myo-inositol phosphate, <8> pH 7, 25 C, in absence of dihexanoylphosphatidylcholine [37]) [37] 1.4 <8> (phosphatidylinositol) [19] Additional information <8, 14, 16> (<16> kinetic data [39,41]; <8> kinetics of butyl-fluorescein myo-inositol phosphate and methyl-fluorescein myo-inositol phosphate cleavage, two-site kinetic model [37]; <14> kinetics, two-site kinetic model [36]) [36, 37, 39, 41] Ki-Value (mM) 2-5 <8> (Ca2+ ) [18] 2-5 <8> (Mg2+ ) [18] 2-5 <8> (Mn2+ ) [18] 2-5 <8> (Zn2+ ) [18] pH-Optimum 4.5-5.5 <6> (<6> enzyme form I, II and III [5]) [5] 5 <4> (<4> hydrolysis of phosphatidylinositol, at 1 mM, low molecular weight enzyme form and high molecular weight enzyme form [1]) [1] 5.4-5.6 <5> (<5> and a second optimum at pH 7.0-7.3 [22]) [22] 5.5 <2, 4, 5, 10> (<4> hydrolysis of phosphatidylinositol, at 0.3 mM, low molecular weight enzyme form [1]; <5> lymphocytes contain multiple enzyme form with activity at pH 5.5 and a single enzyme form with maximum activity at pH 7.0 [4]; <2,4> 2 optima: at pH 5.5 and at pH 7.4 [6,7]) [1, 4, 6, 7, 9] 5.5-7 <4> (<4> hydrolysis of phosphatidylinositol 4,5-diphosphate, at 1 mM Ca2+ , low molecular weight enzyme form [1]) [1] 5.6 <7> [15] 5.9-6.6 <11> [17] 6 <4> (<4> hydrolysis of phosphatidylinositol 4,5-diphosphate, at 3 mM Ca2+ , low molecular weight enzyme form [1]) [1] 6-7 <3> [16]

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Phosphatidylinositol diacylglycerol-lyase

6-9 <16> (<16> wild-type PI-PLC, in the absence of 1 M Cl- [41]) [41] 6.6 <4> [20] 7 <3, 5, 8, 15, 16> (<8,16> assay at [32,37,40]; <5> lymphocytes contain multiple enzyme forms with activity at pH 5.5 and a single enzyme form with maximum activity at pH 7.0 [4]; <3> membrane enzyme solubilized with sodium cholate, cytoplasmic enzyme [26]) [4, 24, 26, 32, 37, 40] 7-7.2 <14> (<14> assay at [36]) [36] 7-7.3 <5> (<5> and a second optimum at pH 5.4-5.6 [22]) [22] 7-8 <16> (<16> wild-type PI-PLC, in the presence of 1 M Cl- [41]) [41] 7-8.5 <4> [8, 11] 7.1 <16> (<16> cleavage of phosphatidylinositol solubilized in diheptanoyl phosphatidylcholine [34]) [34] 7.2-7.5 <8> [19] 7.4 <2, 4> (<2,4> 2 optima: at pH 5.5 and at pH 7.4 [6,7]) [6, 7] 7.5 <9, 16> (<9,16> assay at [31,33,35]) [14, 31, 33, 35] 8 <3> (<3> membrane enzyme solubilized with octyl glucoside [26]) [26] Additional information <4> (<4> the enzyme exhibits considerable heterogeneity with respect to pH-optima [10]) [10] pH-Range 4.5-7 <7> (<7> pH 4.5: about 50% of maximal activity, pH 7.0: about 40% of maximal activity [15]) [15] 5.3-7.7 <4> (<4> about 45% of maximal activity at pH 5.3 and pH 7.7 [20]) [20] 5.5-8.2 <11> (<11> about 55% of maximal activity at pH 5.5 and pH 8.2 [17]) [17] 6-11 <4> (<4> pH 6.0: about 10% of maximal activity, pH 11.0: about 40% of maximal activity [8]) [8] 8 <16> (<16> cleavage of phosphatidylinositol solubilized in diheptanoyl phosphatidylcholine, drop in activity around pH 8, consistent with the drop in binding affinity for activating surfaces [34]) [34] Additional information <16> (<16> pH-dependence study of wild-type and mutant PI-PLC [41]) [41] Temperature optimum ( C) 25 <8> (<8> assay at [37]) [37] 30 <15> [24] 37 <8, 9> (<8,9> assay at [31,32,33]) [31, 32, 33] 39 <8> (<8> assay at [38]) [38] Temperature range ( C) 20-70 <15> (<15> 20 C: about 80% of maximal activity, 70 C: about 35% of maximal activity [24]) [24]

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Phosphatidylinositol diacylglycerol-lyase

4.6.1.13

4 Enzyme Structure Molecular weight 18000 <3> (<3> membrane enzyme, gel filtration [26]) [26] 23000 <15, 16> (<16> gel filtration [14]; <15> nondenaturing PAGE [24]) [14, 24] 29000 <8> (<8> gel filtration [18,19]) [18, 19] 32900 <14> [36] 34460 <9> (<9> R69C mutant PI-PLC, electrospray ionization mass spectrometry [31]) [31] 40000-43000 <6> (<6> enzyme form II, gel filtration [5]) [5] 58000-72000 <6> (<6> enzyme form III, gel filtration [5]) [5] 105000-120000 <6> (<6> enzyme form I, gel filtration [5]) [5] 250000-271000 <6> (<6> enzyme form IV, gel filtration [5]) [5] Subunits ? <5, 6, 15> (<5> x * 68000, SDS-PAGE [4]; <15> x * 15000, a-peptide, + x * 23000, b-peptide, SDS-PAGE [24]; <6> x * 140000, enzyme form PLC-M2, SDS-PAGE [23]) [4, 23, 24] dimer <6> (<6> 2 * 150000, enzyme form PLC-M1, SDS-PAGE [23]) [23] monomer <3, 8> (<8> 1 * 29000, SDS-PAGE [18]; <3> 1 * 18000, membrane enzyme, SDS-PAGE [26]; <3> 1 * 57000, cytoplasmic enzyme, SDS-PAGE [26]; <8> 1 * 35000 [37]) [18, 26, 37] tetramer <6> (<6> 4 * 150000, enzyme form PLC-M1, SDS-PAGE [23]) [23]

5 Isolation/Preparation/Mutation/Application Source/tissue adrenal medulla <6> [2] blood platelet <3, 10> [3, 9, 16] brain <4, 5, 6> [10, 11, 12, 13, 20, 23] culture medium <8> [45] flower <12> (<12> tip [17]) [17] heart <6> (<6> four enzyme forms: I-IV [5]) [5] kidney <4> (<4> proximal tubule brush border membrane [8]) [8] leaf <13> (<13> outer leaves [17]) [17] lymphocyte <5> (<5> multiple enzyme forms [4]; <5> from mesenteric lymph nodes [22]) [4, 22] skeletal muscle <2, 4> (<4> normal and denervated fast and slow muscles [7]) [6, 7] smooth muscle <4, 7> (<4> vascular [1]; <7> iris [15]) [1, 15] spleen <3> [26] stem <11, 19> (<19> elongating stem [17]) [17]

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4.6.1.13

Phosphatidylinositol diacylglycerol-lyase

Localization cytoplasm <3, 16> (<16> recombinant PI-PLC, expressed in Escherichia coli BL21 [39]) [26, 39] cytosol <3, 4> [7, 16] extracellular <8> [45] membrane <3> [26] microsome <4> [11] sarcolemma <2, 4> [6] Purification <3> [26] <4> (partial, a low molecular weight enzyme form and a high molecular weight enzyme form) [1] <5> [4] <6> (large scale, two enzyme forms: PLC-M1 and PLC-M2) [23] <8> [18, 19, 21] <8> (recombinant wild-type and mutant PI-PLC) [32] <9> (PI-PLC mutants) [33] <9> (R69C mutant PI-PLC) [31] <14> (recombinant PI-PLC) [36] <15> [24] <16> [14, 21] <16> (recombinant PI-PLC) [35, 40] <16> (recombinant wild-type and mutant PI-PLC) [34] <16> (wild-type and mutant PI-PLC) [41] Crystallization <8> [25] <8> (X-ray crystal structure) [32] <8> (in complex with myo-inositol) [30] <14> [29] <14> (recombinant PI-PLC) [36] <16> (wild-type and D274N mutant PI-PLC) [41] Cloning <4> (expression in vascular smooth muscle cells and rat aorta) [1] <8> [32] <8> (expression in Escherichia coli, construction of four vectors for highlevel expression) [21] <14> (PI-PLC gene is part of the virulence gene cluster, overexpression in Escherichia coli MM294) [36] <16> (expression in Escherichia coli MM294) [35] <16> (expression in Escherichia coli, construction of four vectors for highlevel expression) [21] <16> (overexpression in Escherichia coli BL21) [39, 40] <16> (wild-type and mutant PI-PLC, expression in Escherichia coli BL21) [34]

433

Phosphatidylinositol diacylglycerol-lyase

4.6.1.13

<20> (expression in Listeria monocytogenes. Listeria monocytogenes expressing Bacillus anthracis phosphatidylinositol-specific phospholipase C shows significantly decreased efficiencies of ascape from a phagosome and in cellto-cell spread) [43] Engineering D274A <8, 16> (<16> catalytic aspartate mutation, 0.005% of wild-type activity, no activation by exogenous anions [41]; <8> mutation of an catalytic diad residue, mutant with abolished activity, NMR study [32]) [32, 41] D274E <16> (<16> catalytic aspartate mutation, 50% of wild-type activity, no activation by chloride ions [41]) [41] D274G <16> (<16> catalytic aspartate mutation, activation of mutant PI-PLC by exogenous anions, e.g. Cl- [41]) [41] D274N <8, 16> (<16> catalytic aspartate mutation, 40fold decreased activity compared with wild-type enzyme, no activation by chloride ions [41]; <8> mutation of an catalytic diad residue, 4.2% of wild-type activity [32]) [32, 41] D33N/R69D <9> (<9> PI-PLC double mutant, 50fold activation by 1 mM Ca2+ [33]) [33] G238W <16> (<16> study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol [40]) [40] G238W/W242A <16> (<16> double mutant with enhanced activation and affinity for phosphatidylcholine interfaces above that of wild-type PI-PLC [40]) [40] G48W/W47A <16> (<16> double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol [40]) [40] H32A <8> (<8> mutation of an catalytic diad residue, mutant with abolished activity, NMR study [32]) [32] I43W/W47A <16> (<16> double mutant with recovered kinetic interfacial activation, lower specific activity than wild-type PI-PLC [40]) [40] K44A <16> (<16> interfacial binding study [34]) [34] M49W/W47A <16> (<16> double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol [40]) [40] N243W/W242A <16> (<16> double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol [40]) [40] Q45W/W47A <16> (<16> double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol [40]) [40] R69A <9> (<9> mutant is specifically activated by guanidinium hydrochloride [31]; <9> mutation of the catalytic Arg-69, not activated by Ca2+ [33]) [31, 33] R69C <9> (<9> mutation of the catalytic Arg-69, not activated by Ca2+ [33]; <9> site-directed chemical modification of the cysteine residue replacing Arg-69, mutant PI-PLCs featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains [31]) [31, 33] R69D <9, 16> (<16> active site mutant with low specific activity towards phosphatidylinositol, interfacial binding study [34]; <9> reduced activity compared with wild-type enzyme, mutant is activated by Ca2+ , mutation en-

434

4.6.1.13

Phosphatidylinositol diacylglycerol-lyase

gineers a catalytic metal binding site into the calcium-independent PI-PLC leading to enhanced stereoselectivity [33]) [33, 34] R69E <9> (<9> mutation of the catalytic Arg-69, inactive mutant, not activated by Ca2+ [33]) [33] R69G <9> (<9> mutant is specifically activated by guanidinium hydrochloride [31]; <9> mutation of the catalytic Arg-69, not activated by Ca2+ [33]) [31, 33] R69N <9> (<9> mutation of the catalytic Arg-69, not activated by Ca2+ [33]) [33] S236W/W242A <16> (<16> double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol [40]) [40] W178A <16> (<16> mutant with reduced stability and specific activity, study of kinetic activation by micellar phosphatidylcholine [39]) [39] W242A <16> (<16> active enzyme, partitioning of mutant enzyme to vesicles is decreased by more than 10fold, study of kinetic activation by micellar phosphatidylcholine [39]; <16> interfacial binding study [34]; <16> mutant with much weaker binding to interfaces and lower kinetic interfacial activation [40]) [34, 39, 40] W242F <16> (<16> kinetic analysis, binding studies to phosphatidylcholine vesicles [39]) [39] W242I <16> (<16> kinetic analysis, binding studies to phosphatidylcholine vesicles [39]) [39] W280A <16> (<16> mutant with reduced stability, study of kinetic activation by micellar phosphatidylcholine [39]) [39] W47A <16> (<16> active enzyme, partitioning of mutant enzyme to vesicles is decreased by more than 10fold, study of kinetic activation by micellar phosphatidylcholine [39]; <16> interfacial binding study [34]; <16> mutant with much weaker binding to interfaces and lower kinetic interfacial activation [40]) [34, 39, 40] W47A/W242A <16> (<16> double mutant, interfacial binding study [34]; <16> double mutant, no affinity for phospholipid surfaces, no kinetic activation by micellar phosphatidylcholine [39]) [34, 39] W47F <16> (<16> kinetic analysis, binding studies to phosphatidylcholine vesicles [39]) [39] W47I <16> (<16> kinetic analysis, binding studies to phosphatidylcholine vesicles [39]) [39] Additional information <16> (<16> tryptophan rescue mutagenesis, reinsertion of a Trp at a different place in helix B in the W47A mutant or in the loop of the W242A mutant [40]) [40] Application analysis <16> (<16> use of the enzyme and specific antibodies for the enzyme for the examination of the growth inhibition, morphological change and ectoenzyme release of the LLC-PK1 cells from pig, effective for the investigation of the function of the glycosyl-phosphatidylinositol-anchor protein [27]) [27]

435

Phosphatidylinositol diacylglycerol-lyase

4.6.1.13

medicine <14> (<14> PI-PLC is a virulence factor of the animal and human pathogen causing listeriosis [36]) [36] molecular biology <1, 8, 16> (<1,8,16> enzyme is used as a tool in the studies of GPI-anchored proteins [45]) [45]

6 Stability pH-Stability 5-11 <3> (<3> stable above pH 5.0 and below pH 11.0 [26]) [26] Temperature stability 25 <16> (<16> some thermal unfolding of PI-PLC occurs [35]) [35] 30 <16> (<16> Tm -value in presence of 30% isopropanol, myo-inositol enhances thermostability in isopropanol [35]) [35] 41.5 <16> (<16> Tm -value, pH 8, W178A mutant PI-PLC [39]) [39] 47 <16> (<16> Tm -value, pH 8, W280A mutant PI-PLC [39]) [39] 50 <15> (<15> pH 8.0, 30 min, stable below [24]) [24] 53.6 <16> (<16> Tm -value in absence of 30% isopropanol [35]; <16> Tm value, pH 8, W47A mutant PI-PLC [39]) [35, 39] 54.4 <16> (<16> Tm -value, pH 8, wild-type PI-PLC [39]; <16> Tm -value, wild-type PI-PLC [40]) [39, 40] 54.6 <16> (<16> Tm -value, pH 8, W47A/W242A double mutant PI-PLC [39]) [39] 56.2 <16> (<16> Tm -value, pH 8, W242A mutant PI-PLC [39]) [39] Organic solvent stability isopropanol <16> (<16> water-miscible, 30%, activates [40]) [40] isopropanol <16> (<16> water/isopropanol mixture decreases stability, at 4 C protein secondary structure is stable for at least 1 week in 30% isopropanol, Tm -value for PI-PLC thermal denaturation decreases linearly with added isopropanol, in presence of 30% isopropanol the Tm -value decreases from 53.6 to 30 C, myo-inositol enhances thermostability in isopropanol [35]) [35] General stability information <4>, repeated freezing and thawing inactivates [1] <16>, myo-inositol stabilizes [35] Storage stability <5>, -20 C, stable for several month [22] <6>, -20 C, several weeks, enzyme forms PLC-I and PLC-II oligomerize to different degree [23]

436

4.6.1.13

Phosphatidylinositol diacylglycerol-lyase

References [1] Griendling, K.K.; Taubman, M.B.; Akers, M.; Mendlowitz, M.; Alexander, R.W.: Characterization of phosphatidylinositol-specific phospholipase C from cultured vascular smooth muscle cells. J. Biol. Chem., 266, 1549815504 (1991) [2] Zahler, P.; Reist, M.; Pilarska, M.; Rosenheck, K.: Phospholipase C and diacylglycerol lipase activities associated with plasma membranes of chromaffin cells isolated from bovine adrenal medulla. Biochim. Biophys. Acta, 877, 372-379 (1986) [3] Dawson, R.M.C.; Hemington, N.; Irvine, R.F.: The inhibition of diacylglycerol-stimulated intracellular phospholipases by phospholipids with a phosphocholine-containing polar group. A possible physiological role for sphingomyelin. Biochem. J., 230, 61-68 (1985) [4] Carter, H.R.; Smith, A.D.: Partial purification of a phosphatidylinositol phosphodiesterase isolated from lymphocytes. Biochem. Soc. Trans., 13, 1215-1216 (1985) [5] Low, M.G.; Weglicki, W.B.: Resolution of myocardial phospholipase C into several forms with distinct properties. Biochem. J., 215, 325-334 (1983) [6] Shute, J.K.; Smith, M.E.: Phosphatidylinositol phosphodiesterase in isolated plasma membranes of rodent skeletal muscle. Biochem. Soc. Trans., 13, 193-194 (1985) [7] Shute, J.K.; Smith, M.E.: Soluble phosphatidylinositol phosphodiesterase in normal and denervated fast and slow muscles of the rat. Biochem. J., 222, 299-305 (1984) [8] Schwertz, D.W.; Kreisberg, J.I.; Venkatachalam, M.A.: Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase. Arch. Biochem. Biophys., 224, 555-567 (1983) [9] Sies, W.; Lapetina, E.G.: Properties and distribution of phosphatidylinositol-specific phospholipase C in human and horse platelets. Biochim. Biophys. Acta, 752, 329-338 (1983) [10] Hirasawa, K.; Irvine, R.F.; Dawson, R.M.C.: Heterogeneity of the calciumdependent phosphatidylinositol phosphodiesterase in rat brain. Biochem. J., 205, 437-442 (1982) [11] Hirasawa, K.; Irvine, R.F.; Dawson, R.M.C.: The catabolism of phosphatidylinisitol by an EDTA-insensitive phospholipase A1 and calcium-dependent phosphatidylinositol phosphodiesterase in rat brain. Eur. J. Biochem., 120, 53-58 (1981) [12] Hirasawa, K.; Irvine, R.F.; Dawson, R.M.C.: The hydrolysis of phosphatidylinositol monolayers at an air/water interface by the calcium-ion-dependent phosphatidylinositol phosphodiesterase of pig brain. Biochem. J., 193, 607614 (1981) [13] Dawson, R.M.C.; Hemington, N.; Irvine, R.F.: The inhibition and activation of Ca2+ -dependent phosphatidylinositol phosphodiesterase by phospholipids and blood plasma. Eur. J. Biochem., 112, 33-38 (1980)

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[14] Taguchi, R.; Asahi, Y.; Ikezawa, H.: Purification and properties of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis. Biochim. Biophys. Acta, 619, 48-57 (1980) [15] Abdel-Latif, A.A.; Luke, B.; Smith, J.P.: Studies on the properties of a soluble phosphatidylinositol-phosphodiesterase of rabbit iris smooth muscle. Biochim. Biophys. Acta, 614, 425-434 (1980) [16] Mauco, G.; Chap, H.; Douste-Blazy, L.: Characterization and properties of a phosphatidylinositol phosphodiesterase (phospholipase C) from platelet cytosol. FEBS Lett., 100, 367-370 (1979) [17] Irvine, R.F.; Letcher, A.J.; Dawson, R.M.C.: Phosphatidylinositol phosphodiesterase in higher plants. Biochem. J., 192, 279-283 (1980) [18] Sundler, R.; Alberts, A.W.; Vagelos, P.R.: Enzymatic properties of phosphatidylinositol inositolphosphohydrolase from Bacillus cereus. Substrate dilution in detergent-phospholipid micelles and bilayer vesicles. J. Biol. Chem., 253, 4175-4179 (1978) [19] Ikezawa, H.; Yamanegi, M.; Taguchi, R.; Miyashita, T.; Ohyabu, T.: Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity. Biochim. Biophys. Acta, 450, 154-164 (1976) [20] Quinn, P.J.; Barenholz, Y.: A comparison of the activity of phosphatidylinositol phosphodiesterase against substrate in dispersions and as monolayers at the air-water interface. Biochem. J., 149, 199-208 (1975) [21] Koke, J.A.; Yang, M.; Henner, D.J.; Volwerk, J.J.; Griffith, O.H.: High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis. Protein Expr. Purif., 2, 51-58 (1991) [22] Allan, D.; Michell, R.H.: Phosphatidylinositol cleavage catalysed by the soluble fraction from lymphocytes. Activity at pH 5.5 and pH 7.0. Biochem. J., 142, 591-597 (1974) [23] Lee, K.Y.; Ryu, S.H.; Suh, P.G.; Choi, W.C.; Rhee, S.G.: Phospholipase C associated with particulate fractions of bovine brain. Proc. Natl. Acad. Sci. USA, 84, 5540-5544 (1987) [24] Iwasaki, Y.; Niwa, S.; Nakano, H.; Nagasawa, T.; Yamane, T.: Purification and properties of phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus. Biochim. Biophys. Acta, 1214, 221-228 (1994) [25] Bullock, T.L.; Ryan, M.; Kim, S.L.; Remington, S.J.; Griffith, O.H.: Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus. Biophys. J., 64, 784-791 (1993) [26] Roy, G.; Villar, L.M.; Lazaro, I.; Gonzalez, M.; Bootello, A.; Gonzalez-Porque, P.: Purification and properties of membrane and cytosolic phosphatidylinositol-specific phospholipase C from human spleen. J. Biol. Chem., 266, 11495-11501 (1991) [27] Itami, C.; Kimura, Y.; Taguchi, R.; Ikezawa, H.; Nakayashi, T.: Growth inhibition, morphological change, and ectoenzyme release of LLC-PK1 cells by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis. Biosci. Biotechnol. Biochem., 61, 776-781 (1997)

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[28] Vizitiu, D.; Kriste, A.G.; Campbell, A.S.; Thatcher, G.R.J.: Inhibition of phosphatidylinositol-specific phospholipase C: studies on synthetic substrates, inhibitors and a synthetic enzyme. J. Mol. Recognit., 9, 197-209 (1996) [29] Moser, J.; Gerstel, B.; Meyer, J.E.; Chakraborty, T.; Wehland, L.; Heinz, D.W.: Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes. J. Mol. Biol., 273, 269-282 (1997) [30] Heinz, D.W.; Ryan, M.; Bullock, T.L.; Griffith, O.H.: Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol. EMBO J., 14, 3855-3863 (1995) [31] Kravchuk, A.V.; Zhao, L.; Kubiak, R.J.; Bruzik, K.S.; Tsai, M.-D.: Mechanism of phosphatidylinositol-specific phospholipase C: Origin of unusually high nonbridging thio effects. Biochemistry, 40, 5433-5439 (2001) [32] Ryan, M.; Liu, T.; Dahlquist, F.W.; Griffith, O.H.: A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C. Biochemistry, 40, 9743-9750 (2001) [33] Kravchuk, A.V.; Zhao, L.; Bruzik, K.S.; Tsai, M.-D.: Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity. Biochemistry, 42, 2422-2430 (2003) [34] Wehbi, H.; Feng, J.; Kolbeck, J.; Ananthanarayanan, B.; Cho, W.; Roberts, M.F.: Investigating the interfacial binding of bacterial phosphatidylinositol-specific phospholipase C. Biochemistry, 42, 9374-9382 (2003) [35] Wehbi, H.; Feng, J.; Roberts, M.F.: Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity. Biochim. Biophys. Acta, 1613, 15-27 (2003) [36] Ryan, M.; Zaikova, T.O.; Keana, J.F.W.; Goldfine, H.; Griffith, O.H.: Listeria monocytogenes phosphatidylinositol-specific phospholipase C: Activation and allostery. Biophys. Chem., 101-102, 347-358 (2002) [37] Birrell, G.B.; Zaikova, T.O.; Rukavishnikov, A.V.; Keana, J.F.W.; Griffith, O.H.: Allosteric interactions within subsites of a monomeric enzyme: Kinetics of fluorogenic substrates of PI-specific phospholipase C. Biophys. J., 84, 3264-3275 (2003) [38] Villar, A.V.; Goni, F.M.; Alonso, A.: Diacylglycerol effects on phosphatidylinositol-specific phospholipase C activity and vesicle fusion. FEBS Lett., 494, 117-120 (2001) [39] Feng, J.; Wehbi, H.; Roberts, M.F.: Role of tryptophan residues in interfacial binding of phosphatidylinositol-specific phospholipase C. J. Biol. Chem., 277, 19867-19875 (2002) [40] Feng, J.; Bradley, W.D.; Roberts, M.F.: Optimizing the interfacial binding and activity of a bacterial phosphatidylinositol-specific phospholipase C. J. Biol. Chem., 278, 24651-24657 (2003) [41] Zhao, L.; Liao, H; Tsai, M.-D.: The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidyl-

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[42]

[43]

[44] [45] [46]

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inositol-specific phospholipase C can be substituted by a chloride-ion. J. Biol. Chem., 279, 31995-32000 (2004) Poussin, M.A.; Goldfine, H.: Involvement of Listeria monocytogenes phosphatidylinositol-specific phospholipase C and host protein kinase C in permeabilization of the macrophage phagosome. Infect. Immun., 73, 4410-4413 (2005) Wei, Z.; Schnupf, P.; Poussin, M.A.; Zenewicz, L.A.; Shen, H.; Goldfine, H.: Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis. Infect. Immun., 73, 6639-6646 (2005) Zenewicz, L.A.; Wei, Z.; Goldfine, H.; Shen, H.: Phosphatidylinositol-specific phospholipase C of Bacillus anthracis down-modulates the immune response. J. Immunol., 174, 8011-8016 (2005) Ikezawa, H.: Bacterial phosphatidylinositol-specific phospholipase C as membrane-attacking agents and tools for research on GPI-anchored proteins. J. Toxicol. Toxin Rev., 23, 479-508 (2004) Wei, Z.; Zenewicz, L.A.; Goldfine, H.: Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors. Proc. Natl. Acad. Sci. USA, 102, 1292712931 (2005)

Glycosylphosphatidylinositol diacylglycerollyase

4.6.1.14

1 Nomenclature EC number 4.6.1.14 Systematic name 6-(a-d-glucosaminyl)-1-phosphatidyl-1d-myo-inositol 1,2-sn-glycerol lyase [6-(a-d-glucosaminyl)-1d-myo-inositol-1,2-cyclic phosphate-forming] Recommended name glycosylphosphatidylinositol diacylglycerol-lyase Synonyms (glycosyl)phosphatidylinositol-specific phospholipase C EC 3.1.4.47 (formerly) GPI-PLC <2, 4, 6> [18, 19, 20] GPI-specific phospholipase C VSG-lipase glycosyl inositol phospholipid anchor-hydrolyzing enzyme glycosylphosphatidylinositol-phospholipase C glycosylphosphatidylinositol-specific phospholipase C <2, 4, 6> [18, 19, 20] phospholipase C, glycosylphosphatidylinositol variant-surface-glycoprotein phospholipase C variant-surface-glycoprotein-1,2-didecanoyl-sn-phosphatidylinositol inositolphosphohydrolase Additional information CAS registry number 129070-68-4

2 Source Organism <1> <2> <3> <4>

Mus musculus (no sequence specified) [2] Rattus norvegicus (no sequence specified) [5, 6, 18] Saccharomyces cerevisiae (no sequence specified) [5] Trypanosoma brucei (no sequence specified) [1, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20] <5> Arachis hypogaea (no sequence specified) [4,10] <6> Bacillus cereus (no sequence specified) [18]

441

Glycosylphosphatidylinositol diacylglycerol-lyase

4.6.1.14

3 Reaction and Specificity Catalyzed reaction 6-(a-d-glucosaminyl)-1-phosphatidyl-1d-myo-inositol = 6-(a-d-glucosaminyl)-1d-myo-inositol 1,2-cyclic phosphate + 1,2-diacyl-sn-glycerol Reaction type P-O bond cleavage Natural substrates and products S 6-(a-d-glucosaminyl)-1-phosphatidyl-1d-myo-inositol <2, 4, 6> (Reversibility: ?) [18, 19, 20] P 6-(a-d-glucosaminyl)-1d-myo-inositol 1,2-cyclic phosphate + 1,2-diacylsn-glycerol Substrates and products S 1,2-dimyristoyl-sn-phosphatidylinositol + H2 O <4> (<4> poor substrate [1]) (Reversibility: ?) [1] P ? S 6-(a-d-glucosaminyl)-1-phosphatidyl-1d-myo-inositol <2, 4, 6> (<2> the assay contains acetylcholinesterase [18]) (Reversibility: ?) [18, 19, 20] P 6-(a-d-glucosaminyl)-1d-myo-inositol 1,2-cyclic phosphate + 1,2-diacylsn-glycerol S acetylcholinesterase <4, 5> (<5> isolated from bovine erythrocytes, does not act on membrane-bound forms but hydrolyzes the membrane-anchor from solubilized substrate [4]) (Reversibility: ?) [4, 12] P soluble acetylcholinesterase + 1,2-diacylglycerol <5> [4] S lipid A <4> (<4> biological precursor of variant-surface-glycoprotein glycolipid [1]) (Reversibility: ?) [1] P ? S phosphatidylinositol <4, 5> (<4> very poor substrate [3]; <4> at 0.005 mM phosphatidylinositol, hydrolysis is maximal at 0.005% Triton X-100, at 1 mM phosphatidylinositol hydrolysis is maximal at 0.05% Triton X-100 [10]) (Reversibility: ?) [3, 4, 10, 15] P inositol 1,2-cyclic phosphate + inositol phosphate <5> [4] S variant-surface-glycoprotein <1, 2, 3, 4, 5> (<1,4> highly specific [1,2]; <1> isolated from Trypanosoma equiperdum BoTat-1 [2]; <4> substrate is tethered to cell membrane by glycolipid moiety containing 1,2-dimyristoyl-sn-phosphatidylinositol [1]; <4> enzyme activity is stimulated by acid-treatment of the cells [8]; <5> peanut enzyme does not act on membrane-bound forms but hydrolyzes the membrane-anchor from solubilized substrate [4]; <4> phospholipase C-type hydrolysis [1]; <5> isolated from Trypanosoma brucei [4]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17] P 1,2-didecanoylglycerol + soluble variant-surface-glycoprotein <4> (<4> i.e. 1,2-dimyristoylglycerol [1,3]) [1, 3] S Additional information <4, 5> (<4> glucosaminyl(a1 ! 6)inositol is the crucial glycan moiety for substrate recognition [3]; <4> no substrate is 1-

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stearoyl-2-arachidonoyl-sn-phosphatidylinositol [1]; <5> no substrates are phosphatidylcholine, phosphatidylserine, phosphatidylinositol 4monophosphate, phosphatidylinositol 4,5-bisphosphate [4]) (Reversibility: ?) [1, 3, 4] P ? Inhibitors 2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecyl-phosphonic acid <4, 6> (<4,6> GPI-1793 [18]) [18] Ca2+ <4> (<4> weak, 5 mM [1]) [1] cetrimide <1> (<1> i.e. alkyltrimethylammonium bromide, 0.3 mg/ml, neutral enzyme form [2]) [2] EDTA <5> [4] EGTA <1, 5> (<1> Ca2+ reverses, only acidic, not neutral enzyme form [2]) [2, 4] glucosaminyl-a-1,6-2-deoxy-d-myo-inositol <4> (<4> i.e. compound VP615L, more effective than VP-606L [3]) [3] glucosaminyl-a-1,6-d-myo-inositol <4> (<4> i.e. compound VP-606L or 6O-(2-amino-2-deoxy-a-d-glucopyranosyl)-d-myo-inositol [3]) [3] glucosaminyl-a-1,6-d-myo-inositol 1,2-cyclic phosphate <4> (<4> i.e. compound VP-601L, product inhibition [3]) [3] glucosaminyl-a-1,6-d-myo-inositol 1-dodecylphosphonate <4> (<4> i.e. compound VP-604L [3]) [3] glucosaminyl-a-1,6-d-myo-inositol 1-hexylphosphonate <4> (<4> i.e. compound VFT-2 [3]) [3] glucosaminyl-a-1,6-d-myo-inositol 1-phosphate <4> (<4> i.e. compound VP-600L [3]) [3] inositol 1-dodecylphosphonate <4> (<4> i.e. compound VP-602L [3]) [3] KCl <4> (<4> 0.125 M [1]) [1] mannosyl-a-1,4-glucosaminyl-a-1,6-d-myo-inositol <4> (<4> i.e. O-a-dmannopyranosyl-1,4-O-2-amino-2-deoxy-a-d-glucopyranosyl-1,6-d-myoinositol [3]) [3] Mg2+ <4> (<4> weak, 5 mM [1]) [1] N-(N,N-Dimethylcarbamyl)-glucosaminyl-a-1,6-d-myo-inositol <4> (<4> i.e. compound VC-109B, less effective than VP-606L [3]) [3] N-acetylglucosaminyl-a-1,6-d-myo-inositol <4> (<4> i.e. compound VC105B, weak [3]) [3] NEM <4> (<4> 5 mM [1]) [1] NH4 Cl <4> (<4> 0.125 M [1]) [1] Na3 VO4 <5> [4] NaCl <4> (<4> 0.125 M [1]) [1] NaF <5> [4] Nonidet P-40 <1> (<1> above 0.1% w/v, neutral enzyme form [2]) [2] phosphatidylcholine <4> (<4> weak [3]) [3] phosphatidylglycerol <4> [3] phosphatidylinositol <4> [3] phosphatidylserine <4> [3]

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propanolol <5> [4] sulfhydryl reagents <4> [1] Triton X-100 <5> (<5> 0.05-0.5%, activation at 0.02% [4]) [4] Zn2+ <4, 5> (<4> strong, 5 mM [1]) [1, 4, 11] deoxycholate <1> (<1> neutral enzyme form, above 1 mg/ml, activation at 0.5-1 mg/ml [2]) [2] myo-inositol-1,2-cyclo-dodecyl-phosphonic acid <2, 4, 6> (<2,4,6> GPI-2350 [18]) [18] myo-inositol-1-O-dodecylphosphonic acid methylester <4> (<4> GPI-2349 [18]) [18] p-chloromercuriphenylsulfonic acid <4, 5> (<4> strong, 5 mM [1]) [1, 4, 10, 11] Additional information <1, 2, 4, 5> (<5> no inhibition by 1,10-phenanthroline [4]; <4> no inhibition by IAA [1]; <1> no inhibition by Mn2+ [2]; <4> no inhibition by palmitate, myristate, phosphatidylethanolamine, inositol 1phosphate, N-acetylglucosamine, ethanolamine, inositol, glucosamine, mannose, glucosaminyl-a-1,6-2-deoxy-d-myo-inositol 1-phosphate (i.e. VP612L), glucosaminyl-a-1,6-2-deoxy-l-myo-inositol (i.e. VP-614L) [3]; <2> low inhibition by 2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecyl-phosphonic acid (GPI-1793) and very low inhibition by myo-inositol-1-O-dodecylphosphonic acid methylester (GPI-2349) [18]) [1, 2, 3, 4, 18] Activating compounds butanol <1> (<1> activation, 2% v/v, acidic enzyme form, not neutral enzyme form [2]) [2] DTT <4> (<4> activation, 0.025 M [1]) [1] EDTA <4> (<4> activation, 5 mM [1]) [1] EGTA <4> (<4> activation, 5 mM [1]) [1] Triton X-100 <5> (<5> activation, above critical micelle concentration, inhibition from 0.05% to 0.5% [4]) [4] deoxycholate <1> (<1> activation, 0.5-1 mg/ml, neutral enzyme form, not acidic enzyme form, inhibits above 1 mg/ml [2]) [2] Additional information <1, 4> (<1> 2 enzyme forms differing in pH-optima and response to stimulation by butanol and deoxycholate [2]; <4> no activation by aminoglycoside antibiotic geneticin (G418) [3]) [2, 3] Metals, ions Ca2+ <4, 5> (<4> not [1]; <5> activation, Mg2+ cannot replace Ca2+ [4]) [1, 4] Turnover number (min–1) 0.65-1.48 <4> (Variant-surface-glycoprotein, <4> 37 C, deacylated enzyme [14]) [14] 19.8-27.3 <4> (Variant-surface-glycoprotein, <4> 37 C, acylated enzyme [14]) [14] Specific activity (U/mg) 0.45 <4> [11] 1.59 <4> (<4> Mono P fraction [7]) [7] 652 <4> [19]

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Glycosylphosphatidylinositol diacylglycerol-lyase

2000 <4> (<4> Q81E [13]) [13] 9000 <4> (<4> Q81N [13]) [13] 280000 <4> (<4> C80A [13]) [13] Additional information <2> (<2> in high cholesterol-containing detergentsoluble glycolipid-enriched membrane microdomains: 488% of the activity measured in plasma membranes, in low cholesterol-containing detergent-soluble glycolipid-enriched membrane microdomains: 141% of the activity measured in plasma membranes, in non-detergent-soluble glycolipid-enriched membrane microdomains: 29% of the activity measured in plasma membranes [18]) [18] 1100000 <4> (<4> wild-type [13]) [13] 3500000 <4> (<4> C80T [13]) [13] 11000000 <4> (<4> concentrated CM-Sephadex pool [1]) [1] Km-Value (mM) 0.0003 <4> (myristate-labeled variant-surface-glycoprotein, <4> 37 C, pH 8, 50 mM sodium phosphate, Q81N [13]) [13] 0.0004-0.0007 <4> (variant-surface-glycoprotein, <4> pH 8 [11]) [11] 0.0005 <4> (myristate-labeled variant-surface-glycoprotein, <4> 37 C, pH 8, 50 mM sodium phosphate, wild-type [13]) [13] 0.0008 <4> (acetylcholinesterase) [12] 0.0011 <4> (myristate-labeled variant-surface-glycoprotein, <4> 37 C, pH 8, wild-type [13]) [13] 0.0017-0.002 <4> (variant-surface-glycoprotein, <4> 37 C, deacylated enzyme [14]) [14] 0.0018 <4> (myristate-labeled variant-surface-glycoprotein, <4> 37 C, pH 8, C80A [13]) [13] 0.0021 <4> (myristate-labeled variant-surface-glycoprotein, <4> 37 C, pH 8, C80T [13]) [13] 0.0026-0.0027 <4> (variant-surface-glycoprotein, <4> 37 C, acylated enzyme [14]) [14] 0.037 <4> (phosphatidylinositol) [12] Ki-Value (mM) 0.0006 <2> (myo-inositol-1,2-cyclo-dodecyl-phosphonic acid) [18] pH-Optimum 4.5-5 <1> (<1> acidic enzyme form [2]) [2] 5.5-6 <5> [4] 6.5-7 <1> (<1> neutral enzyme form [2]) [2] 7.5-8.5 <4> [1] pH-Range 6.5-9.5 <4> (<4> about half-maximal activity at pH 6.5 and 9.5, little or no activity below pH 6 [1]) [1] Temperature optimum ( C) 37 <1, 4> (<1,4> assay at [1,2,3]) [1, 2, 3]

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Glycosylphosphatidylinositol diacylglycerol-lyase

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4 Enzyme Structure Molecular weight 39000 <4> (<4> PAGE [16]) [16] 47000 <4> (<4> gel filtration, sedimentation equilibrium centrifugation, in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid [1]) [1] Subunits dimer <4> (<4> a2 , 2 * 39000, gel filtration, dominates in presence of 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate [16]) [16] monomer <4> (<4> 1 * 37000, SDS-PAGE [1]; <4> gel filtration, dominates in presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [16]) [1, 16] tetramer <4> (<4> alph4, 4 * 39000, gel filtration, dominates in presence of Nonident P-40 [16]) [16] Posttranslational modification side-chain modification <4> (<4> co- and posttranslational thioacylation with myristate and palmitate [14]) [14, 17]

5 Isolation/Preparation/Mutation/Application Source/tissue adipocyte <2> [5, 6, 18] brain <1> [2] myelin <1> (<1> neutral enzyme form [2]) [2] seed <5> (<5> dry [4]) [4] Localization cytoplasmic membrane <1> (<1> neutral enzyme form [2]) [2] glycosome <4> (<4> in case of mutations on the endosome targeting motif, glycosomal GPI-PLC from Trypanosoma brucei fails to produce the glycosylphosphatidylinositol deficiency in Leishmania major [19]) [19] lysosome <1> (<1> predominantly acidic enzyme form [2]) [2] membrane <1, 4> [1, 2] plasma membrane <2> [18] spheroplast <3> [5] Additional information <1, 4> (<1> subcellular localization in mouse brain membranes [2]; <4> endo-lysosomal system, wild-type, endosomal GPI-PLC from Trypanosoma brucei causes a deficiency of protein glycosylphosphatidylinositols in Leishmania major [19]) [2, 19] Purification <4> (recombinant protein) [7, 11] <4> (solubilized by n-octyl glucoside) [1] <5> (partial) [4]

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Glycosylphosphatidylinositol diacylglycerol-lyase

Cloning <4> (expression in Escherichia coli) [7, 14, 17] <4> (expression in Leishmania major) [19] <4> (expression in Xenopus oocytes) [17] <4> (expression of a teracycline-inducible GPIPLC-gene in conditional knock-out bloodstraem forms and in procyclic cell line) [15] <4> (expression of the PLC elimination construct in Trypanosoma brucei) [9, 12] <4> (overexpression in Escherichia coli) [11] Engineering C184A <4> (<4> activity is not effected [13]; <4> exhibits activity in the range detected for the unmutated protein, deficiency of cell-associated gp63, localization in endo-lysosome [19]) [13, 19] C184S <4> (<4> fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid [11,12]) [11, 12] C24S <4> (<4> fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid [11,12]) [11, 12] C269-270-273S <4> (<4> 33% activity of wild-type enzyme [13]) [13] C269C/C270S/C273S <4> (<4> exhibits activity in the range detected for the unmutated protein, localization in glycosome [19]) [19] C269S <4> (<4> fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid [11,12]) [11, 12] C269S/C270S <4> (<4> exhibits activity in the range detected for the unmutated protein, localization in glycosome [19]) [19] C269S/C273S <4> (<4> exhibits activity in the range detected for the unmutated protein, localization in glycosome [19]) [19] C270S <4> (<4> fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid [11,12]) [11, 12] C270S/C273S <4> (<4> exhibits activity in the range detected for the unmutated protein, localization in glycosome [19]) [19] C273S <4> (<4> fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid [11,12]) [11, 12] C332S <4> (<4> fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid [11,12]) [11, 12] C347A <4> (<4> exhibits activity in the range detected for the unmutated protein, deficiency of cell-associated gp63, localization in endo-lysosome [19]) [19] C347S <4> (<4> fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid [11,12]) [11, 12] C80A <4> (<4> 33% activity of wild-type enzyme, resistant to p-chloromercuriphenylsulfonic acid [13]; <4> exhibits activity in the range detected for the unmutated protein, localization in glycosome [19]) [13, 19] C80F <4> (<4> enzyme is inactive [13]) [13] C80S <4> (<4> fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid [11,12]) [11, 12]

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Glycosylphosphatidylinositol diacylglycerol-lyase

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C80T <4> (<4> 33% activity of wild-type enzyme, resistant to p-chloromercuriphenylsulfonic acid [13]) [13] H34Q <4> (<4> enzyme is totally inactive [11,12]) [11, 12] Q81A <4> (<4> enzyme is inactive [13]) [13] Q81E <4> (<4> enzyme is inactive [13]) [13] Q81G <4> (<4> enzyme is inactive [13]) [13] Q81K <4> (<4> enzyme is inactive [13]) [13] Q81L <4> (<4> inactive [19]; <4> enzyme is inactive [13]) [13, 19] Q81N <4> (<4> specific activity is 500fold decreased [13]) [13] Additional information <4> (<4> tetracyclin-inducible GPIPLC-gene, in conditional knock-out bloodstream forms and in procyclic cell line [15]; <4> PLC elimination construct, which yielded in a PLC null-mutant [9,12]; <4> generation of conditional knock-out bloodstraem forms [15]) [9, 12, 15] Application medicine <4> (<4> the relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host [20]) [20]

6 Stability Temperature stability 37 <4> (<4> 10 min, stable [1]) [1] 50 <4> (<4> 10 min, inactivation [1]) [1] Storage stability <4>, -70 C, in crude membranes, 6 months [1] <4>, -70 C, purified enzyme preparation, at least 2 months [1] <4>, 4 C, 30% loss within 5 days, 10% loss of activity in the presence of 50% glycerol [1]

References [1] Hereld, D.; Krakow, J.L.; Bangs, J.D.; Hart, G.W.; Englund, P.T.: A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein. J. Biol. Chem., 261, 13813-13819 (1986) [2] Fouchier, F.; Baltz, T.; Rougon, G.: Identification of glycosylphosphatidylinositol-specific phospholipases C in mouse brain membranes. Biochem. J., 269, 321-327 (1990) [3] Morris, J.C.; Ping-Sheng, L.; Shen, T.Y.; Mensa-Wilmot, K.: Glycan requirements of glycosylphosphatidylinositol phospholipase C from Trypanosoma brucei. Glucosaminylinositol derivatives inhibit phosphatidylinositol phospholipase C. J. Biol. Chem., 270, 2517-2524 (1995) [4] Butikofer, P.; Brodbeck, U.: Partial purification and characterization of a (glycosyl) inositol phospholipid-specific phospholipase C from peanut. J. Biol. Chem., 268, 17794-17802 (1993)

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Glycosylphosphatidylinositol diacylglycerol-lyase

[5] Mller, G.; Grey, S.; Jung, C.; Bandlow, W.: Insulin-like signaling in yeast: modulation of protein phosphatase 2A, protein kinase A, cAMP-specific phosphodiesterase, and glycosyl-phosphatidylinositol-specific phospholipase C activities. Biochemistry, 39, 1475-1488 (2000) [6] Mller, G.; Deary, E.A.; Korndçrfer, A.; Bandlow, W.: Stimulation of a glycosyl-phosphatidylinositol-specific phospholipase by insulin and the sulfonylurea, glimepiride, in rat adipocytes depends on increased glucose transport. J. Cell Biol., 126, 1267-1276 (1994) [7] Mensa-Wilmot, K.; Morris, J.C.; Al-Qahtani, A.; Englund, P.T.: Purification and use of recombinant glycosylphosphatidylinositol-phospholipase C. Methods Enzymol., 250, 641-655 (1995) [8] Rolin, S.; Hanocq-Quertier, J.; Paturiaux-Hanocq, F.; Nolan, D.; Salmon, D.; Webb, H.; Carrington, M.; Voorheis, P.; Pays, E.: Simultaneous but independent activation of adenylate cyclase and glycosylphosphatidylinositol-phospholipase C under stress conditions in Trypanosoma brucei. J. Biol. Chem., 271, 10844-10852 (1996) [9] Webb, H.; Carnall, N.; Vanhamme, L.; Rolin, S.; Van Den Abbeele, J.; Welburn, S.; Pays, E.; Carrington, M.: The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice. J. Cell. Biol., 139, 103-114 (1997) [10] Buetikofer, P.; Boschung, M.; Brodbeck, U.; Menon, A.K.: Phosphatidylinositol hydrolysis by Trypanosoma brucei glycosylphosphatidylinositol phospholipase C. J. Biol. Chem., 271, 15533-15541 (1996) [11] Carnall, N.; Webb, H.; Carrington, M.: Mutagenesis study of the glycosylphosphatidylinositol phospholipase C of Trypanosoma brucei. Mol. Biochem. Parasitol., 90, 423-432 (1997) [12] Carrington, M.; Carnall, N.; Crow, M.S.; Gaud, A.; Redpath, M.B.; Wasunna, C.L.; Webb, H.: The properties and function of the glycosylphosphatidylinositol-phospholipase C in Trypanosoma brucei. Mol. Biochem. Parasitol., 91, 153-164 (1998) [13] Rashid, M.B.; Russell, M.; Mensa-Wilmot, K.: Roles of Gln81 and Cys80 in catalysis by glycosylphosphatidylinositol-phospholipase C from Trypanosoma brucei. Eur. J. Biochem., 264, 914-920 (1999) [14] Armah, D.A.; Mensa-Wilmot, K.: S-myristoylation of a glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei. J. Biol. Chem., 274, 5931-5938 (1999) [15] Ochatt, C.M.; Buetikofer, P.; Navarro, M.; Wirtz, E.; Boschung, M.; Armah, D.; Cross, G.A.: Conditional expression of glycosylphosphatidylinositol phospholipase C in Trypanosoma brucei. Mol. Biochem. Parasitol., 103, 35-48 (1999) [16] Armah, D.A.; Mensa-Wilmot, K.: Tetramerization of glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei. J. Biol. Chem., 275, 19334-19342 (2000) [17] Paturiaux-Hanocq, F.; Hanocq-Quertier, J.; de Almeida, M.L.; Nolan, D.P.; Pays, A.; Vanhamme, L.; Van den Abbeele, J.; Wasunna, C.L.; Carrington, M.; Pays, E.: A role for the dynamic acylation of a cluster of cysteine residues in regulating the activity of the glycosylphosphatidylinositol-specific

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phospholipase C of Trypanosoma brucei. J. Biol. Chem., 275, 12147-12155 (2000) [18] Mller, G.; Schulz, A.; Wied, S.; Frick, W.: Regulation of lipid raft proteins by glimepiride- and insulin-induced glycosylphosphatidylinositol-specific phospholipase C in rat adipocytes. Biochem. Pharmacol., 69, 761-780 (2005) [19] Zheng, Z.; Butler, K.D.; Tweten, R.K.; Mensa-Wilmot, K.: Endosomes, glycosomes, and glycosylphosphatidylinositol catabolism in Leishmania major. J. Biol. Chem., 279, 42106-42113 (2004) [20] Gruszynski, A.E.; van Deursen, F.J.; Albareda, M.C.; Best, A.; Chaudhary, K.; Cliffe, L.J.; Del Rio, L.; Dunn, J.D.; Ellis, L.; Evans, K.J.; Figueiredo, J.M.; Malmquist, N.A.; Omosun, Y.; Palenchar, J.B.; Prickett, S.; Punkosdy, G.A.; van Dooren, G.; Wang, Q.; Menon, A.K.; Matthews, K.R.; Bangs, J.D.: Regulation of surface coat exchange by differentiating African trypanosomes. Mol. Biochem. Parasitol., 147, 211-223 (2006)

450

FAD-AMP lyase (cyclizing)

4.6.1.15

1 Nomenclature EC number 4.6.1.15 Systematic name FAD AMP-lyase (riboflavin-cyclic-4’,5’-phosphate-forming) Recommended name FAD-AMP lyase (cyclizing) Synonyms FMN cyclase FMN cyclase/dha kinase <1, 2> (<1,2> bifunctional enzyme [3]) [3] Flavine-adenine-dinucleotide cyclase Rivoflavin cyclic phosphate synthase CAS registry number 208349-48-8

2 Source Organism <1> Rattus norvegicus (no sequence specified) [1, 2, 3] <2> Homo sapiens (UNIPROT accession number: Q3LXA3) [3]

3 Reaction and Specificity Catalyzed reaction FAD = AMP + riboflavin cyclic-4’,5’-phosphate Reaction type P-O bond cleavage Natural substrates and products S FAD <1> (Reversibility: ?) [1, 2] P AMP + riboflavin cyclic-4’,5’-phosphate <1> [1, 2] Substrates and products S FAD <1, 2> (Reversibility: ?) [1, 2, 3] P AMP + riboflavin cyclic-4’,5’-phosphate <1> [1, 2]

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FAD-AMP lyase (cyclizing)

4.6.1.15

Inhibitors ADP <1> [2] ATP <1, 2> (<1,2> inhibits the FMN cyclase activity [3]) [2, 3] FAD <1> (<1> concentration higher than 0.1 mM [1]; <1> concentration higher than 0.3 mM [2]) [1, 2] Additional information <1> (<1> twelve nucleotidic compounds tested, not inhibited by FMN, cFMN, dADP, dATP, AMP [2]) [2] Activating compounds Co2+ <1> (<1> activates [2]) [2] Mn2+ <1> (<1> activates [2]) [2] Additional information <1> (<1> thirty-five compounds structurally related to FAD tested [2]) [2] Metals, ions Co2+ <1> (<1> enzyme-activating cation [2]) [2] Mn2+ <1> (<1> enzyme-activating cation [1,2]) [1, 2] Additional information <1> (<1> Mg2+ , Ca2+ , Zn2+ , Ni2+ , Cu2+ , Fe3+ , Li+ , Na+ , K+ not required for activity [2]) [2] Turnover number (min–1) 4.82 <1> (FAD, <1> Mn2+ , pH: 7.5 [2]) [2] 19.3-24.1 <1> (FAD, <1> Mn2+ , pH: 8.3 [2]) [2] Specific activity (U/mg) 0.01 <2> (<2> FMN cyclase activity, lysate supernatant of BL21 cells, pH7.5, 37 C [3]) [3] 13 <1> (<1> p-nitrophenyl-d-TMP as substrate [1]) [1] Km-Value (mM) 0.006-0.008 <1> (FAD) [1] 0.009 <1> (FAD, <1> Mn2+ , pH: 7.5 [2]) [2] 0.036-0.045 <1> (FAD, <1> Mn2+ , pH: 8.3 [2]) [2] 0.09 <1> (FAD, <1> Co2+, pH: 7.5 [2]) [2] Ki-Value (mM) 2.5e-005 <1> (ADP) [2] 5e-005 <1> (ATP) [2] pH-Optimum 7.3 <1> (<1> Co2+ as activating cation [2]) [2] 8.5 <1> (<1> Mn2+ as activating cation [2]) [2] 9 <1> [1] Temperature optimum ( C) 37 <1> [1]

452

4.6.1.15

FAD-AMP lyase (cyclizing)

4 Enzyme Structure Molecular weight 59000 <1> (<1> SDS-PAGE [2]) [2] 100000 <1> (<1> ultrafiltration [2]) [2] 140000 <1> (<1> gel filtration [1,2]) [1, 2] Subunits ? <2> (<2> x * 59400, SDS-PAGE [3]) [3] dimer <1> [2]

5 Isolation/Preparation/Mutation/Application Source/tissue brain <2> [3] liver <1> [1, 2, 3] Localization cytoplasm <1> [1] Purification <1> [3] <1> (chromatography on DEAE-cellulose) [2] <2> [3] Cloning <2> (expression in BL21 cells) [3]

6 Stability Storage stability <1>, -20 C, 1 mg/ml bovine serum albumin [1] <1>, -80 C, TE: buffer 20 mM Tris-HCl and 0.5 mM EDTA, pH 8.2 at 4 C, 10 months [2]

References [1] Fraiz, F.J.; Pinto, R.M.; Costas, M.J.; Avalos, M.; Canales, J.; Cabezas, A.; Cameselle, J.C.: Enzymic formation of riboflavin 4’,5’-cyclic phosphate from FAD: evidence for a specific low-Km FMN cyclase in rat liver. Biochem. J., 330, 881-888 (1998) [2] Cabezas, A.; Pinto, R.M.; Fraiz, F.; Canales, J.; Gonzalez-Santiago, S.; Cameselle, J.C.: Purification, characterization, and substrate and inhibitor structure-activity studies of rat liver FAD-AMP lyase (cyclizing): Preference for FAD and specificity for splitting ribonucleoside diphosphate-X into ribonu-

453

FAD-AMP lyase (cyclizing)

4.6.1.15

cleotide and a five-atom cyclic phosphodiester of X, either a monocyclic compound or a cis-bicyclic phosphodiester-pyranose fusion. Biochemistry, 40, 13710-13722 (2001) [3] Cabezas, A.; Costas, M.J.; Pinto, R.M.; Couto, A.; Cameselle, J.C.: Identification of human and rat FAD-AMP lyase (cyclic FMN forming) as ATP-dependent dihydroxyacetone kinases. Biochem. Biophys. Res. Commun., 338, 16821689 (2005)

454

Sirohydrochlorin cobaltochelatase

4.99.1.3

1 Nomenclature EC number 4.99.1.3 Systematic name cobalt-sirohydrochlorin cobalt-lyase (sirohydrochlorin-forming) Recommended name sirohydrochlorin cobaltochelatase Synonyms CbiK <1, 3, 4, 5, 6, 9, 10> [1, 3, 5, 6] CbiX <2, 3, 8, 12> [2, 4, 7] CbiX0H <3> [7] CbiXL <3, 8> [7] CbiXOH <8> [7] CbiXS <2, 12> [2] anaerobic cobalt chelatase <1> [1, 3] archaeal cobaltochelatase <2, 12> [2] cobalamin cobalt chelatase <3> [4] cobalt chelatase <1, 3, 4, 5, 6, 9, 10> [3, 5] cobaltochelatase <2, 3, 8, 12> [2, 7] sirohydrochlorin cobalt chelatase <3> [4] sirohydrochlorin cobaltochelatase <2, 12> [2] CAS registry number 81295-49-0

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10>

Salmonella typhimurium (no sequence specified) [1, 3, 5, 6] Methanosarcina barkeri (no sequence specified) [2] Bacillus megaterium (no sequence specified) [4, 5, 7] Pseudomonas aeruginosa (no sequence specified) [5] Rhodobacter sphaeroides (no sequence specified) [5] Clostridium acetobutylicum (no sequence specified) [5] no activity in Bacillus subtilis [5] Synechocystis sp. (no sequence specified) [7] Porphyromonas gingivalis (no sequence specified) [5] Clostridium difficile (no sequence specified) [5]

455

Sirohydrochlorin cobaltochelatase

4.99.1.3

<11> no activity in eukaryota [5] <12> Methanobacter thermoautotrophicum (no sequence specified) [2]

3 Reaction and Specificity Catalyzed reaction cobalt-sirohydrochlorin + 2 H+ = sirohydrochlorin + Co2+ Natural substrates and products S precorrin-2 + Co2+ <1> (<1> tetrapyrrole biosynthesis, formation of tetrapyrrole cofactors [3]) (Reversibility: ?) [3] P Co-precorrin-2 + H+ <1> [3] S sirohydrochlorin + Co2+ <1, 3, 4, 5, 6, 9, 10> (<3,6,9,10> cobalamin biosynthesis [4,5]; <4,5> cobalamin biosynthesis, oxygen-independent pathway [5]; <1> cobalamin biosynthesis, oxygen-independent route, CbiK is a biglobal enzyme containing 2 a/b domains, which generate an active site with a deep rectangular cleft at their interface [5]) (Reversibility: ?) [4, 5] P cobaltsirohydrochlorin + H+ <1, 3, 4, 5, 6, 9, 10> [4, 5] S sirohydrochlorin + Co2+ <1, 2, 3, 8, 12> (<3,8> biosynthesis of vitamin B12 [7]; <1> cobalamin and siroheme biosynthesis,cbiK is able to substitute for cysG [6]; <2,12> cobalamin biosynthetic pathway [2]; <1> cobalamin branched biosynthetic pathway [1]) (Reversibility: ?) [1, 2, 6, 7] P cobalt-sirohydrochlorin + H+ <1, 2, 3, 8, 12> [1, 2, 6, 7] S sirohydrochlorin + Fe2+ <2, 12> (<2,12> CbiXS can act as a ferrochelatase in the biosynthesis of siroheme in vivo [2]) (Reversibility: ?) [2] P siroheme + H+ <2, 12> [2] Substrates and products S precorrin-2 + Co2+ <1> (Reversibility: ?) [1] P cobalt-precorrin-2 + H+ <1> [1] S precorrin-2 + Co2+ <1, 4> (<1> tetrapyrrole biosynthesis, formation of tetrapyrrole cofactors [3]) (Reversibility: ?) [3, 5] P Co-precorrin-2 + H+ <1, 4> [3, 5] S sirohydrochlorin + Co2+ <1, 3, 4, 5, 6, 9, 10> (<3,6,9,10> cobalamin biosynthesis [4,5]; <4,5> cobalamin biosynthesis, oxygen-independent pathway [5]; <1> cobalamin biosynthesis, oxygen-independent route, CbiK is a biglobal enzyme containing 2 a/b domains, which generate an active site with a deep rectangular cleft at their interface [5]) (Reversibility: ?) [4, 5] P cobaltsirohydrochlorin + H+ <1, 3, 4, 5, 6, 9, 10> [4, 5] S sirohydrochlorin + Co2+ <1, 2, 3, 8, 12> (<3,8> biosynthesis of vitamin B12 [7]; <1> cobalamin and siroheme biosynthesis,cbiK is able to substitute for cysG [6]; <2,12> cobalamin biosynthetic pathway [2]; <1> cobalamin branched biosynthetic pathway [1]) (Reversibility: ?) [1, 2, 6, 7] P cobalt-sirohydrochlorin + H+ <1, 2, 3, 8, 12> [1, 2, 6, 7] S sirohydrochlorin + Fe2+ <2, 12> (<2,12> CbiXS can act as a ferrochelatase in the biosynthesis of siroheme in vivo [2]) (Reversibility: ?) [2] P siroheme + H+ <2, 12> [2]

456

4.99.1.3

Sirohydrochlorin cobaltochelatase

S Additional information <2, 10, 12> (<10> CbiK fused together with CbiL as a multifunctional protein [5]; <2,12> precorrin-2 is no substrate [2]) (Reversibility: ?) [2, 5] P ? <2, 10, 12> [2, 5] Metals, ions Co2+ <1, 2, 12> (<1> incorporates Co2+ over Fe2+ [3]; <2,12> insertion of cobalt into sirohydrochlorin [2]) [2, 3] Fe2+ <2, 3, 8, 12> (<3,8> iron-sulfur center [7]; <2,12> able to chelate [2]) [2, 7] Ni2+ <12> (<12> able to chelate nickel [2]) [2] Specific activity (U/mg) 0.018 <12> [2] 0.06 <12> (<12> specific activity to chelate nickel [2]) [2] 0.08 <8> (<8> overproduced in Escherichia coli [7]) [7] 0.122 <2> [2] 1.4 <3> (<3> CbiX6H, overproduced in Escherichia coli [7]) [7]

4 Enzyme Structure Molecular weight 14000 <2, 12> (<2,12> gene-predicted molecular mass [2]) [2] 15000 <2> (<2> SDS-PAGE [2]) [2] 17000 <12> (<12> SDS-PAGE [2]) [2] 35000 <3> (<3> SDS-PAGE [7]) [7] 38000 <8> (<8> SDS-PAGE [7]) [7] 40000 <2, 12> (<2,12> gel filtration [2]) [2]

5 Isolation/Preparation/Mutation/Application Purification <1> [1, 5] <2> [2] <3> [7] <3> (purified anearobically) [4] <8> [7] <12> [2] Crystallization <1> [3, 5] <1> (hexagonal crystals are grown using the hanging-drop method, crystals are in space group P6(3)22 with cell dimensions a = b = 128.08 A, c = 85.44 A) [1]

457

Sirohydrochlorin cobaltochelatase

4.99.1.3

Cloning <1> (cloned and overexpressed) [5] <1> (expressed in Escherichia coli) [6] <1> (overexpressed in Escherichia coli BL21(DE3)pLys cells by cloning the gene into pET14b) [1] <2> (overproduced in Escherichia coli) [2] <3> (cloned and overexpressed in Escherichia coli and Bacillus megaterium) [7] <8> (overexpressed in Escherichia coli) [7] <12> (overproduced in Escherichia coli) [2] Engineering C259S <3> (<3> site-directed mutagenesis [7]) [7] C262S <3> (<3> site-directed mutagenesis [7]) [7] H145A/H207A <1> (<1> site-directed mutagenesis [1]) [1] M257L <3> (<3> site-directed mutagenesis [7]) [7]

6 Stability Oxidation stability <4>, requires molecular oxygen [5]

References [1] Schubert, H.L.; Raux, E.; Wilson, K.S.; Warren, M.J.: Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis. Biochemistry, 38, 10660-10669 (1999) [2] Brindley, A.A.; Raux E.; Leech, H.K.; Schubert, H.L.; Warren, M.J.: A story of chelatase evolution: Identification and characterisation of a small 13-15 kDa ’ancestral’ cobaltochelatase (CbiXS) in the Archaea. J. Biol. Chem., 278, 22388-22395 (2003) [3] Schubert, H.L.; Raux, E.; Matthews, M.A.; Phillips, J.D.; Wilson, K.S.; Hill, C.P.; Warren, M.J.: Structural diversity in metal ion chelation and the structure of uroporphyrinogen III synthase. Biochem. Soc. Trans., 30, 595-600 (2002) [4] Leech, H.K.; Raux-Deery, E.; Heathcote, P.; Warren, M.J.: Production of cobalamin and sirohaem in Bacillus megaterium: an investigation into the role of the branchpoint chelatases sirohydrochlorin ferrochelatase (SirB) and sirohydrochlorin cobalt chelatase (CbiX). Biochem. Soc. Trans., 30, 610-613 (2002) [5] Raux, E.; Schubert, H.L.; Warren, M.J.: Biosynthesis of cobalamin (vitamin B12 ): a bacterial conundrum. Cell. Mol. Life Sci., 57, 1880-1893 (2000)

458

4.99.1.3

Sirohydrochlorin cobaltochelatase

[6] Raux, E.; Thermes, C.; Heathcote, P.; Rambach, A.; Warren, M.J.: A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12 ) and siroheme biosynthesis. J. Bacteriol., 179, 3202-3212 (1997) [7] Leech, H.K.; Raux, E.; McLean, K.J.; Munro, A.W.; Robinson, N.J.; Borrelly, G.P.; Malten, M.; Jahn, D.; Rigby, S.E.; Heathcote, P.; Warren, M.J.: Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif. J. Biol. Chem., 278, 41900-41907 (2003)

459

Sirohydrochlorin ferrochelatase

4.99.1.4

1 Nomenclature EC number 4.99.1.4 Systematic name siroheme ferro-lyase (sirohydrochlorin-forming) Recommended name sirohydrochlorin ferrochelatase Synonyms CysG <4> (<4> Salmonella enterica [4]) [4] Met8p <1, 5> (<1,5> Saccharomyces cerevisiae [1,3]) [1, 3] SirB <3> (<3> Bacillus megaterium [2]) [2] sirohydrochlorin ferrochelatase <2> [5] Additional information <1, 5> (<1> member of the class 2 chelatases [3]; <5> Met8p is a class 2 chelatase [1]) [1, 3] CAS registry number 9012-93-5

2 Source Organism <1> <2> <3> <4> <5>

Saccharomyces cerevisiae (no sequence specified) [1, 3] Arabidopsis thaliana (no sequence specified) [5] Bacillus megaterium (no sequence specified) [2] Salmonella enterica (no sequence specified) [4] Saccharomyces cerevisiae (UNIPROT accession number: P15807) [1]

3 Reaction and Specificity Catalyzed reaction sirohydrochlorin + Fe2+ = siroheme + 2 H+ (<1> the enzyme from Pseudomonas chloroaphis contains Ca2+ and protoheme IX, the iron of which must be in the form Fe2+ for activity, the enzyme exhibits a strong preference for aliphatic aldoximes, such as butyraldoxime and acetaldoxime, over aromatic aldoximes, such as pyridine-2-aldoxime, which is a poor substrate, no activity was found with the aromatic aldoximes benzaldoxime and pyridine-4-aldoxime, mechanism [3])

460

4.99.1.4

Sirohydrochlorin ferrochelatase

Natural substrates and products S sirohydrochlorin + Fe2+ <1, 2> (Reversibility: ?) [1, 5] P siroheme + H+ S sirohydrochlorin + Fe2+ <1, 3, 4, 5> (<4> CysG, siroheme biosynthesis [4]; <1> Met8p catalyzes ferrochelation during the biosynthesis of siroheme [3]; <5> Met8p catalyzes the final two steps in the biosynthesis of siroheme involving a b-NAD+ -dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield siroheme [1]; <3> SirB is responsible for the final step in siroheme synthesis [2]) (Reversibility: ?) [1, 2, 3, 4] P siroheme + 2 H+ <1, 3, 4, 5> (<4> sulfur metabolism depends on siroheme [4]) [1, 2, 3, 4] Substrates and products S precorrin-2 + Co2+ <3> (<3> SirB, much lower specific activity than with sirohydrochlorin [2]) (Reversibility: ?) [2] P cobalt-precorrin-2 + 2 H+ <3> [2] S sirohydrochlorin + Co2+ <2> (<2> pH 8.0 [5]) (Reversibility: ?) [5] P cobalt-sirohydrochlorin + H+ S sirohydrochlorin + Co2+ <3, 4, 5> (<3> SirB, lower specificity for cobalt than for iron [2]) (Reversibility: ?) [1, 2, 4] P cobalt-sirohydrochlorin + 2 H+ <3, 4, 5> [1, 2, 4] S sirohydrochlorin + Fe2+ <1, 2> (<2> pH 8.0 [5]) (Reversibility: ?) [1, 5] P siroheme + H+ S sirohydrochlorin + Fe2+ <1, 3, 4, 5> (<4> CysG structure, the multifunctional siroheme synthase CysG synthesizes siroheme from uroporphyrinogen III, CysG contains two structurally independent modules: a bismethyltransferase and a dual-function dehydrogenase-chelatase [4]; <1> Met8p catalyzes ferrochelation during the synthesis of siroheme, both ferrochelation and NAD+ -dependent dehydrogenation of preccorin-2 to produce sirohydrochlorin take place in a single bifunctional active site, Asp-141 participates in both catalytic reactions, which are not linked mechanistically, mechanism [3]; <5> Met8p structure, bifunctional Met8p catalyzes the final two steps in the biosynthesis of siroheme involving a b-NAD+ -dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield siroheme, both catalytic activities share a single active site, Asp-141 functions as a general base and plays an essential role in both dehydrogenase and chelatase processes [1]; <3> SirB, monofunctional ferrochelatase, higher specificity for iron over cobalt [2]; <4> CysG, siroheme biosynthesis [4]; <1> Met8p catalyzes ferrochelation during the biosynthesis of siroheme [3]; <5> Met8p catalyzes the final two steps in the biosynthesis of siroheme involving a b-NAD+ -dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield siroheme [1]; <3> SirB is responsible for the final step in siroheme synthesis [2]) (Reversibility: ?) [1, 2, 3, 4] P siroheme + 2 H+ <1, 3, 4, 5> (<4> sulfur metabolism depends on siroheme [4]) [1, 2, 3, 4]

461

Sirohydrochlorin ferrochelatase

4.99.1.4

Inhibitors Co2+ <2> (<2> inactivates the enzyme at high concentrations [5]) [5] Fe2+ <2> (<2> inactivates the enzyme at high concentrations [5]) [5] Metals, ions Co2+ <2> (<2> inactivates the enzyme at high concentrations [5]) [5] Fe2+ <2> (<2> inactivates the enzyme at high concentrations [5]) [5] Specific activity (U/mg) 0.0034 <3> (<3> pH 8, cobaltochelation of precorrin-2 [2]) [2] 0.0054 <2> (<2> isolated recombinant enzyme, with ferrous iron as substrate [5]) [5] 0.0138 <4> (<4> S128D mutant CysG, cobalt chelation of sirohydrochlorin [4]) [4] 0.0311 <1> (<1> wild-type Met8p [1]) [1] 0.0485 <2> (<2> isolated recombinant enzyme, with cobalt as substrate [5]) [5] 0.0656 <4> (<4> wild-type CysG, cobalt chelation of sirohydrochlorin [4]) [4] 0.243 <4> (<4> S128A mutant CysG, cobalt chelation of sirohydrochlorin [4]) [4] 0.337 <3> (<3> pH 8, cobaltochelation of sirohydrochlorin [2]) [2] Additional information <5> [1] Km-Value (mM) Additional information <2> (<2> it is not possible to measure an accurate KM value for the metals as at low concentration of metal the assay is not sensitive enough, whereas at higher metal ion concentrations the enzyme is inactivated [5]) [5] pH-Optimum 8 <3, 5> (<3,5> assay at [1,2]) [1, 2]

4 Enzyme Structure Subunits homodimer <1, 4, 5> (<1> each monomer is composed of three functional domains, domain structure [3]; <5> three structural domains per monomer, domain structure [1]) [1, 3, 4] monomer <3> (<3> 1 * 33000, SDS-PAGE [2]) [2] Posttranslational modification phosphoprotein <4> (<4> CysG, phosphorylation of Ser-128, plays a regulatory role [4]) [4]

462

4.99.1.4

Sirohydrochlorin ferrochelatase

5 Isolation/Preparation/Mutation/Application Localization chloroplast <2> [5] soluble <3> (<3> recombinant SirB [2]) [2] Purification <1> (recombinant Met8p) [1] <2> (recombinant protein) [5] <3> (recombinant SirB) [2] <4> [4] Crystallization <1> (X-ray crystal structure of Met8p) [3] <1> (X-ray crystal structure of Met8p, hanging drop method) [1] <4> (X-ray crystal structure of CysG) [4] Cloning <1> (MET8 gene) [1] <2> (expression in Escherichia coli) [5] <3> (sirB, expression in Escherichia coli BL21(DE3)pLysS, sirA,B,C operon, amino acid sequence) [2] <4> [4] Engineering D141A <1, 5> (<5> mutant of bifunctional Met8p is completely inactive as both dehydrogenase and ferrochelatase [1]; <1> mutant of bifunctional Met8p is devoid of both dehydrogenase and ferrochelatase activities [3]) [1, 3] G22D <1, 5> (<5> mutant of bifunctional Met8p is completely inactive as dehydrogenase, but functions as ferrochelatase [1]; <1> mutant of bifunctional Met8p is completely inactive as NAD+ -dependent dehydrogenase, but functions as ferrochelatase [3]) [1, 3] H237A <5> (<5> mutant of bifunctional Met8p is active as both dehydrogenase and ferrochelatase [1]) [1] S128A <4> (<4> mutant has higher cobalt chelatase activity than wild-type CysG with sirohydrochlorin as substrate [4]) [4] S128D <4> (<4> mutant has lower cobalt chelatase activity than wild-type CysG with sirohydrochlorin as substrate [4]) [4]

References [1] Schubert, H.L.; Raux, E.; Brindley, A.A.; Leech, H.K.; Wilson, K.S.; Hill, C.P.; Warren, M.J.: The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase. EMBO J., 21, 2068-2075 (2002) [2] Raux, E.; Leech, H.K.; Beck, R.; Schubert, H.L.; Santander, P.J.; Roessner, C.A.; Scott, A.I.; Martens, J.H.; Jahn, D.; Thermes, C.; Rambach, A.; Warren,

463

Sirohydrochlorin ferrochelatase

4.99.1.4

M.J.: Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium. Biochem. J., 370, 505-516 (2003) [3] Schubert, H.L.; Raux, E.; Matthews, M.A.; Phillips, J.D.; Wilson, K.S.; Hill, C.P.; Warren, M.J.: Structural diversity in metal ion chelation and the structure of uroporphyrinogen III synthase. Biochem. Soc. Trans., 30, 595-600 (2002) [4] Stroupe, M.E.; Leech, H.K.; Daniels, D.S.; Warren, M.J.; Getzoff, E.D.: CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis. Nat. Struct. Biol., 10, 1064-1073 (2003) [5] Raux-Deery, E.; Leech, H.K.; Nakrieko, K.A.; McLean, K.J.; Munro, A.W.; Heathcote, P.; Rigby, S.E.; Smith, A.G.; Warren, M.J.: Identification and characterization of the terminal enzyme of siroheme biosynthesis from Arabidopsis thaliana: a plastid-located sirohydrochlorin ferrochelatase containing a 2FE-2S center. J. Biol. Chem., 280, 4713-4721 (2005)

464

Aliphatic aldoxime dehydratase

4.99.1.5

1 Nomenclature EC number 4.99.1.5 Systematic name aliphatic aldoxime hydro-lyase (alophatic-nitrile-forming) Recommended name aliphatic aldoxime dehydratase Synonyms OxdA <1, 2> [1, 3, 4, 5, 6] OxdRG <3> [2] aldoxime dehydratase <2> [1] alkylaldoxime dehydratase <3> [2] CAS registry number 203210-76-8

2 Source Organism <1> Pseudomonas chlororaphis (no sequence specified) [1, 3, 4, 5, 6] <2> Pseudomonas chlororaphis (UNIPROT accession number: Q7WSJ4) [1] <3> Rhodococcus globerulus (UNIPROT accession number: Q76EV4) [2]

3 Reaction and Specificity Catalyzed reaction an aliphatic aldoxime = an aliphatic nitrile + H2 O Reaction type dehydration Natural substrates and products S alkylaldoxime <3> (<3> responsible for the metabolism of alkylaldoxime [2]) (Reversibility: ?) [2] P alkylnitrile + H2 O <3> [2] S Additional information <2> (<2> involved in carbon-nitrogen triple bond synthesis, responsible for the metabolism of aldoxime in vivo [1]) (Reversibility: ?) [1] P ? <2> [1]

465

Aliphatic aldoxime dehydratase

4.99.1.5

Substrates and products S (E)-pyridine-3-aldoxime <3> (<3> not Z-form, poor substrate, 0.78% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P pyridine-3-nitrile + H2 O <3> [2] S (E/Z)-2-phenylpropionaldoxime <3> (<3> 5.23% of the activity with (E/ Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P 2-phenylpropiononitrile + H2 O <3> [2] S (E/Z)-acetaldoxime <2> (Reversibility: ?) [1] P acetonitrile + H2 O <2> [1] S (E/Z)-cyclohexanecarboxaldehyde oxime <3> (<3> best substrate [2]) (Reversibility: ?) [2] P ? <3> [2] S (E/Z)-indoleacetaldoxime <3> (<3> 7.29% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P indoleacetonitrile + H2 O <3> [2] S (E/Z)-isobutyraldoxime <3> (<3> 26.5% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P isobutyronitrile + H2 O <3> [2] S (E/Z)-isocapronaldoxime <3> (<3> 88.3% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P isocaprononitrile + H2 O <3> [2] S (E/Z)-isovaleraldoxime <3> (<3> 58.6% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P isovaleronitrile + H2 O <3> [2] S (E/Z)-mandelaldoxime <3> (<3> 3.09% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P mandelonitrile + H2 O <3> [2] S (E/Z)-n-butyraldoxime <2, 3> (<3> 48.9% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]; <2> best substrate, accepts both the E- and the Z-forms of butyraldoxime as substrates [1]) (Reversibility: ?) [1, 2] P n-butyronitrile + H2 O <2, 3> (<2> butyronitrile does not act as substrate [1]) [1, 2] S (E/Z)-n-capronaldoxime <3> (<3> 64.2% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P n-caprononitrile + H2 O <3> [2] S (E/Z)-n-valeraldoxime <3> (<3> 39.3% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P n-valeronitrile + H2 O <3> [2] S (E/Z)-propionaldoxime <3> (<3> 7.76% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P propiononitrile + H2 O <3> [2] S (E/Z)-pyridine-2-aldoxime <2> (<2> poor substrate [1]) (Reversibility: ?) [1] P pyridine-2-nitrile + H2 O <2> [1]

466

4.99.1.5

Aliphatic aldoxime dehydratase

S (Z)-3-phenylpropionaldoxime <3> (<3> 67.3% of the activity with (E/Z)cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P 3-phenylpropiononitrile + H2 O <3> [2] S (Z)-phenylacetaldoxime <3> (<3> 26.6% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime [2]) (Reversibility: ?) [2] P phenylacetonitrile + H2 O <3> [2] S aldoxime <3> (<3> wide range of substrates, aliphatic aldoximes are more effective substrates than arylalkyl aldoximes, aromatic aldoximes are also dehydrated [2]) (Reversibility: ?) [2] P nitrile + H2 O <3> [2] S aliphatic aldoxime <2> (<2> strong preference of aliphatic aldoximes over aromatic aldoximes [1]) (Reversibility: ?) [1] P aliphatic nitrile + H2 O <2> [1] S alkylaldoxime <3> (<3> responsible for the metabolism of alkylaldoxime [2]) (Reversibility: ?) [2] P alkylnitrile + H2 O <3> [2] S an aliphatic aldoxime <1> (Reversibility: ?) [1] P an aliphatic nitrile + H2 O S butyraldoxime <1> (<1> the substrate binds to heme forming a hexa-coordinate low-spin heme [4]) (Reversibility: ?) [4, 5] P butyronitrile + H2 O S n-butyraldoxime <1> (<1> reaction intermediate of the heme-containing enzyme with a highly oxidized heme that is formed concomitantly upon direct binding of the substrate, heme directly activates the organic substrate [6]) (Reversibility: ?) [6] P n-butyronitrile + H2 O S Additional information <2> (<2> not: butyronitrile, aromatic aldoximes, such as benzaldoxime, isonitrosoacetophenone, and pyridine-4-aldoxime [1]; <2> involved in carbon-nitrogen triple bond synthesis, responsible for the metabolism of aldoxime in vivo [1]) (Reversibility: ?) [1] P ? <2> [1] Inhibitors 5,5’-dithiobis-2-nitrobenzoate <2> (<2> weak inhibition [1]) [1] AgNO3 <2> (<2> very sensitive to, concentration-dependent, 1 mM, 85.8% inhibition [1]) [1] CO <2> (<2> 49% inhibition [1]) [1] Cd2+ <3> (<3> strong inhibition at 1 mM, activates at 0.1 mM [2]) [2] Co2+ <3> (<3> strong inhibition at 1 mM, activates at 0.1 mM [2]) [2] Cu+ <3> (<3> strong inhibition at 1 mM, activates at 0.1 mM [2]) [2] Cu2+ <3> (<3> strong inhibition at 1 mM, activates at 0.1 mM [2]) [2] diethyldithiocarbamate <2> (<2> 1 mM, 40.6% inhibition [1]) [1] guaiacol <3> (<3> slight inhibition [2]) [2] hydroxylamine <2> (<2> concentration-dependent, 1 mM, 100% inhibition [1]) [1] iodoacetate <2> (<2> weak inhibition [1]) [1] KCN <2> (<2> 1 mM, 41.3% inhibition [1]) [1]

467

Aliphatic aldoxime dehydratase

4.99.1.5

miconazole <3> (<3> slight inhibition [2]) [2] N-ethylmaleimide <2> (<2> weak inhibition [1]) [1] nitroblue tetrazolium <3> (<3> slight inhibition [2]) [2] sulfhydryl reagent <3> (<3> 1 mM [2]) [2] Tiron <3> (<3> inhibits dehydration of (Z)-3-phenylpropionaldoxime, but not of (E/Z)-n-valeraldoxime [2]) [2] Zn2+ <3> (<3> strong inhibition at 1 mM, activates at 0.1 mM [2]) [2] digallol <3> (<3> slight inhibition [2]) [2] dimethylphenylenediamine <3> (<3> slight inhibition [2]) [2] heavy metal ion <3> (<3> 1 mM [2]) [2] p-chloromercuribenzoate <2> (<2> weak inhibition [1]) [1] p-phenylenediamine <3> (<3> slight inhibition [2]) [2] phenazine methosulfate <3> (<3> slight inhibition [2]) [2] phenylhydrazine <2, 3> (<2> 1 mM, 71.1% inhibition [1]; <3> inhibits dehydration of (Z)-3-phenylpropionaldoxime, but not of (E/Z)-n-valeraldoxime [2]) [1, 2] tetramethylphenylenediamine <3> (<3> slight inhibition [2]) [2] trimethylhydroquinone <3> (<3> slight inhibition [2]) [2] Additional information <2, 3> (<2> not inhibited by chelating agents, such as 2,2’-dipyridyl, o-phenanthroline, 8-hydroxyquinoline, EDTA, and by phenylmethanesulfonyl fluoride, diisopropyl fluorophosphate [1]; <3> not inhibited by hydrazine, KCN, NaN3 , and NH2 OH [2]) [1, 2] Cofactors/prosthetic groups heme <1> (<1> ferric heme is six-coordinate low spin, whereas the ferrous heme is five-coordinate high-spin. Characterization of the heme environment in OxdA using resonance Raman spectroscopy [3]; <1> heme directly activates the organic substrate [6]; <1> the substrate binds to heme forming a hexa-coordinate low-spin heme [4]) [3,4,5,6] protoheme IX <2, 3> (<2> hemoprotein, the heme iron of the active enzyme is in the ferrous state, carries protoheme IX as the prosthetic group, 0.69 mol heme per mol of subunit [1]; <3> prosthetic group, heme b, heme iron is present in a reduced form, contains 0.37 mol heme per mol of enzyme [2]) [1,2] heme b <3> (<3> prosthetic group, protoheme IX, heme iron is present in a reduced form, contains 0.37 mol heme per mol of enzyme [2]) [2] Additional information <2> (<2> no requirement of FMN [1]) [1] Activating compounds 2-mercaptoethanol <3> (<3> activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 2.13fold [2]) [2] FAD <3> (<3> 1 mM, 1.2fold activation in presence of Na2 S, 4.5fold in absence of Na2 S [2]) [2] FMN <3> (<3> 1 mM, 1.7fold activation in presence of Na2 S, 2.4fold in absence of Na2 S [2]) [2] l-cysteine <3> (<3> activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 2.39fold [2]) [2]

468

4.99.1.5

Aliphatic aldoxime dehydratase

Na2 S <3> (<3> activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 8.35fold [2]) [2] Na2 S2 O4 <2, 3> (<3> activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 8.13fold [2]; <2> requirement of a reducing agent for activity [1]) [1, 2] Na2 S2 O5 <3> (<3> activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 5.84fold [2]) [2] Na2 SO3 <3> (<3> activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 7.51fold [2]) [2] Riboflavin <3> (<3> 1 mM, 1.1fold activation in presence of Na2 S, 4.2fold in absence of Na2 S [2]) [2] cysteamine <3> (<3> activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 3.93fold [2]) [2] duroquinone <3> (<3> activates [2]) [2] reducing reagent <3> (<3> requirement, activates [2]) [2] thioglycerol <3> (<3> activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 4.06fold [2]) [2] vitamin K3 <3> (<3> activates [2]) [2] Additional information <3> (<3> not activated by NaHSO3, Na2 SO4, NaHSO4 or Na2 S2 O7 [2]) [2] Metals, ions Ca2+ <2> (<2> contains 1.58 mol Ca2+ per mol of homodimeric OxdA, may act as another cofactor [1]) [1] Cd2+ <3> (<3> activates at 0.1 mM, strong inhibition at 1 mM [2]) [2] Co2+ <3> (<3> activates at 0.1 mM, strong inhibition at 1 mM [2]) [2] Cu+ <3> (<3> activates at 0.1 mM, strong inhibition at 1 mM [2]) [2] Cu2+ <3> (<3> activates at 0.1 mM, strong inhibition at 1 mM [2]) [2] Fe2+ <2, 3> (<2> contains 1.62 mol iron per mol of homodimeric OxdA, present in the heme molecule, the heme iron of the active enzyme is in the ferrous state [1]; <3> heme iron is present in a reduced form, 1 mM, activates [2]) [1, 2] Fe3+ <3> (<3> 1 mM, activates [2]) [2] Zn2+ <3> (<3> activates at 0.1 mM, strong inhibition at 1 mM [2]) [2] Turnover number (min–1) 5.4 <2> ((E/Z)-pyridine-2-aldoxime, <2> pH 7, 30 C [1]) [1] 324 <2> ((E/Z)-n-butyraldoxime, <2> pH 7, 30 C [1]) [1] 336 <2> ((E/Z)-acetaldoxime, <2> pH 7, 30 C [1]) [1] Specific activity (U/mg) 0.0879 <3> (<3> pH 7, 30 C, wild-type OxdRG, (Z)-phenylacetaldoxime as substrate, in presence of 1 mM Na2 S [2]) [2] 0.758 <2> (<2> pH 7, 30 C, butyraldoxime as substrate, under aerobic conditions [1]) [1] 2.18 <3> (<3> pH 7, 30 C, recombinant OxdRG, (Z)-phenylacetaldoxime as substrate, in presence of 1 mM Na2 S [2]) [2] 197 <2> (<2> pH 7, 30 C, butyraldoxime as substrate, under anaerobic reduced conditions, OxdA reduced by Na2 S2O4 [1]) [1]

469

Aliphatic aldoxime dehydratase

4.99.1.5

Km-Value (mM) 0.25 <2> ((E/Z)-n-butyraldoxime, <2> pH 7, 30 C [1]) [1] 1.13 <3> ((E/Z)-cyclohexanecarboxaldehyde oxime, <3> pH 7, 30 C [2]) [2] 1.13 <3> ((E/Z)-n-valeraldoxime, <3> pH 7, 30 C [2]) [2] 1.4 <3> ((Z)-phenylacetaldoxime, <3> pH 7, 30 C [2]) [2] 1.73 <3> ((E/Z)-n-butyraldoxime, <3> pH 7, 30 C [2]) [2] 2.31 <3> ((Z)-3-phenylpropionaldoxime, <3> pH 7, 30 C [2]) [2] 2.94 <3> ((E/Z)-n-capronaldoxime, <3> pH 7, 30 C [2]) [2] 3.23 <3> ((E/Z)-mandelaldoxime, <3> pH 7, 30 C [2]) [2] 3.4 <2> ((E/Z)-pyridine-2-aldoxime, <2> pH 7, 30 C [1]) [1] 3.91 <3> ((E/Z)-indoleacetaldoxime, <3> pH 7, 30 C [2]) [2] 3.97 <3> ((E/Z)-isovaleraldoxime, <3> pH 7, 30 C [2]) [2] 5.13 <3> ((E/Z)-propionaldoxime, <3> pH 7, 30 C [2]) [2] 5.54 <3> ((E/Z)-isobutyraldoxime, <3> pH 7, 30 C [2]) [2] 6.76 <3> ((E/Z)-isocapronaldoxime, <3> pH 7, 30 C [2]) [2] 11 <2> ((E/Z)-acetaldoxime, <2> pH 7, 30 C [1]) [1] 11.9 <3> ((E/Z)-2-phenylpropionaldoxime, <3> pH 7, 30 C [2]) [2] 20 <3> ((E)-pyridine-3-aldoxime, <3> pH 7, 30 C [2]) [2] Additional information <3> (<3> Km for the activating Na2 S is 0.0408 mM [2]) [2] pH-Optimum 5.5 <2> [1] 8 <3> (<3> 0.1 M potassium phosphate buffer [2]) [2] Additional information <2> (<2> maximum at pH 5.5, another high activity level occurs at pH 9.4 [1]) [1] pH-Range 5-10 <2> (<2> active over a broad pH-range [1]) [1] Temperature optimum ( C) 30 <3> (<3> around, 0.1 M potassium phosphate buffer, pH 8 [2]) [2] 45 <2> [1]

4 Enzyme Structure Molecular weight 76200 <2, 3> (<2> OxdA reduced by Na2 S2 O4, gel filtration [1]; <3> wildtype OxdRG, gel filtration [2]) [1, 2] 76400 <2> (<2> OxdA without reducing conditions, gel filtration [1]) [1] Subunits dimer <3> (<3> 2 * 42000, wild-type OxdRG, SDS-PAGE, 2 * 39892, calculated from the sequence [2]) [2] homodimer <2> (<2> 2 * 40127, calculated from the amino acid sequence, 2 * 38000, SDS-PAGE [1]) [1]

470

4.99.1.5

Aliphatic aldoxime dehydratase

5 Isolation/Preparation/Mutation/Application Purification <1> [5, 6] <1> (recombinant) [3] <2> (recombinant OxdA) [1] <3> (wild-type OxdRG: 104fold, recombinant OxdRG expressed in Escherichia coli JM109: 14.9fold) [2] Cloning <2> (oxdA gene, overexpression in Escherichia coli BL21-Codon-Plus(DE3)RIL, sequencing, gene cluster organisation) [1] <3> (oxd gene, overexpression in Escherichia coli JM109, sequencing, gene cluster organisation) [2] Engineering H169A <1> (<1> the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme [5]) [5] H296A <1> (<1> the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme [5]) [5] H299A <1> (<1> mutant is unable to bind heme [5]; <1> the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme [5]) [5] H320A <1> (<1> mutation does not affect the overall structure of OxdA but causes loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the Raman spectrum for the CO-bound form [5]; <1> the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme [5]) [5] H338A <1> (<1> the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme [5]) [5] Application synthesis <3> (<3> may be a useful biocatalyst for the production of various nitriles from the corresponding aldoximes [2]) [2]

6 Stability Temperature stability 20 <2> (<2> after 15 min preincubation OxdA is reduced anaerobic conditions, 100% of enzyme activity [1]) [1] 25 <2> (<2> after 15 min preincubation OxdA is reduced anaerobic conditions, 94% of enzyme activity [1]) [1] 30 <2> (<2> after 15 min preincubation OxdA is reduced anaerobic conditions, 91% of enzyme activity [1]) [1] 35 <2> (<2> after 15 min preincubation OxdA is reduced anaerobic conditions, 78% of enzyme activity [1]) [1]

by Na2 S2 O4, under by Na2 S2 O4, under by Na2 S2 O4, under by Na2 S2 O4, under

471

Aliphatic aldoxime dehydratase

4.99.1.5

40 <2, 3> (<3> 30 min, 0.1 M potassium phosphate buffer, pH 8, 35% loss of activity [2]; <2> after 15 min preincubation OxdA is reduced by Na2 S2 O4, under anaerobic conditions, 51% of enzyme activity [1]) [1, 2] 45 <2> (<2> after 15 min preincubation OxdA is reduced by Na2 S2 O4, under anaerobic conditions, 14% of enzyme activity [1]) [1] 50 <2> (<2> after 15 min preincubation OxdA is reduced by Na2 S2 O4, under anaerobic conditions, 3.2% of enzyme activity [1]) [1]

References [1] Oinuma, K-I.; Hashimoto, Y.; Konishi, K.; Goda, M.; Noguchi, T.; Higashibata, H.; Kobayashi, M.: Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23: Sequencing, gene expression, purification and characterization. J. Biol. Chem., 278, 29600-29608 (2003) [2] Xie, S.-X.; Kato, Y.; Komeda, H.; Yoshida, S.; Asano, Y.: A gene cluster responsible for alkylaldoxime metabolism coexisting with nitrile hydratase and amidase in Rhodococcus globerulus A-4. Biochemistry, 42, 12056-12066 (2003) [3] Oinuma, K.; Ohta, T.; Konishi, K.; Hashimoto, Y.; Higashibata, H.; Kitagawa, T.; Kobayashi, M.: Heme environment in aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis. FEBS Lett., 568, 44-48 (2004) [4] Oinuma, K.; Kumita, H.; Ohta, T.; Konishi, K.; Hashimoto, Y.; Higashibata, H.; Kitagawa, T.; Shiro, Y.; Kobayashi, M.: Stopped-flow spectrophotometric and resonance Raman analyses of aldoxime dehydratase involved in carbonnitrogen triple bond synthesis. FEBS Lett., 579, 1394-1398 (2005) [5] Konishi, K.; Ishida, K.; Oinuma, K.; Ohta, T.; Hashimoto, Y.; Higashibata, H.; Kitagawa, T.; Kobayashi, M.: Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase. J. Biol. Chem., 279, 47619-47625 (2004) [6] Konishi, K.; Ohta, T.; Oinuma, K.; Hashimoto, Y.; Kitagawa, T.; Kobayashi, M.: Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center. Proc. Natl. Acad. Sci. USA, 103, 564-568 (2006)

472

Indoleacetaldoxime dehydratase

4.99.1.6

1 Nomenclature EC number 4.99.1.6 Systematic name (indol-3-yl)acetaldehyde-oxime hydro-lyase [(indol-3-yl)acetonitrile-forming] Recommended name indoleacetaldoxime dehydratase Synonyms EC 4.2.1.29 (formerly) dehydratase, indoleacetaldoxime indoleacetaldoxime hydro-lyase CAS registry number 9024-27-5

2 Source Organism <1> <2> <3> <4> <5>

Aspergillus niger (no sequence specified) [2] Penicillium chrysogenum (no sequence specified) [2] Fusarium oxysporum (no sequence specified) [2] Musa acuminata (no sequence specified) [2] Gibberella fujikuroi (no sequence specified) [1,2,3,4]

3 Reaction and Specificity Catalyzed reaction 3-indoleacetaldoxime = 3-indoleacetonitrile + H2 O Reaction type elimination Substrates and products S 3-indoleacetaldoxime <1, 2, 3, 4, 5> (Reversibility: ?) [1, 2, 3, 4] P 3-indoleacetonitrile <1, 2, 3, 4, 5> [1, 2, 3, 4] Inhibitors 2,3-dimercaptopropanol <5> (<5> reversal by dehydroascorbic acid, pyridoxal 5’-phosphate or frozen storage [4]) [3, 4]

473

Indoleacetaldoxime dehydratase

4.99.1.6

2-mercaptoethanol <5> [3] 8-hydroxyquinoline <5> (<5> inhibition is partly reversed by ferric citrate, ascorbic acid and dehydroascorbic acid [3]) [3] Ag+ <5> (<5> 1 mM, complete inactivation [3]) [3] Al3+ <5> (<5> 1 mM, complete inactivation [3]) [3] benzaldoxime <5> [3] Cu2+ <5> (<5> 1 mM, complete inactivation [3]) [3] Cys <5> [3] diethyl dithiocarbamate <5> [3] Hg2+ <5> (<5> 1 mM, complete inactivation [3]) [3] KCN <5> (<5> reversed by pyridoxal 5’-phosphate [4]; <5> activates at 0.001-0.1 mM, inhibits at 1-10 mM [3]) [3, 4] mandelaldoxime <5> [3] mercaptoacetic acid <5> (<5> weak [3]) [3] Mo5+ <5> (<5> 1 mM, complete inactivation [3]) [3] NEM <5> (<5> protection by phenylacetaldoxime [4]) [3, 4] NaBH4 <5> (<5> inhibition is partly reversed by pyridoxal-5-phosphate or dehydroascorbic acid [3]) [3] phenylacetaldoxime <5> (<5> competitive [3,4]) [3, 4] phenylpropionaldoxime <5> [3] phenylthiocyanate <5> [4] Zn2+ <5> (<5> 1 mM, complete inactivation [3]) [3] p-hydroxymercuribenzoate <5> [3] tetrahydrofolic acid <5> [4] Cofactors/prosthetic groups pyridoxal 5’-phosphate <5> (<5> required [3]; <5> activates [4]; <5> cofactor [1]) [1,3,4] Activating compounds dehydroascorbic acid <5> (<5> activates [4]) [4] dihydrofolic acid <5> (<5> activates [4]) [4] glutathione <5> (<5> activates [3]) [3] Metals, ions Fe2+ <5> (<5> activates, no activation by Fe3+ [4]) [4] Fe3+ <5> (<5> Ferric citrate promotes activity [3]) [3] KCN <5> (<5> activates at 0.001-0.1 mM, inhibits at 1-10 mM [3]) [3] Specific activity (U/mg) Additional information <5> [3] Km-Value (mM) 0.17 <5> (3-indoleacetaldoxime) [3] pH-Optimum 7 <5> [3]

474

4.99.1.6

Indoleacetaldoxime dehydratase

pH-Range 6-9 <5> (<5> pH 6.0: about 60% of maximal activity, pH 9.0: about 35% of maximal activity [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <4> [2] Purification <5> [3, 4]

6 Stability pH-Stability 6-7 <5> (<5> maximally stable [3]) [3]

References [1] Kumar, S.A.; Mahadevan, S.: 3-Indoleacetaldoxime hydro-lyase: a pyridoxal5’-phosphate activated enzyme. Arch. Biochem. Biophys., 103, 516-518 (1963) [2] Mahadevan, S.: Conversion of 3-indoleacetaldoxime to 3-indoleacetonitrile by plants. Arch. Biochem. Biophys., 100, 557-558 (1963) [3] Shulka, P.S.; Mahadevan, S.: Indoleacetaldoxime hydro-lyase. II. Purification and properties. Arch. Biochem. Biophys., 125, 873-883 (1968) [4] Shulka, P.S.; Mahadevan, S.: Indoleacetaldoxime hydro-lyase (4.2.1.29). III. Further studies on the nature and mode of action of the enzyme. Arch. Biochem. Biophys., 137, 166-174 (1970)

475

Phenylacetaldoxime dehydratase

4.99.1.7

1 Nomenclature EC number 4.99.1.7 Systematic name (Z)-phenylacetaldehyde-oxime hydro-lyase (phenylacetonitrile-forming) Recommended name phenylacetaldoxime dehydratase Synonyms OxdB <1> [4, 7] OxdFG <3> [6] OxdRE <2> [7]

2 Source Organism <1> Bacillus sp. (no sequence specified) [1, 2, 3, 4, 5, 7] <2> Rhodococcus sp. (no sequence specified) [7] <3> Fusarium graminearum (UNIPROT accession number: Q2WG72) [6]

3 Reaction and Specificity Catalyzed reaction (Z)-phenylacetaldehyde oxime = phenylacetonitrile + H2 O (<1> oxidation state of heme controls the coordination structure of a substrate-hem complex, which regulates enzyme activity [4]) Substrates and products S (E/Z)-2-phenylpropionaldoxime <3> (Reversibility: ?) [6] P (E/Z)-2-phenylpropiononitrile + H2 O S (E/Z)-4-phenylbutyraldoxime <1> (<1> 6.8% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1] P phenylbutyronitrile + H2 O S (E/Z)-4-phenylbuyraldoxime <3> (Reversibility: ?) [6] P (E/Z)-4-phenylbuyronitrile + H2 O S (E/Z)-indoleacetaldoxime <3> (Reversibility: ?) [6] P (E/Z)-indoleacetonitrile + H2 O S (E/Z)-indoleacetaldoxime <1> (<1> 18.1% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1]

476

4.99.1.7

Phenylacetaldoxime dehydratase

P indoleacetonitrile + H2 O S (E/Z)-isocapronaldoxime <1> (<1> 29.4% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1] P isocapronitrile + H2 O S (E/Z)-isovaleraldoxime <1, 3> (<1> 20.2% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1, 6] P isovaleronitrile + H2 O S (E/Z)-mandelaldoxime <3> (Reversibility: ?) [6] P (E/Z)-mandeloacetonitrile + H2 O S (E/Z)-n-butyraldoxime <1, 3> (<1> 11% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1, 6] P n-butyronitrile + H2 O S (E/Z)-n-capronaldoxime <3> (Reversibility: ?) [6] P n-caprononitrile + H2 O S (E/Z)-n-capronaldoxime <1> (<1> 57.1% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1] P n-capronitrile + H2 O S (E/Z)-n-valeraldoxime <1, 3> (<1> 39.3% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1, 6] P n-valeronitrile + H2 O S (E/Z)-propionaldoxime <1> (<1> 8.2% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1] P propionitrile + H2 O S (Z)-3-phenylpropionaldoxime <1> (Reversibility: ?) [2] P 3-phenylpropionitrile + H2 O (<1> quantitative yield of product [2]) S (Z)-3-phenylpropionaldoxime <1> (<1> 63% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1] P phenylpropionitrile + H2 O S (Z)-naphthoacetaldoxime <1> (<1> 4.5% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1] P naphthoacetonitrile + H2 O S (Z)-p-chlorophenylacetaldoxime <1> (<1> 7.3% of activity with (Z)-phenylacetaldehyde oxime [1]) (Reversibility: ?) [1] P p-chlorophenylacetonitrile + H2 O S (Z)-p-methoxyphenylacetaldoxime <1> (<1> 6.8% of activity with (Z)phenylacetaldehyde oxime [1]) (Reversibility: ?) [1] P p-methoxyphenylacetonitrile + H2 O S (Z)-phenylacetaldehyde oxime <1, 3> (Reversibility: ?) [1, 3, 4, 6] P phenylacetonitrile + H2 O S (Z)-3-phenylpropionaldoxime <3> (Reversibility: ?) [6] P (Z)-3-phenylpropiononitrile + H2 O Cofactors/prosthetic groups FMN <1> (<1> required [3]; <1> absolute requirement [1]) [1,3] heme <1, 2> (<1> protoheme IX [1]; <1> active state contains ferrous heme [4]; <1> both the Fe(II) and Fe(III) hemes in OxdB bind the substrate, (Z)phenylacetaldehyde oxime, using a different coordination mode. Ferrous

477

Phenylacetaldoxime dehydratase

4.99.1.7

OxdB is active, but ferric OxdB is not [4]; <1,2> ferrous enzyme includes a five-coordinate high-spin heme to which the substrate is bound via its nitrogen atom for the reaction to occur. Although the ferric enzyme is inactive for catalysis, the substrate is bound to the ferric heme via its oxygen atom [7]) [1,4,7] Activating compounds NaN3 <1> (<1> activation [1]) [1] SO23- <1> (<1> activation [1]) [1] Sulfite <1> (<1> substitutes for FMN, to a low degree [1]) [1] Additional information <1> (<1> up to 5fold enhancement of activity with FMN under anaerobic conditions [1]) [1] Metals, ions Fe2+ <1> (<1> activation [1]) [1] Sn2+ <1> (<1> activation [1]) [1] Km-Value (mM) 0.03 <1> ((Z)-phenylacetaldehyde oxime, <1> pH.0, 30 C, aerobic conditions [1]) [1] 0.031 <1> ((Z)-phenylacetaldehyde oxime, <1> pH.0, 30 C, anaerobic conditions [1]) [1] 0.31 <1> ((Z)-phenylacetaldehyde oxime, <1> pH.0, 30 C, aerobic conditions, Na2 SO3 substitutes for FMN [1]) [1] 0.802 <3> ((E/Z)-n-capronaldoxime) [6] 0.846 <1> ((Z)-naphthoacetaldoxime, <1> pH 7.0, 30 C [1]) [1] 0.872 <1> ((Z)-phenylacetaldehyde oxime, <1> pH 7.0, 30 C [1]) [1] 1.24 <1> ((Z)-p-chlorophenylacetaldoxime, <1> pH 7.0, 30 C [1]) [1] 1.36 <1> ((Z)-3-phenylpropionaldoxime, <1> pH 7.0, 30 C [1]) [1] 1.46 <3> ((E/Z)-indoleacetaldoxime) [6] 1.7 <3> ((E/Z)-mandelaldoxime) [6] 1.79 <3> ((E/Z)-4-phenylbuyraldoxime) [6] 2.4 <1> ((E/Z)-indoleacetaldoxime, <1> pH 7.0, 30 C [1]) [1] 2.42 <1> ((E/Z)-n-valeraldoxime, <1> pH 7.0, 30 C [1]) [1] 2.66 <3> ((E/Z)-isovaleraldoxime) [6] 2.76 <3> ((Z)-3-phenylpropionaldoxime) [6] 2.87 <3> ((E/Z)-n-butyraldoxime) [6] 2.98 <1> ((E/Z)-isocapronaldoxime, <1> pH 7.0, 30 C [1]) [1] 3.08 <1> ((Z)-p-methoxyphenylacetaldoxime, <1> pH 7.0, 30 C [1]) [1] 3.52 <3> ((Z)-phenylacetaldehyde oxime) [6] 3.58 <1> ((E/Z)-isovaleraldoxime, <1> pH 7.0, 30 C [1]) [1] 3.71 <3> ((E/Z)-2-phenylpropionaldoxime) [6] 4.32 <1> ((E/Z)-propionaldoxime, <1> pH 7.0, 30 C [1]) [1] 5.24 <1> ((E/Z)-4-phenylbutyraldoxime, <1> pH 7.0, 30 C [1]) [1] 6.12 <1> ((E/Z)-n-capronaldoxime, <1> pH 7.0, 30 C [1]) [1] 10.1 <3> ((E/Z)-n-valeraldoxime) [6] 11.1 <1> ((E/Z)-n-butyraldoxime, <1> pH 7.0, 30 C [1]) [1]

478

4.99.1.7

Phenylacetaldoxime dehydratase

pH-Optimum 5.5 <3> (<3> activity with (Z)-phenylacetaldehyde oxime [6]) [6] 7 <1> [1] 7-8 <1> [4] pH-Range 7-8 <1> [4] 7.8-8.3 <1> [2] Temperature optimum ( C) 25 <3> [6] 30 <1> [1, 2]

4 Enzyme Structure Molecular weight 34100 <3> (<3> gel filtration [6]) [6] 40000 <1> (<1> native PAGE [1]) [1] Subunits monomer <1, 3> (<1> 1 * 40000, SDS-PAGE [1]) [1, 6]

5 Isolation/Preparation/Mutation/Application Localization soluble <1> [1, 3] Purification <1> [1, 7] <1> (partial) [3] <2> [7] Crystallization <3> (His6-tagged enzyme form) [6] Cloning <1> (expression in Escherichia coli) [7] <2> (expression in Escherichia coli) [7] <3> (overexpression in Escherichia coli) [6] Engineering H306A <1> (<1> very low activity, substrate is bound to ferrous heme [4]) [4] Application synthesis <1> (<1> high yield synthesis of 3-phenylpropionitrile from unpurified (E/Z)-3-phenylpropionaldoxime, which is spontaneously formed from 3-phenlypropionaldehyde and hydroxylamine in a butyl acetate/water biphasic system, production of further nitriles from their corresponding al-

479

Phenylacetaldoxime dehydratase

4.99.1.7

doximes [2]; <1> production of enzyme by expression in Escherichia coli and Bacillus subtilis, at 37 C, yield of mainly inactive inclusion bodies, at 30 C, enzyme is largely soluble and active. Enhancement of production by increasing the volume of culture medium [5]) [2, 5]

6 Stability pH-Stability 4.5-8 <3> (<3> stable [6]) [6] 6 <1> (<1> denaturation below [4]) [4] 10 <1> (<1> denaturation above [4]) [4] Temperature stability 20 <3> (<3> pH 7.0, stable below [6]) [6] 45 <1> (<1> stable up to [1]) [1]

References [1] Kato, Y.; Nakamura, K.; Sakiyama, H.; Mayhew, S.G.; Asano, Y.: Novel hemecontaining lyase, phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of the gene. Biochemistry, 39, 800-809 (2000) [2] Xie, S.X.; Kato, Y.; Asano, Y.: High yield synthesis of nitriles by a new enzyme, phenylacetaldoxime dehydratase, from Bacillus sp. strain OxB-1. Biosci. Biotechnol. Biochem., 65, 2666-2672 (2001) [3] Asano, Y.; Kato, Y.: Z-phenylacetaldoxime degradation by a novel aldoxime dehydratase from Bacillus sp. strain OxB-1. FEMS Microbiol. Lett., 158, 185190 (1998) [4] Kobayashi, K.; Yoshioka, S.; Kato, Y.; Asano, Y.; Aono, S.: Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex. J. Biol. Chem., 280, 5486-5490 (2005) [5] Kato, Y.; Asano, Y.: High-level expression of a novel FMN-dependent hemecontaining lyase, phenylacetaldoxime dehydratase of Bacillus sp. strain OxB1, in heterologous hosts. Protein Expr. Purif., 28, 131-139 (2003) [6] Kato, Y.; Asano, Y.: Purification and characterization of aldoxime dehydratase of the head blight fungus, Fusarium graminearum. Biosci. Biotechnol. Biochem., 69, 2254-2257 (2005) [7] Kobayashi, K.; Pal, B.; Yoshioka, S.; Kato, Y.; Asano, Y.; Kitagawa, T.; Aono, S.: Spectroscopic and substrate binding properties of heme-containing aldoxime dehydratases, OxdB and OxdRE. J. Inorg. Biochem., 100, 1069-1074 (2006)

480

Isopenicillin-N epimerase

5.1.1.17

1 Nomenclature EC number 5.1.1.17 Systematic name penicillin-N 5-amino-5-carboxypentanoyl-epimerase Recommended name isopenicillin-N epimerase Synonyms IPN epimerase <3> [11] isopenecillin N-CoA epimerase <3> [12] isopenicillin N epimerase <3> [10, 12] isopenicillin N-CoA epimerase <3> [11] CAS registry number 88201-43-8

2 Source Organism <1> <2> <3> <4> <5>

Streptomyces lactamdurans (no sequence specified) [4] Streptomyces lipmanii (no sequence specified) [1] Acremonium chrysogenum (no sequence specified) [5, 9, 10, 11, 12] Streptomyces clavuligerus (no sequence specified) [2, 3, 6, 8] Nocardia lactamdurans (no sequence specified) [7,9]

3 Reaction and Specificity Catalyzed reaction isopenicillin N = penicillin N Reaction type Epimerization Natural substrates and products S isopenicillin N <1, 2, 3, 4, 5> (<3,5> part of the cephalosporin pathway [7,9]) (Reversibility: r) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] P penicillin N <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6, 7, 8, 9]

481

Isopenicillin-N epimerase

5.1.1.17

Substrates and products S deacetoxycephalosporin C <4> (<4> less than 1% of activity with penicillin N [6]) (Reversibility: ?) [6] P ? S isopenicillin <1, 2, 3, 4, 5> (Reversibility: r) [1, 2, 3, 4, 5, 6, 7, 8, 9] P penicillin N <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6, 7, 8, 9] S isopenicillin N <1, 2, 3, 4, 5> (<3,5> part of the cephalosporin pathway [7,9]) (Reversibility: r) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12] P penicillin N <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6, 7, 8, 9] Inhibitors 4-chloromercuribenzoate <4, 5> (<4> 1 mM, 88% inhibition [6]; <5> 0.1 mM, 36% inibition [7]) [6, 7] ammonium sulfate <5> (<5> 100 mM, 47% inhibition [7]) [7] Cu2+ <5> (<5> 1 mM, complete inhibition [7]) [7] d-cycloserine <4> (<4> 1 mM, 23% inhibition [6]) [6] FAD <4> (<4> 0.5 mM, 80% inhibition [6]) [6] Fe2+ <5> (<5> 0.135 mM, complete inhibition [7]) [7] fluorescamine <4> (<4> inactivation at molar ratios higher than 2 [6]) [6] Hg2+ <5> (<5> 1 mM, complete inhibition [7]) [7] hydroxylamine <4> (<4> 1 mM, 95% inhibition [6]; <4> 10 mM, 68% inhibition, readdition of pyridoxal 5’-phosphate causes an increase in activity to 63% of the control [3]) [3, 6] I- <5> (<5> 1 mM, 27% inhibition [7]) [7] iodoacetamide <4, 5> (<5> 0.1 mM [7]; <4> 1 mM, 28% inhibition [6]; <5> 40% inhibition [7]) [6, 7] iproniazid <4> (<4> 1 mM, 25% inhibition [6]; <4> 10 mM, 33% inhibition [3]) [3, 6] isoniazid <4> (<4> 1 mM, 23% inhibition [6]) [6] N-ethylmaleimide <4, 5> (<5> 0.1 mM [7]; <4> 1 mM, 45% inhibition [6]; <5> 33% inhibition [7]) [6, 7] phenylglyoxal <4> (<4> 1 mM, 33% inhibition [6]) [6] Zn2+ <5> (<5> 1 mM, 50% inhibition [7]) [7] Cofactors/prosthetic groups pyridoxal 5’-phosphate <4, 5> (<4> 1 mol of pyridoxal 5’-phosphate per mol enzyme, 1.5fold increase in activity in the presence of 0.01 mM pyridoxal 5’phosphate [6]; <5> stabilizes enzyme during purification, may be tightly bound to the enzyme [7]; <4> putative cofactor, does not increase enzyme activity in assay system, stabilizes enzyme during purification [3]) [3,6,7,8] Specific activity (U/mg) 0.00009 <1> (<1> activity in cell extracts [4]) [4] 0.000727 <5> [7] 3.85 <4> (<4> isopenicillin N epimerization [6]) [6] 7.77 <4> (<4> penicillin N epimerization [6]) [6] Additional information <3, 4> (<4> 19.53 units, 1 unit is defined as the amount of enzyme which produces 0.001 mg of penicillin N in 1 h [3]; <3>

482

5.1.1.17

Isopenicillin-N epimerase

92.0 units, 1 unit is the activity forming 1 ng of cephalosporin/min in a coupled assay, activity in cell extract [9]; <3> strain C10 (ATCC 48272): 93 ng of cephalosporin C formed per min and mg of protein, transformant strain TMCD2: no activity, transformant strain TMCD26: 223 ng of cephalosporin C formed per min and mg of protein, transformant strain TMCD32: no activity, transformant strain TMCD39: no activity, transformant strain TMCD53: 226 ng of cephalosporin C formed per min and mg of protein, transformant strain TMCD242: 234 ng of cephalosporin C formed per min and mg of protein, transformant strain TMCD474: 202 ng of cephalosporin C formed per min and mg of protein [11]) [3, 9, 11] Km-Value (mM) 0.0024 <4> (pyridoxal 5’-phosphate) [6] 0.27 <5> (isopenicillin N) [7] 0.3 <4> (isopenicillin N) [6] 0.78 <4> (penicillin N) [6] pH-Optimum 7 <5> [7] 7.8-8.3 <4> [6] pH-Range 6.5-8 <5> (<5> strong decrease in activity above pH 8.0 and below pH 6.5 [7]) [7] Temperature optimum ( C) 25 <5> (<5> slightly lower activity at 20 and 30 C [7]) [7] Temperature range ( C) 5-45 <5> (<5> reduced activity below 20 C or above 30 C [7]) [7]

4 Enzyme Structure Molecular weight 50000 <4> (<4> gel filtration [6]) [3, 6] 59000 <5> (<5> gel filtration [7]) [7] 60000 <4> (<4> gel filtration [3]) [3] Subunits monomer <4, 5> (<4> 1 * 60000, SDS-PAGE [3]; <5> 1 * 59000, SDS-PAGE [7]; <4> 1 * 47000, SDS-PAGE [6]) [3, 6, 7]

5 Isolation/Preparation/Mutation/Application Source/tissue mycelium <3> [11]

483

Isopenicillin-N epimerase

5.1.1.17

Purification <4> (ammonium sulfate, DE-52, DEAE-Affi-gel blue, Sephadex G-200, calcium phosphate, Mono Q) [6] <4> (ammonium sulfate, Sephadex G-200, DEAE-trisacryl) [3] <5> (protamine sulfate, ammonium sulfate, Sephadex G-75, Mono Q) [7] Cloning <2> (expression in Penicillium chrysogenum) [1] <3> (Penecillium chrysogenum strains are constructed expressing cefD1 (isopenecillin N-CoA synthetase), cefD2 (isopenecillin N-CoA epimerase), cefEF (deacetylcephalosporin-actyltransferase), and cefG (deacetylcephalosporin C synthetase) genes cloned from Acremonium chrysogenum. HPLC analysis of cell extracts show that transformant TA64, TA71, and TA98 accumulate intracellularly deacetylcephalosporin C and, in the last strain (TA98), also cephalosporin C.) [12] <4> (expression in Penicillium chrysogenum) [2] Application biotechnology <3> (<3> The results indicate that Penecillium chrysogenum can be used as heterologous host for the production of deacetylcephalosporin C [12]) [12] medicine <3> (<3> the conversion of isopenicillin N into penicillin N is part of the cephalosporin biosynthesis pathway. Cephalosporin is one of the most potent b-lactam antibiotics, widely used in the treatment of infectious diseases [10]) [10] synthesis <3> (<3> epimerization is a rate-limiting step in cephalosporin biosynthesis in Acremonium chrysogenum. When epimerase activity is enhanced, there is a clear increase in cephalosporin production. Thus, this method provides a strategy that could easily be applied to improve industrial cephalosporin producing strains [11]) [11]

6 Stability pH-Stability 4.5-9 <5> [7] 5.3-10 <4> (<4> stable at 4 C for 24 h [6]) [6] Temperature stability 20-40 <5> (<5> stable for 2 h [7]) [7] 45 <4> (<4> rapid denaturation above [6]) [6] 60 <4> (<4> complete inactivation after 10 min [6]) [6] General stability information <4>, unstable enzyme [3] <5>, unstable enzyme, pyridoxal 5’-phosphate stabilizes [7] Storage stability <4>, -65 C, 1 week, 61% loss of activity [3] <5>, -70 C, 0.05 mM pyridoxal 5’-phosphate, 17 d, no loss of activity [7]

484

5.1.1.17

Isopenicillin-N epimerase

References [1] Cantwell, C.; Beckmann, R.; Whiteman, P.; Queener, S.W.; Abraham, E.P.: Isolation of deacetoxycephalosporin-c from fermentation broths of Penicillium chrysogenum transformants - construction of a new fungal biosynthetic-pathway. Proc. R. Soc. Lond. B Biol. Sci., 248, 283-289 (1992) [2] Yeh, W.K.; Ghag, S.K.; Queener, S.W.: Enzymes for epimerization of isopenicillin N, ring expansion of penicillin N, and 3’-hydroxylation of deacetoxycephalosporin C. Function, evolution, refolding, and enzyme engineering. Ann. N.Y. Acad. Sci., 672, 396-408 (1992) [3] Jensen, S.E.; Westlake, D.W.S.; Wolfe, S.: Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus. Can. J. Microbiol., 29, 1526-1531 (1983) [4] Castro, J.M.; Liras, P.; Cortes, J.; Martin, J.F.: Regulation of a-aminoadipylcysteinyl-valine, isopenicillin N synthetase, isopenicillin N isomerase and deacetoxycephalosporin C synthetase by nitrogen sources in Streptomyces lactamdurans. Appl. Microbiol. Biotechnol., 22, 32-40 (1985) [5] Ramos, F.R.; Lopez-Nieto, M.J.; Martin, J.F.: Coordinate increase of isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase in a high cephalosporin-producing mutant of Acremonium chrysogenum and simultaneous loss of the three enzymes in a non-producing mutant. FEMS Microbiol. Lett., 35, 123-127 (1986) [6] Usui, S.; Yu, C.A.: Purification and properties of isopenicillin-N epimerase from Streptomyces clavuligerus. Biochim. Biophys. Acta, 999, 78-85 (1989) [7] Laiz, L.; Liras, P.; Castro, J.M.; Martin, J.F.: Purification and characterization of the isopenicillin-N epimerase from Nocardia lactamdurans. J. Gen. Microbiol., 136, 663-671 (1990) [8] Mehta, P.K.; Christen, P.: Homology of 1-aminocyclopropane-1-carboxylate synthase, 8-amino-7-oxononanoate synthase, 2-amino-6-caprolactam racemase, 2,2-dialkylglycine decarboxylase, glutamate-1-semialdehyde 2,1-aminomutase and isopenicillin-N-epimerase with aminotransferases. Biochem. Biophys. Res. Commun., 198, 138-143 (1994) [9] Ullan, R.V.; Casqueiro, J.; Banuelos, O.; Fernandez, F.J.; Gutierrez, S.; Martin, J.F.: A novel epimerization system in fungal secondary metabolism involved in the conversion of isopenicillin N into penicillin N in Acremonium chrysogenum. J. Biol. Chem., 277, 46216-46225 (2002) [10] Martin, J.F.; Ullan, R.V.; Casqueiro, J.: Novel genes involved in cephalosporin biosynthesis: the three-component isopenicillin N epimerase system. Adv. Biochem. Eng./Biotechnol., 88, 91-109 (2004) [11] Ullan, R.V.; Casqueiro, J.; Naranjo, L.; Vaca, I.; Martin, J.F.: Expression of cefD2 and the conversion of isopenicillin N into penicillin N by the twocomponent epimerase system are rate-limiting steps in cephalosporin biosynthesis. Mol. Genet. Genomics, 272, 562-570 (2004) [12] Ullan, R.V.; Campoy, S.; Casqueiro, J.; Fernandez, F.J.; Martin, J.F.: Deacetylcephalosporin C production in Penicillium chrysogenum by expression of the isopenicillin N epimerization, ring expansion, and acetylation genes. Chem. Biol., 14, 329-339 (2007)

485

Serine racemase

5.1.1.18

1 Nomenclature EC number 5.1.1.18 Systematic name serine racemase Recommended name serine racemase Synonyms SRR <8> [5] CAS registry number 77114-08-0

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8>

Mus musculus (no sequence specified) [1, 3, 6, 7, 9, 10, 13, 16, 17, 20] Homo sapiens (no sequence specified) [8, 14, 21] Rattus norvegicus (no sequence specified) [4, 11, 15, 16, 19] Ambystoma tigrinum (no sequence specified) [16] Streptomyces garyphalus (no sequence specified) [2] Mus musculus (UNIPROT accession number: Q9QZX7) [18] Enterococcus gallinarum (no sequence specified) [12] Homo sapiens (UNIPROT accession number: Q6IA55) [5]

3 Reaction and Specificity Catalyzed reaction l-serine = d-serine Reaction type Racemization Natural substrates and products S Additional information <1, 3, 4> (<1> enzyme modulates physiologic regulation of cerebellar granule cell migration [17]; <1,3,4> enzyme product may serve as a ligand for setting the sensitivity of N-methyl-d-aspartate receptors under physiological conditions [16]; <1> main enzyme to

486

5.1.1.18

Serine racemase

synthesize d-serine [1]; <3> role for d-serine in peripheral nerve transduction [4]) (Reversibility: ?) [1, 4, 16, 17] P ? Substrates and products S d-alanine <5> (Reversibility: r) [2] P l-alanine S d-serine <1> (Reversibility: ?) [3, 7] P pyruvate + NH3 S d-serine <1> (<1> a,b-elimination reaction [10]) (Reversibility: ?) [10] P pyruvate + H2 O S d-serine <1, 3, 5> (<1> racemization reaction [10]) (Reversibility: r) [2, 3, 7, 10, 19] P l-serine S l-alanine <5> (Reversibility: r) [2] P d-alanine S l-serine <1> (<1> a,b-elimination reaction [10]) (Reversibility: ?) [10] P pyruvate + NH3 S l-serine <1, 2, 3, 5, 6, 8> (<1,2> racemization reaction [10,21]) (Reversibility: r) [2, 3, 5, 7, 8, 9, 10, 13, 18, 19, 20, 21] P d-serine (<3> highly selctive toward l-serine [19]; <6> specific for synthesis of d-serine [18]) S l-serine <1, 2> (<2> elimination reaction [21]) (Reversibility: ?) [3, 7, 13, 20, 21] P pyruvate + NH3 S l-serine O-sulfate <1> (Reversibility: ?) [3, 13] P O-sulfopyruvate + NH3 S l-serine-O-sulfate <2> (<2> elimination reaction [21]) (Reversibility: ?) [21] P O-sulfopyruvate + NH3 S l-threonine <1> (<1> a,b-elimination reaction [10]) (Reversibility: ?) [10] P 2-oxobutanoate + NH3 S Additional information <1, 2, 3, 4> (<2> specific for l-serine [8]; <1> enzyme modulates physiologic regulation of cerebellar granule cell migration [17]; <1,3,4> enzyme product may serve as a ligand for setting the sensitivity of N-methyl-d-aspartate receptors under physiological conditions [16]; <1> main enzyme to synthesize d-serine [1]; <3> role for dserine in peripheral nerve transduction [4]; <1> racemization and elimination activities reside at the same active site of enzyme. Racemization activity is specific to serine, elimination activity has a broader specificity for l-amino acids with a suitable leaving group at the b-carbon [3]; <1> ratio of elimination reaction/racemization reaction for substrate l-serine is 3.7 [10]; <1> ratio of synthesized pyruvate/d-serine is about 3 [20]) (Reversibility: ?) [1, 3, 4, 8, 10, 16, 17, 20] P ?

487

Serine racemase

5.1.1.18

Inhibitors Co2+ <1> [7] Cu2+ <1> [7] cystamine <1> (<1> 50% inhibition at 0.0081 mM [9]) [9] d-cycloserine <7> (<7> 2 mM, 64% inhibition [12]) [12] d-cysteine <1> (<1> 2 mM, 72% residual activity [6]) [6] dihydroxyfumarate <1> [3] EDTA <1> (<1> strong [20]) [7, 20] Fe2+ <1> (<1> slight inhibition of both activities [7]) [7] glycine <1> (<1> competitive [3]; <1> 2 mM, 20% residual activity [6]) [3, 6] hydroxylamine <3, 5> [2, 19] l-2,3-diaminopropionic acid <1> (<1> 2 mM, 68% residual activity [6]) [6] l-asparagine <1> (<1> 2 mM, 38% residual activity [6]) [3, 6] l-aspartic acid <1> (<1> 2 mM, 53% residual activity [6]) [6] l-cycloserine <1> (<1> 10 mM, 45% inhibition [9]) [9] l-cysteine <1> (<1> 10 mM, 90% inhibition [9]; <1> 2 mM, 58% residual activity [6]) [6, 9] l-cysteine-S-sulfate <1> [3] l-erythro-3-hydroxyaspartate <1> [3] l-homocysteic acid <1> (<1> 2 mM, 59% residual activity [6]) [6] l-serine-O-sulfate <1> (<1> 10 mM, 50% inhibition [9]; <1> 2 mM, 9% residual activity [6]) [6, 9] maleate <1> [3] malonate <1> [3] Ni2+ <1> (<1> slight inhibition of both activities [7]) [7] oxaloacetic acid <1> (<1> 2 mM, 56% residual activity [6]) [6] Zn2+ <1> [7] amino-oxyacetic acid <3, 6> (<6> complete inhibition [18]) [18, 19] b-chloro-l-alanine <1> (<1> 2 mM, 68% residual activity [6]) [6] b-fluoro-d,l-alanine <1> (<1> 2 mM, 81% residual activity [6]) [6] meso-tartrate <1> [3] nitric oxide <2> [14] Additional information <5> (<5> not inhibitory: d-cycloserine [2]) [2] Cofactors/prosthetic groups pyridoxal 5’-phosphate <2, 3, 5, 7, 8> (<5> both isozyme I and II [2]; <8> consensus sequence in protein [5]) [2,5,8,12,19] Activating compounds ADP <1> (<1> less effective than ATP [20]) [13, 20] ATP <1> (<1> 1 mM, decrease of Km -value for racemization by 85%, allosteric mechanism. Inhibitory to l-serine O-sulfate dehydration reaction [13]; <1> and MG2+, up to 5fold stimulation [20]) [10, 13, 20] d-serine <2> (<2> up to 5fold increase in activity [14]) [14] GTP <1> [13]

488

5.1.1.18

Serine racemase

Additional information <1> (<1> activation of enzyme by glutamate neurotransmission involving a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors [17]) [17] Metals, ions Ca2+ <1> (<1> may partially replace Mg2+ [13]; <1> or Mn2+ , required, binding konstant 0.0062 mM. Possible glutamatergic-mediated regulation of enzyme via intracellular calcium concentration [9]) [7, 9, 13] EDTA <1> (<1> complete inhibition, provokes a profound conformational change [9]) [9] Mg2+ <1> (<1> and ATP, up to 5fold stimulation [20]; <1> required by both isoforms A and B [13]; <1> required, both activities [7]) [7, 13, 20] Mn2+ <1> (<1> may partially replace Mg2+ [13]; <1> or Ca2+ , required [9]) [7, 9, 13] Turnover number (min–1) 0.004 <1> (d-serine, <1> wild-type, pH 7.4, 37 C [10]) [10] 0.015 <1> (d-serine, <1> mutant Q155D, racemization, pH 7.4, 37 C [10]) [10] 0.02 <1> (l-serine, <1> wild-type, racemization, pH 7.4, 37 C [10]) [10] 0.028 <1> (d-serine, <1> wild-type, racemization, presence of ATP, pH 7.4, 37 C [10]) [10] 0.042 <1> (l-serine, <1> wild-type, racemization, presence of ATP, pH 7.4, 37 C [10]) [10] 0.045 <1> (l-serine, <1> mutant Q155D, racemization, pH 7.4, 37 C [10]) [10] 0.11 <1> (d-serine, <1> mutant Q155D, racemization, presence of ATP, pH 7.4, 37 C [10]) [10] 0.23 <1> (l-serine, <1> mutant Q155D, presence of ATP, pH 7.4, 37 C [10]) [10] 0.49 <1> (l-serine O-sulfate, <1> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [3]) [3] 3.2 <1> (d-serine, <1> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [3]) [3] 3.8 <1> (l-serine, <1> pH 8.0, 37 C, presence of 1 mM ATP, racemization reaction [3]) [3] 4 <1> (l-serine, <1> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [3]) [3] 14.5 <1> (d-serine, <1> pH 8.0, 37 C, presence of 1 mM ATP, racemization reaction [3]) [3] Specific activity (U/mg) 0.083 <3> (<3> pH 8.0, 37 C [19]) [19] Km-Value (mM) 0.49 <1> (l-serine O-sulfate, <1> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [3]) [3] 1.8 <1> (l-serine, <1> racemization reaction, isoform A, pH 8.6, 37 C [13]) [13]

489

Serine racemase

5.1.1.18

3.2 <1> (d-serine, <1> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [3]) [3] 3.8 <1> (l-serine, <1> pH 8.0, 37 C, presence of 1 mM ATP, racemization reaction [3]) [3] 4 <1> (l-serine, <1> pH 8.0, 37 C, presence of 1 mM ATP, elimination reaction [3]) [3] 4.8 <1> (l-serine, <1> pH 8.1, 37 C [9]) [9] 8 <1> (d-serine, <1> wild-type, racemization, pH 7.4, 37 C [10]) [10] 8.2 <1> (d-serine, <1> wild-type, racemization, presence of ATP, pH 7.4, 37 C [10]) [10] 9 <1> (l-serine, <1> wild-type, racemization, pH 7.4, 37 C [10]; <1> wildtype, racemization, presence of ATP, pH 7.4, 37 C [10]) [10] 9.2 <1> (d-serine, <1> mutant Q155D, racemization, pH 7.4, 37 C [10]; <1> mutant Q155D, racemization, presence of ATP, pH 7.4, 37 C [10]) [10] 10 <1, 3> (l-serine, <3> pH 8.0, 37 C [19]; <1> mutant Q155D, racemization, pH 7.4, 37 C [10]) [10, 19] 10.5 <1> (l-serine, <1> mutant Q155D, racemization, presence of ATP, pH 7.4, 37 C [10]) [10] 13 <1> (l-serine, <1> racemization reaction, isoform A, presence of ATP, pH 8.6, 37 C [13]) [13] 14.5 <1> (d-serine, <1> pH 8.0, 37 C, presence of 1 mM ATP, racemization reaction [3]) [3] 30 <1> (l-serine, <1> pH 8.0, 37 C, racemization reaction [7]) [7] 35 <5> (l-serine) [2] 49 <1> (d-serine, <1> pH 8.0, 37 C, racemization reaction [7]) [7] 60 <3> (d-serine, <3> pH 8.0, 37 C [19]) [19] 75 <1> (d-serine, <1> pH 8.0, 37 C, elimination reaction [7]) [7] 75 <1> (l-serine, <1> pH 8.0, 37 C, elimination reaction [7]) [7] 111 <5> (l-alanine) [2] Ki-Value (mM) 0.043 <1> (l-erythro-3-hydroxyaspartate, <1> pH 8.0, 37 C [3]) [3] 0.071 <1> (malonate, <1> pH 8.0, 37 C [3]) [3] 0.55 <1> (maleate, <1> pH 8.0, 37 C [3]) [3] 0.64 <1> (l-cysteine-S-sulfate, <1> pH 8.0, 37 C [3]) [3] 0.66 <1> (meso-tartrate, <1> pH 8.0, 37 C [3]) [3] 0.69 <1> (dihydroxyfumarate, <1> pH 8.0, 37 C [3]) [3] 1.13 <1> (l-asparagine, <1> pH 8.0, 37 C [3]) [3] 1.64 <1> (glycine, <1> pH 8.0, 37 C [3]) [3] 1.9 <1> (l-aspartic acid, <1> pH 8.6, 37 C [6]) [6] pH-Optimum 7 <3> (<3> 10% of maximum activity [19]) [19] 8-9 <3> [19] 8-9.5 <1> [7] pH-Range 6.5 <1> (<1> negligible activity below [7]) [7]

490

5.1.1.18

Serine racemase

Temperature optimum ( C) 37 <3> [19]

4 Enzyme Structure Molecular weight 55000 <1> (<1> isoforms A and B, gel filtration [13]) [13] 78000 <1> (<1> gel filtration [7]) [7] 82900 <1> (<1> gel filtration [9]) [9] 153000 <1> (<1> gel filtration [9]) [9] Subunits ? <1, 3, 5, 6, 8> (<3> x * 37000, SDS-PAGE [19]; <8> 36000-37000, calculated [5]; <1> x * 36121, MALDI- MS, x * 36123, calculated [3]; <6> x * 36300, calculated, x * 38000, SDS-PAGE [18]; <5> x* 93000, isozyme I, x * 73000, isozyme II, SDS-PAGE [2]) [2, 3, 5, 18, 19] dimer <1> (<1> 2 * 37000, SDS-PAGE [7]; <1> 2 * 37500, SDS-PAGE, dimertetramer equilibrium [9]) [7, 9] tetramer <1> (<1> 4 * 37500, SDS-PAGE, dimer-tetramer equilibrium [9]) [9]

5 Isolation/Preparation/Mutation/Application Source/tissue NT2-N cell <8> (<8> neuronal like cell line [5]) [5] brain <1, 2, 6, 8> (<1> astrocyte glia, activation of enzyme by glutamate neurotransmission involving a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors [17]; <6> glial cells of brain, highest levels of enzyme in astrocyte-enriched cultures [18]; <2> high levels in hippocampus and corpus callosum [8]) [5, 6, 8, 13, 17, 18, 21] brain cortex <1> (<1> increase of enzyme expression after birth [1]) [1] cerebellum <1> (<1> increase of enzyme expression after birth [1]) [1] cerebral cortex <3> (<3> glial culture from [19]) [19] corpus striatum <1> (<1> increase of enzyme expression after birth [1]) [1] epithelium <3> (<3> vestibular sensory epithelium, in transitional cells therein, which are parasensory cells located between the sensory epithelium and the dark cells, and in dark cells [15]) [15] glioblastoma cell <2> (<2> cell line U87, inverse regulation of enzyme activty by d-serine and nitric oxide [14]) [14] heart <8> (<8> peripheral expression in cardiac myocyte [5]) [5] hippocampus <3> (<3> elevated levels of enzyme mRNA in Alzheimerﬀ´s disease [11]) [11] kidney <8> (<8> peripheral expression in convoluted tubules [5]) [5] liver <8> [5] microglia <3> (<3> transcriptional induction by amyloid b-peptide [11]) [11]

491

Serine racemase

5.1.1.18

nerve <3> (<3> Schwann cell and other endoneural components of spinal nerve, lysates of sciatic nerve [4]) [4] retina <1, 3, 4> (<1,3,4> Mller cells and astrocytes in retina [16]) [16] skeletal muscle <8> [5] Localization membrane <7> [12] soluble <3, 6> [18, 19] Purification <1> (both isoforms A and B) [13] <1> (recombinant enzyme expressed in insect cells) [7] <2> (expression in Escherichia coli with N-terminal His-tag, purification protocol from inclusion bodies) [21] <5> (purification of isozyme I, partial purification of isozyme II) [2] Cloning <6> [18] Engineering H152S <1> (<1> ratio of elimination reaction to racemization is 1.4 pared to 3.7 in wild-type [10]) [10] K56G <6> (<6> no enzymic activity [18]) [18] N154F <1> (<1> ratio of elimination reaction to racemization is 0.33 pared to 3.7 in wild-type [10]) [10] P153S <1> (<1> ratio of elimination reaction to racemization is 0.24 pared to 3.7 in wild-type [10]) [10] Q155D <1> (<1> ratio of elimination reaction to racemization is 0.25 pared to 3.7 in wild-type [10]) [10]

comcomcomcom-

Application medicine <3> (<3> elevated levels of enzyme mRNA in Alzheimer´s disease hippocampus [11]) [11]

6 Stability General stability information <3>, 4 C, stable for at least 4 days [19]

References [1] Wang, L.Z.; Zhu, X.Z.: Spatiotemporal relationships among d-serine, serine racemase, and d-amino acid oxidase during mouse postnatal development. Acta Pharmacol. Sin., 24, 965-974 (2003) [2] Svensson, M.L.; Gatenbeck, S.: The presence of two serine racemases in Streptomyces garyphalus, a d-cycloserine producer. Arch. Microbiol., 129, 213-215 (1981)

492

5.1.1.18

Serine racemase

[3] Strisovsky, K.; Jiraskova, J.; Mikulova, A.; Rulisek, L.; Konvalinka, J.: Dual substrate and reaction specificity in mouse serine racemase: identification of high-affinity dicarboxylate substrate and inhibitors and analysis of the b-eliminase activity. Biochemistry, 44, 13091-13100 (2005) [4] Wu, S.; Barger, S.W.; Sims, T.J.: Schwann cell and epineural fibroblast expression of serine racemase. Brain Res., 1020, 161-166 (2004) [5] Xia, M.; Liu, Y.; Figueroa, D.J.; Chiu, C.S.; Wei, N.; Lawlor, A.M.; Lu, P.; Sur, C.; Koblan, K.S.; Connolly, T.M.: Characterization and localization of a human serine racemase. Brain Res. Mol. Brain Res., 125, 96-104 (2004) [6] Dunlop, D.S.; Neidle, A.: Regulation of serine racemase activity by amino acids. Brain Res. Mol. Brain Res., 133, 208-214 (2005) [7] Strisovsky, K.; Jiraskova, J.; Barinka, C.; Majer, P.; Rojas, C.; Slusher, B.S.; Konvalinka, J.: Mouse brain serine racemase catalyzes specific elimination of l-serine to pyruvate. FEBS Lett., 535, 44-48 (2003) [8] De Miranda, J.; Santoro, A.; Engelender, S.; Wolosker, H.: Human serine racemase: molecular cloning, genomic organization and functional analysis. Gene, 256, 183-188 (2000) [9] Cook, S.P.; Galve-Roperh, I.; Martinez del Pozo, A.; Rodriguez-Crespo, I.: Direct calcium binding results in activation of brain serine racemase. J. Biol. Chem., 277, 27782-27792 (2002) [10] Foltyn, V.N.; Bendikov, I.; De Miranda, J.; Panizzutti, R.; Dumin, E.; Shleper, M.; Li, P.; Toney, M.D.; Kartvelishvily, E.; Wolosker, H.: Serine racemase modulates intracellular d-serine levels through an a,b-elimination activity. J. Biol. Chem., 280, 1754-1763 (2005) [11] Wu, S.Z.; Bodles, A.M.; Porter, M.M.; Griffin, W.S.; Basile, A.S.; Barger, S.W.: Induction of serine racemase expression and d-serine release from microglia by amyloid b-peptide. J. Neuroinflammation, 1, 2 (2004) [12] Arias, C.A.; Martin-Martinez, M.; Blundell, T.L.; Arthur, M.; Courvalin, P.; Reynolds, P.E.: Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174. Mol. Microbiol., 31, 1653-1664 (1999) [13] Neidle, A.; Dunlop, D.S.: Allosteric regulation of mouse brain serine racemase. Neurochem. Res., 27, 1719-1724 (2002) [14] Shoji, K.; Mariotto, S.; Ciampa, A.R.; Suzuki, H.: Regulation of serine racemase activity by d-serine and nitric oxide in human glioblastoma cells. Neurosci. Lett., 392, 75-78 (2006) [15] Dememes, D.; Mothet, J.P.; Nicolas, M.T.: Cellular distribution of d-serine, serine racemase and d-amino acid oxidase in the rat vestibular sensory epithelia. Neuroscience, 137, 991-997 (2006) [16] Stevens, E.R.; Esguerra, M.; Kim, P.M.; Newman, E.A.; Snyder, S.H.; Zahs, K.R.; Miller, R.F.: d-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors. Proc. Natl. Acad. Sci. USA, 100, 6789-6794 (2003) [17] Kim, P.M.; Aizawa, H.; Kim, P.S.; Huang, A.S.; Wickramasinghe, S.R.; Kashani, A.H.; Barrow, R.K.; Huganir, R.L.; Ghosh, A.; Snyder, S.H.: Serine racemase: activation by glutamate neurotransmission via glutamate recep-

493

Serine racemase

[18] [19] [20]

[21]

494

5.1.1.18

tor interacting protein and mediation of neuronal migration. Proc. Natl. Acad. Sci. USA, 102, 2105-2110 (2005) Wolosker, H.; Blackshaw, S.; Snyder, S.H.: Serine racemase: a glial enzyme synthesizing d-serine to regulate glutamate-N-methyl-d-aspartate neurotransmission. Proc. Natl. Acad. Sci. USA, 96, 13409-13414 (1999) Wolosker, H.; Sheth, K.N.; Takahashi, M.; Mothet, J.P.; Brady, R.O., Jr.; Ferris, C.D.; Snyder, S.H.: Purification of serine racemase: biosynthesis of the neuromodulator d-serine. Proc. Natl. Acad. Sci. USA, 96, 721-725 (1999) De Miranda, J.; Panizzutti, R.; Foltyn, V.N.; Wolosker, H.: Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyld-aspartate (NMDA) receptor coagonist d-serine. Proc. Natl. Acad. Sci. USA, 99, 14542-14547 (2002) Nagayoshi, C.; Ishibashi, M.; Kita, Y.; Matsuoka, M.; Nishimoto, I.; Tokunaga, M.: Expression, refolding and characterization of human brain serine racemase in Escherichia coli with N-terminal His-tag. Protein Pept. Lett., 12, 487-490 (2005)

Maltose epimerase

5.1.3.21

1 Nomenclature EC number 5.1.3.21 Systematic name maltose 1-epimerase Recommended name maltose epimerase CAS registry number 166799-98-0

2 Source Organism <1> Lactobacillus brevis (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction a-maltose = b-maltose Reaction type isomerization Substrates and products S a-d-glucose <1> (<1> at 26% of the activity with b-maltose [1]) (Reversibility: r) [1] P b-d-glucose S a-lactose <1> (<1> at 5% of the activity with b-maltose [1]) (Reversibility: ?) [1] P b-lactose S b-d-glucose <1> (<1> at 27% of the activity with b-maltose [1]) (Reversibility: r) [1] P a-d-glucose S b-maltose <1> (Reversibility: r) [1] P a-maltose <1> [1] S b-cellobiose <1> (<1> at 4% of the activity with b-maltose [1]) (Reversibility: ?) [1] P a-cellobiose

495

Maltose epimerase

5.1.3.21

Inhibitors Cu2+ <1> [1] Hg2+ <1> [1] Specific activity (U/mg) 1710 <1> [1] Km-Value (mM) 2.2 <1> (b-maltose) [1] pH-Optimum 6.5-7 <1> [1] pH-Range 5-8.5 <1> (<1> more than 80% of activity in the pH-range pH 5.0-8.5 [1]) [1] Temperature optimum ( C) 40 <1> [1]

4 Enzyme Structure Molecular weight 43000 <1> (<1> gel filtration [1]) [1] Subunits monomer <1> (<1> 1 * 45000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> [1]

6 Stability Temperature stability 45 <1> (<1> 10 min, 50 mM phosphate buffer, stable [1]) [1] 60 <1> (<1> 10 min, 50 mM phosphate buffer, 50% loss of activity [1]) [1]

References [1] Shirokane, Y.; Suzuki, M.: A novel enzyme, maltose 1-epimerase from Lactobacillus brevis IFO 3345. FEBS Lett., 367, 177-179 (1995)

496

L-Ribulose-5-phosphate

3-epimerase

5.1.3.22

1 Nomenclature EC number 5.1.3.22 Systematic name l-ribulose-5-phosphate 3-epimerase Recommended name l-ribulose-5-phosphate 3-epimerase Synonyms l-xylulose 5-phosphate 3-epimerase <1> [1] SgaU <1> [1] UlaE <1> [1] CAS registry number 9024-19-5

2 Source Organism <1> Escherichia coli (no sequence specified) (<1> large subunit [1]) [1]

3 Reaction and Specificity Catalyzed reaction l-ribulose 5-phosphate = l-xylulose 5-phosphate Natural substrates and products S l-ribulose 5-phosphate <1> (<1> along with EC 4.1.1.83, 3-dehydro-l-gulonate-6-phosphate decarboxylase, this enzyme is involved in a pathway for the utilization of l-ascorbate by Escherichia coli [1]) (Reversibility: ?) [1] P l-xylulose 5-phosphate Substrates and products S l-ribulose 5-phosphate <1> (<1> along with EC 4.1.1.83, 3-dehydro-l-gulonate-6-phosphate decarboxylase, this enzyme is involved in a pathway for the utilization of l-ascorbate by Escherichia coli [1]) (Reversibility: ?) [1] P l-xylulose 5-phosphate

497

L-Ribulose-5-phosphate

3-epimerase

5.1.3.22

5 Isolation/Preparation/Mutation/Application Purification <1> (His-tagged protein) [1] Cloning <1> (expression in Escherichia coli) [1]

References [1] Yew, W.S.; Gerlt, J.A.: Utilization of l-ascorbate by Escherichia coli K-12: assignments of functions to products of the yjf-sga and yia-sgb operons. J. Bacteriol., 184, 302-306 (2002)

498

UDP-2,3-Diacetamido-2,3-dideoxyglucuronic acid 2-epimerase

5.1.3.23

1 Nomenclature EC number 5.1.3.23 Systematic name 2,3-diacetamido-2,3-dideoxy-a-d-glucuronate 2-epimerase Recommended name UDP-2,3-diacetamido-2,3-dideoxyglucuronic acid 2-epimerase Synonyms 2,3-diacetamido-2,3-dideoxy-a-d-glucuronic acid 2-epimerase <1, 2> [2] UDP-2,3-diacetamido-2,3-dideoxy-a-d-glucuronic acid 2-epimerases <1, 2> [2] UDP-GlcNAc3NAcA 2-epimerase <1, 2> [2] UDP-a-d-GlcNAc3NAcA 2-epimerase <1, 2> [2] WbpI <1> [2] WlbD <2> [1, 2]

2 Source Organism <1> Pseudomonas aeruginosa (no sequence specified) [2, 3] <2> Bordetella pertussis (no sequence specified) [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction UDP-2,3-diacetamido-2,3-dideoxy-a-d-glucuronate = UDP-2,3-diacetamido2,3-dideoxy-a-d-mannuronate Natural substrates and products S UDP-2,3-diacetamido-2,3-dideoxy-a-d-glucuronate <1, 2> (<1> the enzyme participates in the biosynthetic pathway for UDP-a-d-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-a-d-mannuronic acid), an important precursor of the B-band lipopolysaccharide of Pseudomonas aeroginosa serotype O5 [2]; <2> the enzyme participates in the the biosynthetic pathway for band-A trisaccharide of Bordetella pertussis [2]) (Reversibility: ?) [2] P UDP-2,3-diacetamido-2,3-dideoxy-a-d-mannuronate

499

UDP-2,3-Diacetamido-2,3-dideoxyglucuronic acid 2-epimerase

5.1.3.23

Substrates and products S UDP-2,3-diacetamido-2,3-dideoxy-a-d-glucuronate <1, 2> (<1> the enzyme participates in the biosynthetic pathway for UDP-a-d-ManNAc3 NAcA (UDP-2,3-diacetamido-2,3-dideoxy-a-d-mannuronic acid), an important precursor of the B-band lipopolysaccharide of Pseudomonas aeroginosa serotype O5 [2]; <2> the enzyme participates in the the biosynthetic pathway for band-A trisaccharide of Bordetella pertussis [2]; <2> he enzyme is highly specific as UDP-a-d-GlcNAc, UDP-a-d-GlcNAcA (UDP-2acetamido-2-deoxy-a-d-glucuronic acid) and UDP-a-d-GlcNAc3 NAc (UDP-2,3-diacetamido-2,3-dideoxy-a-d-glucose) cannot act as substrates [2]; <1> the enzyme is highly specific as UDP-a-d-GlcNAc, UDP-a-dGlcNAcA (UDP-2-acetamido-2-deoxy-a-d-glucuronic acid) and UDP-ad-GlcNAc3 NAc (UDP-2,3-diacetamido-2,3-dideoxy-a-d-glucose) cannot act as substrates [2]) (Reversibility: ?) [2] P UDP-2,3-diacetamido-2,3-dideoxy-a-d-mannuronate pH-Optimum 6 <1, 2> [2] Temperature optimum ( C) 30-37 <1, 2> [2]

4 Enzyme Structure Subunits ? <1, 2> (<1> x * 40300, His6-WbpI, SDS-PAGE [2]; <2> x * 42600, His6WlbD, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Purification <1> (recombinant) [2] <2> [1] <2> (recombinant) [2] Crystallization <2> (crystals of the mutant Gln339Arg wlbD enzyme obtained by sittingdrop vapour diffusion, uncomplexed Gln339Arg and UDP-GlcNAc complex. Space group as P2(1)2(1)2(1), with unit-cell parameters a = 78, b = 91, c = 125 , a = 78 , b = 91 , c = 90 . The asymmetric unit contains two monomers and 53% solvent) [1] Cloning <1> (His6-WbpI is overexpressed in Escherichia coli RosettaTM cells) [2] <2> (His6-WlbD is overexpressed in Escherichia coli RosettaTM cells) [2] <2> (overexpression in Escherichia coli) [1]

500

5.1.3.23

UDP-2,3-Diacetamido-2,3-dideoxyglucuronic acid 2-epimerase

6 Stability Storage stability <1>, His6-WbpI stored at -20 C in the presence of 25% glycerol retains activity for at least 4 weeks [2] <2>, His6-WlbD stored at -20 C in the presence of 25% glycerol retains activity for at least 4 weeks [2]

References [1] Sri Kannathasan, V.; Staines, A.G.; Dong, C.J.; Field, R.A.; Preston, A.G.; Maskell, D.J.; Naismith, J.H.: Overexpression, purification, crystallization and data collection on the Bordetella pertussis wlbD gene product, a putative UDP-GlcNAc 2’-epimerase. Acta Crystallogr. Sect. D, 57, 1310-1312 (2001) [2] Westman, E.L.; McNally, D.J.; Rejzek, M.; Miller, W.L.; Kannathasan, V.S.; Preston, A.; Maskell, D.J.; Field, R.A.; Brisson, J.-R.; Lam, J.S.: Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-a -d-glucuronic acid 2-epimerases from respiratory pathogens. Biochem. J., 405, 123-130 (2007) [3] Westman, E.L.; McNally, D.J.; Rejzek, M.; Miller, W.L.; Kannathasan, V.S.; Preston, A.; Maskell, D.; Field, R.A.; Brisson, J.-R.; Lam, J.S.: Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-a -d-glucuronic acid 2-epimerases from respiratory pathogens. [Erratum to document cited in CA147:229240]. Biochem. J., 405, 625 (2007)

501

Polyenoic fatty acid isomerase

5.3.3.13

1 Nomenclature EC number 5.3.3.13 Systematic name (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate D8;11 -D7;8 -isomerase Recommended name polyenoic fatty acid isomerase Synonyms PAI <1, 4> (<4> polyenoic fatty acid isomerase from Propionibacterium acnes [8,9]) [7, 8, 9] PFI eicosapentaenoate cis-D5;8;11;14;17 -eicosapentaenoate cis-D5 -trans-D7;9 -cisD14;17 -isomerase polyenoic fatty acid isomerase <4> [8, 9] polyunsaturated fatty acid isomerase <2> [2] CAS registry number 159002-84-3

2 Source Organism <1> <2> <3> <4>

Propionibacterium acnes (no sequence specified) [7] Ptilota filicina (no sequence specified) [1, 2, 3, 4, 5] Ptilota filicina (UNIPROT accession number: Q8W257) [6] Propionibacterium acnes (UNIPROT accession number: Q6A8X5) [8, 9]

3 Reaction and Specificity Catalyzed reaction (5Z,8Z,11Z,14Z,17Z)-icosapentaenoate = (5Z,7E,9E,14Z,17Z)-icosapentaenoate (<2> mechansim of hydrogen transfers in the isomerization reaction including a diene intermediate [3]; <2> regio- and stereochemistry of the isomerization reaction, mechanism [1]; <4> double bond isomerization [8,9]) Reaction type isomerization

502

5.3.3.13

Polyenoic fatty acid isomerase

Natural substrates and products S (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate <2> (<2> likely the native substrate, biosynthesis of conjugated triene-containing fatty acids [1]) (Reversibility: ?) [1] P (5Z,7E,9E,14Z,17Z)-eicosapentaenoate S Additional information <3> (<3> PFI may be part of a stress tolerance mechanism [6]) (Reversibility: ?) [6] P ? Substrates and products S (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexenoate <2> (<2> excellent substrate [3]) (Reversibility: ?) [3] P (4Z,7Z,9E,11E,16Z,19Z)-docosahexenoate S (5Z,8Z,11Z)-eicosatrienoate <2> (Reversibility: ?) [3] P ? S (5Z,8Z,11Z,14Z)-eicosatetraenoate <2> (<2> arachidonate, next best substrate after (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate, the C11 olefinic position becomes protonated by a solvent-derived proton, mechanism [1]; <2> arachidonic acid [3]) (Reversibility: ?) [1, 3] P (5Z,7E,9E,14Z)-eicosatetraenoate S (5Z,8Z,11Z,14Z)-eicosatetraenoate <3> (<3> arachidonic acid, conversion to the conjugated triene [6]) (Reversibility: ?) [6] P ? S (5Z,8Z,11Z,14Z)-eicosatetraenoyl methyl ester <2> (<2> methyl ester of arachidonate [1]) (Reversibility: ?) [1] P (5Z,7E,9E,14Z,17Z)-eicosapentaenoyl methyl ester S (5Z,8Z,11Z,14Z)-eicosatetraenoyl-N-ethanolamide <2> (<2> anandamide [2,3,5]; <2> arachidonoylethanolamide [1]) (Reversibility: ?) [1, 2, 3, 5] P (5Z,7E,9E,14Z)-eicosatetraenoyl-N-ethanolamide (<2> conjugated triene anandamide, cannabimimetic substance [5]; <2> termed CTA, i.e. conjugated triene anandamide, cannabimimetic substance [2]) S (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate <2, 3> (<2> likely the native substrate, biosynthesis of conjugated triene-containing fatty acids [1]; <2> best substrate, likely the native substrate, mechanism [1]; <3> the preferred substrates are highly unsaturated free fatty acids such as eicosapentaenoic acid [6]) (Reversibility: ?) [1, 3, 5, 6] P (5Z,7E,9E,14Z,17Z)-eicosapentaenoate S (6Z,9Z,12Z)-octadecatrienoate <2> (<2> g-linolenate, PFI intramolecularly transfers the bis-allylic pro-S hydrogen from the C11 position to the C13 position, the bis-allylic pro-R hydrogen at C8 in g-linolenate is lost to the solvent [1]; <2> g-linolenic acid [3]) (Reversibility: ?) [1, 3] P (6Z,8E,10E)-octadecatrienoate S (6Z,9Z,12Z)-octatrienoic acid <4> (<4> linolenic acid [8,9]) (Reversibility: ?) [8, 9] P (6Z,10E,12Z)-octatrienoic acid S (7Z,10Z,13Z,16Z)-docosatetraenoate <2> (<2> adrenic acid [3]) (Reversibility: ?) [3]

503

Polyenoic fatty acid isomerase

5.3.3.13

P ? S (8Z,11Z,14Z)-eicosatrienoate <2> (<2> dihomo-g-linolenic acid, formation of two products: a conjugated triene and a conjugated diene [3]) (Reversibility: ?) [3] P ? S (9Z,12Z)-octadecadienoic acid <4> (<4> linoleic acid [8,9]) (Reversibility: ?) [8, 9] P (10E,12Z)-octadecadienoic acid (<4> conjugated linoleic acid, double bond isomerization [8]; <4> conjugated linoleic acid, double bound isomerization [9]) S (9Z,12Z,15Z)-octadecatrienoate <2> (<2> a-linolenate, one-fifth as fast as g-linolenate [1]; <2> a-linolenic acid [3]) (Reversibility: ?) [1, 3] P ? S linoleic acid <1> (Reversibility: ?) [7] P (10E,12Z)-octadeca-10,12-dienoic acid S polyenoic fatty acid <2, 3> (<3> PFI catalyzes the isomerization of a wide range of substrates containing three ore more methylene interrupted olefins into a Z,E,E conjugated triene functionality [6]; <2> PFI is capable of isomerizing the methylene interrupted olefins of a wide range of polyenoic fatty acids into a conjugated triene functionality [2]) (Reversibility: ?) [2, 6] P ? S polyunsaturated fatty acid <2> (<2> PFI catalyzes the formation of conjugated trienes from a variety of polyunsaturated fatty acid precursors, regio- and stereochemistry, enzyme may recognize the protonated from of the substrate [1]) (Reversibility: ?) [1] P ? S Additional information <2, 3> (<3> PFI may be part of a stress tolerance mechanism [6]; <2> not: linoleic acid [1]; <2> substrate binding site, PFI preferentially orients the substrate in the catalytic site with respect to the methyl terminus and likely reacts, preferentially, with the protonated form of the substrate, not: linoleic acid [3]) (Reversibility: ?) [1, 3, 6] P ? Inhibitors Additional information <2> (<2> not inhibited by EDTA, o-phenanthroline, acetylsalicylic acid, baicalein, dipyridamole, eicosatetraynoic acid, esculetin, indomethacin, naproxen, NDGA or SKF-525A [1]) [1] Cofactors/prosthetic groups FAD <4> [9] flavin <3> (<3> bound to PFI, cDNA contains a flavin-binding motif near the mature N-terminus [6]) [6] Additional information <2, 4> (<2> no requirement for molecular oxygen [1]; <4> flavin is a putative cofactor, either FAD or FMN [8]) [1,8]

504

5.3.3.13

Polyenoic fatty acid isomerase

Turnover number (min–1) 11.6 <2> ((5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate, <2> assuming one catalytic site per enzyme molecule [3]) [3] 41 <2> ((5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate, <2> pH 7.2, assuming one catalytic site per enzyme molecule [1]) [1] Specific activity (U/mg) 1.06 <2> (<2> pH 7.2, arachidonate as substrate [1]) [1] Additional information <2> [3] Km-Value (mM) 0.0019 <2> ((4Z,7Z,10Z,13Z,16Z,19Z)-docosahexenoate, <2> pH 7, 22 C [3]) [3] 0.0096 <2> ((5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate, <2> pH 7, 22 C [3]) [3] 0.0152 <2> ((5Z,8Z,11Z,14Z)-eicosatetraenoyl-N-ethanolamide, <2> pH 9, 22 C [3]) [3] 0.017 <2> ((7Z,10Z,13Z,16Z)-docosatetraenoate, <2> pH 7, 22 C [3]) [3] 0.0175 <2> ((5Z,8Z,11Z,14Z)-eicosatetraenoyl-N-ethanolamide, <2> pH 7, 22 C [3]) [3] 0.0255 <2> ((5Z,8Z,11Z,14Z)-eicosatetraenoate, <2> pH 7, 22 C [3]) [3] 0.0328 <2> ((5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate, <2> pH 7.2 [1]) [1] 0.0399 <2> ((5Z,8Z,11Z,14Z)-eicosatetraenoate, <2> pH 7.2 [1]) [1] 0.0528 <2> ((5Z,8Z,11Z)-eicosatrienoate, <2> pH 7, 22 C [3]) [3] 0.15 <2> ((6Z,9Z,12Z)-octadecatrienoate, <2> pH 7.2 [1]) [1, 3] 1.4 <1> (Linoleic acid) [7] Additional information <2> (<2> study of the effect of the pH on kinetic parameters with several substrates [3]) [3] pH-Optimum 4.5 <2> [1] 6 <2> (<2> below [3]) [3] 6.5 <2> (<2> assay at [2,5]) [2, 5] 7.2 <2> (<2> assay at [6]) [6] 7.5 <1> [7] pH-Range 4-8 <2> (<2> there is a 30% increase in rate of product formation from pH 8 to 6.5, from pH 6.5 to 4.5 the apparent velocity increases 2.4fold, below pH 4 the activity slowly decreases [1]) [1] Additional information <2> (<2> pH-dependence profile of PFI, catalytically active over a wide pH-range with (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate as substrate [3]) [3] Temperature optimum ( C) 22 <2, 3> (<2,3> assay at [3,6]) [3, 6]

505

Polyenoic fatty acid isomerase

5.3.3.13

4 Enzyme Structure Molecular weight 125000 <3> (<3> sedimentation equilibrium utracentrifugation [6]) [6] 174000 <2> (<2> gel filtration [1]) [1] Subunits ? <2> (<2> x * 61000, SDS-PAGE [3]) [3] homodimer <3> (<3> 2 * 61000, SDS-PAGE, 2 * 55900, sequence calculation including the signal peptide, 2 * 53600, sequence calculation without the signal peptide [6]) [6] trimer <2> (<2> 3 * 61000, SDS-PAGE, 3 * 58119, mass spectrometry [1]) [1] Posttranslational modification glycoprotein <3> (<3> native PFI is N-glycosylated [6]) [6] proteolytic modification <3> (<3> translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation [6]) [6]

5 Isolation/Preparation/Mutation/Application Purification <2> [2, 3, 5, 6] <2> (102fold) [1] <4> (of the recombinant protein by glutathione sepharose chromatography) [8, 9] Crystallization <4> (in two forms) [8] <4> (six crystal structures in apo and product-bound forms, by single isomorphous replacement with anomalous scattering, SIRAS) [9] Cloning <1> (expression in Escherichia coli, in Saccharomyces cerevisiae and in transgenic tabacco cells) [7] <2> (cDNA, expression in Arabidopsis thaliana) [6] <4> (overexpression in Escherichia coli as a GST-fusion protein) [8, 9]

6 Stability pH-Stability 4-10 <2> (<2> PFI is relatively stable at these pH extremes [3]) [3] Storage stability <2, 3>, -20 C, purified PFI, months, stable [3, 6]

506

5.3.3.13

Polyenoic fatty acid isomerase

References [1] Wise, M.L.; Hamberg, M.; and Gerwick, W.H.: Biosynthesis of conjugated fatty acids by a novel isomerase from the red marine alga Ptilota filicina. Biochemistry, 33, 15223-15232 (1994) [2] Wise, M.L.; Soderstrom, K.; Murray, T.F.; Gerwick, W.H.: Synthesis and cannabinoid receptor binding activity of conjugated triene anandamide, a novel eicosanoid. Experientia, 52, 88-92 (1996) [3] Wise, M.L.; Rossi, J.; Gerwick, W.H.: Binding site characterization of polyenoic fatty-acid isomerase from the marine alga Ptilota filicina. Biochemistry, 36, 2985-2992 (1997) [4] Zheng, W.; Wise, M.L.; Wyrick, A.; Metz, J.G.; Yuan, L.; Gerwick, W.H.: Polyenoic fatty-acid isomerase from the marine red alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA. Arch. Biochem. Biophys., 402, 11-20 (2002) [5] Gerwick, W.H.; Wise, M.L.; Soderstrom, K.; Murray, T.F.: Biosynthesis and cannabinoid receptor affinity of the novel eicosanoid, conjugated triene anandamide. Adv. Exp. Med. Biol., 407, 329-334 (1997) [6] Zheng, W.; Wise, M.L.; Wyrick, A.; Metz, J.G.; Yuan, L.; Gerwick, W.H.: Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA. Arch. Biochem. Biophys., 401, 11-20 (2002) [7] Hornung, E.; Krueger, C.; Pernstich, C.; Gipmans, M.; Porzel, A.; Feussner, I.: Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds. Biochim. Biophys. Acta, 1738, 105-114 (2005) [8] Liavonchanka, A.; Hornung, E.; Feussner, I.; Rudolph, M.: In-house SIRAS phasing of the polyunsaturated fatty-acid isomerase from Propionibacterium acnes. Acta Crystallogr. Sect. F, 62, 153-156 (2006) [9] Liavonchanka, A.; Hornung, E.; Feussner, I.; Rudolph, M.G.: Structure and mechanism of the Propionibacterium acnes polyunsaturated fatty acid isomerase. Proc. Natl. Acad. Sci. USA, 103, 2576-2581 (2006)

507

trans-2-Decenoyl-[acyl-carrier protein] isomerase

5.3.3.14

1 Nomenclature EC number 5.3.3.14 Systematic name decenoyl-[acyl-carrier-protein] D2 -trans-D3 -cis-isomerase Recommended name trans-2-decenoyl-[acyl-carrier protein] isomerase Synonyms FabM <2> [9] CAS registry number 9030-80-2

2 Source Organism <1> <2> <3> <4>

Escherichia coli (no sequence specified) [1, 2, 3, 4, 6, 8, 10] Streptococcus pneumoniae (no sequence specified) [9] Streptococcus mutans (no sequence specified) [7] Escherichia coli (UNIPROT accession number: P0A6Q3) [5]

3 Reaction and Specificity Catalyzed reaction trans-dec-2-enoyl-[acyl-carrier-protein] = cis-dec-3-enoyl-[acyl-carrier-protein] (<1> both enzyme activities are carried out by the same active site carrying a His and a Asp residue in an hydrophobic environment [10]; <1> reaction of EC 4.2.1.60 and isomerization reaction, both catalyzed by the enzyme, share the common intermediate, enzyme-bound a,b-decenoate [8]) Substrates and products S cis-3-decenoyl-N-acetylcysteamine <1> (Reversibility: r) [2] P trans-2-decenoyl-N-acetylcysteamine S cis-b,g-decenoyl-N-acetylcysteamine <1> (Reversibility: ?) [1] P ? S trans-2-decenoyl-(acyl-carrier-protein) <1> (<1> enzyme is equally active with acyl carrier protein derived from Escherichia coli, spinach or a protein A:acyl carrier protein fusion protein. With acyl carrier protein

508

5.3.3.14

P S P S P S P S

P

trans-2-Decenoyl-[acyl-carrier protein] isomerase

derived from Escherichia coli or from spinach, equilibrium results in equal amounts of trans-3- or cis-2-decenoyl-(acyl-carrier-protein), regardless of the initial substrate. With the fusion protein, yield is about 17% cis-3- and 49% trans-2-decenoyl-(acyl-carrier-protein) [6]) (Reversibility: r) [6] cis-3-decanoyl-(acyl-carrier-protein) trans-2-decenoyl-N-acetylcysteamine <1> (Reversibility: r) [6] cis-3-decenoyl-N-acetylcysteamine trans-2-decenoyl-N-acetylcysteamine <1, 2> (Reversibility: r) [8, 9] cis-3-decanoyl-N-acetylcysteamine trans-2-octenoyl-N-acetylcysteamine <2> (Reversibility: ?) [9] cis-3-octenoyl-N-acetylcysteamine Additional information <1, 2> (<1> enzyme activity depends more on acyl chain length than acyl carrier protein structure or origin [6]; <2> enzyme does not catalyze dehydration reaction of EC 4.2.1.60 [9]) (Reversibility: ?) [6, 9] ?

Inhibitors 3-decynoyl-N-acetylcysteamine <1> (<1> covalent modification of ative site residue H70 [2]; <1> irreversible inactivation of both dehydrase activity of EC 4.2.1.60 and isomerase activity of enzyme, inhibiton occurs through isomerzation to the allene and subsequent covalent modification of enzyme [4]; <1> strong inhibition of both dehydrase activity of EC 4.2.1.60 and isomerase activity of enzyme [3]) [2, 3, 4] 3-dodecynoyl-N-acetylcysteamine <1> [3] 3-nonynoyl-N-acetylcysteamine <1> (<1> noncompetitive [3]) [3] 3-octynoyl-N-acetylcysteamine <1> (<1> 24% inhibition at 0.005 mM [3]) [3] 3-undecynoyl-N-acetylcysteamine <1> [3] p-chloromercuribenzoate <1> (<1> 0.5 mM, 35% inhibition [1]) [1] Additional information <1> (<1> not inhibitory: iodoacetic acid, chloroacetic acid, iodoacetamide, N-ethylmaleimide [1]) [1] Turnover number (min–1) 15 <1> (cis-3-decenoyl-N-acetylcysteamine, <1> pH 9.0, 30 C [2]) [2] Specific activity (U/mg) 0.000087 <2> (<2> pH 7.0 [9]) [9] Additional information <2> (<2> direct assay for enzyme activity based on substrate analogues [9]) [9] Km-Value (mM) 0.5 <1> (cis-b,g-decenoyl-N-acetylcysteamine, <1> pH 7.0 [1]) [1] Additional information <1> (<1> Km -value gradually decreases with decreasing pH-value [2]) [2]

509

trans-2-Decenoyl-[acyl-carrier protein] isomerase

5.3.3.14

Ki-Value (mM) 0.00013 <1> (3-Decynoyl-N-acetylcysteamine, <1> pH 7.0, 30 C, isomerase acitivity [3]) [3] 0.00019 <1> (3-undecynoyl-N-acetylcysteamine, <1> pH 7.0, 30 C, isomerase acitivity [3]) [3] 0.00023 <1> (3-Nonynoyl-N-acetylcysteamine, <1> pH 7.0, 30 C, isomerase acitivity [3]) [3] 0.00075 <1> (3-Dodecynoyl-N-acetylcysteamine, <1> pH 7.0, 30 C, isomerase acitivity [3]) [3] pH-Optimum 7.95 <1> (<1> assay at [8]) [8] pH-Range 7.3-10.2 <1> (<1> more than 50% of activity maximum within this range [2]) [2]

4 Enzyme Structure Molecular weight 27500 <1> (<1> sucrose density sedimentation [1]) [1] 28000 <1> (<1> gel filtration [1]) [1] 36000 <1> (<1> gel filtration [2]) [2] 129000 <2> (<2> gel filtration [9]) [9] Subunits ? <4> (<4> x * 18800, calculated [5]) [5] dimer <1> (<1> 2 * 18000, SDS-PAGE [2]; <1> crystallization data [10]) [2, 10] tetramer <2> (<2> 4 * 31000, SDS-PAGE [9]) [9]

5 Isolation/Preparation/Mutation/Application Purification <1> [1] <4> (simplified purification protocol) [5] Crystallization <1> (free enzyme and modified by inhibitor 3-decynoyl-N-acteylcysteamine) [10] Cloning <4> [5] Engineering Additional information <3> (<3> enzyme insertion mutant, no production of unsaturated fatty acids by mutant strain. Strain is extremely sensitive to low pH-values and exhibits reduced glycolyic capability and altered membrane composition, resulting in higher impermeability to protons [7]) [7]

510

5.3.3.14

trans-2-Decenoyl-[acyl-carrier protein] isomerase

References [1] Kass, L.R.; Brock, D.J.H.; Bloch, K.: b-Hydroxydecanoyl thioester dehydrase. J. Biol. Chem., 242, 4418-4431 (1967) [2] Helmkamp, G.H.; Bloch, K.: b-Hydroxydecanoyl thioester dehydrase. Studies on molecular structure and active site. J. Biol. Chem., 244, 6014-6022 (1969) [3] Helmkamp, G.; Rando, R.R.; Brock, D.J.H.; Bloch, K.: b-Hydroxydecanoyl thioester dehydrase. J. Biol. Chem., 243, 3229-3231 (1968) [4] Endo, K.; Helmkamp, G.M.; Bloch, K.: Mode of inhibition of b-hydroxydecanoyl thioester dehydrase by 3-decynoyl-N-acetylcysteamine. J. Biol. Chem., 245, 4293-4296 (1970) [5] Cronan, J.E.; Li, W.B.; Coleman, R.; Narasimhan, M.; De Mendoza, D.; Schwab, J.M.: Derived amino acid sequence and identification of active site residues of Escherichia coli b-hydroxydecanoyl thioester dehydrase. J. Biol. Chem., 263, 4641-4646 (1988) [6] Guerra, D.J.; Browse, J.A.: Escherichia coli b-hydroxydecanoyl thioester dehydrase reacts with native C10 acyl-acyl-carrier proteins of plant and bacterial origin. Arch. Biochem. Biophys., 280, 336-345 (1990) [7] Fozo, E.M.; Quivey, R.G., Jr.: The fabM gene product of Streptococcus mutans is responsible for the synthesis of monounsaturated fatty acids and is necessary for survival at low pH. J. Bacteriol., 186, 4152-4158 (2004) [8] Rando, R.R.; Bloch, K.: Mechanism of action of b-hydroxydecanoyl thioester dehydrase. J. Biol. Chem., 243, 5627-5634 (1968) [9] Marrakchi, H.; Choi, K.H.; Rock, C.O.: A new mechanism for anaerobic unsaturated fatty acid formation in Streptococcus pneumoniae. J. Biol. Chem., 277, 44809-44816 (2002) [10] Leesong, M.; Henderson, B.S.; Gillig, J.R.; Schwab, J.M.; Smith, J.L.: Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: two catalytic activities in one active site. Structure, 4, 253-264 (1996)

511

Ascopyrone tautomerase

5.3.3.15

1 Nomenclature EC number 5.3.3.15 Systematic name 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose D3 -D1 -isomerase Recommended name ascopyrone tautomerase Synonyms 1,5-anhydro-d-glycero-hex-3-en-2-ulose tautomerase <1> [1] APM tautomerase <1> [1] APTM <1> [1] APTM1 <2> [2] APTM2 <2> [2] ascopyrone P tautomerase <1> [1] ascopyrone intramolecular oxidoreductase <1> [1] ascopyrone isomerase <1> [1] ascopyrone tautomerase <1> [1]

2 Source Organism <1> Anthracobia melaloma (no sequence specified) [1] <2> Anthracobia melanoma (no sequence specified) [2]

3 Reaction and Specificity Catalyzed reaction 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose = 1,5-anhydro-4-deoxy-dglycero-hex-1-en-3-ulose Natural substrates and products S 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose <1, 2> (<1> i.e. ascopyrone M. This enzyme catalyses one of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5anhydro-d-fructose [1]; <2> i.e. ascopyrone M. This enzyme catalyses one of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydro-d-fructose. The other enzymes involved in this pathway are EC 4.2.1.110 (aldos-2-ulose dehydra-

512

5.3.3.15

Ascopyrone tautomerase

tase), EC 4.2.1.111 (1,5-anhydro-d-fructose dehydratase) and EC 4.2.2.13 (exo-(1,4)-a-d-glucan lyase) [2]) (Reversibility: ir) [1, 2] P 1,5-anhydro-4-deoxy-d-glycero-hex-1-en-3-ulose (<1> i.e. ascopyrone P [1]; <2> i.e. ascopyrone P. Ascopyrone P is an anti-oxidant [2]) Substrates and products S 1,5-anhydro-4-deoxy-d-glycero-hex-3-en-2-ulose <1, 2> (<1> i.e. ascopyrone M. This enzyme catalyses one of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydrod-fructose [1]; <2> i.e. ascopyrone M. This enzyme catalyses one of the steps in the anhydrofructose pathway, which leads to the degradation of glycogen and starch via 1,5-anhydro-d-fructose. The other enzymes involved in this pathway are EC 4.2.1.110 (aldos-2-ulose dehydratase), EC 4.2.1.111 (1,5-anhydro-d-fructose dehydratase) and EC 4.2.2.13 (exo-(1,4)-a-d-glucan lyase) [2]; <1,2> i.e. ascopyrone M [1,2]) (Reversibility: ir) [1, 2] P 1,5-anhydro-4-deoxy-d-glycero-hex-1-en-3-ulose (<1,2> i.e. ascopyrone P [1,2]; <2> i.e. ascopyrone P. Ascopyrone P is an anti-oxidant [2]) Specific activity (U/mg) 3873 <1> [1] pH-Optimum 5.5 <2> (<2> optimal range: 5.0-6.0 [2]) [2]

4 Enzyme Structure Molecular weight 140000 <1> (<1> gel filtration [1]) [1] Subunits dimer <1, 2> (<1> 2 * 60000, SDS-PAGE [1]; <2> 2 * 60000 [2]) [1, 2]

5 Isolation/Preparation/Mutation/Application Purification <1> [1]

References [1] Yu, S.; Refdahl, C.; Lundt, I.: Enzymatic description of the anhydrofructose pathway of glycogen degradation; I. Identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and a-1,4-glucan lyase in the fungus Anthracobia melaloma. Biochim. Biophys. Acta, 1672, 120-129 (2004) [2] Yu, S.; Fiskesund, R.: The anhydrofructose pathway and its possible role in stress response and signaling. Biochim. Biophys. Acta, 1760, 1314-1322 (2006)

513

Capsanthin/capsorubin synthase

5.3.99.8

1 Nomenclature EC number 5.3.99.8 Systematic name violaxanthin-capsorubin isomerase (ketone-forming) Recommended name capsanthin/capsorubin synthase Synonyms CCS <1> [5] CCS ketoxanthophyll synthase <1> [4] capsanthin-capsorubin synthase <1> [4, 5] CAS registry number 162032-85-1

2 Source Organism <1> Capsicum annuum (no sequence specified) [1, 2, 3, 4, 5]

3 Reaction and Specificity Catalyzed reaction antheraxanthin = capsanthin violaxanthin = capsorubin Reaction type intramolecular oxidoreduction isomerization Natural substrates and products S Additional information <1> (<1> relation of molecular genetics of the y locus in pepper to capsanthin-capsorubin synthase and to fruit color [5]; <1> the capsanthin-capsorubin synthase gene is activated specifically during the final stages of Capsicum fruit ripening. The yellow phenotype results from a deletion of the capsanthin-capsorubin synthase gene [4]) (Reversibility: ?) [4, 5] P ?

514

5.3.99.8

Capsanthin/capsorubin synthase

Substrates and products S antheraxanthin <1> (Reversibility: ?) [3] P capsanthin S violaxanthin <1> (Reversibility: ?) [3] P capsorubin S Additional information <1> (<1> relation of molecular genetics of the y locus in pepper to capsanthin-capsorubin synthase and to fruit color [5]; <1> the capsanthin-capsorubin synthase gene is activated specifically during the final stages of Capsicum fruit ripening. The yellow phenotype results from a deletion of the capsanthin-capsorubin synthase gene [4]) (Reversibility: ?) [4, 5] P ? Specific activity (U/mg) 0.0073 <1> [3]

4 Enzyme Structure Molecular weight 60000 <1> (<1> gel filtration [3]) [3] Subunits monomer <1> (<1> 1 * 50000, SDS-PAGE [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue fruit <1> (<1> the gene is specifically expressed during chromoplast development in fruits accumulating ketocarotenoids, but not in mutants impaired in this biosynthetic step [3]) [3, 4, 5] pericarp <1> (<1> disc [3]) [3] Localization chloroplast <1> [3] Purification <1> [3] Cloning <1> [3] <1> (cDNA encoding capsanthin-capsorubin synthase is placed under the transcriptional control of a tobamovirus subgenomic promoter. Leaves from transfeceted plants expressing capsanthin-capsorubin synthase develop an orange phenotype and accumulate high levels of capsanthin. This phenomenon is associated with thylakoid membrane distortion and reduction of grana stacking) [2]

515

Capsanthin/capsorubin synthase

5.3.99.8

<1> (the promoter of Capsicum annum capsanthin/capsorubin synthase is strongly upregulated during tomato ripening of transgenic tomato plants that produce fruits of the climacteric type (characterized by an increase in respiration and ethylene production). Induction occurs at the mature green stage preceding ripening. Ethylene positively influences the expression of the gene in tomato. Water loss strongly induces the promoter) [1]

References [1] Kuntz, M.; Chen, H.C.; Simkin, A.J.; Romer, S.; Shipton, C.A.; Drake, R.; Schuch, W.; Bramley, P.M.: Upregulation of two ripening-related genes from a non-climacteric plant (pepper) in a transgenic climacteric plant (tomato). Plant J., 13, 351-361 (1998) [2] Kumagai, M.H.; Keller, Y.; Bouvier, F.; Clary, D.; Camara, B.: Functional integration of non-native carotenoids into chloroplasts by viral-derived expression of capsanthin-capsorubin synthase in Nicotiana benthamiana. Plant J., 14, 305-315 (1998) [3] Bouvier, F.; Hugueney, P.; d’Harlingue, A.; Kuntz, M.; Camara, B.: Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid. Plant J., 6, 45-54 (1994) [4] Lefebvre, V.; Kuntz, M.; Camara, B.; Palloix, A.: The capsanthin-capsorubin synthase gene: a candidate gene for the y locus controlling the red fruit colour in pepper. Plant Mol. Biol., 36, 785-789 (1998) [5] Popovsky, S.; Paran, I.: Molecular genetics of the y locus in pepper: its relation to capsanthin-capsorubin synthase and to fruit color. Theor. Appl. Genet., 101, 86-89 (2000)

516

Neoxanthin synthase

5.3.99.9

1 Nomenclature EC number 5.3.99.9 Systematic name violaxanthin-neoxanthin isomerase (epoxide-opening) Recommended name neoxanthin synthase CAS registry number 318960-21-3

2 Source Organism <1> Solanum tuberosum (no sequence specified) [2] <2> Lycopersicon esculentum (UNIPROT accession number: Q9LWA6) [1]

3 Reaction and Specificity Catalyzed reaction violaxanthin = neoxanthin

5 Isolation/Preparation/Mutation/Application Source/tissue fruit <2> (<2> mature green, pink and red [1]) [1] seedling <2> [1] Cloning <1> (plant protoplasts obtained from Arabidopsis thaliana tissue culture and Nicotiana tabacum leaves are transformed with the full length cDNA under control of the 35S CaMV promoter by electroporation and PEG) [2] <2> (expression in Escherichia coli or transfection of Nicotiana benthamiana leaves) [1]

517

Neoxanthin synthase

5.3.99.9

References [1] Bouvier, F.; D’Harlingue, A.; Backhaus, R.A.; Kumagai, M.H.; Camara, B.: Identification of neoxanthin synthase as a carotenoid cyclase paralog. Eur. J. Biochem., 267, 6346-6352 (2000) [2] Al-Babili, S.; Hugueney, P.; Schledz, M.; Welsch, R.; Frohnmeyer, H.; Laule, O.; Beyer, P.: Identification of a novel gene coding for neoxanthin synthase from Solanum tuberosum. FEBS Lett., 485, 168-172 (2000)

518

Phosphoglucosamine mutase

5.4.2.10

1 Nomenclature EC number 5.4.2.10 Systematic name a-d-glucosamine 1,6-phosphomutase Recommended name phosphoglucosamine mutase Synonyms aminodeoxyglucose phosphate phosphomutase glucosamine phosphomutase CAS registry number 9031-92-9

2 Source Organism <1> <2> <3> <4>

Staphylococcus aureus (no sequence specified) [4] Escherichia coli (no sequence specified) [2, 3, 6] Pseudomonas aeruginosa (no sequence specified) [1] Helicobacter pylori (no sequence specified) [5]

3 Reaction and Specificity Catalyzed reaction d-glucosamine 1-phosphate = d-glucosamine 6-phosphate Reaction type isomerization Natural substrates and products S d-glucoamine 6-phosphate <1, 2, 3, 4> (<2> ping-pong bi-bi reaction mechanism [2,3]; <2> two basically different reaction sequences, 1. phosphate group transfer yielding the intermediate d-GlcN-1,6-diphosphate, 2. conversion to GlcN-1-phosphate [3]; <1,2,3,4> involved in formation of cell-wall precursor UDP-N-acetylglucosamine [1,2,3,4,5,6]) (Reversibility: r) [1, 2, 3, 4, 5, 6] P d-glucosamine 1-phosphate <1, 2, 3, 4> [1, 2, 3, 4, 5, 6]

519

Phosphoglucosamine mutase

S P S P

d-glucosamine d-glucosamine d-glucosamine d-glucosamine

5.4.2.10

1,6-diphosphate <2> (Reversibility: r) [3] 6-phosphate <2> [3] 1-phosphate <2> (Reversibility: r) [3] 6-phosphate <2> [3]

Substrates and products S d-mannose 1-phosphate <3> (<3> 5fold lower activity than phosphoglucosamine mutase activity [1]) (Reversibility: ?) [1] P d-mannose 6-phosphate <3> [1] S d-glucoamine 6-phosphate <1, 2, 3, 4> (<2> ping-pong bi-bi reaction mechanism [2,3]; <2> two basically different reaction sequences, 1. phosphate group transfer yielding the intermediate d-GlcN-1,6-diphosphate, 2. conversion to GlcN-1-phosphate [3]; <1,2,3,4> involved in formation of cell-wall precursor UDP-N-acetylglucosamine [1,2,3,4,5,6]) (Reversibility: r) [1, 2, 3, 4, 5, 6] P d-glucosamine 1-phosphate <1, 2, 3, 4> [1, 2, 3, 4, 5, 6] S d-glucosamine 1,6-diphosphate <2> (Reversibility: r) [3] P d-glucosamine 6-phosphate <2> [3] S d-glucosamine 1-phosphate <2> (Reversibility: r) [3] P d-glucosamine 6-phosphate <2> [3] S d-glucose 1-phosphate <2, 3> (<2> in the presence of Glc-1,6-diphosphate, 1400fold lower activity than for d-glucosamine 1-phosphate or dglucosamine 1,6-diphosphate, ping-pong reaction mechanism [3]; <3> 50fold lower activity than phosphoglucosamine mutase activity [1]) (Reversibility: ?) [1, 3] P d-glucose 6-phosphate <2, 3> [1, 3] Inhibitors Ca2+ <2> (<2> 80% reduced activity [2]) [2] EDTA <2> (<2> abolishes autophosphorylation and catalytic activity [2]) [2] Mn2+ <2> (<2> 80% reduced activity [2]) [2] Zn2+ <2> (<2> efficient autophosphorylation, but absence of catalytic activity [2]) [2] Activating compounds d-glucosamine 1,6-diphosphate <2> (<2> retains the enzyme in an active and phosphorylated form [2,3]) [2, 3] d-glucose 1,6-diphosphate <1, 2> (<2> retains the enzyme in an active and phosphorylated form [3]; <1> 100fold activation [4]; <2> 20fold activation at 0.7 mM [6]) [3, 4, 6] Metals, ions Mg2+ <2> (<2> essential for autophosphorylation and catalytic activity [2]) [2] Turnover number (min–1) 0.0055 <2> (d-glucose 1-phosphate) [3] 0.0138 <2> (d-glucose 1-phosphate, <2> single-mutation S100T [3]) [3]

520

5.4.2.10

Phosphoglucosamine mutase

2.42 <2> (d-glucosamine 6-phosphate, <2> 6 x His-tagged recombinant enzyme [3]) [3] 7.9 <2> (d-glucosamine 6-phosphate) [3] Specific activity (U/mg) 0.007 <2> (<2> substrate: d-glucose 6-phosphate [3]) [3] 0.06 <3> (<3> substrate: d-glucose 1-phosphate [1]) [1] 0.1 <1> [4] 0.55 <3> (<3> substrate: d-mannose 1-phosphate [1]) [1] 0.95 <4> [5] 2.5 <3> (<3> substrate: d-glucosamine 6-phosphate [1]) [1] 2.59 <2> [6] 3 <2> (<2> 6x His-tagged recombinant enzyme [3]) [3] 10 <2> (<2> substrate: d-glucosamine 6-phosphate [3]) [3] Km-Value (mM) 0.05 <2> (d-glucosamine 6-phosphate) [3] 0.08 <2> (d-glucosamine 1-phosphate) [3] 0.08 <2> (d-glucose 1-phosphate, <2> single mutation S100T [3]) [3] 0.08 <2> (d-glucosamine 1,6-diphosphate) [3] 0.5 <2> (d-glucose 1,6-diphosphate) [3] 0.65 <2> (d-glucose 1-phosphate) [3]

4 Enzyme Structure Subunits trimer <2> (<2> 3 * 47412, gel filtration, mass spectrometry [3]) [3] Posttranslational modification phosphoprotein <2> (<2> the only phosphogroup at Ser102 [3]; <2> in vitro autophosphorylation at S102 in the presence of [g-32 P]ATP [2]) [2, 3, 6]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <2> [2, 3, 6] Purification <1> (to homogeneity, recombinant enzyme) [4] <2> (near homogeneity) [3, 6] <2> (near homogeneity, recombinant enzyme) [2, 3] <3> (near homogeneity, recombinant enzyme) [1] <4> (near homogeneity, recombinant enzyme) [5] Cloning <1> (overexpression in Escherichia coli) [4] <2> (overexpression in Escherichia coli) [2, 3, 6]

521

Phosphoglucosamine mutase

5.4.2.10

<3> (overexpression in Escherichia coli) [1] <4> (overexpression in Escherichia coli) [5] Engineering S100A <2> (<2> 50fold less active [2]; <2> very low activity [3]) [2, 3] S100T <2> (<2> contributes to the specificity towards sugar- or aminosugarphosphates, much more efficient in catalysis of the phosphoglucose mutase reaction [3]) [3] S102A <2> (<2> completely inactive, loss of autophosphorylation ability [2]; <2> site of phosphorylation, no activity [3]) [2, 3]

References [1] Tavares, I. M.; Jolly, L.; Pompeo, F.; Leitao, J.H.; Fialho, A.M.; Sa-Correia, I.; Mengin-Lecreulx, D.: Identification of the Pseudomonas aeruginosa glmM gene, encoding phosphoglucosamine mutase. J. Bacteriol., 182, 4453-4457 (2000) [2] Jolly, L.; Pompeo, F.; van Heijenoort, J.; Fassy, F.; Mengin-Lecreulx, D.: Autophosphorylation of phosphoglucosamine mutase from Escherichia coli. J. Bacteriol., 182, 1280-1285 (2000) [3] Jolly, L.; Ferrari, P.; Blanot, D.; van Heijenoort, J.; Fassy, F.; Mengin-Lecreulx, D.: Reaction mechanism of phosphoglucosamine mutase from Escherichia coli. Eur. J. Biochem., 262, 202-210 (1999) [4] Jolly, L.; Wu, S.; van Heijenoort, J.; De Lencastre, H.; Mengin-Lcreulx, D.; Tomasz, A.: The femR315 gene from Staphylococcus aureus, the interruption of which results in reduced methicillin resistance, encodes a phophoglucosamine mutase. J. Bacteriol., 179, 5321-5325 (1997) [5] De Reuse, H.; Labigne, A.; Mengin-Lecreulx, D.: The Helicobacter pylori ureC gene codes for a phosphoglucosamine mutase. J. Bacteriol., 179, 34883493 (1997) [6] Mengin-Lecreulx, D.; van Heijenoort, J.: Characterization of the essential gene glmM encoding phosphoglucosamine mutase in Escherichia coli. J. Biol. Chem., 271, 32-39 (1996)

522

(Hydroxyamino)benzene mutase

5.4.4.1

1 Nomenclature EC number 5.4.4.1 Systematic name (hydroxyamino)benzene hydroxymutase Recommended name (hydroxyamino)benzene mutase Synonyms HAB mutase hydroxylaminobenzene hydroxymutase hydroxylaminobenzene mutase CAS registry number 261765-91-7

2 Source Organism <1> Pseudomonas pseudoalcaligenes (no sequence specified) [1, 2, 3, 4, 5]

3 Reaction and Specificity Catalyzed reaction (hydroxyamino)benzene = 2-aminophenol (<1> intramolecular hydroxyl transfer [3,4]; <1> proposed intramolecular rearrangment mechanism, different from the nonenzymatic Bamberger reaction which proceeds via intermolecular hydroxyl transfer [4]) Reaction type dismutation Substrates and products S (hydroxyamino)benzene <1> (<1> highly selective for the production of the ortho-isomer [1,2]) (Reversibility: ?) [1, 2] P 2-aminophenol <1> (<1> highly selective for the production of the orthoisomer [1,2]) [1, 2] S 4-hydoxyaminobiphenyl ether <1> (Reversibility: ?) [1] P 2-amino-5-phenoxyphenol <1> [1]

523

(Hydroxyamino)benzene mutase

5.4.4.1

S 4-hydroxyylaminobenzoate <1> (Reversibility: ?) [4] P 4-amino-3-hydroxybenzoate <1> [4] Inhibitors Triton X-100 <1> (<1> 0.5% [3]) [3] Zn2+ <1> (<1> 1 mM, 70% inhibition [3]) [3] Specific activity (U/mg) 38 <1> [3] Km-Value (mM) 0.15 <1> ((hydroxylamino)benzene, <1> at pH 7.0 [3]) [3] pH-Range 6.5-9.5 <1> (<1> 56% and 59% of maximal activity at pH 6.5 and pH 9.5 respectively [3]) [3]

4 Enzyme Structure Molecular weight Additional information <1> (<1> mutase forms high-molecular-mass complexes which elute with the void-volume, mutase tends to associate even in the presence of SDS [3]) [3]

5 Isolation/Preparation/Mutation/Application Localization Additional information <1> (<1> not an integral membrane protein [2]) [2] Purification <1> (Hitrap-SP/Hitrap-Q, filtration/precipitation, Cu2+ -chelating chromatography) [3] <1> (recombinant HabB and HabA) [2] <1> (recombinant enzyme) [1] Cloning <1> (expression of HabB mutase in Escherichia coli) [1] <1> (expression of habA and habB in Escherichia coli) [2] Application synthesis <1> (<1> expression of enzyme plus nitrobenzene nitroreductase in Escherichia coli. Rapid and stoichiometric conversion of nitrobenzene to 2-aminophenol, of 2-nitroacetophenone to 2-amino-3-hydroxyacetophenone, and of 3-nitroacetophenone to 3-amino-2-hydroxyacetophenone, as well as further conversions. Final yields of aminophenols after extraction and recovery are over 64% [5]) [5]

524

5.4.4.1

(Hydroxyamino)benzene mutase

6 Stability Temperature stability 60 <1> (<1> recombinant HabA, inactivation above, 50% loss of activity at 70 C after 10 min [2]) [2] 85 <1> (<1> recombinant HabB, no loss of activity after 10 min [2]) [2] General stability information <1>, mutase in crude extracts is stable in the presence of 2% SDS, the partially purified enzyme loses 50% activity in the presence of 0.1% SDS after 30 sec [3]

References [1] Nadeau, L.J.; He, Z.; Spain, J.C.: Production of 2-amino-5-phenoxyphenol from 4-nitrobiphenyl ether using nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45. J. Ind. Microbiol. Biotechnol., 24, 301-305 (2000) [2] Davis, J.K.; Paoli, G.C.; He, Z.; Nadeau, L.J.; Somerville, C.C.; Spain, J.C.: Sequence analysis and initial characterization of two isozymes of hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45. Appl. Environ. Microbiol., 66, 2965-2971 (2000) [3] He, Z.; Nadeau, L.J.; Spain, J.C.: Characterization of hydroxylaminobenzene mutase from pNBZ139 cloned from Pseudomonas pseudoalcaligenes JS45: a highly associated SDS-stable enzyme catalyzing an intramolecular transfer of hydroxy groups. Eur. J. Biochem., 267, 1110-1116 (2000) [4] Nadeau, L.J.; He, Z.; Spain, J.C.: Bacterial conversion of hydroxylamino aromatic compounds by both lyase and mutase enzymes involves intramolecular transfer of hydroxyl groups. Appl. Environ. Microbiol., 69, 2786-2793 (2003) [5] Kadiyala, V.; Nadeau, L.J.; Spain, J.C.: Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols. Appl. Environ. Microbiol., 69, 6520-6526 (2003)

525

Isochorismate synthase

1 Nomenclature EC number 5.4.4.2 Systematic name isochorismate hydroxymutase Recommended name isochorismate synthase Synonyms amonabactin EC 5.4.99.6 Eds16 ICS <1, 4, 5, 16> [15, 16] ICS1 gene product <1, 16> [15] IcsI isochorismate mutase isochorismic synthase MbtI <9> [14] PchA <7> [13] Sid2 synthase, isochorismate isochorismate synthase <1, 4, 16> [15] CAS registry number 37318-53-9

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10>

526

Cyanidium caldarium (no sequence specified) [15] Bacillus subtilis (no sequence specified) [4] Escherichia coli (no sequence specified) [2, 3, 6, 10, 12] Glycine max (no sequence specified) [15] Arabidopsis thaliana (no sequence specified) [16] Aerobacter aerogenes (no sequence specified) [1,11] Pseudomonas aeruginosa (no sequence specified) [13] Enterobacter aerogenes (no sequence specified) [6] Mycobacterium tuberculosis (no sequence specified) [14] Flavobacterium sp. (no sequence specified) [5, 6]

5.4.4.2

5.4.4.2

<11> <12> <13> <14> <15> <16>

isochorismate synthase

Galium mollugo (no sequence specified) [8, 9] Morinda citrifolia (no sequence specified) [7] Rubia tinctorum (no sequence specified) [7] Morinda lucida (no sequence specified) [8] Galium uliginosum (no sequence specified) [8] Arabidopsis thaliana (UNIPROT accession number: Q9S7H8) [15]

3 Reaction and Specificity Catalyzed reaction chorismate = isochorismate Reaction type group transfer isomerization Natural substrates and products S chorismate <2, 3> (<3> the first enzyme involved in the biosynthesis of the powerful iron-chelating agent enterobactin [2]; <3> first committed step in the biosynthesis of menaquinone [3]; <2> two different isochorismate mutases, one is involved in the biosynthesis of the respiratory chain component menaquinone, MedF, and the other is involved in the synthesis of siderophore 2,3-dihydroxybenzoate, DhbC [4]; <3> enzyme catalyzes the pivotal step in enterobactin and menaquinone biosynthesis [12]) (Reversibility: ?) [2, 3, 4, 12] P ? S chorismate <1, 4, 5, 7, 16> (<7> first enzyme of pyochelin biosynthesis [13]; <16> alternative pathway to produce salicyl acid in response to pathogens [15]; <5> involved in biosynthesis of salicylic acid [16]) (Reversibility: r) [13, 15, 16] P isochorismate <7> [13] S Additional information <9> (<9> the enzyme is essential for siderohore biosynthesis, first step in salicylate production [14]) (Reversibility: ?) [14] P ? Substrates and products S chorismate <2, 3> (<3> the first enzyme involved in the biosynthesis of the powerful iron-chelating agent enterobactin [2]; <3> first committed step in the biosynthesis of menaquinone [3]; <2> two different isochorismate mutases, one is involved in the biosynthesis of the respiratory chain component menaquinone, MedF, and the other is involved in the synthesis of siderophore 2,3-dihydroxybenzoate, DhbC [4]; <3> enzyme catalyzes the pivotal step in enterobactin and menaquinone biosynthesis [12]) (Reversibility: ?) [2, 3, 4, 12] P isochorismate

527

isochorismate synthase

5.4.4.2

S chorismate <1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16> (<6, 10> r [1, 5]; <3> not reversible, isochorismate synthase involved in enterobactin biosynthesis, MenF [3]; <3> r, isochorismate synthase involved in menaquinone biosynthesis, EntC [2]; <7> first enzyme of pyochelin biosynthesis [13]; <16> alternative pathway to produce salicyl acid in response to pathogens [15]; <5> involved in biosynthesis of salicylic acid [16]) (Reversibility: r) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16] P isochorismate <2, 3, 6, 7, 8, 10, 11, 12, 13, 14, 15> [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] S Additional information <9> (<9> the enzyme is essential for siderohore biosynthesis, first step in salicylate production [14]) (Reversibility: ?) [14] P ? Inhibitors Cu2+ <3> [3] EDTA <6> [1] Hg2+ <3> [3] K+ <3> [3] Mg2+ <3> (<3> above 1 mM [3]) [3] NEM <3> [3] Activating compounds 2-mercaptoethanol <3> (<3> stimulates, 2.25fold stimulation at 20-30 mM [3]) [3] dithiothreitol <3> (<3> stimulates, 2fold stimulation at 1 mM [3]) [3] Metals, ions Mg2+ <3, 6, 7, 10, 11> (<10,11> absolute requirement [5,8,9]; <6> stimulates, optimal concentration: 2-5 mM [1]; <10> optimal concentration for soluble enzyme: 5-20 mM. Optimal concentration for enzyme immobilized on CNBr-Sepharose or alkylamine glass: 2.5-5 mM. Activity of the soluble enzyme without Mg2+ is 5.5% of the activity without Mg2+ [5]; <3> sharp increase in activity, optimal concentration: 1 mM, inhibition above 1 mM [3]; <7> strictly required for activity [13]) [1, 3, 5, 8, 9, 13] Turnover number (min–1) 0.72 <7> (chorismate) [13] 1.33 <3> (chorismate, <3> in absence of 2-mercaptoethanol [3]) [3] 1.8 <3> (isochorismate) [2] 2.88 <3> (chorismate) [2] 2.93 <3> (chorismate, <3> in presence of 2-mercaptoethanol [3]) [3] 6.08 <7> (chorismate) [13] Specific activity (U/mg) 0.0017 <7> [13] 0.808 <3> [3] Additional information <10, 12, 13, 15> (<12,13> HPLC assay allows a rapid and accurate determination [7]) [6, 7, 8]

528

5.4.4.2

isochorismate synthase

Km-Value (mM) 0.0045 <7> (chorismate) [13] 0.005 <3> (isochorismate) [2] 0.014 <3> (chorismate) [2] 0.076 <10> (isochorismate, <10> enzyme immobilized on alkylamine glass [5]) [5] 0.171 <10> (isochorismate, <10> enzyme immobilized on CNBr-Sepharose [5]) [5] 0.195 <3> (chorismate) [3] 0.254 <10> (isochorismate, <10> soluble enzyme [5,6]) [5, 6] 0.27 <10> (chorismate, <10> enzyme immobilized on CNBr-Sepharose [5]) [5] 0.313 <10> (chorismate, <10> enzyme immobilized on alkylamine glass [5]) [5] 0.35 <10> (chorismate, <10> soluble enzyme [5,6]) [5, 6] 0.675 <11> (isochorismate) [9] 0.807 <11> (chorismate) [8, 9] pH-Optimum 6.5-9 <3> (<3> about 20% of maximal activity at pH 6.5 and pH 9.0 [3]) [3] 7 <7> [13] 7.4-8.5 <10> (<10> enzyme immobilized on alkylamine glass [5]) [5] 7.5-8 <3> [3] 7.6-8.2 <10> (<10> enzyme immobilized on CNBr-Sepharose [5]) [5] 7.8-7.9 <11> [8] 8 <6, 10, 11> (<10> soluble enzyme [5]) [1, 5, 9] pH-Range 5.5-8.5 <6> (<6> pH 5.5: about 20% of maximal activity, pH 8.5: about 90% of maximal activity [1]) [1] 5.5-8.8 <11> (<11> about 50% of maximal activity at pH 5.5 and at pH 8.8 [9]) [9] Temperature optimum ( C) 25 <11> [8] 37 <3> [3] 42 <10> (<10> soluble enzyme [5]) [5] 60 <10> (<10> enzyme immobilized on CNBr-Sepharose or alkylamine glass [5]) [5] Temperature range ( C) 15-50 <3> (<3> 15 C: 72% of maximal activity, 20 C: about 74% of maximal activity, 30 C or 40 C: about 80% of maximal activity, 50 C: 42% of maximal activity [3]) [3]

529

isochorismate synthase

5.4.4.2

4 Enzyme Structure Molecular weight 42000 <3> (<3> isochorismate synthase involved in menaquinone biosynthesis, EntC, gel filtration [2]) [2] 45000 <10> (<10> gel filtration [6]) [6] 48000-50000 <7> (<7> gel filtration [13]) [13] 50000 <7> (<7> native PAGE [13]) [13] 98000 <3> (<3> isochorismate synthase involved in enterobactin biosynthesis, MenF, gel filtration [3]) [3] Additional information <3> (<3> identification and sequencing of the gene menF that encodes the enzyme that is responsible for menaquinone biosynthesis. The sequence of MenF is 23.5% identical and 57.8% similar to that of EntC [12]) [12] Subunits ? <3> (<3> x * 48777, isochorismate synthase involved in menaquinone biosynthesis, calculation from nucleotide sequence [10]; <3> x * 49000, isochorismate synthase involved in menaquinone biosynthesis, SDS-PAGE [10]) [10] dimer <3> (<3> 2 * 48000, isochorismate synthase involved in enterobactin biosynthesis, MenF, SDS-PAGE [3]) [3] monomer <3, 7, 9, 10> (<7> 1 * 50000, SDS-PAGE [13]; <3> 1 * 43000, isochorismate synthase involved in menaquinone biosynthesis, EntC, SDS-PAGE [2]; <10> 1 * 36240, SDS-PAGE, a protein with MW 32770 also detected by SDS-PAGE is generated from the 36240 MW protein during the purification procedure [6]; <9> in solution [14]) [2, 6, 13, 14]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <11, 12, 13, 14, 15> [7, 8, 9] leaf <5> (<5> activity is increased in O3-exposed leaves [16]) [16] Localization chloroplast <1, 16> [15] Purification <3> [2, 3] <7> (DEAE-Sepharose, Phenyl-Sepharose, Mono Q) [13] <9> (recombinant) [14] <10> [6] <11> [9] <11> (partial) [8]

530

5.4.4.2

isochorismate synthase

Crystallization <9> (sitting-drop vapour diffusion, crystals diffrect to a maximum resolution of 1.8 A. They belong to space group O2(1)2(1)2(1) with unit-cell parameters a = 51.8 A, b = 163.4 A, c = 194.9 A) [14] Cloning <3> [2, 10] <9> (overexpression in Escherichia coli BL21(DE3)) [14] <16> [15]

6 Stability Temperature stability 4 <10> (<10> half-life of the enzyme immobilized on alkylamine glass: 210 days. Half-life of enzyme immobilized on CNBr Sepharose: 110 days [5]) [5] 37 <10> (<10> 40 h, immobilized enzyme, stable [5]) [5] General stability information <10>, stability of the enzyme is greatly increased by immobilization [5] <11>, no activity in presence of 20 mM ascorbate or 5 mM thiourea. 43% decrease in activity in presence of 10 mM Cys [9] <12, 13>, glycerol, EDTA and dithiothreitol stabilize [7] Storage stability <3>, -20 C, 40% loss of activity after 5 days. 1% bovine serum albumin, 10% glycerol, or 20% dimethyl sulfoxide stabilize for up to 10 days [3] <11>, 4 C or -20 C, 50% loss of activity after 4 days [9] <13>, -80 C, stable for at least 2 months [7]

References [1] Young, I.G.; Gibson, F.: Regulation of the enzymes involved in the biosynthesis of 2,3-dihydroxybenzoic acid in Aerobacter aerogenes and Escherichia coli. Biochim. Biophys. Acta, 177, 401-411 (1969) [2] Liu, J.; Quinn, N.; Berchthold, G.A.; Walsh, C.T.: Overexpression, purification, and characterization of isochorismate synthase (EnrC), the first enzyme involved in the biosynthesis of Enterobactin from Chorismate. Biochemistry, 29, 1417-1425 (1990) [3] Daruwala, R.; Bhattacharyya, D.K.; Kwon, O.; Meganathan, R.: Menaquinone (vitamin K2 ) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli. J. Bacteriol., 179, 3133-3138 (1997) [4] Rowland, B.M.; Taber, H.W.: Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosynthesis of menaquinone and 2,3-dihydroxybenzoate. J. Bacteriol., 178, 854-861 (1996)

531

isochorismate synthase

5.4.4.2

[5] Schaaf, P.M.M.; Heide, L.E.; Leistner, E.W.; Tani, Y.; El-Olemy, M.M.: Immobilization of isochorismate hydroxymutase. Comparison of native versus immobilized enzyme. J. Nat. Prod., 56, 1304-1312 (1993) [6] Schaaf, P.M.; Heide, L.E.; Leistner, E.W.; Tani, Y.; Karas, M.; Deutzmann, R.: Properties of isochorismate hydroxymutase from Flavobacterium K3-15. J. Nat. Prod., 56, 1294-1303 (1993) [7] Poulsen, C.; van der Heijden, R.; Verpoorte, R.: Assay of isochorismate synthase from plant cell cultures by high-performance liquid chromatography. Phytochemistry, 30, 2873-2876 (1991) [8] Leduc, C.; Ruhnau, P.; Leistner, E.: Isochorismate hydroxymutase from Rubiaceae cell suspension culture. Plant Cell Rep., 10, 334-337 (1991) [9] Leduc, C.; Birgel, R.; Muller, R.; Leistner, E.: Isochorismate hydroxymutase from a cell-suspension culture of Galium mollugo L.. Planta, 202, 206-210 (1997) [10] Daruwala, R.; Kwon, O.; Meganathan, R.; Hudspeth, M.E.S.: A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene. FEMS Microbiol. Lett., 140, 159-163 (1996) [11] Zamir, L.O.; Devor, K.A.; Jensen, R.A.; Tiberio, R.; Sauriol, F.; Mamer, O.: Biosynthesis of isochorismate in Klebsiella pneumonia: origin of O-2. Can. J. Microbiol., 37, 276-280 (1991) [12] Muller, R.; Dahm, C.; Schulte, G.; Leistner, E.: An isochorismate hydroxymutase isogene in Escherichia coli. FEBS Lett., 378, 131-134 (1996) [13] Gaille, C.; Reimmann, C.; Haas, D.: Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of Pseudomonas aeruginosa. J. Biol. Chem., 278, 16893-16898 (2003) [14] Harrison, A.J.; Ramsay, R.J.; Baker, E.N.; Lott, J.S.: Crystallization and preliminary x-ray crystallographic analysis of MbtI, a protein essential for siderophore biosynthesis in Mycobacterium tuberculosis. Acta Crystallogr. Sect. F, F61, 121-123 (2005) [15] Wildermuth, M.C.; Dewdney, J.; Wu, G.; Ausubel, F.M.: Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature, 414, 562-565 (2001) [16] Ogawa, D.; Nakajima, N.; Sano, T.; Tamaoki, M.; Aono, M.; Kubo, A.; Kanna, M.; Ioki, M.; Kamada, H.; Saji, H.: Salicylic acid accumulation under O3 exposure is regulated by ethylene in tobacco plants. Plant Cell Physiol., 46, 1062-1072 (2005)

532

3-(Hydroxyamino)phenol mutase

5.4.4.3

1 Nomenclature EC number 5.4.4.3 Systematic name 3-(hydroxyamino)phenol hydroxymutase Recommended name 3-(hydroxyamino)phenol mutase Synonyms 3-hydroxylaminophenol mutase 3HAP mutase CAS registry number 224427-05-8

2 Source Organism <1> Ralstonia eutropha (no sequence specified) (<1> mitochondrial enzyme [1]) [1]

3 Reaction and Specificity Catalyzed reaction 3-hydroxyaminophenol = aminohydroquinone (<1> enzyme requires neither a cofactor nor oxygen for the enzymatic reaction, enzyme catalyzes a Bamberg-type rearrangement [1]) Natural substrates and products S 3-hydroxyaminophenol <1> (<1> the enzyme is involved in the degradative pathway of 3-nitrophenol [1]) (Reversibility: ?) [1] P aminohydroquinone Substrates and products S 2-chloro-5-hydroxylaminophenol <1> (Reversibility: ?) [1] P 2-amino-5-chlorohydroquinone <1> [1] S 3-hydroxyaminophenol <1> (<1> the enzymatic reaction is regiospecific: the enzyme directs the formation of aminohydroquinone exclusively, whereas the chemical reaction forms the isomeric 4-aminocatechol [1];

533

3-(Hydroxyamino)phenol mutase

P S P S P S P

5.4.4.3

<1> the enzyme is involved in the degradative pathway of 3-nitrophenol [1]) (Reversibility: ?) [1] aminohydroquinone 4-hydroxyaminotoluene <1> (Reversibility: ?) [1] 6-amino-m-cresol <1> [1] hydroxylaminobenzene <1> (Reversibility: ?) [1] 2-aminophenol + 4-aminophenol <1> [1] Additional information <1> (<1> not: 4-hydroxylaminobenzoate [1]) (Reversibility: ?) [1] ?

Inhibitors 1,10-phenanthroline <1> (<1> weak [1]) [1] 3-nitrosophenol <1> (<1> autoxidation product of substrate 3-hydroxyaminophenol, inhibition can be partly reversed by addition of 2 mM DTT and/or 10 mM hydroxylamine [1]) [1] AgNO3 <1> (<1> at 1 mM [1]) [1] H2 O2 <1> (<1> at 0.0003 mM [1]) [1] l-cysteine <1> (<1> 39% loss of activity after 90 min, 83% loss of activity after 240 min [1]) [1] Additional information <1> (<1> not: EDTA, tiron, 10 mM DTT, 0.026 mM NaBH4 , 0.1 mM KOCN, 0.1 mM NaN3 , Co2+, Cu2+ , Fe2+ , Fe3+ , Mg2+ , Mn2+ , Ni2+ , Zn2+ , NaNO2, hydroxylamine [1]) [1] Metals, ions Additional information <1> (<1> metal cations play no role in the reaction mechanism [1]) [1] Specific activity (U/mg) 4.7 <1> [1] Km-Value (mM) 0.1 <1> (3-hydroxyaminophenol, <1> pH 7.0, on ice [1]) [1] pH-Optimum 6.5 <1> [1]

4 Enzyme Structure Subunits ? <1> (<1> x * 62000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue culture condition:3-nitrophenol grown-cell <1> [1] culture condition:succinate grown-cell <1> [1]

534

5.4.4.3

3-(Hydroxyamino)phenol mutase

Purification <1> [1]

6 Stability Temperature stability 60 <1> (<1> 1 min: 72% loss of activity, 8 min: complete loss of activity [1]) [1] Storage stability <1>, -70 C, 7% loss of activity, after 30 days [1] <1>, storage on ice, 13% loss of activity after 14 days [1] <1>, storage on ice, 37% loss of activity after 38 days [1]

References [1] Schenzle, A.; Lenke, H.; Spain, J.C.; Knackmuss, H.J.: 3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 catalyzes a Bamberger rearrangement. J. Bacteriol., 181, 1444-1450 (1999)

535

Squalene-hopene cyclase

5.4.99.17

1 Nomenclature EC number 5.4.99.17 Systematic name squalene mutase (cyclizing) Recommended name squalene-hopene cyclase Synonyms SHC <4> [20, 22, 23, 24, 25, 26] cyclase, squalene-hopanoid CAS registry number 76600-69-6

2 Source Organism <1> <2> <3> <4>

Rhodopseudomonas palustris (no sequence specified) [9] Zymomonas mobilis (no sequence specified) [3, 15] Methylococcus capsulatus (no sequence specified) [17] Alicyclobacillus acidocaldarius (no sequence specified) [1, 2, 4, 5, 6, 7, 8, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27] <5> Bacillus acidocaldarius (no sequence specified) [11, 16]

3 Reaction and Specificity Catalyzed reaction squalene + H2 O = hopan-22-ol squalene = hop-22(29)-ene Reaction type cyclization Substrates and products S (3S)-2,3-oxidosqualene <4> (Reversibility: ?) [25] P lanosterol S (E,E,E,E)-2,6,10,14,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26]

536

5.4.99.17

Squalene-hopene cyclase

P 8-(4,8-dimethyl-nona-3,7-dienyl)-1,1,4a,8a-tetramethyl-7-methylene-tetradecahydro-phenanthrene (<4> low conversion, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,18,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P 1,1,4a,10a,10b-pentamethyl-8-methylene-7-(4-methyl-pent-3-enyl)-octadecahydro-chrysene (<4> one of the three major products, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,18,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P 1,1,4a,6a,8,10a-hexamethyl-7-(4-methyl-pent-3-enyl)1,2,3,4,4a,4b,5,6,6a,7,8,9,10,10a,12,12a-hexadecahydro-chrysene (<4> one of the three major products, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,18,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P 1,1,4a,8,10a,10b-hexamethyl-7-(4-methyl-pent-3-enyl)1,2,3,4,4a,4b,5,6,8,9,10,10a,10b,11,12,12a-hexadecahydro-chrysene (<4> one of the three major products, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,18,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P 2,4a,4b,7,7,10a-hexamethyl-1-(4-methyl-pent-3-enyl)-octadecahydrochrysen-2-ol (<4> minor product, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,18,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P 2-(3a,5a,5b,8,8,11a-hexamethyl-icosahydro-cyclopenta[a]chrysen-3-yl)propan-2-ol (<4> minor product, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,18,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P a-3-isopropenyl-3a,5a,5b,8,8,11a-hexamethyl-icosahydro-cyclopenta[a]chrysene (<4> minor product, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,18,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P b-3-isopropenyl-3a,5a,5b,8,8,11a-hexamethyl-icosahydro-cyclopenta[a]chrysene (<4> minor product, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,23-pentamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P 2-(5a,5b,8,8,11a-pentamethyl-icosahydro-cyclopenta[a]chrysen-3-yl)-propan-2-ol (<4> one of the three major products, yield 19.8%, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,23-pentamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26] P 3-isopropenyl-5a,5b,8,8,11a-pentamethyl-icosahydro-cyclopenta[a]chrysene (<4> one of the three major products, yield 29.4%, NMR spectroscopic analysis [26]) S (E,E,E,E)-2,6,10,15,23-pentamethyltetracosa-2,6,10,14,18,22-hexaene <4> (Reversibility: ?) [26]

537

Squalene-hopene cyclase

5.4.99.17

P 3-isopropenyl-5a,5b,8,8,13b-pentamethyl-icosahydro-cyclopenta[a]chrysene (<4> one of the three major products, yield 34.8%, NMR spectroscopic analysis [26]) S 2,3-oxidosqualene + H2 O <4> (<4> mutant enzymes produce hop-22(29)en-3-ols [2]) (Reversibility: ?) [2, 10, 13] P ? S 3-(farnesyldimethylallyl)indole <4> (Reversibility: ?) [27] P ? (<4> conversion into a 2:1 mixture of a tetracyclic and a pentacyclic product [27]) S squalene <4> (Reversibility: ?) [22] P ? S squalene <4> (Reversibility: ?) [24, 25] P hopene S squalene <1, 4, 5> (<5> products are formed in a molar ratio of hopene:hopanol, 5:1 [16]) (Reversibility: ?) [2, 5, 7, 8, 9, 10, 12, 13, 14, 16, 20] P hop-22(29)-ene <1, 4> [2, 5, 7, 9, 12, 14] S squalene + H2 O <1, 4> (Reversibility: ?) [2, 7, 8, 9, 10, 12, 13, 14, 20] P hopan-22-ol <1, 4> [2, 7, 9, 12, 14] S Additional information <2, 4> (<4> mutations lead to altered product pattern [5,6,7,14]; <4> overview of cyclization products of wild type and mutant enzymes [8,12]; <2> enzyme produces a wide variety of products due to lack of specificity [3]; <4> (E,E,E,E)-2,6,11,14,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene: no detectable enzymatic activity [26]; <4> no enzymatic cyclization of 3-(geranylgeranyl)indole [27]) (Reversibility: ?) [3, 5, 6, 7, 8, 12, 14, 26, 27] P ? Inhibitors (18E)-29-methylidenehexanor-2,3-oxidosqualene <4> (<4> IC50 0.2 microMol, pH 6.0, 55 C [24]) [24] (1E,3E,7E,11E)-15,16-epoxy-8,12,16-trimethyl-1-methylthio-1,3,7,11-heptadecatetraene <4> (<4> IC50 1 microMol, pH 6.0, 55 C [24]) [24] (1E,3E,7E,11E,15E)-19,20-epoxy-7,12,16,20-tetramethyl-1-methylthio1,3,7,11,15-heneicosapentaene <4> (<4> IC50 1.4 microMol, pH 6.0, 55 C, not time-dependency up to 10fold higher concentration than IC50 [24]) [24] (1Z,3E,7E,11E)-15,16-epoxy-8,12,16-trimethyl-1-methylthio-1,3,7,11-heptadecatetraene <4> (<4> IC50 4 microMol, pH 6.0, 55 C [24]) [24] (1Z,3E,7E,11E,15E)-19,20-epoxy-7,12,16,20-tetramethyl-1-methylthio1,3,7,11,15-heneicosapentaene <4> (<4> IC50 1.8 microMol, pH 6.0, 55 C, not time-dependency up to 10fold higher concentration than IC50 [24]) [24] (3E,7E,11E)-15,16-epoxy-8,12,16-trimethyl-1-phenylthio-1,3,7,11-heptadecatetraene <4> (<4> IC50 2.2 microMol, pH 6.0, 55 C [24]) [24] (4-(2-[(allyl-cyclopropyl-amino)-methyl]-cyclopropylmethoxy)-phenyl)-(4bromo-phenyl)-methanone <4> (<4> IC50 59 nM [23]) [23] (4-[6-(allyl-methyl-amino)-hexyloxy]-2-fluoro-phenyl)-(4-bromo-phenyl)methanone <4> (<4> IC50 60 nM [23]) [23]

538

5.4.99.17

Squalene-hopene cyclase

(4-[6-(allyl-methyl-amino)-hexyloxy]-phenyl)-(4-bromo-phenyl)-methanone <4> (<4> IC50 96 nM [23]) [23] (4-bromo-phenyl)-(4-(2-[(cyclopropyl-methyl-amino)-methyl]-cyclopropylmethoxy)-2-fluoro-phenyl)-methanone <4> (<4> IC50 50 nM [23]) [23] (4-bromo-phenyl)-(4-(2-[(cyclopropyl-methyl-amino)-methyl]-cyclopropylmethoxy)-phenyl)-methanone <4> (<4> IC50 62 nM [23]) [23] (4-bromo-phenyl)-(4-[4-(cyclopropyl-methyl-amino)-but-2-enyloxy]-phenyl)-methanone <4> (<4> IC50 18 nM [23]) [23] (4-bromo-phenyl)-(4-[6-(cyclopropyl-methyl-amino)-hexyloxy]-2-fluorophenyl)-methanone <4> (<4> IC50 38 nM [23]) [23] (4-chloro-phenyl)-(4-[4-(4,5-dihydro-oxazol-2-yl)-benzylidene]-piperidin-1yl)-methanone <4> (<4> IC50 2800 nM [23]) [23] (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate <5> (<5> 99% inhibition at 1 mM [16]; <5> i.e. AMO 1618, competitive inhibition [11]) [11, 16] (5E,9E)-13,14-epoxy-6,10,14-trimethyl-1-phenylthio-1,5,9-pentadecatriene <4> (<4> IC50 7 microMol, pH 6.0, 55 C [24]) [24] (5E,9E,13E)-17,18-epoxy-5,10,14,18-tetramethyl-1-phenylthio-1,5,9,13-nonadecatetraene <4> (<4> IC50 3 microMol, pH 6.0, 55 C [24]) [24] 1-(4-(4-[(4-chloro-phenoxycarbonyl)-methyl-amino]-cyclohexyl)-benzyl)-1hydroxy-piperidinium <4> (<4> IC50 123 nM [23]) [23] 3-(10’-(allylmethylamino)decanoyl)chroman-2,4-dione <4> (<4> IC50 100 microMol [22]) [22] 3-carboxy-4-nitrophenyl-dithio-1,1’,2-trisnorsqualene <4> (<4> covalently modifies C435 [10]) [10] 4’-[4-(allyl-methyl-amino)-but-2-enyloxy]-biphenyl-4-yl-(4-bromo-phenyl)methanone <4> (<4> IC50 29 nM [23]) [23] 4-[4-(allyl-methyl-amino)-but-2-enyloxy]-phenyl-(4-bromo-phenyl)-methanone <4> (<4> IC50 40 nM [23]) [23] 4-[6-(allyl-methyl-amino)-hexyloxy]-piperidin-1-yl-(4-fluoro-phenyl)-methanone <4> (<4> IC50 1200 nM [23]) [23] 4-[6-(cyclopropyl-methyl-amino)-hexyloxy]-piperidin-1-yl-(4-fluoro-phenyl)-methanone <4> (<4> IC50 760 nM [23]) [23] 7-(10’-(dimethylamino-N-decyloxy))chromen-2-one <4> (<4> IC50 5 microMol [22]) [22] 7-(10-(allylmethylamino)-decyloxy)chromen-2-one <4> (<4> IC50 2 microMol [22]) [22] 7-(4’-(N,N,N’-trimethylethylethylendiamino)-but-2-ynyloxy)chromen-2-one <4> (<4> not active at 100 microMol [22]) [22] 7-(4’-(N-diethylamino)-but-2-ynyloxy)chromen-2-one <4> (<4> IC50 5 microMol [22]) [22] 7-(4’-(N-pyrrolidyn)-but-2-ynyloxy)chromen-2-one <4> (<4> IC50 5 microMol [22]) [22] 7-(4’-allylmethylamino-but-2-ynyloxy)chromen-2-one <4> (<4> IC50 0.75 microMol [22]) [22] 7-(6’-(benzylamino-hexyloxy))chromen-2-one <4> (<4> IC50 8 microMol [22]) [22]

539

Squalene-hopene cyclase

5.4.99.17

7-(6-(allylmethylamino)-hexyloxy)chromen-2-one <4> (<4> IC50 4-5 microMol [22]) [22] 7-(8’-(dimethylamino-N-octyloxy))chromen-2-one <4> (<4> IC50 5-7 microMol [22]) [22] 7-(morpholinyl-N-hexyloxy)chromen-2-one <4> (<4> IC50 6 microMol [22]) [22] 7-(morpholinyl-N-octyloxy)chromen-2-one <4> (<4> IC50 7 microMol [22]) [22] 7-(piperidinyl-N-hexyloxy)chromen-2-one <4> (<4> IC50 8 microMol [22]) [22] Cu2+ <5> (<5> slight inhibition at 1 mM [11]) [11] diethyldicarbonate <5> (<5> 92% inhibition at 5 mM [16]) [16] farnesol <5> (<5> inhibition at 0.1 mM [11]) [11] Fe2+ <5> (<5> slight inhibition at 1 mM [11]) [11] HECAMEG <1> (<1> 80% inactivation compared to CHAPS [9]) [9] N,N-dimethyldodecylamine N-oxide <4> (<4> forms a complex with the enzyme [8]) [8] N-ethylmaleimide <5> (<5> 65% inhibition at 5 mM, 20% inhibition at 1 mM [16]) [16] N-dodecyliodoacetamide <4> (<4> IC50 wild-type > 200 microMol, quintuple mutant > 200 microMol, sextuple mutant > 200 microMol, pH 6.0, 50 C [25]) [25] N-squalenyliodoacetamide <4> (<4> IC50 wild-type > 200 microMol, quintuple mutant > 200 microMol, sextuple mutant 50 microMol, pH 6.0, 50 C [25]) [25] Ro 48-8071 <4> (<4> IC50 0.35 microMol [22]) [22] sodium dodecylsulfate <5> (<5> strong inhibition [11]) [11] sodium taurodeoxycholate <5> (<5> under 0.15% and above 0.25% [11]) [11] taurodeoxycholate <1> (<1> 80% inactivation compared to CHAPS [9]) [9] Triton-X100 <1> (<1> 96% inhibition [9]) [9] Zn2+ <5> (<5> slight inhibition at 5 mM [11]) [11] Zwittergent <1> (<1> 80% inactivation compared to CHAPS [9]) [9] allyl-(4-[3-(4-bromo-phenyl)-5-fluoro-1-methyl-1H-indazol-6-yloxy]-but-2enyl)-methyl-amine <4> (<4> IC50 281 nM [23]; <4> IC50 332 nM [23]) [23] allyl-(4-[3-(4-bromo-phenyl)-benzo[b]thiophen-6-yloxy]-butyl)-methylamine <4> (<4> IC50 75 nM [23]) [23] allyl-(4-[3-(4-bromo-phenyl)-benzo[d]isoxazol-6-yloxy]-but-2-enyl)-amine <4> (<4> IC50 49 nM [23]) [23] allyl-(4-[3-(4-bromo-phenyl)-benzofuran-6-yloxy]-but-2-enyl)-methyl-amine <4> (<4> IC50 23 nM [23]) [23] allyl-(4-[4-(6-bromo-benzo[d]isothiazol-3-yl)-phenoxy]-but-2-enyl)-methylamine <4> (<4> IC50 130 nM [23]) [23] allyl-(6-[1-(4-bromo-phenyl)-isoquinolin-6-yloxy]-hexyl)-methyl-amine <4> (<4> IC50 186 nM [23]) [23] allyl-(6-[3-(4-bromo-phenyl)-1-methyl-1H-indazol-6-yloxy]-hexyl)-methylamine <4> (<4> IC50 289 nM [23]) [23]

540

5.4.99.17

Squalene-hopene cyclase

allyl-(6-[3-(4-bromo-phenyl)-1H-indazol-6-yloxy]-hexyl)-methyl-amine <4> (<4> IC50 180 nM [23]) [23] allyl-(6-[3-(4-bromo-phenyl)-benzo[d]isothiazol-6-yloxy]-hexyl)-methylamine <4> (<4> IC50 306 nM [23]) [23] allyl-(6-[3-(4-bromo-phenyl)-benzo[d]isoxazol-6-yloxy]-hexyl)-methylamine <4> (<4> IC50 75 nM [23]) [23] allyl-(6-[3-(4-bromo-phenyl)-benzofuran-6-yloxy]-hexyl)-methyl-amine <4> (<4> IC50 80 nM [23]) [23] allyl-(6-[4-(4-bromo-phenyl)-1H-benzo[d][1,2]oxazin-7-yloxy]-hexyl)methyl-amine <4> (<4> IC50 172 nM [23]) [23] allyl-(6-[4-(6-bromo-benzo[d]isothiazol-3-yl)-phenoxy]-hexyl)-methylamine <4> (<4> IC50 141 nM [23]) [23] azasqualene <5> (<5> inhibition at 0.001 mM [11]) [11] dodecyldimethylamine N-oxide <5> (<5> competitive inhibition [11]) [11] dodecyltrimethylammonium bromide <5> (<5> competitive inhibition, 50% inhibition at 0.0001 mM [11]) [11] methyl-[4-(4-piperidin-1-ylmethyl-phenyl)-cyclohexyl]-carbamic acid 4chloro-phenyl ester <4> (<4> IC50 406 nM [23]) [23] octylthiogucopyranoside <1> (<1> complete inactivation [9]) [9] p-chloromercuribenzenesulfonic acid <5> (<5> 96% inhibition at 1 mM [16]) [16] squalene-maleimide <4> (<4> time-dependent inhibitor [10]) [10] Additional information <3, 4, 5> (<4> vinyl sulfide and ketene dithioacetal derivates of truncated 2,3-ocidosqualene interact with active site of the enzyme [1]; <4> effect of thiol-modifying inhibitors on mutant enzymes [10]; <4> sulfur-substituted oxidosqualene analogues serve as inhibitors [8]; <3> several detergents have inhibitory effect [17]; <4> sulfur-containing analogues of 2,3-oxidosqualene inhibit enzyme activity, 50% inhibition at concentrations in the nanomolar range [19]; <5> inhibition by n-alkyldimethylammoniumhalides with alkyl chain lengths between 12 and 18 C atoms, inhibition increases with decreasing chain length [11]; <4> inhibitors designed as cholesterol-lowering agents, for 11 inhibitors the structures of the enzymeinhibitor complexes were determined by X-ray crystallography [23]) [1, 8, 10, 11, 17, 19, 23] Activating compounds ethanol <2> (<2> 1.6fold increase of activity when added to the enzyme test system at a concentration of 6% [15]) [15] propanol <2> (<2> 1.6fold increase of activity when added to the enzyme test system at a concentration of 6% [15]) [15] sodium taurodeoxycholate <5> (<5> 1.5fold activation at 0.16% [11]) [11] Turnover number (min–1) Additional information <4> (<4> wild type and mutant enzymes [8, 10, 12, 14]) [8, 10, 12, 14]

541

Squalene-hopene cyclase

5.4.99.17

Specific activity (U/mg) 2.27e-006 <2> (<2> crude extract [15]) [15] 0.1487 <1> [9] 0.345 <5> [11] Additional information <4> (<4> specific activities for wild type and mutant enzymes [4,8,12,13]) [4, 8, 12, 13] Km-Value (mM) 0.001 <4> ((3S)-2,3-oxidosqualene, <4> quintuple mutant, 50 C, pH 6.0 [25]) [25] 0.0015 <4> ((3S)-2,3-oxidosqualene, <4> wild-type, 50 C, pH 6.0 [25]) [25] 0.0023 <4> ((3S)-2,3-oxidosqualene, <4> sextuple mutant, 50 C, pH 6.0 [25]) [25] 0.003 <5> (squalene) [11, 16] 0.01 <4> (squalene, <4> quintuple mutant, 50 C, pH 6.0 [25]) [25] 0.0132 <4> (squalene, <4> wild-type, 50 C, pH 6.0 [25]) [25] 0.0162 <4> (squalene, <4> wild-type, 45 C, pH 6.0 [20]) [20] 0.0167 <4> (squalene, <4> mutant T41A, 45 C, pH 6.0 [20]) [13, 20] 0.0169 <4> (squalene, <4> mutant W133A, 45 C, pH 6.0 [20]) [20] 0.017 <4> (squalene, <4> wild type enzyme and D376E mutant [4]) [4] 0.0189 <4> (squalene, <4> mutant E93A, 45 C, pH 6.0 [20]) [20] 0.0213 <4> (squalene, <4> mutant E45A, 45 C, pH 6.0 [20]) [20] 0.0255 <4> (squalene, <4> mutant R127Q, 45 C, pH 6.0 [20]) [20] 0.102 <4> (squalene, <4> mutant Q262A, 45 C, pH 6.0 [20]) [20] 0.156 <4> (squalene, <4> mutant Q262G, 45 C, pH 6.0 [20]) [20] 0.185 <4> (squalene, <4> mutant P263A, 45 C, pH 6.0 [20]) [20] 0.197 <4> (squalene, <4> mutantY267A, 45 C, pH 6.0 [20]) [20] 0.237 <4> (squalene, <4> mutant P263G, 45 C, pH 6.0 [20]) [20] 0.816 <4> (squalene, <4> mutant F434A, 45 C, pH 6.0 [20]) [20] 0.955 <4> (squalene, <4> mutant F437A, 45 C, pH 6.0 [20]) [20] Additional information <4> (<4> Km for wild type and mutant enzymes [2, 8, 10, 12, 13, 14]; <4> squalene sextuple mutant: no activity [25]) [2, 8, 10, 12, 13, 14, 25] Ki-Value (mM) 0.00014 <5> (dodecyldimethylamine N-oxide) [11] 0.00032 <5> (dodecyltrimethylammonium bromide) [11] 0.00054 <5> ((5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate) [11] Additional information <4> (<4> Ki for sulfur-substituted oxidosqualene analogues [8,19]) [8, 19] pH-Optimum 6 <4, 5> (<4> F605A mutant [7]) [7, 11, 16] 6.5 <1> [9] 6.8 <3> [17]

542

5.4.99.17

Squalene-hopene cyclase

pH-Range 4.8-8 <3> (<3> 50% activity at pH 8, very low activity at pH 4.8 [17]) [17] 5-8 <1> (<1> no activity below pH 5 or above pH 8 [9]) [9] Temperature optimum ( C) 30 <1> [9] 35-50 <4> (<4> mutant Q262G [20]) [20] 40-50 <3> [17] 45 <4> (<4> mutant F437A0 [20]) [20] 45-55 <4> (<4> mutant Q262A [20]) [20] 50 <4> (<4> Y612F mutant [6]; <4> F605A mutant [7]; <4> mutant E45A [20]; <4> mutant P263A [20]; <4> mutant P263G [20]; <4> mutant R127Q [20]) [6, 7, 20] 50-60 <4> (<4> mutantY267A [20]) [20] 55 <4> (<4> W258L mutant [13]; <4> mutant E93A [20]; <4> mutant W133A [20]) [13, 20] 60 <4, 5> (<4> wild-type [20]; <4> wild type enzyme [6,7]; <4> mutant F434A [20]; <4> mutant T41A [20]) [6, 7, 11, 13, 16, 20] Additional information <4> (<4> wild type and mutant enzymes [2, 6, 8, 12, 13, 14]) [2, 6, 8, 12, 13, 14]

4 Enzyme Structure Molecular weight 70000 <1> (<1> SDS-PAGE [9]) [9] 72000 <4> (<4> SDS-PAGE [4,19]) [4, 19] 74000 <3> (<3> calculated from amino acid sequence [17]) [17] 75000 <5> (<5> SDS-PAGE [16]) [16] 150000 <5> (<5> gel filtration [11]) [11] Subunits dimer <5> (<5> 2 * 75000, SDS-PAGE, enzyme forms aggregates in absence of detergent [11]) [11]

5 Isolation/Preparation/Mutation/Application Localization cytoplasmic membrane <5> [11] membrane <1, 2, 3, 4, 5> [6, 9, 15, 16, 17, 18] Purification <1> (2000-fold) [9] <4> [7, 13, 20, 25, 27] <4> (ion exchange and gel filtration) [21] <4> (wild type and mutants, homogeneity) [4] <5> [16] <5> (homogeneity) [11]

543

Squalene-hopene cyclase

5.4.99.17

Crystallization <4> [7] <4> (cocrystallization with 2-azasqualene) [21] <4> (complexes with 11 human oxidosqualene cyclase inhibitors produced by cocrystallization, elucidation of the structures by X-ray diffraction analyses) [23] <4> (resolution of 2.0 ) [18] Cloning <3> [17] <4> [2, 4, 5, 6, 7, 8, 10, 12, 13, 27] <4> (expression in Escherichia coli) [21, 22, 26] <4> (expression in Escherichia coli BL21) [20] <4> (expression in Escherichia coli JM 105) [23, 25] Engineering C25S/C50S/C435S/C455S/C537S <4> (<4> quintuple mutant [25]) [25] C25S/C50S/D376C/C435S/C455S/C537S <4> (<4> sextuple mutant [25]) [25] D376E <4> (<4> 10% enzyme activity [4]) [4] D376E/D377E <4> (<4> no enzyme activity [4]) [4] D376G <4> (<4> 0.2% activity when enzyme concentration is increased to 100fold [4]) [4] D376Q <4> (<4> no enzyme activity [4]) [4] D376R <4> (<4> no enzyme activity [4]) [4] D377C/V380E/V381A <4> (<4> no detectable cyclization of squalene [2]) [2] D377E <4> (<4> 0.2% activity when enzyme concentration is increased to 100fold [4]) [4] D377G <4> (<4> 0.2% activity when enzyme concentration is increased to 100fold [4]) [4] D377Q <4> (<4> 0.2% activity when enzyme concentration is increased to 100fold [4]) [4] D377R <4> (<4> no enzyme activity [4]) [4] E45A <4> (<4> reduced enzyme activity [2]; <4> mutation located around the “back waters“ [20]) [2, 20] E45D <4> (<4> reduced enzyme activity [2]) [2] E45K <4> (<4> no enzyme activity [2]) [2] E45Q <4> (<4> slightly increased enzyme activity [2]) [2] E93A <4> (<4> mutation located around the “back waters“ [20]) [20] F434A <4> (<4> mutation near the substrate channel [20]) [20] F437A <4> (<4> mutation near the substrate channel [20]) [20] F605A <4> (<4> altered product pattern [7]) [7] P263A <4> (<4> mutation located between C29 of the hopanyl cation and the “front water“ [20]) [20] P263G <4> (<4> mutation located between C29 of the hopanyl cation and the “front water“ [20]) [20] Q262A <4> (<4> mutation located between C29 of the hopanyl cation and the “front water“ [20]) [20]

544

5.4.99.17

Squalene-hopene cyclase

Q262G <4> (<4> mutation located between C29 of the hopanyl cation and the “front water“ [20]) [20] R127Q <4> (<4> mutation located around the “back waters“ [20]) [20] T41A <4> (<4> mutation located around the “back waters“ [20]) [20] W133A <4> (<4> mutation located around the “back waters“ [20]) [20] W23V <4> (<4> same activity and optimal temperature as wild type enzyme [13]) [13] W258L <4> (<4> 60% of wild type activity, lower temperature optimum [13]) [13] W406V <4> (<4> no enzyme activity [13]) [13] W417A <4> (<4> no enzyme activity [13]) [13] W485V <4> (<4> same activity and optimal temperature as wild type enzyme [13]) [13] W522V <4> (<4> same activity and optimal temperature as wild type enzyme [13]) [13] W533A <4> (<4> same activity and optimal temperature as wild type enzyme [13]) [13] W591L <4> (<4> same activity and optimal temperature as wild type enzyme [13]) [13] W78S <4> (<4> same activity and optimal temperature as wild type enzyme [13]) [13] Y267A <4> (<4> mutation near the substrate channel [20]) [20] Y495F <4> (<4> reduced enzyme activity, wild-type product pattern [6]) [6] Y609F <4> (<4> wild type activity, altered product pattern [6]) [6] Y612F <4> (<4> reduced enzyme activity, wild-type product pattern [6]) [6] Additional information <4> (<4> overview [8]; <4> modification of critically located Cys residues [10]; <4> various mutations of conserved amino acid residues [12]; <4> mutations of Y609, Y495, Y612 and Y420 lead to an altered product pattern, compared to wild-type enzyme [5]) [5, 8, 10, 12]

6 Stability Temperature stability 60 <4> (<4> wild type enzyme: 50% loss of activity after 120 min, D376E mutant: 50% loss of activity after 100 min [4]) [4] 70 <4> (<4> stable for 10 min [6]) [6] Storage stability <1>, -20 C, stable for weeks [9] <1>, 6 C, stable for several days [9]

545

Squalene-hopene cyclase

5.4.99.17

References [1] Ceruti, M.; Balliano, G.; Rocco, F.; Milla, P.; Arpicco, S.; Cattel, L.; Viola, F.: Vinyl sulfide derivatives of truncated oxidosqualene as selective inhibitors of oxidosqualene and squalene-hopene cyclases. Lipids, 36, 629-636 (2001) [2] Dang, T.; Prestwich, G.D.: Site-directed mutagenesis of squalene-hopene cyclase: altered substrate specificity and product distribution. Chem. Biol., 7, 643-649 (2000) [3] Douka, E.; Koukkou, A.; Drainas, C.; Grosdemange-Billiard, C.; Rohmer, M.: Structural diversity of the triterpenic hydrocarbons from the bacterium Zymomonas mobilis: the signature of defective squalene cyclization by the squalene/hopene cyclase. FEMS Microbiol. Lett., 199, 247-251 (2001) [4] Feil, C.; Suessmuth, R.; Jung, G.; Poralla, K.: Site-directed mutagenesis of putative active-site residues in squalene-hopene cyclase. Eur. J. Biochem., 242, 51-55 (1996) [5] Full, C.: Bicyclic triterpenes as new main products of squalene-hopene cyclase by mutation at conserved tyrosine residues. FEBS Lett., 509, 361-364 (2001) [6] Full, C.; Poralla, K.: Conserved Tyr residues determine functions of Alicyclobacillus acidocaldarius squalene-hopene cyclase. FEMS Microbiol. Lett., 183, 221-224 (2000) [7] Hoshino, T.; Kouda, M.; Abe, T.; Sato, T.: Functional analysis of Phe605, a conserved aromatic amino acid in squalene-hopene cyclases. Chem. Commun., 2000, 1485-1486 (2000) [8] Hoshino, T.; Sato, T.: Squalene-hopene cyclase: catalytic mechanism and substrate recognition. Chem. Commun., 2000, 291-301 (2002) [9] Kleemann, G.; Kellner, R.; Poralla, K.: Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris, a purple non-sulfur bacterium producing hopanoids and tetrahymanol. Biochim. Biophys. Acta, 1210, 317-320 (1994) [10] Milla, P.; Lenhart, A.; Grosa, G.; Viola, F.; Weihofen, W.A.; Schulz, G.E.; Balliano, G.: Thiol-modifying inhibitors for understanding squalene cyclase function. Eur. J. Biochem., 269, 2108-2116 (2002) [11] Ochs, D.; Tappe, C.H.; Gaertner, P.; Kellner, R.; Poralla, K.: Properties of purified squalene-hopene cyclase from Bacillus acidocaldarius. Eur. J. Biochem., 194, 75-80 (1990) [12] Sato, T.; Hoshino, T.: Functional analysis of the DXDDTA motif in squalenehopene cyclase by site-directed mutagenesis experiments: initiation site of the polycyclization reaction and stabilization site of the carbocation intermediate of the initially cyclized A-ring. Biosci. Biotechnol. Biochem., 63, 2189-2198 (1999) [13] Sato, T.; Hoshino, T.: Kinetic studies on the function of all the conserved tryptophans involved inside and outside the QW motifs of squalene-hopene cyclase: stabilizing effect of the protein structure against thermal denaturation. Biosci. Biotechnol. Biochem., 63, 1171-1180 (1999) [14] Sato, T.; Sasahara, S.; Yamakami, T.; Hoshino, T.: Functional analyses of Tyr420 and Leu607 of Alicyclobacillus acidocaldarius squalene-hopene cy-

546

5.4.99.17

[15]

[16] [17] [18] [19] [20] [21] [22] [23]

[24]

[25] [26]

[27]

Squalene-hopene cyclase

clase. Neoachillapentaene, a novel triterpene with the 1,5,6-trimethylcyclohexene moiety produced through folding of the constrained boat structure. Biosci. Biotechnol. Biochem., 66, 1660-1670 (2002) Schmidt, A.; Bringer-Meyer, S.; Poralla, K.; Sahm, H.: Influence of ethanol on the activities of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase and squalene-hopene-cyclase in Zymomonas mobilis. Appl. Microbiol. Biotechnol., 30, 170-175 (1989) Seckler, B.; Poralla, K.: Characterization and partial purification of squalene-hopene cyclase from Bacillus acidocaldarius. Biochim. Biophys. Acta, 881, 356-363 (1986) Tippelt, A.; Jahnke, L.; Poralla, K.: Squalene-hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids. Biochim. Biophys. Acta, 1391, 223-232 (1998) Wendt, K.U.; Lenhart, A.; Schulz, G.E.: The structure of the membrane protein squalene-hopene cyclase at 2.0 resolution. J. Mol. Biol., 286, 175-187 (1999) Zheng, Y.F.; Abe, I.; Prestwich, G.D.: Inhibition kinetics and affinity labeling of bacterial squalene:hopene cyclase by thia-substituted analogs of 2,3-oxidosqualene. Biochemistry, 37, 5981-5987 (1998) Sato, T.; Kouda, M.; Hoshino, T.: Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius. Biosci. Biotechnol. Biochem., 68, 728-738 (2004) Reinert, D.J.; Balliano, G.; Schulz, G.E.: Conversion of squalene to the pentacarbocyclic hopene. Chem. Biol., 11, 121-126 (2004) Cravotto, G.; Balliano, G.; Tagliapietra, S.; Palmisano, G.; Penoni, A.: Umbelliferone aminoalkyl derivatives, a new class of squalene-hopene cyclase inhibitors. Eur. J. Med. Chem., 39, 917-924 (2004) Lenhart, A.; Reinert, D.J.; Aebi, J.D.; Dehmlow, H.; Morand, O.H.; Schulz, G.E.: Binding structures and potencies of oxidosqualene cyclase inhibitors with the homologous squalene-hopene cyclase. J. Med. Chem., 46, 20832092 (2003) Rocco, F.; Bosso, S.O.; Viola, F.; Milla, P.; Roma, G.; Grossi, G.; Ceruti, M.: Conjugated methyl sulfide and phenyl sulfide derivatives of oxidosqualene as inhibitors of oxidosqualene and squalene-hopene cyclases. Lipids, 38, 201-207 (2003) Ceruti, M.; Balliano, G.; Rocco, F.; Lenhart, A.; Schulz, G.E.; Castelli, F.; Milla, P.: Synthesis and biological activity of new iodoacetamide derivatives on mutants of squalene-hopene cyclase. Lipids, 40, 729-735 (2005) Nakano, S.; Ohashi, S.; Hoshino, T.: Squalene-hopene cyclase: insight into the role of the methyl group on the squalene backbone upon the polycyclization cascade. Enzymatic cyclization products of squalene analogs lacking a 26-methyl group and possessing a methyl group at C7 or C11 . Org. Biomol. Chem., 2, 2012-2022 (2004) Tanaka, H.; Noguchi, H.; Abe, I.: Enzymatic formation of indole-containing unnatural cyclic polyprenoids by bacterial squalene:hopene cyclase. Org. Lett., 7, 5873-5876 (2005)

547

5-(Carboxyamino)imidazole ribonucleotide mutase

5.4.99.18

1 Nomenclature EC number 5.4.99.18 Systematic name 5-carboxyamino-1-(5-phospho-d-ribosyl)imidazole carboxymutase Recommended name 5-(carboxyamino)imidazole ribonucleotide mutase Synonyms N5 -CAIR mutase <3> [6] N5 -carboxyaminoimidazole ribonucleotide mutase <3> [6] PurE <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6, 7, 8, 9] CAS registry number 255379-40-9

2 Source Organism <1> <2> <3> <4> <5>

Escherichia coli (no sequence specified) [1, 2, 4, 7, 9] Acetobacter aceti (no sequence specified) [3] Sulfolobus solfataricus (no sequence specified) [6] Brevibacterium ammoniagenes (no sequence specified) [5] Thermotoga maritima (no sequence specified) [8]

3 Reaction and Specificity Catalyzed reaction 5-carboxyamino-1-(5-phospho-d-ribosyl)imidazole = 5-amino-1-(5-phospho-d-ribosyl)imidazole-4-carboxylate Substrates and products S 5-carboxyamino-1-(5-phospho-d-ribosyl)imidazole <1> (<1> the carbamate carboxylate is directly transferred to generate 5-amino-1-(5-phospho-d-ribosyl)imidazole-4-carboxylate, without equilibration with HCO-3 and CO2 in solution [2]) (Reversibility: r) [2, 4] P 5-amino-1-(5-phospho-d-ribosyl)imidazole-4-carboxylate

548

5.4.99.18

5-(Carboxyamino)imidazole ribonucleotide mutase

Specific activity (U/mg) Additional information <1> [1]

4 Enzyme Structure Molecular weight 126000 <1> (<1> gel filtration [1]) [1] 136000 <1> (<1> sucrose gradient ultracentrifugation [1]) [1] Subunits octamer <1> (<1> 8 * 17000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> [1, 9] Crystallization <1> (hanging-drop vapor-diffusion method, crystals grown in the presence of 4-carboxaminoimidazole ribonucleotide belong to space group P2(1)2(1)2(1), with unit cell parameters a = 86.92, b = 94.55 and c = 149.96 . The 1.5 crystal structure reveals an octameric structure with 422 symmetry) [9] <2> (hanging-drop method, crystals grow best at 295 K with the optimized mother-liquor conditions of 21-23% PEG 4 K, 0.19 M ammonium acetate and 90 mM citrate, pH 5.25-5.5. Crystals belong to space group I422, with unitcell parameters a = 99.25, c = 164.81 ) [3] <5> (nanodroplet vapor diffusion method, 1.77 resolution, unit cell parameters: a = b = 103.25 , c = 65.45 , a = b = 90) [8] Cloning <1> [7] <3> [6] <4> [5]

References [1] Meyer, E.; Leonard, N.J.; Bhat, B.; Stubbe, J.; Smith, J.M.: Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway. Biochemistry, 31, 5022-5032 (1992) [2] Meyer, E.; Kappock, T.J.; Osuji, C.; Stubbe, J.: Evidence for the Direct Transfer of the carboxylate of N5 -carboxyaminoimidazole ribonucleotide (N5 CAIR) to generate 4-carboxy-5-aminoimidazole ribonucleotide catalyzed by

549

5-(Carboxyamino)imidazole ribonucleotide mutase

5.4.99.18

Escherichia coli PurE, an N5 -CAIR mutase. Biochemistry, 38, 3012-3018 (1999) [3] Settembre, E.C.; Chittuluru, J.R.; Mill, C.P.; Kappock, T.J.; Ealick, S.E.: Acidophilic adaptations in the structure of Acetobacter aceti N5 -carboxyaminoimidazole ribonucleotide mutase (PurE). Acta Crystallogr. Sect. D, 60, 17531760 (2004) [4] Mueller, E.J.; Meyer, E.; Rudolph, J.; Davisson, V.J.; Stubbe, J.: N5 -carboxyaminoimidazole ribonucleotide: evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli. Biochemistry, 33, 2269-2278 (1994) [5] Chung, S.O.; Lee, J.H.; Lee, S.Y.; Lee, D.S.: Genomic organization of purK and purE in Brevibacterium ammoniagenes ATCC 6872: purE locus provides a clue for genomic evolution. FEMS Microbiol. Lett., 137, 265-268 (1996) [6] Sorensen, I.S.; Dandanell, G.: Identification and sequence analysis of Sulfolobus solfataricus purE and purK genes. FEMS Microbiol. Lett., 154, 173-180 (1997) [7] Watanabe, W.; Sampei, G.; Aiba, A.; Mizobuchi, K.: Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5’-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis. J. Bacteriol., 171, 198-204 (1989) [8] Schwarzenbacher, R.; Jaroszewski, L.; von Delft, F.; et al.: Crystal structure of a phosphoribosylaminoimidazole mutase PurE from Thermotoga maritima at 1.77 A resolution. Proteins, 55, 474-478 (2004) [9] Mathews, I.I.; Kappock, T.J.; Stubbe, J.; Ealick, S.E.: Crystal structure of Escherichia coli PurE, an unusual mutase in the purine biosynthetic pathway. Structure, 7, 1395-1406 (1999)

550

Copalyl diphosphate synthase

5.5.1.12

1 Nomenclature EC number 5.5.1.12 Systematic name (+)-copalyl-diphosphate lyase (decyclizing) Recommended name copalyl diphosphate synthase Synonyms (-)-abietadiene synthase abietadiene cyclase cyclase, abietadiene CAS registry number 157972-08-2

2 Source Organism <1> <2> <3> <4>

Abies grandis (no sequence specified) [1, 2, 3, 4, 5, 6, 7, 8, 9] Cucurbita maxima (no sequence specified) [10] Gibberella fujikuroi (no sequence specified) [11] Pinus contorta (no sequence specified) [1, 2]

3 Reaction and Specificity Catalyzed reaction geranylgeranyl diphosphate = (+)-copalyl diphosphate Reaction type intramolecular cyclization Natural substrates and products S geranylgeranyl diphosphate <1, 2, 3, 4> (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] P (+)-copalyl diphosphate Substrates and products S farnesyl diphosphate <1> (Reversibility: ?) [4] P (E)-b-farnesene

551

Copalyl diphosphate synthase

5.5.1.12

S geranylgeranyl diphosphate <1, 2, 3, 4> (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] P (+)-copalyl diphosphate S geranylgeranyl diphosphate <1> (Reversibility: ?) [2] P (-)-abietadiene Inhibitors 14,15-dihydro-15-aza-geranylgeranyl diphosphate <1> [5, 6] diethyl dicarbonate <1, 4> [1] Fe2+ <1, 4> (<1,4> at concentrations about 0.1 mM [1]) [1] geranylgeranyl diphosphate <1> (<1> substrate inhibition [4]) [4, 5, 6] Mn2+ <1, 4> (<1,4> at concentrations about 0.1 mM [1]) [1] norpimarenylamine <1> [7] p-hydroxymercuribenzoate <1> [1] Metals, ions Fe2+ <1, 4> [1] Mg2+ <1, 4> (<1,4> maximal activity at 2.5 mM [1]; <1> 10 mM selectively enabled the conversion of copalyl diphosphate [6]) [1, 6] Mn2+ <1, 4> [1] Turnover number (min–1) 0.02 <1> (farnesyl diphosphate) [4] 0.1 <1> (geranylgeranyl diphosphate, <1> D405N, Y520A [6]) [6] 0.2 <1> (geranylgeranyl diphosphate, <1> D402N, R365A, R454A [6]) [6] 0.5 <1> (geranylgeranyl diphosphate, <1> R411A [6]) [6] 0.75 <1> (geranylgeranyl diphosphate) [4] 2.2 <1> (geranylgeranyl diphosphate) [5, 6] Specific activity (U/mg) 0.0214 <1> [1] Km-Value (mM) 0.0004 <1> (geranylgeranyl diphosphate, <1> W358A [6]) [6] 0.0006 <1> (geranylgeranyl diphosphate, <1> D405N [6]) [6] 0.0008 <1> (geranylgeranyl diphosphate, <1> D361A [6]) [6] 0.001 <1> (geranylgeranyl diphosphate, <1> D402A, D402E, D405E, R454A [6]) [6] 0.002 <1> (geranylgeranyl diphosphate, <1> E499A, R411A, R365A [6]) [6] 0.003 <1> (geranylgeranyl diphosphate, <1> wild-type, D405A, Y520A [6]) [4, 5, 6] 0.007 <1> (geranylgeranyl diphosphate, <1> D404A [6]) [6] pH-Optimum 7.2 <1> (<1> with geranylgeranyl diphosphate as substrate [6]) [6]

552

5.5.1.12

Copalyl diphosphate synthase

4 Enzyme Structure Molecular weight 80000 <1, 4> (<1,4> gel filtration [1]) [1, 2, 4] 90000 <1> (<1> gel filtration [5]) [5] Subunits monomer <1, 4> (<1,4> 1 * 80000, SDS-PAGE [1]) [1, 4, 5]

5 Isolation/Preparation/Mutation/Application Source/tissue seed <2> [10] Localization plastid <2> [10] soluble <1> [4] Purification <1> [1] <4> [1] Cloning <1> (expression in Escherichia coli) [2, 3, 4, 5, 6, 7, 9] <2> (expression in Escherichia coli) [10] Engineering D361A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D402A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D402E <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D402N <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D404A <1> (<1> unreactive with geranylgeranyl diphosphate [5]) [5, 6] D404E <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D404N <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D405A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D405E <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D405N <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] D621A <1> (<1> unreactive with (+)-copalyl diphosphate [5]) [5, 7]

553

Copalyl diphosphate synthase

5.5.1.12

D625A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] D766A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] D845A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] D:107-868 <1> (<1> lower turnover with copalyl diphosphate than wild-type [9]) [9] D:85-849 <1> (<1> no turnover with copalyl diphosphate [9]) [9] E499A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] E589A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] E699A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] E773A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] E778A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] N765A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] R365A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] R411A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] R454A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] R584A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] R586A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] R762A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] S721A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] T617A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] T769A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] T848A <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] W358A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6] Y520A <1> (<1> lower turnover with geranylgeranyl diphosphate than wildtype [6]) [6]

554

5.5.1.12

Copalyl diphosphate synthase

Y841F <1> (<1> no effect of geranylgeranyl diphosphate reaction, but lower turnover with copalyl diphosphate than wild-type [7]) [7] rAS:D96A <1> (<1> nearly the same turnover with copalyl diphosphate like wild-type [9]) [9] rAS:K86A/R87A <1> (<1> lower turnover with copalyl diphosphate than wild-type [9]) [9]

References [1] LeFever, R.E.; Vogel, B.S.; Croteau, R.: Diterpenoid resin acid biosynthesis in conifers: enzymic cyclization of geranylgeranyl pyrophosphate to abietadiene, the precursor of abietic acid. Arch. Biochem. Biophys., 313, 139-149 (1994) [2] Ravn, M.M.; Coates, R.M.; Jetter, R.; Croteau, R.B.: Stereospecific intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to (-)-abietadiene catalyzed by recombinant cyclase from grand fir (Abies grandis). Chem. Commun., 1998, 21-22 (1998) [3] Ravn, M.M.; Coates, R.M.; Flory, J.E.; Peters, R.J.; Croteau, R.: Stereochemistry of the cyclization-rearrangement of (+)-copalyl diphosphate to (-)-abietadiene catalyzed by recombinant abietadiene synthase from Abies grandis. Org. Lett., 2, 573-576 (2000) [4] Peters, R.J.; Flory, J.E.; Jetter, R.; Ravn, M.M.; Lee, H.J.; Coates, R.M.; Croteau, R.B.: Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the “pseudomature“ recombinant enzyme. Biochemistry, 39, 15592-15602 (2000) [5] Peters, R.J.; Ravn, M.M.; Coates, R.M.; Croteau, R.B.: Bifunctional abietadiene synthase: Free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites. J. Am. Chem. Soc., 123, 89748978 (2001) [6] Peters, R.J.; Croteau, R.B.: Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. Biochemistry, 41, 1836-1842 (2002) [7] Peters, R.J.; Croteau, R.B.: Abietadiene synthase catalysis: mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement. Proc. Natl. Acad. Sci. USA, 99, 580-584 (2002) [8] Ravn, M.M.; Peters, R.J.; Coates, R.M.; Croteau, R.: Mechanism of abietadiene synthase catalysis: stereochemistry and stabilization of the cryptic pimarenyl carbocation intermediates. J. Am. Chem. Soc., 124, 6998-7006 (2002) [9] Peters, R.J.; Carter, O.A.; Zhang, Y.; Matthews, B.W.; Croteau, R.B.: Bifunctional abietadiene synthase: mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations. Biochemistry, 42, 2700-2707 (2003)

555

Copalyl diphosphate synthase

5.5.1.12

[10] Smith, M.W.; Yamaguchi, S.; Ait-Ali, T.; Kamiya, Y.: The first step of gibberellin biosynthesis in pumpkin is catalyzed by at least two copalyl diphosphate synthases encoded by differentially regulated genes. Plant Physiol., 118, 1411-1419 (1998) [11] Tudzynski, B.; Kawaide, H.; Kamiya, Y.: Gibberellin biosynthesis in Gibberella fujikuroi: cloning and characterization of the copalyl diphosphate synthase gene. Curr. Genet., 34, 234-240 (1998)

556

Ent-copalyl diphosphate synthase

5.5.1.13

1 Nomenclature EC number 5.5.1.13 Systematic name ent-copalyl-diphosphate lyase (decyclizing) Recommended name ent-copalyl diphosphate synthase Synonyms GfCPS/KS <7> (<7> the bifunctional enzyme [10]) [10] OsCPS1 <5> [11] OsCPS1ent <5> [13] OsCPS2ent <10> [13] OsCyc2 <11> [11] ent-copylyl diphosphate synthase/ent-kaurene synthase <7> [10] ent-kaurene synthase A ent-kaurene synthetase A CAS registry number 9055-64-5 (in Chemical Abstracts not distinguished from EC 4.2.3.19)

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11>

Triticum aestivum (no sequence specified) [5, 7] Pisum sativum (no sequence specified) [5, 7, 9] Zea mays (no sequence specified) [12] Helianthus annuus (no sequence specified) (<4> SULT1B2 [4]) [4] Oryza sativa (no sequence specified) [11,13] Cucurbita maxima (no sequence specified) (<6> FaGT2 gene [5,6,8]) [5,6,8] Gibberella fujikuroi (no sequence specified) [2,10] Marah macrocarpus (no sequence specified) [3,4] Phaeosphaeria sp. (no sequence specified) [1,8] Oryza sativa (UNIPROT accession number: Q5MQ85) [13] Oryza sativa (UNIPROT accession number: Q68CL9) [11]

557

Ent-copalyl diphosphate synthase

5.5.1.13

3 Reaction and Specificity Catalyzed reaction geranylgeranyl diphosphate = ent-copalyl diphosphate Reaction type intramolecular lyase Natural substrates and products S copalyl diphosphate <7> (<7> bifunctional enzyme [2]) (Reversibility: ?) [2] P ent-kaurene <7> [2] S geranylgeranyl diphosphate <2, 7> (<2,7> gibberellin biosynthesis [2,9]) (Reversibility: ?) [2, 9] P copalyl diphosphate <2, 7> [2, 9] S geranylgeranyl diphosphate <3, 5, 10, 11> (<5> OsCPS1 participates in biosynthesis of gibberelins [11]; <5> OsCPS1ent normally operates in biosynthesis of gibberellin [13]; <10> OsCPS2ent is involved in secondary metabolism producing defensive phytochemicals. OsCPS2ent mRNA is specifically induced in leaves prior to production of the corresponding phytoalexins. The transcriptional control of OsCPS2ent seems to be an important means of regulating defensive phytochemical biosynthesis [13]; <11> OsCyc2 is possibly involved in phytoalexin biosynthesis [11]; <3> transcript levels of the An2 gene encoding copalyl diphosphate synthase are strongly up-regulated by attack by Fusarium graminearum [12]) (Reversibility: ?) [11, 12, 13] P ent-copalyl diphosphate Substrates and products S copalyl diphosphate <4, 7, 8, 9> (<7,9> bifunctional enzyme [1,2,8]) (Reversibility: ?) [1, 2, 4, 8] P ent-kaurene <4, 7, 8, 9> [1, 2, 4, 8] S geranylgeranyl diphosphate <1, 2, 4, 6, 8> (Reversibility: ?) [4, 5] P ent-kaurene <1, 2, 4, 6, 8> (<6> gives ent-kaurene + an unidentified compound [5]) [4, 5] S geranylgeranyl diphosphate <2, 6, 7, 8, 9> (<2,7> gibberellin biosynthesis [2,9]) (Reversibility: ?) [1, 2, 3, 6, 8, 9] P copalyl diphosphate <2, 6, 7, 8, 9> [1, 2, 3, 6, 8, 9] S geranylgeranyl diphosphate <3, 5, 10, 11> (<5> OsCPS1 participates in biosynthesis of gibberelins [11]; <5> OsCPS1ent normally operates in biosynthesis of gibberellin [13]; <10> OsCPS2ent is involved in secondary metabolism producing defensive phytochemicals. OsCPS2ent mRNA is specifically induced in leaves prior to production of the corresponding phytoalexins. The transcriptional control of OsCPS2ent seems to be an important means of regulating defensive phytochemical biosynthesis [13]; <11> OsCyc2 is possibly involved in phytoalexin biosynthesis [11]; <3> transcript levels of the An2 gene encoding copalyl diphosphate

558

5.5.1.13

Ent-copalyl diphosphate synthase

synthase are strongly up-regulated by attack by Fusarium graminearum [12]) (Reversibility: ?) [11, 12, 13] P ent-copalyl diphosphate Inhibitors 2’-isopropyl-4’-(trimethylammonium chloride)-5’-methylphenyl-piperidine1-carboxylate <9> (<9> AMO-1618, a quaternary ammonium salt, leads to total enzyme inhibition at 0.001 mM [1]) [1] EDTA <9> (<9> inhibits activity in cell free extract containing the enzyme [8]) [8] Additional information <6> (<6> substrate inhibition [8]; <6> inhibitory effects on partially purified enzyme of 14 quaternary ammonium iodides at 3 different concentrations are reported [6]) [6, 8] Turnover number (min–1) 0.00117 <9> (copalyl diphosphate, <9> at pH 8 [8]) [8] 0.0308 <9> (geranylgeranyl diphosphate, <9> at pH 8 [8]) [8] 0.035 <6> (geranylgeranyl diphosphate, <6> at pH 8 [8]) [8] Specific activity (U/mg) 0.00075 <9> (<9> at pH 8 [8]) [8] Additional information <4, 8> (<4,8> specific activity per mg of protein for different plant organs [4]) [4] Km-Value (mM) 0.00023 <9> (copalyl diphosphate, <9> at pH 8 [8]) [8] 0.00873 <9> (geranylgeranyl diphosphate, <9> at pH 8 [8]) [8] 0.0169 <6> (geranylgeranyl diphosphate, <6> at pH 8 [8]) [8]

4 Enzyme Structure Molecular weight 75000 <8> (<8> sedimentation velocity [3]) [3] 83000 <8> (<8> gel filtration [3]) [3] 92000 <2> (<2> calculated from amino acid sequence [9]) [9] 106000 <9> (<9> calculated from amino acid sequence [1,8]) [1, 8] 107000 <7> (<7> calculated from amino acid sequence [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue cotyledon <4, 8> [4] endosperm <6, 8> [3, 5] hypocotyl <4, 8> [4] leaf <10> (<10> OsCPS2ent mRNA is specifically induced in leaves prior to production of the corresponding phytoalexins [13]) [13] root <4, 8> [4]

559

Ent-copalyl diphosphate synthase

5.5.1.13

Localization chloroplast stroma <1, 2> [5] Purification <1> (partial, using ammonium sulfate fractionation, column chromatography on Sephadex G25) [5, 7] <2> (partial, GST recombinant protein) [9] <2> (partial, using ammonium sulfate fractionation, column chromatography on Sephadex G25) [5, 7] <4> (partial, using ammonium sulfate fractionation) [4] <6> (partial, gradient centrifugation) [5] <7> (recombinant enzyme, using Sepharose 4B glutathione affinity resin) [2] <8> (partial, using ammonium sulfate fractionation) [4] <8> (using ammonium sulfate fractionation, Sephadex A-50 chromatography and PAGE electrophoresis) [3] <9> (cell free extract) [8] <9> (recombinant enzyme, using Sepharose 4B glutathione affinity resin and SDS PAGE electrophoresis) [1, 8] Cloning <2> (expression in Escherichia coli of a recombinant GST fused enzyme) [9] <5> (OsCPS1ent, expression in Escherichia coli BL21) [13] <6> (expression in Escherichia coli of a recombinant GST fused enzyme) [6, 8] <7> (expression in Escherichia coli of a recombinant GST fused enzyme) [2] <7> (overexpression in Arabidopsis) [10] <9> (expression in Escherichia coli of a recombinant GST fused enzyme) [1, 8] <10> (OsCPS2ent, expression in Escherichia coli BL21) [13] Engineering D320A <9> (<9> the mutation leads to complete enzyme activity [8]) [8] D656A <9> (<9> the mutation causes a small reduction in activity [8]) [8]

References [1] Kawaide, H.; Imai, R.; Sassa, T.; Kamiya, Y.: ent-Kaurene synthetase from the fungus Phaeosphaeria sp. L487. cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase in fungal gibberellin biosynthesis. J. Biol. Chem., 272, 21706-21712 (1997) [2] Toyomasu, T.; Kawaide, H.; Ishizaki, A.; Shinoda, S.; Otsuka, M.; Mitsuhashi, W.; Sassa, T.: Cloning of a full-length cDNA encoding ent-kaurene synthase from Gibberella fujikuroi: functional analysis of a bifunctional diterpene cyclase. Biosci. Biotechnol. Biochem., 64, 660-664 (2000) [3] Duncan, J.D.; West, C.A.: Properties of kaurene synthetase from Marah macrocarpus endosperm: evidence for the participation of separate but interacting enzymes. Plant Physiol., 68, 1128-1134 (1981)

560

5.5.1.13

Ent-copalyl diphosphate synthase

[4] Shen-Miller, J.; West, C.A.: Distribution and ent-kaurene synthetase in Helianthus annuus and Marah macrocarpus. Phytochemistry, 24, 461-464 (1985) [5] Aach, H.; Boese, G.; Graebe, J.E.: ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localization of ent-kaurene synthetase in plastids of three species. Planta, 197, 333-342 (1995) [6] Saito, T.; Yamane, H.; Sakurai, A.; Murofushi, N.; Takahashi, N.; Kamiya, Y.: Inhibition of ent-kaurene synthase by quaternary ammonium growth retardants. Biosci. Biotechnol. Biochem., 60, 1040-1042 (1996) [7] Aach, H.; Bode, H.; Robinson, D.G.; Graebe, J.E.: ent-Kaurene synthase is located in proplastids of meristematic shoot tissues. Planta, 202, 211-219 (1997) [8] Kawaide, H.; Sassa, T.; Kamiya, Y.: Functional analysis of the two interacting cyclase domains in ent-kaurene synthase from the fungus Phaeosphaeria sp. L487 and a comparison with cyclases from higher plants. J. Biol. Chem., 275, 2276-2280 (2000) [9] Ait-Ali, T.; Swain, S.M.; Reid, J.B.; Sun, T.; Kamiya, Y.: The LS locus of pea encodes the gibberellin biosynthesis enzyme ent-kaurene synthase A. Plant J., 11, 443-454 (1997) [10] Otsuka, M.; Kenmoku, H.; Ogawa, M.; Okada, K.; Mitsuhashi, W.; Sassa, T.; Kamiya, Y.; Toyomasu, T.; Yamaguchi, S.: Emission of ent-kaurene, a diterpenoid hydrocarbon precursor for gibberellins, into the headspace from plants. Plant Cell Physiol., 45, 1129-1138 (2004) [11] Otomo, K.; Kenmoku, H.; Oikawa, H.; Konig, W.A.; Toshima, H.; Mitsuhashi, W.; Yamane, H.; Sassa, T.; Toyomasu, T.: Biological functions of entand syn-copalyl diphosphate synthases in rice: key enzymes for the branch point of gibberellin and phytoalexin biosynthesis. Plant J., 39, 886-893 (2004) [12] Harris, L.J.; Saparno, A.; Johnston, A.; Prisic, S.; Xu, M.; Allard, S.; Kathiresan, A.; Ouellet, T.; Peters, R.J.: The maize An2 gene is induced by Fusarium attack and encodes an ent-copalyl diphosphate synthase. Plant Mol. Biol., 59, 881-894 (2005) [13] Prisic, S.; Xu, M.; Wilderman, P.R.; Peters, R.J.: Rice contains two disparate ent-copalyl diphosphate synthases with distinct metabolic functions. Plant Physiol., 136, 4228-4236 (2004)

561

Aspartate-tRNAAsn ligase

6.1.1.23

1 Nomenclature EC number 6.1.1.23 Systematic name l-aspartate:tRNAAsx ligase (AMP-forming) Recommended name aspartate-tRNAAsn ligase Synonyms AspRS <3> [5] aspartic acid translase aspartyl ribonucleic synthetase aspartyl-tRNA synthetase <3> [5, 6] aspartyl-transfer RNA synthetase aspartyl-transfer ribonucleic acid synthetase EC 6.1.1.12 (formerly) ND-AspRS <1, 3, 4, 5, 6, 9> [4, 10, 11, 12] synthetase, aspartyl-transfer ribonucleate aspartate tRNA synthetase aspartyl tRNA synthetase aspartyl-transfer ribonucleate synthetase non-discriminating aspartyl-tRNA synthetase <9> [12] nondiscriminating AspRS <3> [5, 6] nondiscriminating aspartyl-tRNA synthetase <1, 3, 4, 5, 6, 8> [4, 8, 9, 10, 11] CAS registry number 9027-32-1

2 Source Organism <1> Chlamydia trachomatis (no sequence specified) [4] <2> Thermus thermophilus (no sequence specified) [3] <3> Pseudomonas aeruginosa (no sequence specified) (<3> subunit of benzoyl-CoA reductase, gene name: bzdA [5]) [5, 6, 11] <4> Helicobacter pylori (no sequence specified) [10] <5> Deinococcus radiodurans (no sequence specified) [1, 4, 7] <6> Halobacterium salinarium (no sequence specified) [4]

562

Aspartate-tRNAAsn ligase

6.1.1.23

<7> <8> <9> <10>

Pyrococcus kodakaraensis (no sequence specified) [2] Sulfolobus tokodaii (no sequence specified) [8,9] Halobacterium salinarum (UNIPROT accession number: O07683) [12] no activity in Sulfolobus tokodaii, strain 7 [8, 9]

3 Reaction and Specificity Catalyzed reaction ATP + l-aspartate + tRNAAsx = AMP + diphosphate + aspartyl-tRNAAsx (<3> residues H31 and G83 are important determinants for substrate specificity towards tRNA substrates, molecular modeling [6]; <8> substrate anticodon, GUC for aspartate and GUU for asparagine, recognition site structure and mechanism [8]; <3> via reaction intermediate l-aspartyl adenylate [11]) Reaction type Aminoacylation Esterification Natural substrates and products S ATP + Asp + tRNAAsn <1, 5, 6> (<6> while large amounts of Asp-tRNAAsn are detrimental to Escherichia coli with trypA34 missense mutation, a smaller amount supports protein synthesis and allows the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase [4]; <1,5> while large amounts pf Asp-tRNAAsn are detrimental to Escherichia coli with trypA34 missense mutation, a smaller amount supports protein synthesis and allows the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase [4]) (Reversibility: ?) [4] P AMP + diphosphate + aspartyl-tRNAAsn S ATP + l-aspartate + tRNAAsn <3> (<3> the product l-aspartyl-tRNAAsn is transamidated by amidotransferase to form Asn-tRNAAsn . Synthesis of Asn-tRNA via the indirect pathway [5]) (Reversibility: ?) [5] P AMP + diphosphate + l-aspartyl-tRNAAsn S ATP + l-aspartate + tRNAAsn <3, 4, 8, 9> (<8> dual activity of the NDAspRS since an asparaginyl-tRNA synthetase is missing in Sulfolobus tokodaii strain 7 [8,9]; <9> reaction via transamidation mechanism [12]) (Reversibility: ?) [6, 8, 9, 10, 12] P AMP + diphosphate + aspartyl-tRNAAsn S ATP + l-aspartate + tRNAAsp <2, 5, 7> (Reversibility: ?) [1, 2, 3] P AMP + diphosphate + Asp-tRNAAsp S ATP + l-aspartate + tRNAAsp <3, 4, 8, 9> (Reversibility: ?) [6, 8, 9, 10, 11, 12] P AMP + diphosphate + aspartyl-tRNAAsp S Additional information <4, 9> (<4> in bacteria that lack AsnRS, AspRS is nondiscriminating and generates both Asp-tRNAAsp and the noncanonical, misacylated Asp-tRNAAsn , this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNAAsn /Glu-tRNAGln amido-

563

Aspartate-tRNAAsn ligase

6.1.1.23

transferase, EC 6.3.5.6, increasing tRNAAsp specificity in an ND-AspRS diminishes in vivo toxicity [10]; <9> the tRNA-dependent transamidation pathway is the essential route for Asn-tRNAAsn formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on NDAspRS, an enzyme with relaxed tRNA specificity, to form Asp-tRNAAsn , the misacylated tRNA is then converted to Asn-tRNAAsn by the action of an Asp-tRNAAsn amidotransferase, EC 6.3.5.6 [12]) (Reversibility: ?) [10, 12] P ? Substrates and products S ATP + Asp + tRNAAsn <1, 5, 6> (<6> while large amounts of Asp-tRNAAsn are detrimental to Escherichia coli with trypA34 missense mutation, a smaller amount supports protein synthesis and allows the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase [4]; <1,5> while large amounts pf Asp-tRNAAsn are detrimental to Escherichia coli with trypA34 missense mutation, a smaller amount supports protein synthesis and allows the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase [4]) (Reversibility: ?) [4] P AMP + diphosphate + aspartyl-tRNAAsn S ATP + l-aspartate + tRNAAsn <2> (<2> only the enzyme AspRS2 aspartylates tRNAAsn [3]) (Reversibility: ?) [3] P AMP + diphosphate + Asp-tRNAAsn S ATP + l-aspartate + tRNAAsn <3, 5> (<3> the product l-aspartyltRNAAsn is transamidated by amidotransferase to form Asn-tRNAAsn . Synthesis of Asn-tRNA via the indirect pathway [5]; <5> aspartyl-tRNA synthetase requires a conserved proline, P77, in the anticodon-binding loop for tRNA(Asn) recognition in vivo. Wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp [7]; <3> two residues in the anticodon recognition domain of the aspartyl-tRNA synthetase, H31 and G83, are individually implicated in the recognition of tRNAAsn [6]) (Reversibility: ?) [5, 6, 7] P AMP + diphosphate + l-aspartyl-tRNAAsn S ATP + l-aspartate + tRNAAsn <3, 4, 8, 9> (<8> dual activity of the NDAspRS since an asparaginyl-tRNA synthetase is missing in Sulfolobus tokodaii strain 7 [8,9]; <9> reaction via transamidation mechanism [12]; <9> in vivo expressed Halobacterium salinarum tRNAAsn , reaction via transamidation mechanism [12]; <4> recombinantly produced tRNA substrate [10]; <3> tRNA substrate from Escherichia coli [6]) (Reversibility: ?) [6, 8, 9, 10, 12] P AMP + diphosphate + aspartyl-tRNAAsn S ATP + l-aspartate + tRNAAsp <2, 5, 7> (Reversibility: ?) [1, 2, 3] P AMP + diphosphate + Asp-tRNAAsp S ATP + l-aspartate + tRNAAsp <3> (<3> preparation of recombinant tRNA substrates from Pseudomonas aeruginosa and Saccharomyces cerevisiae,

564

Aspartate-tRNAAsn ligase

6.1.1.23

P S

P S

P S

P

overview, activity with two variants C36U and C38U of yeast tRNAAsp [11]) (Reversibility: ?) [11] AMP + diphosphate + aspartyl-tRNAAsx ATP + l-aspartate + tRNAAsp <5> (<5> aspartyl-tRNA synthetase requires a conserved proline, P77, in the anticodon-binding loop for tRNA(Asn) recognition in vivo. Wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp [7]) (Reversibility: ?) [7] AMP + diphosphate + l-aspartyl-tRNAAsp ATP + l-aspartate + tRNAAsp <3, 4, 8, 9> (<9> in vivo expressed Halobacterium salinarum tRNAAsp [12]; <4> recombinantly produced tRNA substrate [10]; <3> tRNA substrate from Escherichia coli [6]) (Reversibility: ?) [6, 8, 9, 10, 11, 12] AMP + diphosphate + aspartyl-tRNAAsp Additional information <3, 4, 8, 9> (<4> in bacteria that lack AsnRS, AspRS is nondiscriminating and generates both Asp-tRNAAsp and the noncanonical, misacylated Asp-tRNAAsn , this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNAAsn /Glu-tRNAGln amidotransferase, EC 6.3.5.6, increasing tRNAAsp specificity in an NDAspRS diminishes in vivo toxicity [10]; <9> the tRNA-dependent transamidation pathway is the essential route for Asn-tRNAAsn formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on ND-AspRS, an enzyme with relaxed tRNA specificity, to form AsptRNAAsn , the misacylated tRNA is then converted to Asn-tRNAAsn by the action of an Asp-tRNAAsn amidotransferase, EC 6.3.5.6 [12]; <9> the Halobacterium salinarum enzyme is unable to use Escherichia coli tRNA as substrate [12]; <8> the purified protein has the ability to catalyze the aspartylation of hydroxylamine through an aspartyl-AMP intermediate [9]; <4> tRNA anticodon binding site structures, overview, Helicobacter pylori AspRS is a nondiscriminating enzyme that aminoacylates both tRNAAsp and tRNAAsn , ND-AspRS is 1.7times more efficient at aminoacylating tRNAAsp over tRNAAsn [10]; <3> two residues in the anticodon recognition domain of the enzyme are individually implicated in the recognition of tRNAAsn , nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs, EC 6.1.1.12, possess a leucine at position 31 and a residue other than a glycine at position 83 [6]) (Reversibility: ?) [6, 9, 10, 12] ?

Inhibitors l-aspartol adenylate <3> (<3> i.e. Asp-ol-AMP, a stable analogue of the natural reaction intermediate l-aspartyl adenylate, biphasic, competitive inhibition, differential inhibition of tRNAAsp and tRNAAsn aspartylation by the enzyme, overview [11]) [11] Cofactors/prosthetic groups ATP <3, 4, 8, 9> [6, 8, 9, 10, 11, 12]

565

Aspartate-tRNAAsn ligase

6.1.1.23

Activating compounds ATP <7> [2] Metals, ions Mg2+ <3, 4, 7, 8, 9> (<7> 3 mol per mol of enzyme [2]) [2, 6, 8, 9, 10, 11, 12] NaCl <9> (<9> required at 0.1-3 M, salt dependence profile, overview [12]) [12] Turnover number (min–1) 0.014 <4> (tRNAAsn , <4> pH 7.5, recombinant wild-type enzyme [10]) [10] 0.015 <4> (tRNAAsn , <4> pH 7.5, recombinant mutant L86M enzyme [10]) [10] 0.021 <4> (tRNAAsp , <4> pH 7.5, recombinant mutant L86M enzyme [10]) [10] 0.022 <4> (tRNAAsp , <4> pH 7.5, recombinant wild-type enzyme [10]) [10] 0.026 <4> (tRNAAsn , <4> pH 7.5, recombinant mutant L81N enzyme [10]) [10] 0.028 <4> (tRNAAsp , <4> pH 7.5, recombinant mutant L81N enzyme [10]) [10] 0.092 <2> (tRNAAsn , <2> AspRS2 [3]) [3] 0.14 <5> (tRNAAsp , <5> 37 C, pH 7.0, mutant enzyme P77K/H28Q [7]) [7] 0.15 <5> (tRNAAsp , <5> 37 C, pH 7.0, wild-type enzyme [7]) [7] 0.17 <5> (tRNAAsp , <5> 37 C, pH 7.0, mutant enzyme H28Q [7]) [7] 0.22 <5> (tRNAAsn , <5> 37 C, pH 7.0, mutant enzyme H28Q [7]; <5> 37 C, pH 7.0, mutant enzyme P77K/H28Q [7]) [7] 0.24 <2> (tRNAAsp , <2> AspRS2 [3]) [3] 0.28 <5> (tRNAAsp , <5> 37 C, pH 7.0, mutant enzyme P77K [7]) [7] 0.51 <5> (tRNAAsn , <5> 37 C, pH 7.0, wild-type enzym [7]) [7] 0.6 <5> (tRNAAsn , <5> 37 C, pH 7.0, mutant enzyme P77K [7]) [7] 7.2 <2> (tRNAAsp , <2> AspRS1 [3]) [3] Additional information <4> (<4> the wild-type enzyme has a kcat for tRNAAsp that is 60% higher than that of tRNAAsn [10]) [10] Specific activity (U/mg) 32.1 <2> [3] Additional information <3> (<3> activities of wild-type and mutant enzymes with different tRNA substrates [6]) [6] Km-Value (mM) 3e-005 <2> (tRNAAsp , <2> AspRS1 [3]) [3] 6e-005 <2> (tRNAAsn , <2> AspRS2 [3]) [3] 7e-005 <2> (tRNAAsp , <2> AspRS2 [3]) [3] 0.0008 <5> (tRNAAsp , <5> 37 C, pH 7.0, mutant enzyme P77K [7]; <5> 37 C, pH 7.0, wild-type enzyme [7]) [7] 0.0013 <5> (tRNAAsp , <5> 37 C, pH 7.0, mutant enzyme P77K/H28Q [7]) [7] 0.0018 <5> (tRNAAsp , <5> 37 C, pH 7.0, mutant enzyme H28Q [7]) [7] 0.002 <5> (tRNAAsn , <5> 37 C, pH 7.0, wild-type enzym [7]) [7] 0.0035 <5> (tRNAAsn , <5> 37 C, pH 7.0, mutant enzyme P77K/H28Q [7]) [7] 0.0038 <5> (tRNAAsn , <5> 37 C, pH 7.0, mutant enzyme P77K [7]) [7]

566

6.1.1.23

Aspartate-tRNAAsn ligase

0.0045 <5> (tRNAAsn , <5> 37 C, pH 7.0, mutant enzyme H28Q [7]) [7] 0.005 <2> (Aspartate, <2> AspRS2 [3]) [3] 0.03 <2> (Aspartate, <2> AspRS1 [3]) [3] 0.033 <2> (ATP, <2> AspRS2 [3]) [3] 0.28 <2> (ATP, <2> AspRS1 [3]) [3] 0.77 <4> (tRNAAsp , <4> pH 7.5, recombinant wild-type enzyme [10]) [10] 0.83 <4> (tRNAAsn , <4> pH 7.5, recombinant wild-type enzyme [10]) [10] 0.85 <4> (tRNAAsp , <4> pH 7.5, recombinant mutant L81N enzyme [10]) [10] 0.87 <4> (tRNAAsp , <4> pH 7.5, recombinant mutant L86M enzyme [10]) [10] 1.87 <4> (tRNAAsn , <4> pH 7.5, recombinant mutant L81N enzyme [10]) [10] 2.23 <4> (tRNAAsn , <4> pH 7.5, recombinant mutant L86M enzyme [10]) [10] Additional information <3, 4> (<4> the L81N/L86M mutant does not follow Michaelis-Menten kinetics [10]) [6, 10] Ki-Value (mM) 0.041 <3> (l-aspartol adenylate, <3> tRNAAsp aspartylation [11]) [11] 0.215 <3> (l-aspartol adenylate, <3> tRNAAsn aspartylation [11]) [11] Additional information <3> (<3> inhibition kinetics [11]) [11] pH-Optimum 7.2 <9> (<9> aminoacylation assay at [12]) [12] 7.5 <3, 4, 8> (<3,4,8> assay at [6,9,10]) [6, 9, 10] Temperature optimum ( C) 37 <3, 9> (<3,9> assay at [6,12]) [6, 12] 57 <8> [9]

4 Enzyme Structure Subunits ? <8> (<8> x * 49074, sequence calculation [9]) [9] Additional information <8> (<8> structural comparison of the nondiscriminating AspRS with structures of discriminating AspRSs, EC 6.1.1.12, highly conserved catalytic domain, but different tRNA binding site in the N-terminal domain, overview [8]) [8]

5 Isolation/Preparation/Mutation/Application Purification <2> (recombinant enzyme) [3] <3> (recombinant His-tagged enzyme from strain ADD1976 by nickel affinity chromatography) [11] <4> (recombinant His-tagged AsnRS from Escherichia coli strain DH5a by nickel affinity chromatography) [10]

567

Aspartate-tRNAAsn ligase

6.1.1.23

<7> (recombinant enzyme) [2] <8> (recombinant enzyme from Escherichia coli strain BL21(DE3)) [8] <8> (recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography) [9] <9> (recombinant His-tagged wild-type and mutant ND-AspRS in Escherichia coli strain trpA34 by nickel affinity chromatography) [12] Crystallization <7> (hanging drop technique, 24 C, 5 days in Crystal Screen I or II, solutions containing either polyethylene glycol or ethylene glycol) [2] <8> (purified recombinant enzyme, 5-8 mg/ml in 20 mM Tris-HCl buffer, pH 7.0, hanging-drop vapour-diffusion method, 26 C, from 0.002 ml protein solution is mixed with 0.002 ml of reservoir solution containing 100 mM sodium HEPES buffer, pH 7.5, containing 100 mM NaCl and 1.6 M (NH4 )2 SO4, equilibration against 0.7 ml reservoir solution, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement) [9] <8> (purified recombinant enzyme, X-ray diffraction structure determination and analysis at 2.3 A resolution) [8] Cloning <1> (Escherichia coli trypA34 missense mutant transformed with heterologous ND-aspS gene) [4] <2> (overexpression in Escherichia coli) [3] <3> (expression of C-terminally His-tagged enzyme in strain ADD1976) [11] <3> (gene aspS, DNA and amino acid sequence determination and analysis, expression of His-tagged wild-type and mutant enzymes) [6] <3> (overproduced from the cloned gene in Pseudomonas aeruginosa) [5] <4> (overexpression of His-tagged AsnRS in Escherichia coli strain DH5a, the enzyme is toxic when heterologously overexpressed in Escherichia coli, because of sequestration of tRNAAsn as Asp-tRNAAsn , this toxicity is rescued upon coexpression of the Helicobacter pylori Asp/Glu-Adt, EC 6.3.5.6) [10] <5> (Escherichia coli trypA34 missense mutant transformed with heterologous ND-aspS gene) [4] <5> (expression in Escherichia coli) [1] <6> (Escherichia coli trypA34 missense mutant transformed with heterologous ND-aspS gene) [4] <7> (expression in JM101Tr cells) [2] <8> (overexpression in Escherichia coli strain BL21(DE3)) [8, 9] <9> (gene aspS, phylogenetic tree, expression of His-tagged wild-type and mutant ND-AspRS in Escherichia coli strain trpA34, carrying a D60N mutation in trpA leading to tryptophan auxotrophy, co-expression of tRNAAsn leading to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNAAsn ) [12]

568

6.1.1.23

Aspartate-tRNAAsn ligase

Engineering G83K <3> (<3> aspartylation reaction of the mutant enzyme is 55% as fast as the wild-type enzyme [6]; <3> site-directed mutagenesis, the mutation increases the specificity of tRNAAsp charging over that of tRNAAsn by 4.2fold [6]) [6] H26A <9> (<9> site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn , altered tRNA substrate specificity compared to the wild-type enzyme, overview [12]) [12] H26A/P84A <9> (<9> site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn , altered tRNA substrate specificity compared to the wild-type enzyme, overview [12]) [12] H26A/P84K <9> (<9> site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn , altered tRNA substrate specificity compared to the wild-type enzyme, overview [12]) [12] H26Q <9> (<9> site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn , altered tRNA substrate specificity compared to the wild-type enzyme, overview [12]) [12] H26Q/P84A <9> (<9> site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn , altered tRNA substrate specificity compared to the wild-type enzyme, overview [12]) [12] H26Q/P84K <9> (<9> site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn , altered tRNA substrate specificity compared to the wild-type enzyme, overview [12]) [12] H28Q <5> (<5> wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp . Mutation H28Q leads to a reverse tRNA preference [7]) [7] H31L <3> (<3> aspartylation reaction of the mutant enzyme is 84% as fast as the wild-type enzyme [6]; <3> aspartylation reaction of the mutant enzyme is 92% as fast as the wild-type enzyme [6]; <3> site-directed mutagenesis, the mutation increases the specificity of tRNAAsp charging over that of tRNAAsn by 3.5fold [6]) [6] H77K/H28Q <5> (<5> wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp . Mutation P77K/H28Q leads to a reverse tRNA preference [7]) [7] L81N <4> (<4> site-directed mutagenesis, the mutation in the anticodon binding domain doubles the kcat for tRNAAsn as compared to the wild-type enzyme [10]) [10] L81N/L86M <4> (<4> site-directed mutagenesis, the mutation in the anticodon binding domain alters the tRNA specificity as compared to the wild-type enzyme, the L81N/L86M mutant does not follow Michaelis-Menten kinetics [10]) [10]

569

Aspartate-tRNAAsn ligase

6.1.1.23

L86M <4> (<4> site-directed mutagenesis, the mutation in the anticodon binding domain alters the tRNA specificity as compared to the wild-type enzyme [10]) [10] P77K <5> (<5> wild-type enzyme shows a slight preference to tRNAAsn over tRNAAsp . Mutation P77K leads to a reverse tRNA preference and a 3fold increase in specificity for tRNAASp over tRNAAsn [7]) [7] P84A <9> (<9> site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn , altered tRAN substrate specificity compared to the wild-type enzyme, overview [12]) [12] P84K <9> (<9> site-directed mutagenesis, mutation the amino acid located in the AspRS anticodon binding domain limits the specificity of this nondiscriminating enzyme towards tRNAAsn , altered tRAN substrate specificity compared to the wild-type enzyme, overview [12]) [12] Additional information <4> (<4> mutations in the anticodon binding domain of Helicobacter pylori ND-AspRS, e.g. at L81, L86, N82, and M87, reduce this enzyme’s ability to misacylate tRNAAsn and enhance tRNAAsp specificity, in a manner that correlates with the toxicity of the enzyme in Escherichia coli, overview [10]) [10]

6 Stability General stability information <9>, ND-AspRS is stable in low and high salt [12] Storage stability <4>, -20 C, purified recombinant enzyme, in 50% glycerol, and 33 mM phosphate, pH 7.4, 3 mM Tris-HCl, 1.5 mM 2-mercaptoethanol, and 0.5 mM phenylmethanesulfonyl fluoride, stable for months [10]

References [1] Curnow, A.W.; Tumbula, D.L.; Pelaschier, J.T.; Min, B.; Sçll, D.: GlutamyltRNAGln amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis. Proc. Natl. Acad. Sci. USA, 95, 12838-12843 (1998) [2] Schmitt, E.; Moulinier, L.; Fujiwara, S.; Imanaka, T.; Thierry, J.C.; Moras, D.: Crystal structure of aspartyl-tRNA synthetase from Pyrococcus kodakaraensis KOD: archaeon specificity and catalytic mechanism of adenylate formation. EMBO J., 17, 5227-5237 (1998) [3] Becker, H.D.; Roy, H.; Moulinier, L.; Mazauric, M.H.; Keith, G.; Kern, D.: Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase. Biochemistry, 39, 3216-3230 (2000) [4] Min, B.; Kitabatake, M.; Polycarpo, C.; Pelaschier, J.; Raczniak, G.; Ruan, B.; Kobayashi, H.; Namgoong, S.; Soll, D.: Protein synthesis in Escherichia coli with mischarged tRNA. J. Bacteriol., 185, 3524-3526 (2003)

570

6.1.1.23

Aspartate-tRNAAsn ligase

[5] Akochy, P.M.; Bernard, D.; Roy, P.H.; Lapointe, J.: Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1. J. Bacteriol., 186, 767-776 (2004) [6] Bernard, D.; Akochy, P.M.; Beaulieu, D.; Lapointe, J.; Roy, P.H.: Two residues in the anticodon recognition domain of the aspartyl-tRNA synthetase from Pseudomonas aeruginosa are individually implicated in the recognition of tRNAAsn . J. Bacteriol., 188, 269-274 (2006) [7] Feng, L.; Yuan, J.; Toogood, H.; Tumbula-Hansen, D.; Soll, D.: AspartyltRNA synthetase requires a conserved proline in the anticodon-binding loop for tRNA(Asn) recognition in vivo. J. Biol. Chem., 280, 20638-20641 (2005) [8] Sato, Y.; Maeda, Y.; Shimizu, S.; Hossain, M.T.; Ubukata, S.; Suzuki, K.; Sekiguchi, T.; Takenaka, A.: Structure of the nondiscriminating aspartyltRNA synthetase from the crenarchaeon Sulfolobus tokodaii strain 7 reveals the recognition mechanism for two different tRNA anticodons. Acta Crystallogr. Sect. D, 63, 1042-1047 (2007) [9] Suzuki, K.; Sato, Y.; Maeda, Y.; Shimizu, S.; Hossain, M.T.; Ubukata, S.; Sekiguchi, T.; Takenaka, A.: Crystallization and preliminary X-ray crystallographic study of a putative aspartyl-tRNA synthetase from the crenarchaeon Sulfolobus tokodaii strain 7. Acta Crystallogr. Sect. F, 63, 608-612 (2007) [10] Chuawong, P.; Hendrickson, T.L.: The nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity. Biochemistry, 45, 8079-8087 (2006) [11] Bernard, D.; Akochy, P.M.; Bernier, S.; Fisette, O.; Brousseau, O.C.; Chenevert, R.; Roy, P.H.; Lapointe, J.: Inhibition by l-aspartol adenylate of a nondiscriminating aspartyl-tRNA synthetase reveals differences between the interactions of its active site with tRNAAsp and tRNAAsn . J. Enzyme Inhib. Med. Chem., 22, 77-82 (2007) [12] Cardoso, A.M.; Polycarpo, C.; Martins, O.B.; Soell, D.: A non-discriminating aspartyl-tRNA synthetase from Halobacterium salinarum. RNA Biol., 3, 110-114 (2006)

571

Glutamate-tRNAGln ligase

6.1.1.24

1 Nomenclature EC number 6.1.1.24 Systematic name l-glutamate:tRNAGlx ligase (AMP-forming) Recommended name glutamate-tRNAGln ligase Synonyms GluRS GluRS1 <7> [10] GluRS2 <6, 7> [7, 9, 10] ND-GluRS <8> [11] non-discriminating glutamyl-tRNA synthetase <8> [11] nondiscriminating glutamyl-tRNA synthetase CAS registry number 9068-76-2

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8>

Rhizobium meliloti (no sequence specified) [4] Bacillus subtilis (no sequence specified) [2, 4, 5, 6, 8] Scenedesmus obliquus (no sequence specified) [3] Geobacillus stearothermophilus (no sequence specified) [4] Hordeum vulgare (no sequence specified) [1] Helicobacter pylori (no sequence specified) [7,9,10] Acidithiobacillus ferrooxidans (no sequence specified) [7,10] Thermosynechococcus elongatus (no sequence specified) [11]

3 Reaction and Specificity Catalyzed reaction ATP + l-glutamate + tRNAGlx = AMP + diphosphate + l-glutamyl-tRNAGlx (<8> active site structure and substrate recognition [11]) ATP + l-glutamate + tRNAGlx = AMP + diphosphate + glutamyl-tRNAGlx

572

6.1.1.24

Glutamate-tRNAGln ligase

Reaction type Aminoacylation Natural substrates and products S ATP + l-glutamate + tRNAGln <8> (<8> tRNAGln is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase [11]) (Reversibility: ?) [11] P AMP + diphosphate + l-glutamyl-tRNAGln S ATP + l-glutamate + tRNAGlu <8> (Reversibility: ?) [11] P AMP + diphosphate + l-glutamyl-tRNAGlu S ATP + l-glutamate + tRNAGlx <1, 2, 3> (<3> the enzyme is capable of mischarging plastidal tRNAGln from barley with glutamate as well as regularly charges the plastidal tRNAGlu from Scenedesmus. The mischarged glutamyl-tRNAGln is subsequently amidated by glutamyl-tRNA amidotransferase to form the glutaminyl-tRNAGln required for plastidal protein biosynthesis [3]; <1> the enzyme aminoacylates both tRNAGlu and tRNAGln because Rhizobium meliloti contains no glutaminyl-tRNAGln ligase [4]; <2> the enzyme is responsible for the in vivo aminoacylation of both tRNAGlu and tRNAGln in Bacillus subtilis [5]) (Reversibility: ?) [3, 4, 5] P AMP + diphosphate + Glu-tRNAGlx <1, 2, 3> [3, 4, 5] Substrates and products S ATP + Glu + tRNAGlu <7> (<7> GluRS1 [10]) (Reversibility: ?) [10] P AMP + diphosphate + l-glutamyl-tRNAGlu S ATP + Glu + tRNAGln <2, 6, 7> (<7> GluRS1 charges tRNAGln (CUG) [10]; <6> GluRS2 [9]; <6> GluRS2 is specific solely for tRNAGln [10]; <7> GluRS2 preferetially charges tRNAGln (UUG) [10]) (Reversibility: ?) [7, 8, 9, 10] P AMP + diphosphate + l-glutamyl-tRNAGln S ATP + l-glutamate + tRNAGln <1, 2, 3, 4, 5> (<5> the enzyme efficiently charges E. coli tRNAGlu and both tRNAGlu and tRNAGln from chloroplasts, no activity with the two E. coli tRNAGln species [1]; <3> the enzyme is capable of mischarging plastidal tRNAGln from barley with glutamate as well as regularly charges the plastidal tRNAGlu from Scenedesmus. The mischarged glutamyl-tRNAGln is subsequently amidated by glutamyl-tRNA amidotransferase to form the glutaminyl-tRNAGln required for plastidal protein biosynthesis [3]; <2> the enzyme aminoacylates tRNAGlu and tRNASGln in Bacillus subtilis and efficiently misacylates E. coli tRNA1Gln in vitro [5]) (Reversibility: ?) [1, 2, 3, 4, 5, 6] P AMP + diphosphate + Glu-tRNAGln <1, 2, 3, 4> [2, 3, 4] S ATP + l-glutamate + tRNAGln <8> (<8> tRNAGln is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase [11]) (Reversibility: ?) [11] P AMP + diphosphate + l-glutamyl-tRNAGln S ATP + l-glutamate + tRNAGlu <1, 2, 3, 4, 5> (<5> the enzyme efficiently charges E. coli tRNAGlu and both tRNAGlu and tRNAGln from chloroplasts, no activity with the two E. coli tRNAGln species [1]; <2> the enzyme ami-

573

Glutamate-tRNAGln ligase

P S P S

P S

P

noacylates tRNAGlu and tRNASGln in Bacillus subtilis and efficiently misacylates E. coli tRNA1Gln in vitro, not tRNA2Gln from E. coli or tRNAGlu from E. coli [5]; <3> the enzyme is capable of mischarging plastidal tRNAGln from barley with glutamate as well as regularly charges the plastidal tRNAGlu from Scenedesmus. The mischarged glutamyl-tRNAGln is subsequently amidated by glutamyl-tRNA amidotransferase to form the glutaminyl-tRNAGln required for plastidal protein biosynthesis [3]; <2> the enzyme interacts with the G64-C50 or G64-U50 in the Ty stem of its tRNA substrate [5]; <2> major recognition element for the enzyme is U at the 34th position of both tRNA1Gln from Bacillus subtilis and tRNA1Gln from E. coli as a modified form [2]) (Reversibility: ?) [1, 2, 3, 4, 5, 6] AMP + diphosphate + Glu-tRNAGlu <1, 2, 3, 4, 5> [1, 2, 3, 4, 5, 6] ATP + l-glutamate + tRNAGlu <8> (<8> preferred tRNA substrate [11]) (Reversibility: ?) [11] AMP + diphosphate + l-glutamyl-tRNAGlu ATP + l-glutamate + tRNAGlx <1, 2, 3> (<3> the enzyme is capable of mischarging plastidal tRNAGln from barley with glutamate as well as regularly charges the plastidal tRNAGlu from Scenedesmus. The mischarged glutamyl-tRNAGln is subsequently amidated by glutamyl-tRNA amidotransferase to form the glutaminyl-tRNAGln required for plastidal protein biosynthesis [3]; <1> the enzyme aminoacylates both tRNAGlu and tRNAGln because Rhizobium meliloti contains no glutaminyl-tRNAGln ligase [4]; <2> the enzyme is responsible for the in vivo aminoacylation of both tRNAGlu and tRNAGln in Bacillus subtilis [5]) (Reversibility: ?) [3, 4, 5] AMP + diphosphate + Glu-tRNAGlx <1, 2, 3> [3, 4, 5] Additional information <8> (<8> the non-discriminating enzyme charges both tRNAGlu and tRNAGln with glutamate, anticodons of tRNAGlu , 34C/ UUC36, and tRNAGln , 34C/UUG36, differ only in base 36, residue Gly366 is responsible for allowing both cytosine and the bulkier purine base G36 of tRNAGln to be tolerated, glutamate recognition structure, overview [11]) (Reversibility: ?) [11] ?

Cofactors/prosthetic groups ATP <8> [11] Metals, ions Mg2+ <8> [11] Turnover number (min–1) 0.036 <8> (tRNAGln ) [11] 0.04 <2> (tRNAGln , <2> homologous tRNAGln [5]) [5] 0.05 <2> (tRNAGlu , <2> homologous tRNAGlu [5]) [5] 0.07 <2> (tRNAGln , <2> tRNAGln from E. coli [5]) [5] 0.1 <8> (tRNAGlu ) [11]

574

6.1.1.24

6.1.1.24

Glutamate-tRNAGln ligase

Specific activity (U/mg) 0.16 <2> [6] Km-Value (mM) 0.00045 <2> (tRNAGln , <2> homologous tRNAGln , in absence factor b [5]) [5] 0.00065 <2> (tRNAGln , <2> homologous tRNAGln , in presence factor b [5]) [5] 0.00079 <8> (tRNAGlu ) [11] 0.00083 <2> (tRNAGlu , <2> homologous tRNAGlu , in absence factor b [5]) [5] 0.0014 <2> (tRNAGlu , <2> homologous tRNAGlu , in presence factor b [5]) [5] 0.0015 <2> (tRNAGln , <2> tRNAGln [5]) [5] 0.0037 <8> (tRNAGln ) [11] Additional information <8> (<8> kinetics [11]) [11]

of regulatory of regulatory of regulatory of regulatory

4 Enzyme Structure Subunits ? <1> (<1> x * 54166, calculation from nucleotide sequence [4]) [4] Additional information <8> (<8> structure comparison of non-discriminating GluRS versus the discriminating GluRS, EC 6.1.1.17, overview [11]) [11]

5 Isolation/Preparation/Mutation/Application Localization chloroplast <5> [1] plastid <3> [3] Purification <2> [6] <3> [3] <8> (recombinant C-terminally and N-terminally His6-tagged ND-GluRS from Escherichia coli strain BL21 by nickel affinity and anion exchange chromatography, and gel filtration to homogeneity) [11] Crystallization <8> (GluRS in complex with glutamate, hanging drop vapor diffusion at 20 C, 0.003 ml protein solution containing 3 mg/ml protein in 20 mM HEPES, pH 7.9, 20 mM NaCl, 10 mM DTT, 0.25 mM zinc acetate and 0.25 mM MgCl2 , is mixed with 0.003 ml reservoir solution containing 740 mM sodium citrate, 140 mM citric acid, pH 5.8, and 10 mM DTT, 1-2 weeks, cryoprotection using 25% v/v of a 50% w/v trehalose solution, X-ray diffraction structure determination and analysis at 2.45 A resolution) [11]

575

Glutamate-tRNAGln ligase

6.1.1.24

Cloning <1> [4] <2> [4] <2> (Bacillus subtilis GluRS-dependent Glu-tRNAGln formation may cause growth inhibition in the transformed Escherichia coli strain, possibly due to abnormal protein synthesis) [8] <2> (the gene is cloned with its sigmaA promoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 for Escherichia coli and bacillus subtilis. Transformation of Bacillus subtilis with this recombinant plasmidleads to a 30fold increase of glutamyl-tRNA synthetase specific activity in crude extracts. Transformation of Escherichia coli with this plasmid gives no recombinants. The presence of Bacillus subtilis glutamyl-tRNA synthetase is lethal for Escherichia coli, probably because this enzyme glutamylates tRNA1Gln in vivo as it does in vitro) [6] <4> [4] <6> (expression in Escherichia coli) [7] <7> (expression in Escherichia coli) [7] <8> (expression of C-terminally and N-terminally His6-tagged ND-GluRS in Escherichia coli strain BL21) [11] Engineering E334R/G417T/ <6> (<6> mutant of GluRS2 specifically and more robustly aminoacylates tRNAGlu 1 instead of tRNAGln [9]) [9] G417T <6> (<6> mutant GluRS2 shows weak activity towards tRNAGlu 1 [9]) [9]

References [1] Schçn, A.; Sçll, D.: tRNA specificity of a mischarging aminoacyl-tRNA synthetase: glutamyl-tRNA synthetase from barley chloroplasts. FEBS Lett., 228, 241-244 (1988) [2] Kim, S.I.; Soll, D.: Major identity element of glutamine tRNAs from Bacillus subtilis and Escherichia coli in the reaction with B. subtilis glutamyl-tRNA synthetase. Mol. Cells, 8, 459-465 (1998) [3] Vothknecht, U.C.; Doernemann, D.: Charging of both, plastidial tRNAGlx and tRNAGlu with glutamate and subsequent amidation of the misacylated tRNAGln by a glutamyl-tRNA amidotransferase in the unicellular green alga Scenedesmus obliquus, mutant C-2A’. Z. Naturforsch. C, 50, 789-795 (1995) [4] Freist, W.; Gauss, D.H.; Soell, D.; Lapointe, J.: Glutamyl-tRNA synthetase. Biol. Chem., 378, 1313-1329 (1997) [5] Lapointe, J.; Duplain, L.; Proulx, M.: A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln in Bacillus subtilis and efficiently misacylates Escherichia coli tRNA1Gln in vitro. J. Bacteriol., 165, 88-93 (1986) [6] Pelchat, M.; Lacoste, L.; Yang, F.; Lapointe, J.: Overproduction of the Bacillus subtilis glutamyl-tRNA synthetase in its host and its toxicity to Escherichia coli. Can. J. Microbiol., 44, 378-381 (1998)

576

6.1.1.24

Glutamate-tRNAGln ligase

[7] Nunez, H.; Lefimil, C.; Min, B.; Soll, D.; Orellana, O.: In vivo formation of glutamyl-tRNA(Gln) in Escherichia coli by heterologous glutamyl-tRNA synthetases. FEBS Lett., 557, 133-135 (2004) [8] Baick, J.W.; Yoon, J.H.; Namgoong, S.; Soll, D.; Kim, S.I.; Eom, S.H.; Hong, K.W.: Growth inhibition of Escherichia coli during heterologous expression of Bacillus subtilis glutamyl-tRNA synthetase that catalyzes the formation of mischarged glutamyl-tRNA1 Gln. J. Microbiol., 42, 111-116 (2004) [9] Lee, J.; Hendrickson, T.L.: Divergent anticodon recognition in contrasting glutamyl-tRNA synthetases. J. Mol. Biol., 344, 1167-1174 (2004) [10] Salazar, J.C.; Ahel, I.; Orellana, O.; Tumbula-Hansen, D.; Krieger, R.; Daniels, L.; Soll, D.: Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates. Proc. Natl. Acad. Sci. USA, 100, 13863-13868 (2003) [11] Schulze, J.O.; Masoumi, A.; Nickel, D.; Jahn, M.; Jahn, D.; Schubert, W.D.; Heinz, D.W.: Crystal structure of a non-discriminating glutamyl-tRNA synthetase. J. Mol. Biol., 361, 888-897 (2006)

577

Lysine-tRNAPyl ligase

6.1.1.25

1 Nomenclature EC number 6.1.1.25 Systematic name l-lysine:tRNAPyl ligase (AMP-forming) Recommended name lysine-tRNAPyl ligase Synonyms LysRS1 <1, 2, 3> [4] LysRS2 <1, 2> [4] Lysyl-tRNA synthetase <2> [3] class I lysyl-tRNA synthetase <2, 3> [4] class II lysyl-tRNA synthetase <2> [4] Additional information <1, 2, 3> (<1,2,3> cf. EC 6.1.1.6 [4]) [4] CAS registry number 782472-12-2

2 Source Organism <1> Methanosarcina barkeri (no sequence specified) [1, 2, 3, 4] <2> Methanosarcina acetivorans (no sequence specified) [3, 4] <3> Methanosarcina barkeri (UNIPROT accession number: Q9C4B7) [4]

3 Reaction and Specificity Catalyzed reaction ATP + l-lysine + tRNAPyl = AMP + diphosphate + l-lysyl-tRNAPyl Natural substrates and products S ATP + l-lysine + tRNAPyl <1, 2, 3> (Reversibility: ?) [1, 2, 4] P AMP + diphosphate + l-lysyl-tRNAPyl S ATP + l-lysine + tRNAPyl <1> (<1> pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthe-

578

6.1.1.25

Lysine-tRNAPyl ligase

tase that charges the pylT-derived tRNA with lysine but is not closely related to EC 6.1.1.6. Charging a tRNACUA with lysine is a likely first step in translation UAG amber codons as pyrrolysine in certain methanogens. Synthesis of pyrrolysyl-tRNACUA may be achieved by condensation of the e nitrogen of lys-tRNACUA and carboxyl group of (4R,5R)-4-substitutedpyrroline-5-carboxylate, allowing direct translation of the UAG codon as pyrrolysine [1]) (Reversibility: ?) [1] P AMP diphosphate + l-lysyl-tRNAPyl <1> [1] S Additional information <1, 2, 3> (<2> LysRS1 and LysRS2 together form lysyl-tRNAPyl , a potential intermediate to pyrrolysyl-tRNAPyl , in an alternative pathway of pyrrolysyl-tRNAPyl formation, evolution and biological function, overview [4]; <1> LysRS1 and LysRS2 together form lysyltRNAPyl , a potential intermediate to pyrrolysyl-tRNAPyl , in an alternative pathway of pyrrolysyl-tRNAPyl formation, Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl to ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon, overview [4]; <3> LysRS1 and LysRS2 together, synergistically, form lysyl-tRNAPyl , a potential intermediate to pyrrolysyl-tRNAPyl , in an alternative pathway of pyrrolysyl-tRNAPyl formation, association of LysRS1 with growth on methylamine, but not an essential role for LysRS1/LysRS2 in the genetic encoding of pyrrolysine, evolution and biologic function, overview [4]) (Reversibility: ?) [4] P ? Substrates and products S ATP + l-lysine + tRNAPyl <1> (<1> pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to EC 6.1.1.6. Charging a tRNACUA with lysine is a likely first step in translation UAG amber codons as pyrrolysine in certain methanogens. Synthesis of pyrrolysyl-tRNACUA may be achieved by condensation of the e nitrogen of lys-tRNACUA and carboxyl group of (4R,5R)-4-substitutedpyrroline-5-carboxylate, allowing direct translation of the UAG codon as pyrrolysine [1]) (Reversibility: ?) [1] P AMP diphosphate + l-lysyl-tRNAPyl <1> [1] S ATP + l-lysine + tRNAPyl <1, 2, 3> (<1> specific for Lys. Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to EC 6.1.1.6. Charging a tRNACUA with lysine is a likely first step in translation UAG amber codons as pyrrolysine in certain

579

Lysine-tRNAPyl ligase

6.1.1.25

methanogens. Synthesis of pyrrolysyl-tRNACUA may be achieved by condensation of the e nitrogen of lys-tRNACUA and carboxyl group of (4R,5R)-4-substituted-pyrroline-5-carboxylate, allowing direct translation of the UAG codon as pyrrolysine [1]; <3> LysRS2 has no preference for either tRNA substrate, and lack of a functional LysRS1 does not compromise aminoacylation of tRNALys [4]; <1> the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNAPyl with lysine [4]) (Reversibility: ?) [1, 2, 3, 4] P AMP + diphosphate + l-lysyl-tRNAPyl <1, 2> [1, 3] S Additional information <1, 2, 3> (<2> LysRS1 and LysRS2 together form lysyl-tRNAPyl , a potential intermediate to pyrrolysyl-tRNAPyl , in an alternative pathway of pyrrolysyl-tRNAPyl formation, evolution and biological function, overview [4]; <1> LysRS1 and LysRS2 together form lysyltRNAPyl , a potential intermediate to pyrrolysyl-tRNAPyl , in an alternative pathway of pyrrolysyl-tRNAPyl formation, Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl to ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon, overview [4]; <3> LysRS1 and LysRS2 together, synergistically, form lysyl-tRNAPyl , a potential intermediate to pyrrolysyl-tRNAPyl , in an alternative pathway of pyrrolysyl-tRNAPyl formation, association of LysRS1 with growth on methylamine, but not an essential role for LysRS1/LysRS2 in the genetic encoding of pyrrolysine, evolution and biologic function, overview [4]; <1> structure of endogenous tRNAPyl l, overview [4]) (Reversibility: ?) [4] P ? Cofactors/prosthetic groups ATP <1, 2, 3> [4] Activating compounds class I LysRS enzyme <1> (<1> both the class I and II enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Class I and II LysRS proteins form a ternary complex with tRNAPyl , with the aminoacylation activity residing in the class II enzyme [3]) [3] class II LysRS enzyme <1> (<1> both the class I and II enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Class I and II LysRS proteins form a ternary complex with tRNAPyl , with the aminoacylation activity residing in the class II enzyme [3]) [3] Metals, ions Mg2+ <1, 2, 3> [4] Turnover number (min–1) 96 <1> (Lys) [1] Additional information <1> (<1> the enzyme charges tRNACUA with lysine at a rate of 18 per sec [1]) [1]

580

6.1.1.25

Lysine-tRNAPyl ligase

Km-Value (mM) 0.0022 <1> (Lys) [1] pH-Optimum 7.2 <1> (<1> assay at [4]) [4] Temperature optimum ( C) 37 <1> (<1> assay at [4]) [4]

4 Enzyme Structure Subunits ? <1> (<1> x * 49000 [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> (recombinant His-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography) [4] Cloning <1> (co-expression of His-tagged LysRS1 and LysRS2 in Escherichia coli strain BL21(DE3)) [4] <1> (expression in Escherichia coli as a 49000 Da protein with a hexahistidine tag) [1] <1> (pylS gene cloned into pET15b, pET20b or pCYB11 and pGX2T vectors for protein expression in Escherichia coli and into pRS413 for expression in yeast) [3] <2> (pylS gene cloned into pET15b, pET20b or pCYB11 and pGX2T vectors for protein expression in Escherichia coli and into pRS413 for expression in yeast) [3] <3> (gene lysK, co-expression with lysS in Escherichia coli) [4] Engineering Additional information <1, 2, 3> (<3> construction of lysS or lysK deletion strains, which both are compromised for charging of tRNAPyl , Escherichia coli carrying pEC02 and pAMSK, and thereby bearing lysS and lysK in addition to pylT and mtmB1, does not produce the UAG-translation product of mtmB1 in the presence and absence of 1 mM exogenous pyrrolysine or 1 mM lysine, comparison of pylS or lysK/lysS to support pylT-dependent amber suppression in Escherichia coli, overview [4]; <1,2> construction of strains bearing deletions of lysK or lysS, the mutants grow normally on methanol and methylamines with wild-type levels of monomethylamine methyltransferase and aminoacyltRNAPyl , the lysK and lysS genes cannot replace pylS in a recombinant system employing tRNAPyl for UAG suppression [4]) [4]

581

Lysine-tRNAPyl ligase

6.1.1.25

References [1] Srinivasan, G.; James, C.M.; Krzycki, J.A.: Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science, 296, 14591462 (2002) [2] Hao, B.; Gong, W.; Ferguson, T.K.; James, C.M.; Krzycki, J.A.; Chan, M.K.: A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science, 1459, 1462-1466 (2002) [3] Hao, B.; Gong, W.; Ferguson, Polycarpo, C.; Ambrogelly, A.; Ruan, B.; Tumbula-Hansen, D.; Ataide, S.F.; Ishitani, R.; Yokoyama, S.; Nureki, O.; Ibba, M.; Soell, D.: Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases. Mol. Cell, 12, 287-294 (2003) [4] Mahapatra, A.; Srinivasan, G.; Richter, K.B.; Meyer, A.; Lienard, T.; Zhang, J.K.; Zhao, G.; Kang, P.T.; Chan, M.; Gottschalk, G.; Metcalf, W.W.; Krzycki, J.A.: Class I and class II lysyl-tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp.. Mol. Microbiol., 64, 1306-1318 (2007)

582

Pyrrolysine-tRNAPyl ligase

6.1.1.26

1 Nomenclature EC number 6.1.1.26 Systematic name l-pyrrolysine:tRNAPyl ligase (AMP-forming) Recommended name pyrrolysine-tRNAPyl ligase Synonyms PylRS <1, 2, 4, 5> [1, 2, 3, 6, 8] PylS <1, 3> [4, 5] class II aminoacyl-tRNA synthetase <1, 3> [4, 5] pyrrolysyl-tRNA synthetase <1, 2, 4, 5> [1, 2, 3, 6, 7, 8] Additional information <1> (<1> cf. EC 6.1.1.25 [7]) [7]

2 Source Organism <1> <2> <3> <4> <5>

Methanosarcina barkeri (no sequence specified) [2, 3, 5, 7] Methanosarcina mazei (no sequence specified) [1, 8] Methanosarcina acetivorans (no sequence specified) [4] Desulfitobacterium hafniense (no sequence specified) [3, 6] Methanosarcina thermophila (UNIPROT accession number: Q1L6A3) [3]

3 Reaction and Specificity Catalyzed reaction ATP + l-pyrrolysine + tRNAPyl = AMP + diphosphate + l-pyrrolysyl-tRNAPyl Natural substrates and products S ATP + l-pyrrolysine + tRNAPyl <1, 2, 3, 4, 5> (<1> direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo [5]; <1> Pyl-tRNAPyl insertion at UAG, a specialized mRNA motif is not essential for stopcodon recoding, unlike for selenocysteine incorporation [2]; <2,4> pyrrolysyltRNA synthetase PylRS attaches l-pyrrolysine to its cognate tRNA, the special amber suppressor tRNAPyl , encoded by gene pylT [6,8]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] P AMP + diphosphate + l-pyrrolysyl-tRNAPyl

583

Pyrrolysine-tRNAPyl ligase

6.1.1.26

S Additional information <1, 2, 5> (<2> enzyme evolution study, PylRS can be placed in the aminoacyl-tRNA synthetase tree as the last known synthetase that evolved for genetic code expansion, pyrrolysine arose before the last universal common ancestral state [8]; <1> Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl to ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon, l-pyrrolysine is found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA, overview [7]; <1> pyrrolysine is required in e.g. methylamine methyltransferase MtmB, overview [5]; <5> the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo [3]; <1> while pyrrolysine is the natural substrate of PylRS, lysine is not recognized by the enzyme [2]) (Reversibility: ?) [2, 3, 5, 7, 8] P ? Substrates and products S ATP + l-pyrrolysine + tRNAPyl <1, 2, 3, 4, 5> (<1> direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo [5]; <1> Pyl-tRNAPyl insertion at UAG, a specialized mRNA motif is not essential for stopcodon recoding, unlike for selenocysteine incorporation [2]; <2,4> pyrrolysyltRNA synthetase PylRS attaches l-pyrrolysine to its cognate tRNA, the special amber suppressor tRNAPyl , encoded by gene pylT [6,8]; <1> direct charging of tRNA(CUA), isolated from Methanosarcina acetivorans strain C2A, with pyrrolysine in vitro and in vivo, PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNACUA in vitro in reactions specific for pyrrolysine [5]; <4> PylRS binds tRNA predominantly along the phosphate backbone of the T-loop, the d-stem and the acceptor stem, while no significant contacts with the anticodon arm occur, the tRNAPyl anticodon is not important for recognition by bacterial PylRS, overview [6]; <2> substrate binding, PylRS utilizes a deep hydrophobic pocket for recognition of the Pyl side chain [8]; <5> the archaeal enzyme does not distinguish between archaeal and bacterial tRNAPyl species, substrate specificity, overview [3]; <1> the archaeal enzyme does not distinguish between archaeal and bacterial tRNAPyl species, substrate specificity, overview, residues from the PylRS amino-terminal domain affect activity in vivo [3]; <4> the bacterial PylRS displays a clear preference for the homologous cognate tRNA, substrate specificity, overview [3]; <3> the enzyme is specific for l-pyrrolysine [4]; <1> the enzyme uses a special amber suppressor tRNA, tRNAPyl , that presumably recognizes this UAG codon, and does not accept l-lysine or tRNALys as substrates, direct transfer of l-pyrroslysine to the UAG codon of tRNAPyl , overview [7]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] P AMP + diphosphate + l-pyrrolysyl-tRNAPyl (<2> pyrrolysine-AMPbinds in a deep hydrophobic pocket, with its position coordinated by a hydrogen-bonding network with PylRS, binding structure, overview [8])

584

6.1.1.26

Pyrrolysine-tRNAPyl ligase

S ATP + N-e-d-prolyl-l-lysine + tRNAPyl <1> (Reversibility: ?) [2] P AMP + diphosphate + N-e-d-prolyl-l-lysyl-tRNAPyl S ATP + N-e-cyclopentyloxycarbonyl-l-lysine + tRNAPyl <1, 4> (<4> substrate specificity, ability of 24 mutant tRNA species to be aminoacylated by the enzyme with the pyrrolysine analog N-e-cyclopentyloxycarbonyl-llysine, overview, the discriminator base G73 and the first base pair G1C72 in the acceptor stem are major identity elements [6]) (Reversibility: ?) [2, 6] P AMP + diphosphate + N-e-cyclopentyloxycarbonyl-l-lysyl-tRNAPyl S Additional information <1, 2, 4, 5> (<2> enzyme evolution study, PylRS can be placed in the aminoacyl-tRNA synthetase tree as the last known synthetase that evolved for genetic code expansion, pyrrolysine arose before the last universal common ancestral state [8]; <1> Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl to ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon, l-pyrrolysine is found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA, overview [7]; <1> pyrrolysine is required in e.g. methylamine methyltransferase MtmB, overview [5]; <5> the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo [3]; <1> while pyrrolysine is the natural substrate of PylRS, lysine is not recognized by the enzyme [2]; <4> enzyme activity with wild-type and different mutant tRNAPyls, overview, 3-labeled tRNAPyl footprinting with S1 and T1 nuclease digestions, overview [6]; <1> structure of endogenous tRNAPyl , overview [7]; <2> substrate-binding specificity of PylRS, overview [8]; <1> synthetic l-pyrrolysine is attached as a free molecule to tRNACUA by PylS, an archaeal class II aminoacyl-tRNA synthetase, inability of recombinant PylS-His6 to synthesize lysyltRNACUA [5]) (Reversibility: ?) [2, 3, 5, 6, 7, 8] P ? Cofactors/prosthetic groups ATP <1, 2, 3, 4, 5> [1,2,3,4,5,6,7,8] Metals, ions Mg2+ <1, 2, 3, 4> [2, 3, 4, 5, 6, 7, 8] Turnover number (min–1) 0.19 <4> (tRNAPyl , <4> pH 7.2, 37 C, wild-type tRNAPyl [6]) [6] Km-Value (mM) 0.00015-0.00098 <1> (<1> equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview [3]) [3] 0.00016-0.001 <5> (<5> equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview [3]) [3] 0.0029 <4> (tRNAPyl , <4> pH 7.2, 37 C, wild-type tRNAPyl [6]) [6]

585

Pyrrolysine-tRNAPyl ligase

6.1.1.26

0.0053-0.0069 <4> (<4> equilibrium binding analysis, dissociation constants of the enzyme with tRNAPyl from different species, overview [3]) [3] 0.053 <1> (pyrrolysine, <1> pH 7.2, 37 C, recombinant enzyme [2]) [2] 0.5 <1> (N-e-d-prolyl-l-lysine, <1> pH 7.2, 37 C, recombinant enzyme [2]) [2] 0.67 <1> (N-e-cyclopentyloxycarbonyl-l-lysine, <1> pH 7.2, 37 C, recombinant enzyme [2]) [2] Additional information <4> (<4> kinetics of pyrrolysylation of several mutant tRNAPyls , overview [6]) [6] pH-Optimum 7.2 <1, 4, 5> (<1,4,5> assay at [2,3,5,6,7]) [2, 3, 5, 6, 7] Temperature optimum ( C) 37 <1, 4, 5> (<1,4,5> assay at [2,3,5,6,7]) [2, 3, 5, 6, 7]

4 Enzyme Structure Subunits ? <2> (<2> x * 51000, full-length enzyme, SDS-PAGE, x * 33000, recombinant N-terminally truncated enzyme form PylRS(c270), SDS-PAGE [1]) [1] Additional information <1, 2, 4, 5> (<2> PylRS is mainly composed of two domains: the N-terminal RNA-binding domain and the C-terminal aaRS catalytic domain [1]; <4> the PylRS sequence can be subdivided into three regions: the highly conserved class II aaRS catalytic core domain at the carboxy-terminal, the unique amino-terminal domain, and a highly variable region linking these two domains [3]; <1> the PylRS sequence can be subdivided into three regions: the highly conserved class II aaRS catalytic core domain at the carboxy-terminal, the unique amino-terminal domain, and a highly variable region linking these two domains, residues from the PylRS amino-terminal domain affect activity in vivo [3]; <5> the PylRS sequence can be subdivided into three regions: the highly conserved class II aaRS catalytic core domain at the carboxy-terminal, the unique amino-terminal domain, and a highly variable region linking these two domains, the aminoterminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo [3]) [1, 3]

5 Isolation/Preparation/Mutation/Application Purification <1> (recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography) [7] <1> (recombinant His-tagged wild-type and mutant enzymes from Escherichia coli BL21(DE3)) [3] <1> (recombinant His6-tagged PylS from Escherichia coli strain BL21(DE3) by nickel affinity chromatography) [5]

586

6.1.1.26

Pyrrolysine-tRNAPyl ligase

<2> (recombinant His-tagged N-terminally truncated enzyme form PylRS(c270) from Escherichia coli strain BL21(DE3) to homogeneity by affinity chromatography, hydrophobic interaction chromatography, and adsorption chromatography) [1] <2> (recombinant N-terminally His-tagged catalytic domain of the enzyme from Escherichia coli by two different steps of affinity chromatography, and gel filtration) [8] <4> (recombinant His-tagged enzyme from Escherichia coli BL21(DE3)) [3] <4> (recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography) [6] <5> (recombinant His-tagged enzyme from Escherichia coli BL21(DE3)) [3] Crystallization <2> (purified recombinant His-tagged N-terminally truncated enzyme form PylRS(c270) in complex with an ATP analogue AMP-PNP, hanging drop vapour diffusion method, in 100 mM sodium cacodylate, pH 6.8, containing 0.25 M NaCl, 5 mM MgSO4 and 5% w/v PEG 4000, 20 C, hexagonal crystals, X-ray diffraction structure determination and analysis at 1.9-2.6 A resolution) [1] <2> (purified recombinant N-terminally His-tagged catalytic domain of PylRS complexed with either AMP-PNP, pyrrolysine-AMP plus pyrophosphate, or the pyrrolysine analogue N-e-[(cylopentyloxy)carbonyl]-l-lysine plus ATP, vapour diffusion method, 10 mg/ml protein in 100 mM Tris, pH 7.0-8.0, 8-14% PEG 2000 monomethyl ether, 10 mM pyrrolysine, and 10 mM AMP-PNP or other ligands, overnight at 16 C, stabilization and cryoprotection by 5 mM EDTA, 10 mM AMP-PNP, 5 mM MgCl2 , 30% ethylen glycol, and additional 2% PEG, hexagonal-shaped crystals, X-ray diffraction structure determination and analysis at 1.8 A resolution) [8] Cloning <1> (functional co-expression with tRNAPyl in Escherichia coli, the recombinant enzyme is active with substrate analogues N-e-d-prolyl-l-lysine and Ne-cyclopentyloxycarbonyl-l-lysine) [2] <1> (gene pylS, expression as His6-tagged enzyme in Escherichia coli strain BL21 (DE3), addition of pyrrolysine to Escherichia coli cells expressing pylT, encoding tRNACUA , and pylS results in the translation of UAG in vivo as a sense codon, inability of PylS-His6 to synthesize lysyltRNACUA , co-expression with gene mtmB1) [5] <1> (gene pylS, expression of His-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3)) [3] <1> (gene pylS, expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3)) [7] <2> (gene pylS, phylogenetic analysis and phylogeny of subclass IIc aaRSs, overview, expression of the N-terminally His-tagged catalytic domain of the enzyme in Escherichia coli) [8] <2> (overexpression of the N-terminally truncated enzyme form PylRS(c270) as N-terminally His-tagged protein in Escherichia coli strain BL21(DE3), ex-

587

Pyrrolysine-tRNAPyl ligase

6.1.1.26

pression of selenomethionine-labeled enzyme in Escherichia coli strain B834(DE3)) [1] <4> (gene pylS, expression in Escherichia coli BL21(DE3) as His-tagged enzyme) [3] <4> (gene pylS, expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3)) [6] <5> (gene pylS, DNA and amino acid sequence determination and analysis, expression in Escherichia coli BL21(DE3) as His-tagged enzyme) [3] Engineering D2A <1> (<1> site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme [3]) [3] D2A/K4A <1> (<1> site-directed mutagenesis, the mutant shows almost completely reduced activity compared to the wild-type enzyme [3]) [3] D7A <1> (<1> site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme [3]) [3] G21L <1> (<1> site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme [3]) [3] H24A <1> (<1> site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme [3]) [3] I26G <1> (<1> site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme [3]) [3] K3A <1> (<1> site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme [3]) [3] K4A <1> (<1> site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme [3]) [3] R19A <1> (<1> site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme [3]) [3] S11A <1> (<1> site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme [3]) [3] S11A/T13A <1> (<1> site-directed mutagenesis, the mutant shows almost completely reduced activity compared to the wild-type enzyme [3]) [3] S18A <1> (<1> site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme [3]) [3] T13A <1> (<1> site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme [3]) [3] W16A <1> (<1> site-directed mutagenesis, the mutant shows 35% reduced activity compared to the wild-type enzyme [3]) [3] Additional information <1, 3> (<1> construction of several truncation mutants, which show 90-99% reduced activity compared to the wild-type enzyme, overview [3]; <3> in vivo ability of lysK/lysS to replace pylS for tRNAPyl -dependent amber suppression in a recombinant system, overview [4]; <1> the genetic code of Escherichia coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins [5]) [3, 4, 5]

588

6.1.1.26

Pyrrolysine-tRNAPyl ligase

References [1] Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Nureki, O.; Yokoyama, S.: Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of pyrrolysyl-tRNA synthetase from the methanogenic archaeon Methanosarcina mazei. Acta Crystallogr. Sect. F, 62, 1031-1033 (2006) [2] Polycarpo, C.R.; Herring, S.; Berube, A.; Wood, J.L.; Soll, D.; Ambrogelly, A.: Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett., 580, 6695-6700 (2006) [3] Herring, S.; Ambrogelly, A.; Gundllapalli, S.; O’Donoghue, P.; Polycarpo, C.R.; Soll, D.: The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity. FEBS Lett., 581, 31973203 (2007) [4] Mahapatra, A.; Srinivasan, G.; Richter, K.B.; Meyer, A.; Lienard, T.; Zhang, J.K.; Zhao, G.; Kang, P.T.; Chan, M.; Gottschalk, G.; Metcalf, W.W.; Krzycki, J.A.: Class I and class II lysyl-tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp.. Mol. Microbiol., 64, 1306-1318 (2007) [5] Blight, S.K.; Larue, R.C.; Mahapatra, A.; Longstaff, D.G.; Chang, E.; Zhao, G.; Kang, P.T.; Green-Church, K.B.; Chan, M.K.; Krzycki, J.A.: Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo. Nature, 431, 333-335 (2004) [6] Herring, S.; Ambrogelly, A.; Polycarpo, C.R.; Soll, D.: Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase. Nucleic Acids Res., 35, 1270-1278 (2007) [7] Polycarpo, C.; Ambrogelly, A.; Berube, A.; Winbush, S.M.; McCloskey, J.A.; Crain, P.F.; Wood, J.L.; Soll, D.: An aminoacyl-tRNA synthetase that specifically activates pyrrolysine. Proc. Natl. Acad. Sci. USA, 101, 12450-12454 (2004) [8] Kavran, J.M.; Gundllapalli, S.; O’Donoghue, P.; Englert, M.; Soll, D.; Steitz, T.A.: Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation. Proc. Natl. Acad. Sci. USA, 104, 11268-11273 (2007)

589

trans-Feruloyl-CoA synthase

6.2.1.34

1 Nomenclature EC number 6.2.1.34 Systematic name trans-ferulate:CoASH ligase (ATP-hydrolysing) Recommended name trans-feruloyl-CoA synthase Synonyms ferulate-CoA ligase trans-feruloyl-CoA synthetase

2 Source Organism <1> Pseudomonas fluorescens (no sequence specified) [1]

3 Reaction and Specificity Catalyzed reaction ferulic acid + CoASH + ATP = trans-feruloyl-CoA + products of ATP breakdown Reaction type Formation of thioester Natural substrates and products S ferulic acid + CoASH + ATP <1> (<1> enzyme is involved in ferulic acid metabolism, inducible enzyme [1]) (Reversibility: ?) [1] P trans-feruloyl-CoA + products of ATP breakdown <1> [1] Substrates and products S ferulic acid + CoASH + ATP <1> (<1> enzyme is involved in ferulic acid metabolism, inducible enzyme [1]) (Reversibility: ?) [1] P trans-feruloyl-CoA + products of ATP breakdown <1> [1] Metals, ions Mg2+ <1> (<1> required [1]) [1]

590

6.2.1.34

trans-Feruloyl-CoA synthase

References [1] Narbad, A.; Gasson, M.J.: Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly isolated strain of Pseudomonas fluorescens. Microbiology, 144, 1397-1405 (1998)

591

Adenosylcobinamide-phosphate synthase

6.3.1.10

1 Nomenclature EC number 6.3.1.10 Systematic name adenosylcobyric acid:(R)-1-aminopropan-2-yl phosphate ligase (ADP-forming) Recommended name adenosylcobinamide-phosphate synthase Synonyms AdoCbi-P synthase <1, 2> [3, 4] CbiB <1, 2> [3, 4]

2 Source Organism <1> Salmonella enterica (no sequence specified) [1, 2, 4] <2> Halobacterium sp. (no sequence specified) [3]

3 Reaction and Specificity Catalyzed reaction ATP + adenosylcobyric acid + (R)-1-aminopropan-2-yl phosphate = ADP + phosphate + adenosylcobinamide phosphate (<2> this enzyme forms part of the anaerobic cobalamin biosynthesis pathway, one of the substrates for this reaction, (R)-1-aminopropan-2-yl phosphate, is produced by CobD, EC 4.1.1.81, threonine phosphate decarboxylase [3]) ATP + adenosylcobyric acid + (R)-1-aminopropan-2-ol = ADP + phosphate + adenosylcobinamide Reaction type C-N bond formation ligation Natural substrates and products S ATP + adenosylcobyric acid + (R)-1-aminopropan-2-ol <1> (Reversibility: ?) [2] P ADP + phosphate + adenosylcobinamide

592

6.3.1.10

Adenosylcobinamide-phosphate synthase

S ATP + adenosylcobyric acid + (R)-1-aminopropan-2-yl phosphate <1> (Reversibility: ?) [2] P ADP + phosphate + adenosylcobinamide phosphate S Additional information <1, 2> (<2> CbiB is involved in a salvaging pathway for cobinamide [3]; <1> CbiB is involved in the anaerobic cobalamin/ cobinamide biosynthesis pathway [1]) (Reversibility: ?) [1, 3] P ? Substrates and products S ATP + adenosylcobyric acid + (R)-1-aminopropan-2-ol <1> (Reversibility: ?) [2] P ADP + phosphate + adenosylcobinamide S ATP + adenosylcobyric acid + (R)-1-aminopropan-2-yl phosphate <1, 2> (<1> amide bond formation, the substrate for CbiB is not aminopropanol but its phosphate [1]) (Reversibility: ?) [1, 2, 3] P ADP + phosphate + adenosylcobinamide phosphate S ATP + ethanolamine phosphate <1> (Reversibility: ?) [4] P norcobalamin + ? S Additional information <1, 2> (<2> CbiB is involved in a salvaging pathway for cobinamide [3]; <1> CbiB is involved in the anaerobic cobalamin/ cobinamide biosynthesis pathway [1]; <1> l-threonine-phosphate is not a substrate [4]) (Reversibility: ?) [1, 3, 4] P ?

5 Isolation/Preparation/Mutation/Application Cloning <2> (cbiB gene, complementation studies) [3] Engineering Additional information <2> (<2> the cbiB in-frame deletion mutant strain JE6791 does not salvage cobinamide [3]) [3]

References [1] Warren, M.J.; Raux, E.; Schubert, H.L.; Escalante-Semerena, J.C.: The biosynthesis of adenosylcobalamin (vitamin B12 ). Nat. Prod. Rep., 19, 390-412 (2002) [2] Cheong, C.G.; Bauer, C.B.; Brushaber, K.R.; Escalante-Semerena, J.C.; Rayment, I.: Three-dimensional structure of the l-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica. Biochemistry, 41, 4798-4808 (2002)

593

Adenosylcobinamide-phosphate synthase

6.3.1.10

[3] Woodson, J.D.; Zayas, C.L.; Escalante-Semerena, J.C.: A new pathway for salvaging the coenzyme B12 precursor cobinamide in archaea requires cobinamide-phosphate synthase (CbiB) enzyme activity. J. Bacteriol., 185, 71937201 (2003) [4] Zayas, C.L.; Claas, K.; Escalante-Semerena, J.C.: The CbiB protein of Salmonella enterica is an integral membrane protein involved in the last step of the de novo corrin ring biosynthetic pathway. J. Bacteriol., 189, 7697-7708 (2007)

594

Glutamate-putrescine ligase

6.3.1.11

1 Nomenclature EC number 6.3.1.11 Systematic name l-glutamate:putrescine ligase (ADP-forming) Recommended name glutamate-putrescine ligase Synonyms YcjK <1> [1] g-Glu-Put synthetase <1> [1] g-glutamylputrescine synthetase <1> [1]

2 Source Organism <1> Escherichia coli (no sequence specified) (<1> large subunit [1]) [1]

3 Reaction and Specificity Catalyzed reaction ATP + l-glutamate + putrescine = ADP + phosphate + g-l-glutamylputrescine Reaction type acid amide formation Natural substrates and products S ATP + glutamate + putrescine <1> (<1> the enzyme is involved in a putrescine utilization pathway [1]) (Reversibility: ?) [1] P ADP + phosphate + g-l-glutamylputrescine Substrates and products S ATP + l-glutamate + putrescine <1> (Reversibility: ?) [1] P ADP + phosphate + g-l-glutamylputrescine S ATP + glutamate + putrescine <1> (<1> the enzyme is involved in a putrescine utilization pathway [1]) (Reversibility: ?) [1] P ADP + phosphate + g-l-glutamylputrescine

595

Glutamate-putrescine ligase

6.3.1.11

Metals, ions Mg2+ <1> (<1> required [1]) [1]

References [1] Kurihara, S.; Oda, S.; Kato, K.; Kim, H.G.; Koyanagi, T.; Kumagai, H.; Suzuki, H.: A novel putrescine utilization pathway involves g-glutamylated intermediates of Escherichia coli K-12. J. Biol. Chem., 280, 4602-4608 (2005)

596

D-Aspartate

ligase

6.3.1.12

1 Nomenclature EC number 6.3.1.12 Systematic name d-aspartate:[b-GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-dAla)]n ligase (ADP-forming) Recommended name d-aspartate ligase Synonyms Aslfm <1> [2] d-aspartic acid-activating enzyme <1> [2] UDP-MurNAc-pentapeptide:d-aspartate ligase <1> [2] aslA <4> [4]

2 Source Organism <1> <2> <3> <4>

Streptococcus faecalis (no sequence specified) [1, 2] Lactobacillus casei (no sequence specified) [1] Enterococcus faecium (no sequence specified) [3] Lactococcus lactis (no sequence specified) [4]

3 Reaction and Specificity Catalyzed reaction ATP + d-aspartate + [b-GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-dAla-d-Ala)]n = [b-GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-6-N-(b-d-Asp)l-Lys-d-Ala-d-Ala)]n + ADP + phosphate Reaction type acid amide formation Natural substrates and products S ATP + d-aspartate + [b-GlcNAc-(1,4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lysd-Ala-d-Ala)]n <3> (<3> the enzyme is responsible for the addition of d-aspartic acid onto the peptidoglycan precursor. specifically ligates the b-carboxylate of d-Asp to the e-amino group of l-Lys in the nucleotide precursor UDP-N-acetylmuramyl-pentapeptide [3]) (Reversibility: ?) [3]

597

D-Aspartate

ligase

6.3.1.12

P [b-GlcNAc-(1,4)-Mur2Ac(oyl-l-Ala-g-d-Glu-6-N-(?-d-Asp)-l-Lys-d-Alad-Ala)]n + ADP + phosphate Substrates and products S ATP + d-aspartate + [b-GlcNAc-(1,4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lysd-Ala-d-Ala)]n <1, 2, 3, 4> (<1,2> strictly specific for ATP. No activity with CTP, GTP, TTP and UTP [1]; <3> the enzyme is highly specific for d-aspartate, as l-aspartate, d-glutamate, d-alanine, d-iso-asparagine and d-malic acid are not substrates [3]) (Reversibility: ?) [1, 3, 4] P [b-GlcNAc-(1,4)-Mur2Ac(oyl-l-Ala-g-d-Glu-6-N-(b-d-Asp)-l-Lys-d-Alad-Ala)]n + ADP + phosphate S ATP + d-aspartate + [b-GlcNAc-(1,4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lysd-Ala-d-Ala)]n <3> (<3> the enzyme is responsible for the addition of d-aspartic acid onto the peptidoglycan precursor. specifically ligates the b-carboxylate of d-Asp to the e-amino group of l-Lys in the nucleotide precursor UDP-N-acetylmuramyl-pentapeptide [3]) (Reversibility: ?) [3] P [b-GlcNAc-(1,4)-Mur2Ac(oyl-l-Ala-g-d-Glu-6-N-(b-d-Asp)-l-Lys-d-Alad-Ala)]n + ADP + phosphate Metals, ions Mg2+ <1, 3> (<3> required [3]; <1> a divalent cation is required for activity. Mg2+ and Mn2+ are approximately equally effective. Optimal concentration of Mg2+ is 50 mM or greater [1]) [1, 3] Mn2+ <1> (<1> a divalent cation is required for activity. Mg2+ and Mn2+ are approximately equally effective. Optimal concentration of Mg2+ is 20 mM [1]) [1] Km-Value (mM) 0.6 <1> (ATP) [1] 2 <1> (d-Aspartate) [1] pH-Optimum 7.5 <1> [1] pH-Range 6.5-8.5 <1> (<1> pH 6.5: about 40% of maximal activity, pH 8.5: about 70% of maximal activity [1]) [1]

5 Isolation/Preparation/Mutation/Application Localization membrane <1> (<1> attached to [2]) [1, 2] Purification <1> [1, 2] <2> [1] <3> [3]

598

6.3.1.12

D-Aspartate

ligase

Cloning <3> [3] Application medicine <3, 4> (<3> the enzyme appears as an attractive target for the development of narrow spectrum antibiotics active against multiresistant Enterococcus faecium [3]; <4> the enzyme is a potential target for specific antimicrobials [4]) [3, 4]

6 Stability Storage stability <1>, -15 C, precipitation with ammonium sulfate, stable for at least several weeks [1]

References [1] Staudenbauer, W.; Strominger, J.L.: Biosynthesis of the peptidoglycan of bacterial cell walls. XXII. Activation of d-aspartic acid for incorporation into peptidoglycan. J. Biol. Chem., 247, 5095-5102 (1972) [2] Staudenbauer, W.; Willoughby, E.; Strominger, J.L.: Further studies of the daspartic acid-activating enzyme of Streptococcus faecalis and its attachment to the membrane. J. Biol. Chem., 247, 5289-5296 (1972) [3] Bellais, S.; Arthur, M.; Dubost, L.; Hugonnet, J.E.; Gutmann, L.; van Heijenoort, J.; Legrand, R.; Brouard, J.P.; Rice, L.; Mainardi, J.L.: Aslfm, the daspartate ligase responsible for the addition of d-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium. J. Biol. Chem., 281, 1158611594 (2006) [4] Veiga, P.; Piquet, S.; Maisons, A.; Furlan, S.; Courtin, P.; Chapot-Chartier, M.P.; Kulakauskas, S.: Identification of an essential gene responsible for dAsp incorporation in the Lactococcus lactis peptidoglycan crossbridge. Mol. Microbiol., 62, 1713-1724 (2006)

599

N-(5-Amino-5-carboxypentanoyl)-L-cysteinylD-valine synthase

6.3.2.26

1 Nomenclature EC number 6.3.2.26 Systematic name l-2-aminohexanedioate:l-cysteine:l-valine ligase (AMP-forming, valine-inverting) Recommended name N-(5-amino-5-carboxypentanoyl)-l-cysteinyl-d-valine synthase Synonyms ACV synthetase d-(a-aminoadipyl)cysteinylvaline synthetase l-(a-aminoadipyl)-l-cysteinyl-d-valine synthetase l-d-(a-aminoadipoyl)-l-cysteinyl-d-valine synthetase d-(L-a-aminoadipoyl)-l-cysteinyl-d-valine synthetase d-l-(a-aminoadipyl)-l-cysteinyl-d-valine synthetase synthetase, d-(a-aminoadipyl)cysteinylvaline CAS registry number 57219-73-5

2 Source Organism <1> Aspergillus nidulans (no sequence specified) [3, 13, 14] <2> Penicillium chrysogenum (no sequence specified) [12, 13, 14] <3> Acremonium chrysogenum (no sequence specified) [1, 2, 4, 5, 6, 7, 8, 13, 14] <4> Streptomyces clavuligerus (no sequence specified) [5, 6, 9, 11, 14] <5> Paecilomyces persicinus (no sequence specified) [13] <6> Lysobacter lactamgenus (no sequence specified) [13] <7> Nocardia lactamdurans (no sequence specified) [10,13]

3 Reaction and Specificity Catalyzed reaction 3 ATP + l-2-aminohexanedioate + l-cysteine + l-valine + H2 O = 3 AMP + 3 diphosphate + N-[l-5-amino-5-carboxypentanoyl]-l-cysteinyl-d-valine

600

6.3.2.26

N-(5-Amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase

Reaction type carboxylic acid amid formation peptide bond formation Substrates and products S 6-oxopiperidine 2-carboxylic acid + l-cysteine + l-valine + ATP <7> (Reversibility: ?) [10] P N-(5-amino-5-carboxypentanoyl)-l-cysteinyl-d-valine + AMP + diphosphate S dl-valine + l-O-(methylserine) + l-2-aminohexanedioate + ATP <3> (Reversibility: ?) [5, 8] P l-O-(methylserinyl)-d-valine + l-O-(methylserinyl)-d-valine + AMP + diphosphate S l-2-aminoadipate + l-cystathionine + l-valine + ATP <7> (Reversibility: ?) [10] P N-(5-amino-5-carboxypentanoyl)-l-cysteinyl-d-valine + AMP + diphosphate S l-2-aminoadipate + l-cysteine + l-allo-isoleucine + ATP <3, 4> (Reversibility: ?) [5, 9] P l-d-(aminoadipoyl)-l-cysteinyl-d-allo-isoleucine + AMP + diphosphate S l-2-aminoadipate + l-cysteine + l-isoleucine + ATP <4> (Reversibility: ?) [9] P l-d-(aminoadipoyl)-l-cysteinyl-d-isoleucine + AMP + diphosphate S l-2-aminoadipate + l-cysteine + l-valine + ATP <1, 2, 3, 4, 5, 6, 7> (Reversibility: ir) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14] P N-(5-amino-5-carboxypentanoyl)-l-cysteinyl-d-valine + AMP + diphosphate S l-2-aminoadipate + l-homocysteine + l-valine + ATP <4> (Reversibility: ?) [9] P N-(5-amino-5-carboxypentanoyl)-l-homocysteinyl-d-valine + AMP + diphosphate S l-2-aminoadipate + allylglycine + l-valine + ATP <3, 4> (Reversibility: ?) [5] P l-d-(aminoadipoyl)-l-allylglycinyl-d-valine + AMP + diphosphate S l-2-aminoadipate + vinylglycine + l-valine + ATP <3, 4> (Reversibility: ?) [5] P l-d-(aminoadipoyl)-l-vinylglycinyl-d-valine + AMP + diphosphate S l-glutamate + l-cysteine + l-valine + ATP <4> (Reversibility: ?) [9] P l-glutamyl-l-cysteinyl-d-valine + AMP + diphosphate S S-carboxymethylcysteine + l-cysteine + l-valine + ATP <3, 4> (Reversibility: ?) [5, 9] P l-S-carboxymethylcysteinyl-l-cysteinyl-d-valine + AMP + diphosphate Inhibitors 5,5’-dithiobis-2-nitrobenzoate <4> (<4> 1 mM [9]) [9] 5,5’-dithiobis-2-nitrobenzoate <4> (<4> 1 mM [9]) [9] AMP <4> (<4> 5 mM [9]) [9] ATP <4> (<4> at concentrations about 5 mM [9]) [9]

601

N-(5-Amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase

6.3.2.26

d-glucose <3> (<3> crude extract is inhibited due to deprivation of ATP via sugar metabolism [6]) [6] diphosphate <4> (<4> 5 mM [9]) [9] dithiothreitol <2> [12] EDTA <4> (<4> 10 mM [9]) [9] glyceraldehyde <3, 4> [6] glyceraldehyde-3-phosphate <3, 4> (<3,4> reacts with l-cysteinee [6]) [6] iodoacetamide <4> (<4> 1 mM [9]) [9] N-ethylmaleimide <4> (<4> 1 mM [9]) [9] pyridoxal 5’-phosphate <4> (<4> 2 mM [9]) [9] bis-d-(l-a-aminoadipyl)-l-cysteinyl-d-valine <2> [12] Cofactors/prosthetic groups ATP <1, 2, 3, 4, 7> (<2> optimal concentration: 5 mM [12]; <4> optimal concentration: 1.5-5 mM [9]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12] Metals, ions Mg2+ <2, 3, 4> (<3,4> optimal concentration: 10 mM [4,9]; <2> optimal concentration: 20 mM [12]) [4, 6, 8, 9, 11, 12] Mn2+ <3> (<3> optimal concentration: 10 mM [4]) [4] Turnover number (min–1) 0.0833 <3> (ATP) [2] 0.133 <3> (cysteine, <3> under saturating conditions [2]) [2] Specific activity (U/mg) 0.0113 <4> [9] 55.71 <7> [10] Km-Value (mM) 0.026 <3> (l-cysteine) [4] 0.045 <2> (l-aminoadipate) [12] 0.08 <2> (l-cysteine) [12] 0.08 <2> (l-valine) [12] 0.12 <4> (l-cysteine) [9] 0.17 <3> (l-aminoadipate) [4] 0.3 <4> (l-valine) [9] 0.34 <3> (l-valine) [4] 0.63 <4> (l-aminoadipate) [9] pH-Optimum 7.5 <3> (<3> assay at [4]) [4] 7.8 <2> (<2> in MOPS buffer [12]) [12] 8-8.5 <4> [9] 8.4 <2> (<2> in Tris buffer [12]) [12] pH-Range 7-9 <4> [9]

602

6.3.2.26

N-(5-Amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase

Temperature optimum ( C) 25 <3> (<3> assay at [4]) [4] 27 <3> (<3> assay at [5]) [5] 29.5 <4> [9] Temperature range ( C) 4-32 <4> [9]

4 Enzyme Structure Molecular weight 220000 <1> (<1> gel filtration [14]) [14] 404100 <7> (<7> calculation from amino acid sequence [13]) [13] 411500 <6> (<6> calculation from amino acid sequence [13]) [13] 414800 <3> (<3> calculation from amino acid sequence [13]) [13] 422500 <1> (<1> calculation from DNA-sequence, PAGE and gel filtration [3]) [3] 424100 <2> (<2> calculation from amino acid sequence [13]) [13] 425000 <3> (<3> gel filtration [2]) [2] 430000 <7> (<7> gel filtration [10]) [10] 470000 <2> (<2> gradient PAGE [12]) [12] Subunits monomer <1, 2, 3, 7> (<3> 1 * 400000, SDS-PAGE [2]; <1> 1 * 220000, SDSPAGE [14]; <7> 1 * 430000, SDS-PAGE [10]; <2> 1 * 470000, SDS-PAGE [12]) [2, 10, 12, 14] Posttranslational modification Glycoprotein <1> [3]

5 Isolation/Preparation/Mutation/Application Source/tissue mycelium <3> [1] Purification <1> [3] <2> [12] <3> [1] <4> [11] <7> [10] Cloning <1> (expression in Escherichia coli) [3] <7> (overexpression in Streptomyces lividans) [10]

603

N-(5-Amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase

6.3.2.26

6 Stability pH-Stability 7 <4> (<4> very unstable below pH 7 [9]) [9] 8.5 <4> (<4> very unstable above pH 8.5 [9]) [9] Temperature stability 4 <4> (<4> half of activity after 70 h [9]) [9, 11] 25 <4> (<4> half of activity after 10 h [9]; <4> half of activity after 8 h [11]) [9, 11] 32 <4> (<4> half of activity after 10 min [9,11]) [9, 11] 34 <4> (<4> the mixture of ATP 5 mM, Mg2+ 5 mM, valine 5 mM prevents the thermal inactivation, 92% activity after 10 min [11]; <4> the mixture of ATP 5 mM, DDT 3 mM, Mg2+ 5 mM, aminoadipate 5 mM, cysteine 1 mM and valine 5 mM prevents the thermal inactivation, 91% activity after 10 min [11]) [11] Organic solvent stability dithiothreitol <4> (<4> 8 mM leads to a 10% higher enzyme activity than at 3 mM [9]; <4> stability of crude enzyme is increased by dithiothreitol [11]) [9, 11] General stability information <3>, glycerol stabilizes enzyme activity [4] Storage stability <2>, -80 C, several months [12] <3>, -80 C, about 15% of activity after 10 months [5] <3>, -80 C, no loss in activity after 6 weeks [5]

References [1] Kallow, W.; Neuhof, T.; Arezi, B.; Jungblut, P.; von Doehren, H.: Penicillin biosynthesis: intermediates of biosynthesis of d-l-a-aminoadipyl-l-cysteinyl-d-valine formed by ACV synthetase from Acremonium chrysogenum. FEBS Lett., 414, 74-78 (1997) [2] Kallow, W.; Von Dohren, H.; Kleinkauf, H.: Penicillin biosynthesis: energy requirement for tripeptide precursor formation by d-(l-a-aminoadipyl)-lcysteinyl-d-valine synthetase from Acremonium chrysogenum. Biochemistry, 37, 5947-5952 (1998) [3] MacCabe, A.P.; Van Liempt, H.; Palissa, H.; Unkles, S.E.; Riach, M.B.R.; Pfeifer, E.; Von Doehren, H.; Kinghorn, J.R.: d-(l-a-Aminoadipyl)-l-cysteinyld-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway. J. Biol. Chem., 266, 12646-12654 (1991) [4] Banko, G.; Demain, A.L.; Wolfe, S.: d-(l-a-Aminoadipyl)-l-cysteinyl-d-valine synthetase (ACV synthetase): a multifunctional enzyme with broad

604

6.3.2.26

[5]

[6] [7] [8]

[9] [10]

[11] [12] [13]

[14]

N-(5-Amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase

substrate specificity for the synthesis of penicillin and cephalosporin precursors. J. Am. Chem. Soc., 109, 2858-2860 (1987) Baldwin, J.E.; Shiau, C.Y.; Byford, M.F.; Schofield, C.J.: Substrate specificity of l-d-(a-aminoadipoyl)-l-cysteinyl-d-valine synthetase from Cephalosporium acremonium: demonstration of the structure of several unnatural tripeptide products. Biochem. J., 301, 367-372 (1994) Zhang, J.; Demain, A.L.: Regulation of ACV synthetase activity in the blactam biosynthetic pathway by carbon sources and their metabolites. Arch. Microbiol., 158, 364-369 (1992) Shiau, C.Y.; Byford, M.F.; Baldwin, J.E.; Schofield, C.J.: Molecular mechanism of the multifunctional enzyme l-d-(a-aminoadipoyl)-l-cysteinyl-d-valine (ACV) synthetase. Biochem. Soc. Trans., 23, 629S (1995) Shiau, C.Y.; Byford, M.F.; Aplin, R.T.; Baldwin, J.E.; Schofield, C.J.: l-d-(aAminoadipoyl)-l-cysteinyl-d-valine synthetase: thioesterification of valine is not obligatory for peptide bond formation. Biochemistry, 36, 8798-8806 (1997) Zhang, J.; Wolfe, S.; Demain, A.L.: Biochemical studies on the activity of d(l-a-aminoadipyl)-l-cysteinyl-d-valine synthetase from Streptomyces clavuligerus. Biochem. J., 283, 691-698 (1992) Coque, J.J.R.; De la Fuente, J.L.; Liras, P.; Martin, J.F.: Overexpression of the Nocardia lactamdurans a-aminoadipyl-cysteinyl-valine synthetase in Streptomyces lividans. The purified multienzyme uses cystathionine and 6-oxopiperidine 2-carboxylate as substrates for synthesis of the tripeptide. Eur. J. Biochem., 242, 264-270 (1996) Zhang, J.; Demain, A.L.: In vitro stabilization of ACV synthetase activity from Streptomyces clavuligerus. Appl. Biochem. Biotechnol., 37, 97-110 (1992) Theilgaard, H.B.A.; Kristiansen, K.N.; Henriksen, C.M.; Nielsen, J.: Purification and characterization of d-(l-a-aminoadipyl)-l-cysteinyl-d-valine synthetase from Penicillium chrysogenum. Biochem. J., 327, 185-191 (1997) Aharonowitz, Y.; Bergmeyer, J.; Cantoral, J.M.; Cohen, G.; Demain, A.L.; Fink, U.; Kinghorn, J.; Kleinkauf, H.; MacCabe, A.; et al.: d-(l-a-Aminoadipyl)-l-cysteinyl-d-valine synthetase, the multienzyme integrating the four primary reactions in b-lactam biosynthesis, as a model peptide synthetase. Bio/Technology, 11, 807-810 (1993) Byford, M.F.; Baldwin, J.E.; Shiau, C.Y.; Schofield, C.J.: The mechanism of ACV synthetase. Chem. Rev., 97, 2631-2649 (1997)

605

Aerobactin synthase

6.3.2.27

1 Nomenclature EC number 6.3.2.27 Systematic name citrate:N6 -acetyl-N6 -l-lysine ligase (ADP-forming) Recommended name aerobactin synthase Synonyms iucA <2> [4] CAS registry number 94047-30-0

2 Source Organism <1> Aerobacter aerogenes (no sequence specified) [1, 2, 3] <2> Vibrio hollisae (UNIPROT accession number: Q4H462) [4]

3 Reaction and Specificity Catalyzed reaction 4 ATP + citrate + N6 -acetyl-N6 -hydroxy-l-lysine + 2 H2 O = 4 ADP + 4 phosphate + aerobactin Reaction type ligation Natural substrates and products S ATP + citrate + N6 -acetyl-N6 -hydroxylysine <1> (<1> synthesis of aerobactin that has a high affinity for Fe3+ [1,2,3]) (Reversibility: ?) [1, 2, 3] P ADP + phosphate + aerobactin <1> [1, 2, 3] Substrates and products S ATP + citrate + N6 -acetyl-N6 -hydroxylysine <1> (<1> 4 mol ATP required for formation of 1 mol aerobactin [2]; <1> synthesis of aerobactin that has a high affinity for Fe3+ [1,2,3]) (Reversibility: ?) [1, 2, 3] P ADP + phosphate + aerobactin <1> [1, 2, 3] S ATP + citrate + N6 -acetyllysine <1> (Reversibility: ?) [3]

606

6.3.2.27

Aerobactin synthase

? ATP + citrate + N6 -butyryl-N6 -hydroxylysine <1> (Reversibility: ?) [3] ? ATP + citrate + N6 -succinyl-N6 -hydroxylysine <1> (Reversibility: ?) [3] ? ATP + tricarballylic acid + N6 -acetyl-N6 -hydroxylysine <1> (Reversibility: ?) [3] P ?

P S P S P S

Inhibitors 3-methyl-3-hydroxyglutaric acid <1> [3] glutaric acid <1> [3] N-ethylmaleimide <1> [1] succinic acid <1> [3] Cofactors/prosthetic groups ATP <1> [1,2,3] Metals, ions Mg2+ <1> (<1> required [1]) [1] Specific activity (U/mg) 0.072 <1> (<1> purified enzyme [1]) [1]

5 Isolation/Preparation/Mutation/Application Localization soluble <1> [1] Purification <1> (partial) [1, 2]

6 Stability Temperature stability 4 <1> (<1> complete loss of activity within 72 h [1]) [1]

References [1] Appanna, D.L.; Grundy, B.J.; Szczepan, E.W.; Viswanatha, T.: Aerobactin synthesis in a cell-free system of Aerobacter aerogenes 62-1. Biochim. Biophys. Acta, 801, 437-443 (1984) [2] Appanna, V.D.; Viswanatha, T.: High-performance liquid chromatography method for monitoring aerobactin synthetase activity. J. Chromatogr., 363, 323-328 (1986)

607

Aerobactin synthase

6.3.2.27

[3] Appanna, V.D.; Viswanatha, T.: Effects of some substrate analogs on aerobactin synthetase from Aerobacter aerogenes 62-1. FEBS Lett., 202, 107-110 (1986) [4] Suzuki, K.; Tanabe, T.; Moon, Y.; Funahashi, T.; Nakao, H.; Narimatsu, S.; Yamamoto, S.: Identification and transcriptional organization of aerobactin transport and biosynthesis cluster genes of Vibrio hollisae. Res. Microbiol., 157, 730-740 (2006)

608

L-Amino-acid

a-ligase

6.3.2.28

1 Nomenclature EC number 6.3.2.28 Systematic name l-amino acid:l-amino acid ligase (ADP-forming) Recommended name l-amino-acid a-ligase Synonyms YwfE <1> [1]

2 Source Organism <1> Bacillus subtilis (no sequence specified) [1, 2]

3 Reaction and Specificity Catalyzed reaction ATP + an l-amino acid + an l-amino acid = ADP + phosphate + l-aminoacyl-l-amino acid

5 Isolation/Preparation/Mutation/Application Application synthesis <1> (<1> fermentative production of l-alanyl-l-glutamine by a metabolically engineered Escherichia coli strain expressing l-amino acid aligase [2]) [2]

References [1] Tabata, K.; Ikeda, H.; Hashimoto, S.: ywf in Bacillus subtilis codes for a novel enzyme, l-amino acid ligase. J. Bacteriol., 187, 5195-5202 (2005) [2] Tabata, K.; Hashimoto, S.: Fermentative production of l-alanyl-l-glutamine by a metabolically engineered Escherichia coli strain expressing l-amino acid a-ligase. Appl. Environ. Microbiol., 73, 6378-6385 (2007)

609

Cyanophycin synthase (L-aspartate-adding)

6.3.2.29

1 Nomenclature EC number 6.3.2.29 Systematic name cyanophycin:l-aspartate ligase (ADP-forming) Recommended name cyanophycin synthase (l-aspartate-adding) Synonyms CGP synthetase <2, 3> [12] CphA <6, 7, 8, 9> [1, 2, 3, 4, 6] CphA1 <2> [8] CphA2 <2> [8] CphANE1 <11> [11] cyanophycin synthetase <1, 2, 4, 5, 6, 7, 8, 9> [1, 2, 3, 4, 5, 6, 7, 8, 9] CAS registry number 131554-17-1

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11>

610

Acinetobacter calcoaceticus (no sequence specified) [9] Anabaena sp. (no sequence specified) [8, 12] Synechocystis sp. (no sequence specified) [12, 13] Synechocystis sp. PCC 6803 (no sequence specified) [7] Anabaena variabilis (UNIPROT accession number: O86109) [5] Synechococcus sp. MA19 (UNIPROT accession number: Q8VTA5) [2] Cyanothece sp. ATCC 51142 (UNIPROT accession number: Q9KGY4) [4] Anabaena variabilis ATCC 29413 (no sequence specified) [6] Synechocystis sp. PCC 6308 (UNIPROT accession number: P73833) [1,3] Acinetobacter sp. (UNIPROT accession number: Q6FCQ7) [10] Nostoc ellipsosporum (UNIPROT accession number: Q0H8A5) [11]

6.3.2.29

Cyanophycin synthase (L-aspartate-adding)

3 Reaction and Specificity Catalyzed reaction ATP + [l-Asp(4-l-Arg)]n + l-Asp = ADP + phosphate + [l-Asp(4-l-Arg)]n l-Asp (<7> i.e. the first reaction of cyanophycin synthesis, the second reaction is catalysed by the enzymes other active centre [4]) Reaction type Carboxylic acid amide formation Natural substrates and products S [l-Asp(4-l-Arg)]n + l-Asp + ATP <8> (<8> a small amount of cyanophycin is required as a primer [6]) (Reversibility: ?) [6] P [l-Asp(4-l-Arg)]n -Asp + ADP + phosphate (<8> no formation of AMP, [l-Asp(4-l-Arg)]n -Asp is the substrate for the second reaction catalysed by cyanophycin synthase, EC 6.3.2.30 [6]) S Additional information <1> (<1> without l-arginine 1.1% activity compared to the reaction mixture containing both substrates, no activity using l-lysine instead of l-arginine, no activity without addition of small amounts of cyanophycin as a primer for synthesis [9]) (Reversibility: ?) [9] P ? Substrates and products S ATP + [l-Asp(4-l-Arg)]n + l-Asp <11> (Reversibility: ?) [11] P [l-Asp(4-l-Arg)]n -l-Asp + ADP + phosphate S l-aspartic acid + ATP <8> (Reversibility: ?) [6] P poly-l-aspartic acid + ADP + phosphate S [l-Asp(4-l-Arg)]n + l-Asp + ATP <8> (<8> a small amount of cyanophycin is required as a primer [6]) (Reversibility: ?) [6] P [l-Asp(4-l-Arg)]n -Asp + ADP + phosphate (<8> no formation of AMP, [l-Asp(4-l-Arg)]n -Asp is the substrate for the second reaction catalysed by cyanophycin synthase, EC 6.3.2.B2 [6]) S [l-Asp(4-l-Arg)]n -Asp + l-Arg + ATP <1, 2, 4, 5, 6, 8, 9> (<2> study of expression profiles of the genes cphA1 and cphA2 and their dependence on the type of nitrogen supply in the medium [8]; <9> normal assay conditions [1]; <4> study determined the effect of different light and nutrition conditions on cyanophycin granule formation [7]; <5,8,9> [l-Asp(4l-Arg)]n -Asp is a cyanophycin molecule with a C-terminal l-Asp residue that is not linked to an l-Arg residue via its b-carboxy group, this intermediate is produced in the first reaction catalysed by cyanophycin synthase [3,5,6]) (Reversibility: ?) [1, 2, 3, 5, 6, 7, 8, 9] P [l-Asp(4-l-Arg)]nþ1 + ADP + phosphate (<5,8,9> no formation of AMP [3,5,6]) S Additional information <1, 6, 7, 8, 9> (<1> without l-arginine 1.1% activity compared to the reaction mixture containing both substrates, no activity using l-lysine instead of l-arginine, no activity without addition of small amounts of cyanophycin as a primer for synthesis [9]; <7> cya-

611

Cyanophycin synthase (L-aspartate-adding)

6.3.2.29

nophycin accumulation is studied, comparison of nitrogen-fixing and non-nitrogen-fixing cyanobacteria [4]; <6> negligible activities when larginine is omitted, no activity when l-arginine is replaced by l-glutamic acid, citrulline, ornithine, arginine amide, agmatine, or norvaline [2]; <9> no activity without l-arginine, with l-lysine instead of l-arginine there is 15% activity compared to the normal conditions [1]; <8> no product is formed if the peptide primer is blocked at the C-terminus or if larginine is solely supplied as substrate [6]) (Reversibility: ?) [1, 2, 4, 6, 9] P ? Activating compounds K+ <5> [5] Mg2+ <5> [5] sulfhydryl reagents <5> [5] Metals, ions Mg2+ <1, 5, 9> (<5> as Mg-ATP [5]) [1, 5, 9] Specific activity (U/mg) 0.00221 <5> (<5> at 28 C, pH 8.2 [5]) [5] 0.0091 <9> (<9> enzyme isolated from Synechocystis cells, 28 C [3]) [3] 0.027 <9> (<9> recombinant enzyme isolated from Escherichia coli [3]) [3] 0.113 <6> (<6> 30 C, pH 8.2, 123fold purification [2]) [2] 0.238 <9> (<9> after 72fold purification, 28 C, pH 8.2 [1]) [1] 0.265 <1> (<1> 30 C, pH 8.2 [9]) [9] Km-Value (mM) 0.015 <8> (l-arginine, <8> 28 C, pH 8.2 [6]) [6] 0.038 <1> (ATP, <1> 30 C, pH 8.2, for the l-arginine-ATP binding site alone [9]) [9] 0.047 <1> (l-arginine, <1> 30 C, pH 8.2 [9]) [9] 0.049 <9> (l-arginine, <9> 28 C, pH 8.2 [1]) [1] 0.2 <9> (ATP, <9> 28 C, pH 8.2 [1]) [1] 0.278 <1> (ATP, <1> 30 C, pH 8.2, for the complete reaction [9]) [9] pH-Optimum 8.2 <9> (<9> Tris buffer [1]) [1] 8.5 <1> (<1> 30 C [9]) [9] pH-Range 6.2-9.7 <9> [1] Temperature optimum ( C) 50 <6, 9> (<9> enzyme becomes inactive after 30 min incubation at this temperature [1]) [1, 2]

612

6.3.2.29

Cyanophycin synthase (L-aspartate-adding)

4 Enzyme Structure Molecular weight 100000 <6> (<6> monomer, SDS-PAGE [2]) [2] 100600 <6> (<6> monomer, calculated from the amino acid sequence [2]) [2] 230000 <5> (<5> gel filtration [5]) [5] 240000 <9> (<9> gel filtration [1]) [1] Subunits dimer <5, 9> (<9> 2 * 90000, SDS-PAGE [1]; <5> 2 * 100000, SDS-PAGE [5]) [1, 5]

5 Isolation/Preparation/Mutation/Application Purification <1> (anion-exchange chromatography, gel filtration, affinity precipitation with cyanophycin, 69fold purification) [9] <5> (dye-ligand, gel filtration and ion-exchange chromatography, enriched about 4500fold) [5] <6> [2] <8> [6] <9> (from Synechocystis and from recombinant Escherichia coli cells) [1, 3] Cloning <1> (expression in Escherichia coli DH1 cells) [9] <5> (expression in Escherichia coli DH5a) [5] <6> (expression in Escherichia coli TOP10 cells) [2] <8> (expression in Escherichia coli BL21(DE3)) [6] <9> (expression in Escherichia coli TOP10 cells) [1, 3] <11> (engineered cyanophycin synthetase (CphA) from Nostoc ellipsosporum confers enhanced CphA activity and cyanophycin accumulation to Escherichia coli) [11] Engineering Additional information <11> (<11> two truncated CphAs, lacking 31 (CphANE1del96) or 59 (CphANE1del180) amino acids of the C-terminal region, are derived from cphANE1 by deleting 96 or 180 bp from its 3 region through the introduction of stop codons. In comparison to the wild-type gene, cphANE1del96 conferrs about 2.1-2.2fold higher enzyme activity on Escherichia coli. These cells accumulate about twofold more cyanophycin than cells expressing the wild-type gene [11]) [11]

613

Cyanophycin synthase (L-aspartate-adding)

6.3.2.29

6 Stability Temperature stability 28 <9> (<9> still active after 30 min incubation [1]) [1] 50-60 <6> (<6> preincubation for 30 min in this temperature range resulted in 84% to 72% activity compared to preincubation at 30 C [2]) [2] General stability information <5>, purified enzyme is unstable at both 0 C and -70 C [5] Storage stability <1>, at 7 C the purified enzyme is stable for about one week, thereafter the activity decreases rapidly, storage at -20 C in the presence of 10% (w/v) DMSO or 50% (w/v) glycerol does not improve the stability significantly [9]

References [1] Aboulmagd, E.; Oppermann-Sanio, F.B.; Steinbchel, A.: Purification of Synechocystis sp. strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity. Appl. Environ. Microbiol., 67, 2176-2182 (2001) [2] Hai, T.; Oppermann-Sanio, F.B.; Steinbchel, A.: Molecular characterization of a thermostable cyanophycin synthetase from the thermophilic cyanobacterium Synechococcus sp. strain MA19 and in vitro synthesis of cyanophycin and related polyamides. Appl. Environ. Microbiol., 68, 93-101 (2002) [3] Aboulmagd, E.; Oppermann-Sanio, F.B.; Steinbchel, A.: Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Arch. Microbiol., 174, 297-306 (2000) [4] Li H.; Sherman, D.M.; Bao, S.; Sherman, L.A.: Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria. Arch. Microbiol., 176, 9-18 (2001) [5] Ziegler, K.; Diener, A.; Herpin, C.; Richter, R.; Deutzmann, R.; Lockau, W.: Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-l-poly-laspartate (cyanophycin). Eur. J. Biochem., 254, 154-159 (1998) [6] Berg, H.; Ziegler, K.; Piotukh, K.; Baier, K.; Lockau, W.; Volkmer-Engert, R.: Biosynthesis of the cyanobacterial reserve polymer multi-l-arginyl-poly-laspartic acid (cyanophycin). Eur. J. Biochem., 267, 5561-5570 (2000) [7] Allen, M.M.; Hutchison, F.; Weathers, P.J.: Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308. J. Bacteriol., 141, 687-693 (1980) [8] Picossi, S.; Valadares, A.; Flores, E.; Herrero, A.: Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer. J. Biol. Chem., 279, 11582-11592 (2004) [9] Krehenbrink, M.; Steinbchel, A.: Partial purification and characterization of a non-cyanobacterial cyanophycin synthetase from Acinetobacter cal-

614

6.3.2.29

[10] [11]

[12]

[13]

Cyanophycin synthase (L-aspartate-adding)

coaceticus strain ADP1 with regard to substrate specificity, substrate affinity and binding to cyanophycin. Microbiology, 150, 2599-2608 (2004) Elbahloul, Y.; Steinbuechel, A.: Engineering the genotype of Acinetobacter sp. strain ADP1 to enhance biosynthesis of cyanophycin. Appl. Environ. Microbiol., 72, 1410-1419 (2006) Hai, T.; Frey, K.M.; Steinbuechel, A.: Engineered cyanophycin synthetase (CphA) from Nostoc ellipsosporum confers enhanced CphA activity and cyanophycin accumulation to Escherichia coli. Appl. Environ. Microbiol., 72, 7652-7660 (2006) Diniz, S.C.; Voss, I.; Steinbuechel, A.: Optimization of cyanophycin production in recombinant strains of Pseudomonas putida and Ralstonia eutropha employing elementary mode analysis and statistical experimental design. Biotechnol. Bioeng., 93, 698-717 (2006) Kolodny, N.H.; Bauer, D.; Bryce, K.; Klucevsek, K.; Lane, A.; Medeiros, L.; Mercer, W.; Moin, S.; Park, D.; Petersen, J.; Wright, J.; Yuen, C.; Wolfson, A.J.; Allen, M.M.: Effect of nitrogen source on cyanophycin synthesis in Synechocystis sp. strain PCC 6308. J. Bacteriol., 188, 934-940 (2006)

615

Cyanophycin synthase (L-arginine-adding)

6.3.2.30

1 Nomenclature EC number 6.3.2.30 Systematic name cyanophycin:l-arginine ligase (ADP-forming) Recommended name cyanophycin synthase (l-arginine-adding) Synonyms CGP synthetase <3, 4> [13] CphA <6, 7, 10, 11> [1, 2, 3, 4, 6] CphA1 <9> [8] CphA2 <9> [8] CphANE1 <12> [11] cyanophycin synthetase <1, 5, 6, 7, 8, 9, 10, 11> [1, 2, 3, 4, 5, 6, 7, 8, 9] CAS registry number 131554-17-1

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11> <12>

616

Acinetobacter calcoaceticus (no sequence specified) [9] Acinetobacter sp. (no sequence specified) [10] Anabaena sp. (no sequence specified) [12, 13] Synechocystis sp. (no sequence specified) [13, 14] Anabaena variabilis (UNIPROT accession number: O86109) [5] Anabaena variabilis sp. ATCC 29413 (no sequence specified) [6] Synechocystis sp. strain PCC 6308 (UNIPROT accession number: P73833) [1,3] Synechocystis sp. PCC 6308 (no sequence specified) [7] Anabaena sp. PCC 7120 (no sequence specified) [8] Synechococcus sp. MA19 (UNIPROT accession number: Q8VTA5) [2] Cyanothece sp. ATCC 51142 (UNIPROT accession number: Q9KGY4) [4] Nostoc ellipsosporum (UNIPROT accession number: Q0H8A5) [11]

6.3.2.30

Cyanophycin synthase (L-arginine-adding)

3 Reaction and Specificity Catalyzed reaction ATP + [l-Asp(4-l-Arg)]n + l-Arg = ADP + phosphate + [l-Asp(4-l-Arg)]n l-Asp (<11> i.e. the first reaction of cyanophycin synthesis, the second reaction is catalysed by acivity EC 6.3.2.29 [4]; <6> i.e. the first reaction of cyanophycin synthesis, the second reaction is catalysed by activity EC 6.3.2.29, sequence analysis suggests that cyanophycin synthase has two ATP binding sites corresponding to two active sites [6]; <10> i.e. the first reaction of cyanophycin synthesis, the second reaction is catalysed by the activity EC 6.3.2.29 [2]; <1,5,7,8> i.e. the first reaction of cyanophycin synthesis, the second reaction is catalysed by the enzymes other active centre [1,3,5,7,9]) Reaction type Carboxylic acid amide formation Natural substrates and products S [l-Asp(4-l-Arg)]n + l-Arg + ATP <1, 6, 9> (<6> a small amount of cyanophycin is required as a primer [6]; <9> study of expression profiles of the genes cphA1 and cphA2 and their dependence on the type of nitrogen supply in the medium [8]) (Reversibility: ?) [6, 8, 9] P [l-Asp(4-l-Arg)]n -Asp + ADP + phosphate (<6> no formation of AMP, [l-Asp(4-l-Arg)]n -Asp is the substrate for the second reaction catalysed by cyanophycin synthase [6]; <9> [l-Asp(4-l-Arg)]n -Asp is the substrate for the second reaction catalysed by cyanophycin synthase [8]) S Additional information <1> (<1> without l-aspartic acid 9.2% activity compared to the reaction mixture containing both substrates, no activity using l-glutamic acid instead of l-aspartic acid, no activity without addition of small amounts of cyanophycin as a primer for synthesis [9]) (Reversibility: ?) [9] P ? Substrates and products S ATP + [l-Asp(4-l-Arg)]n -l-Asp + l-Arg <12> (<12> l-Lys cannot replace l-Arg in reaction with CphANE1 or CphANE1del96 [11]) (Reversibility: ?) [11] P [l-Asp(4-l-Arg)]n+1 + ADP + phosphate S l-aspartic acid + ATP <6> (Reversibility: ?) [6] P poly-l-aspartic acid + ADP + phosphate S [l-Asp(4-l-Arg)]n + l-Arg + ATP <1, 5, 6, 7, 8, 9, 10> (<5,6,7> a small amount of cyanophycin is required as a primer [3,5,6]; <9> study of expression profiles of the genes cphA1 and cphA2 and their dependence on the type of nitrogen supply in the medium [8]; <6> a short peptide is required as a primer, the primer peptide is elongated at its C-terminus [6]; <7> normal assay conditions [1]; <8> study of the effect of different light and nutrition conditions on cyanophycin granule formation [7]) (Reversibility: ?) [1, 2, 3, 5, 6, 7, 8, 9]

617

Cyanophycin synthase (L-arginine-adding)

6.3.2.30

P [l-Asp(4-l-Arg)]n -Asp + ADP + phosphate (<6> no formation of AMP, [l-Asp(4-l-Arg)]n -Asp is the substrate for the second reaction catalysed by cyanophycin synthase [6]; <5> no formation of AMP, [l-Asp(4-lArg)]n -Asp is the substrate for the second reaction catalysed by cyanophycin synthase, EC 6.3.2.29 [5]; <9> [l-Asp(4-l-Arg)]n -Asp is the substrate for the second reaction catalysed by cyanophycin synthase [8]; <7> no formation of AMP, [l-Asp(3-l-Arg)]n -Asp is the substrate for the second reaction catalysed by cyanophycin synthase [3]) S Additional information <1, 6, 7, 10, 11> (<1> without l-aspartic acid 9.2% activity compared to the reaction mixture containing both substrates, no activity using l-glutamic acid instead of l-aspartic acid, no activity without addition of small amounts of cyanophycin as a primer for synthesis [9]; <11> cyanophycin accumulation is studied, comparison of nitrogen-fixing and non-nitrogen-fixing cyanobacteria [4]; <10> negligible activities when l-aspartic acid is omitted [2]; <7> no activity without l-aspartic acid, l-glutamic acid cannot substitute for l-aspartic acid [1]; <6> no product is formed if the peptide primer is blocked at the Cterminus or if l-arginine is solely supplied as substrate [6]) (Reversibility: ?) [1, 2, 4, 6, 9] P ? Activating compounds K+ <5> [5] Mg2+ <5> [5] Sulfhydryl reagents <5> [5] Metals, ions Mg2+ <1, 5, 7> (<5> as Mg-ATP [5]) [1, 5, 9] Specific activity (U/mg) 0.00221 <5> (<5> at pH 8.2 and 28 C [5]) [5] 0.113 <10> (<10> 30 C, pH 8.2, 123fold purification [2]) [2] 0.238 <7> (<7> after 72fold purification, 28 C, pH 8.2 [1]) [1] 0.265 <1> (<1> 30 C, pH 8.2 [9]) [9] Km-Value (mM) 0.2 <7> (ATP, <7> 28 C, pH 8.2 [1]) [1] 0.21 <1> (ATP, <1> 30 C, pH 8.2, for the l-aspartic acid-ATP binding site alone [9]) [9] 0.24 <1> (l-Aspartic acid, <1> 30 C, pH 8.2 [9]) [9] 0.278 <1> (ATP, <1> 30 C, pH 8.2, for the complete reaction [9]) [9] 0.45 <7> (l-Aspartic acid, <7> 28 C, pH 8.2 [1]) [1] pH-Optimum 8.2 <7> (<7> Tris buffer [1]) [1] 8.5 <1> (<1> 30 C [9]) [9] pH-Range 6.2-9.7 <7> [1]

618

6.3.2.30

Cyanophycin synthase (L-arginine-adding)

Temperature optimum ( C) 50 <7, 10> (<7> enzyme becomes inactive after 30 min incubation at this temperature [1]) [1, 2]

4 Enzyme Structure Molecular weight 100000 <10> (<10> monomer, SDS-PAGE [2]) [2] 100600 <10> (<10> monomer, calculated from the amino acid sequence [2]) [2] 230000 <5> (<5> gel filtration [5]) [5] 240000 <7> (<7> gel filtration [1]) [1] Subunits dimer <5, 7> (<7> 2 * 90000, SDS-PAGE [1]; <5> 2 * 100000, SDS-PAGE [5]) [1, 5]

5 Isolation/Preparation/Mutation/Application Purification <1> (anion-exchange chromatography, gel filtration, affinity precipitation with cyanophycin, 69fold purification) [9] <5> (dye-ligand, gel filtration and ion-exchange chromatography, enriched about 4500fold) [5] <6> [6] <7> (from Synechocystis and from recombinant Escherichia coli cells) [1, 3] <10> [2] Cloning <1> (expression in Escherichia coli DH1 cells) [9] <5> (expression in Escherichia coli DH5a) [5] <6> (expression in Escherichia coli BL21(DE3)) [6] <7> (expression in Escherichia coli TOP10 cells) [1, 3] <10> (expression in Escherichia coli TOP10 cells) [2] <12> (engineered cyanophycin synthetase (CphA) from Nostoc ellipsosporum confers enhanced CphA activity and cyanophycin accumulation to Escherichia coli) [11] Engineering Additional information <12> (<12> two truncated CphAs, lacking 31 (CphANE1del96) or 59 (CphANE1del180) amino acids of the C-terminal region, are derived from cphANE1 by deleting 96 or 180 bp from its 3 region through the introduction of stop codons. In comparison to the wild-type gene, cphANE1del96 conferrs about 2.1-2.2fold higher enzyme activity on Escherichia coli. These cells accumulate about twofold more cyanophycin than cells expressing the wild-type gene [11]) [11]

619

Cyanophycin synthase (L-arginine-adding)

6.3.2.30

6 Stability Temperature stability 28 <7> (<7> still active after 30 min incubation [1]) [1] 50-60 <10> (<10> preincubation for 30 min in this temperature range results in 84% to 72% activity compared to preincubation at 30 C [2]) [2] Additional information <5> (<5> purified enzyme is unstable at both 0 C and -70 C [5]) [5] General stability information <5>, purified enzyme is unstable at both 0 C and -70 C [5] Storage stability <1>, at 7 C the purified enzyme is stable for about one week, thereafter the activity decreases rapidly, storage at -20 C in the presence of 10% (w/v) DMSO or 50% (w/v) glycerol does not improve the stability significantly [9]

References [1] Aboulmagd, E.; Oppermann-Sanio, F.B.; Steinbchel, A.: Purification of Synechocystis sp. strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity. Appl. Environ. Microbiol., 67, 2176-2182 (2001) [2] Hai, T.; Oppermann-Sanio, F.B.; Steinbchel, A.: Molecular characterization of a thermostable cyanophycin synthetase from the thermophilic cyanobacterium Synechococcus sp. strain MA19 and in vitro synthesis of cyanophycin and related polyamides. Appl. Environ. Microbiol., 68, 93-101 (2002) [3] Aboulmagd, E.; Oppermann-Sanio, F.B.; Steinbchel, A.: Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Arch. Microbiol., 174, 297-306 (2000) [4] Li H.; Sherman, D.M.; Bao, S.; Sherman, L.A.: Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria. Arch. Microbiol., 176, 9-18 (2001) [5] Ziegler, K.; Diener, A.; Herpin, C.; Richter, R.; Deutzmann, R.; Lockau, W.: Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-l-poly-laspartate (cyanophycin). Eur. J. Biochem., 254, 154-159 (1998) [6] Berg, H.; Ziegler, K.; Piotukh, K.; Baier, K.; Lockau, W.; Volkmer-Engert, R.: Biosynthesis of the cyanobacterial reserve polymer multi-l-arginyl-poly-laspartic acid (cyanophycin). Eur. J. Biochem., 267, 5561-5570 (2000) [7] Allen, M.M.; Hutchison, F.; Weathers, P.J.: Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308. J. Bacteriol., 141, 687-693 (1980) [8] Picossi, S.; Valadares, A.; Flores, E.; Herrero, A.: Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer. J. Biol. Chem., 279, 11582-11592 (2004)

620

6.3.2.30

Cyanophycin synthase (L-arginine-adding)

[9] Krehenbrink, M.; Steinbchel, A.: Partial purification and characterization of a non-cyanobacterial cyanophycin synthetase from Acinetobacter calcoaceticus strain ADP1 with regard to substrate specificity, substrate affinity and binding to cyanophycin. Microbiology, 150, 2599-2608 (2004) [10] Elbahloul, Y.; Steinbuechel, A.: Engineering the genotype of Acinetobacter sp. strain ADP1 to enhance biosynthesis of cyanophycin. Appl. Environ. Microbiol., 72, 1410-1419 (2006) [11] Hai, T.; Frey, K.M.; Steinbuechel, A.: Engineered cyanophycin synthetase (CphA) from Nostoc ellipsosporum confers enhanced CphA activity and cyanophycin accumulation to Escherichia coli. Appl. Environ. Microbiol., 72, 7652-7660 (2006) [12] Koop, A.; Voss, I.; Thesing, A.; Kohl, H.; Reichelt, R.; Steinbuechel, A.: Identification and localization of cyanophycin in bacteria cells via imaging of the nitrogen distribution using energy-filtering transmission electron microscopy. Biomacromolecules, 8, 2675-2683 (2007) [13] Diniz, S.C.; Voss, I.; Steinbuechel, A.: Optimization of cyanophycin production in recombinant strains of Pseudomonas putida and Ralstonia eutropha employing elementary mode analysis and statistical experimental design. Biotechnol. Bioeng., 93, 698-717 (2006) [14] Kolodny, N.H.; Bauer, D.; Bryce, K.; Klucevsek, K.; Lane, A.; Medeiros, L.; Mercer, W.; Moin, S.; Park, D.; Petersen, J.; Wright, J.; Yuen, C.; Wolfson, A.J.; Allen, M.M.: Effect of nitrogen source on cyanophycin synthesis in Synechocystis sp. strain PCC 6308. J. Bacteriol., 188, 934-940 (2006)

621

(Carboxyethyl)arginine b-lactam-synthase

6.3.3.4

1 Nomenclature EC number 6.3.3.4 Systematic name l-N2 -(2-carboxyethyl)arginine cyclo-ligase (AMP-forming) Recommended name (carboxyethyl)arginine b-lactam-synthase Synonyms b-lactam-forming enzyme synthetase, b-lactam CAS registry number 68247-54-1

2 Source Organism <1> Streptomyces clavuligerus (no sequence specified) [1, 2, 3, 4, 5, 6]

3 Reaction and Specificity Catalyzed reaction ATP + l-N2 -(2-carboxyethyl)arginine = AMP + diphosphate + deoxyamidinoproclavaminate (<1> overview [6]; <1> it has been proposed that l-N2 -(2carboxyethyl)arginine is first converted into an acyl-AMP by reaction with ATP and loss of diphosphate, and that the b-lactam ring is then formed by the intramolecular attack of the b-nitrogen on the activated carboxy group, in a sequential ordered bi-ter kinetic mechanism [2]) Reaction type cyclization formation of cyclic amides Natural substrates and products S N2 -(carboxyethyl)-l-arginine + ATP <1> (<1> early step in clavulanic acid biosynthesis [2]; <1> overview on biological role [6]) (Reversibility: ?) [1, 2, 6] P AMP + diphosphate + deoxyamidinoproclavaminate

622

6.3.3.4

(Carboxyethyl)arginine b-lactam-synthase

Substrates and products S N2 -(carboxyethyl)-l-arginine + ATP <1> (<1> mechanism [2]; <1> early step in clavulanic acid biosynthesis [2]; <1> overview on biological role [6]) (Reversibility: ?) [1, 2, 6] P AMP + diphosphate + deoxyamidinoproclavaminate <1> [1] Inhibitors diphosphate <1> (<1> product inhibition [2]) [2] N2 -(carboxymethyl)-l-arginine <1> (<1> competitive to N2 -(carboxyethyl)l-arginine [2]) [2] deoxyamidinoproclavaminate <1> (<1> product inhibition [2]) [2] Cofactors/prosthetic groups ATP <1> (<1> ATP-binding site [6]) [1,3,5,6] Metals, ions Mg2+ <1> (<1> requirement [1]; <1> single Mg2+ ion in the active site [4]; <1> two Mg2+ ions in the active site [3]) [1, 3, 4, 5] Km-Value (mM) 0.153 <1> (ATP, <1> pH 7.5 [2]) [2] 0.22 <1> (N2 -(carboxyethyl)-l-arginine, <1> pH 7.5 [2]) [2]

4 Enzyme Structure Subunits ? <1> (<1> x * 56000, SDS-PAGE [5]; <1> x * 54500, deduced from gene sequence [6]) [5, 6]

5 Isolation/Preparation/Mutation/Application Purification <1> (recombinant protein expressed in E. coli) [2] Crystallization <1> [3, 4] Cloning <1> [5] <1> (homologous to Class B asparagine synthases) [1] Engineering Additional information <1> (<1> disruption mutant, complete loss of clavulanic acid production, accumulation of N2 -(carboxyethyl)-l-arginine, but no effect on synthesis of penicillin or cephamycin [1]) [1]

623

(Carboxyethyl)arginine b-lactam-synthase

6.3.3.4

6 Stability Storage stability <1>, -20 C, 50 mm Tris-HCl, 10% glycerol, 2 mM dithiothreitol, 0.01 mM EDTA, pH 7.5, stable [2]

References [1] Bachmann, B.O.; Li, R.; Townsend, C.A.: b-Lactam synthetase: a new biosynthetic enzyme. Proc. Natl. Acad. Sci. USA, 95, 9082-9086 (1998) [2] Bachmann, B.O.; Townsend, C.A.: Kinetic mechanism of the b-Lactam synthetase of Streptomyces clavuligerus. Biochemistry, 39, 11187-11193 (2000) [3] Miller, M.T.; Bachmann, B.O.; Townsend, C.A.; Rosenzweig, A.C.: The catalytic cycle of b-lactam synthetase observed by X-ray crystallographic snapshots. Proc. Natl. Acad. Sci. USA, 99, 14752-14757 (2002) [4] Miller, M.T.; Bachmann, B.O.; Townsend, C.A.; Rosenzweig, A.C.: Structure of b-lactam synthetase reveals how to synthesize antibiotics instead of asparagine. Nat. Struct. Biol., 8, 684-689 (2001) [5] McNaughton, H.J.; Thirkettle, J.E.; Zhang, Z.; Schofield, C.J.; Jensen, S.E.; Barton, B.; Greaves, P.: b-Lactam synthetase: implications for b-lactamase evolution. Chem. Commun., 1998, 2325-2326 (1998) [6] Townsend, C.A.: New reactions in clavulanic acid biosynthesis. Curr. Opin. Chem. Biol., 6, 583-589 (2002)

624

5-(Carboxyamino)imidazole ribonucleotide synthase

6.3.4.18

1 Nomenclature EC number 6.3.4.18 Systematic name 5-amino-1-(5-phospho-d-ribosyl)imidazole:carbon-dioxide forming)

ligase

(ADP-

Recommended name 5-(carboxyamino)imidazole ribonucleotide synthase Synonyms N5 -CAIR synthetase <2> [5] N5 -carboxyaminoimidazole ribonucleotide synthetase <2> [5] PurK <1, 2, 3> [1, 2, 3, 4, 5, 6, 7]

2 Source Organism <1> Escherichia coli (no sequence specified) (<1> large subunit [7]) [1, 2, 3, 6, 7] <2> Sulfolobus solfataricus (no sequence specified) [5] <3> Brevibacterium ammoniagenes (no sequence specified) [4]

3 Reaction and Specificity Catalyzed reaction ATP + 5-amino-1-(5-phospho-d-ribosyl)imidazole + HCO-3 = ADP + phosphate + 5-carboxyamino-1-(5-phospho-d-ribosyl)imidazole Reaction type carboxylation Natural substrates and products S Additional information <1> (<1> the purEK operon is regulated by the purR gene product [7]) (Reversibility: ?) [7] P ? Substrates and products S ATP + 5-amino-1-(5-phospho-d-ribosyl)imidazole + HCO3- <1> (Reversibility: ?) [2]

625

5-(Carboxyamino)imidazole ribonucleotide synthase

6.3.4.18

P ADP + phosphate + 5-carboxyamino-1-(5-phospho-d-ribosyl)imidazole S Additional information <1> (<1> the purEK operon is regulated by the purR gene product [7]) (Reversibility: ?) [7] P ?

4 Enzyme Structure Molecular weight 78000 <1> (<1> sucrose gradient ultracentrifugation [1]) [1] 79000 <1> (<1> gel filtration [1]) [1] Subunits dimer <1> (<1> 2 * 39000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> [1] Crystallization <1> (hanging drop vapour diffusion of native enzyme, crystals of the native enzyme belong to space group C222(1) with unit cell dimensions of a = 93.4 , b = 95.2 and c = 120.6 . Crystals of the enzyme/MgADP complex are grown by batch methods at room temperature with poly(ethylene glycol)8000 as precipitant. The crystals belong to the space group P1 with unit cell dimensions of a = 60.6 , b = 92.1 , c = 102.6 , a = 66.1, b = 82.7 and g = 81.8 ) [3] Cloning <1> [6] <2> [5] <3> [4]

References [1] Meyer, E.; Leonard, N.J.; Bhat, B.; Stubbe, J.; Smith, J.M.: Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway. Biochemistry, 31, 5022-5032 (1992) [2] Mueller, E.J.; Meyer, E.; Rudolph, J.; Davisson, V.J.; Stubbe, J.: N5 -carboxyaminoimidazole ribonucleotide: evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli. Biochemistry, 33, 2269-2278 (1994) [3] Thoden, J.B.; Kappock, T.J.; Stubbe, J.; Holden, H.M.: Three-dimensional structure of N5 -carboxyaminoimidazole ribonucleotide synthetase: a mem-

626

6.3.4.18

5-(Carboxyamino)imidazole ribonucleotide synthase

ber of the ATP grasp protein superfamily. Biochemistry, 38, 15480-15492 (1999) [4] Chung, S.O.; Lee, J.H.; Lee, S.Y.; Lee, D.S.: Genomic organization of purK and purE in Brevibacterium ammoniagenes ATCC 6872: purE locus provides a clue for genomic evolution. FEMS Microbiol. Lett., 137, 265-268 (1996) [5] Sorensen, I.S.; Dandanell, G.: Identification and sequence analysis of Sulfolobus solfataricus purE and purK genes. FEMS Microbiol. Lett., 154, 173-180 (1997) [6] Watanabe, W.; Sampei, G.; Aiba, A.; Mizobuchi, K.: Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5’-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis. J. Bacteriol., 171, 198-204 (1989) [7] Tiedeman, A.A.; Keyhani, J.; Kamholz, J.; Daum, H.A., 3rd; Gots, J.S.; Smith, J.M.: Nucleotide sequence analysis of the purEK operon encoding 5’-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12. J. Bacteriol., 171, 205-212 (1989)

627

Asparaginyl-tRNA synthase (glutaminehydrolysing)

6.3.5.6

1 Nomenclature EC number 6.3.5.6 Systematic name Asp-tRNAAsn :l-glutamine amido-ligase (ADP-forming) Recommended name asparaginyl-tRNA synthase (glutamine-hydrolysing) Synonyms AdT <6> [8] AsnRS <1, 5> [13] Asp-AdT <7> [15] Asp-tRNAAsn amidotransferase <7> [15] Asp/Glu-Adt <8> [9] asparaginyl-tRNA synthetase <1, 5> [13] GatCAB <8, 16> [10, 11] asparaginyl-transfer RNA synthetase <1, 5> [13] aspartyl-tRNAAsn amidotransferase glutamine-dependent Asp-tRNAAsn /Glu-tRNAGln amidotransferase <8> [9] tRNA-dependent amidotransferase <4, 6> [12, 14] CAS registry number 37211-76-0

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11>

628

Brugia malayi (no sequence specified) [13] Chlamydia trachomatis (no sequence specified) [4, 7] Bacillus subtilis (no sequence specified) [1, 2, 6, 7] Thermus thermophilus (no sequence specified) [2, 5, 6, 14] Homo sapiens (no sequence specified) [13] Pseudomonas aeruginosa (no sequence specified) (<6> subunit of benzoyl-CoA reductase, gene name: bzdA [8]) [8,12] Halobacterium salinarum (no sequence specified) [15] Helicobacter pylori (no sequence specified) [7,9,11] Haloferax volcanii (no sequence specified) [1] Sulfolobus solfataricus (no sequence specified) [7] Methanococcus jannaschii (no sequence specified) [6, 7]

6.3.5.6

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

<12> <13> <14> <15> <16>

Archaeoglobus fulgidus (no sequence specified) [6, 7] Deinococcus radiodurans (no sequence specified) [1, 2, 5, 6, 7] Halobacterium salinarium (no sequence specified) [7] Aeropyrum pernix (no sequence specified) [6, 7] Methanothermobacter thermautotrophicus (no sequence specified) (<16> fragment of Ssp [7]) [3, 6, 7, 10] <17> Methanosarcina mazei (no sequence specified) [7] <18> Sulfolobus tokodaii (no sequence specified) [7]

3 Reaction and Specificity Catalyzed reaction ATP + Asp-tRNAAsn + l-glutamine = ADP + phosphate + Asn-tRNAAsn + lglutamate Reaction type transamidation Natural substrates and products S ATP + aspartyl-tRNAAsn + l-glutamine <7, 8> (Reversibility: ?) [9, 15] P ADP + phosphate + asparaginyl-tRNAAsn + l-glutamate S Asp-tRNAAsn + ? <2, 4, 8, 13, 16> (<4> in vivo only Asn-tRNAAsn formation occurs, since Thermus possesses a mischarging Asp-tRNA synthetase, but lacks a mischarging Glu-tRNA synthetase, and cellular activity of AdTs only depends upon presence or lack of the mischarging amino acyl-tRNA synthetases, explaining why those enzymes have not restricted their specificity [2]; <2> tRNA-dependent transamidation pathway of Asn-tRNA formation, which is required for protein synthesis or under certain metabolic situations for asparagine synthesis [4]; <4,13> AdT is related to the biosynthesis of asparagine rather than to provide charged tRNA for protein biosynthesis [6]; <4,13> tRNA-dependent transamidation pathway of Asn-tRNA formation, only biosynthetic route to asparagine is via Asn-tRNA [5,7]; <16> the mischarging Asp-tRNA synthetase correlates with the absence in the genome of Asn-tRNA synthetase and the presence of Asp-tRNAAsn amidotransferase, employed by the transamidation pathway [3]; <13> gatCAB genes encoded Asp-AdT may be responsible for biosynthesis of asparagine, glutamine as amide source [1]; <2,8> it is likely that the dual specificity amidotransferase serves in Asn-tRNA and Gln-tRNA formation in vivo [4,7]; <13> in vivo only Asn-tRNAAsn formation occurs [2,7]; <4,13,16> enzyme of the two-step pathway for the synthesis of Asn-tRNA that requires misacylated Asp-tRNAAsn as intermediate, which is generated by a Asp-tRNA synthetase [1,2,3,5]; <13> in vivo only Asn-tRNAAsn formation, because the substrate availability of AsptRNAAsn allows only one final product [1]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7] P Asn-tRNAAsn + ? <2, 4, 8, 13, 16> [1, 2, 3, 4, 5, 6, 7]

629

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

6.3.5.6

S Glu-tRNAGln + ? <2, 8> (<2,8> it is likely that the dual specificity amidotransferase serves in Asn-tRNA and Gln-tRNA formation in vivo [4,7]; <2> tRNA-dependent transamidation pathway of Gln-tRNA formation, which is required for protein synthesis or under certain metabolic situations for glutamine synthesis [4]) (Reversibility: ?) [4, 7] P Gln-tRNAGln + ? <2, 8> [4, 7] S Additional information <1, 5> (<5> human cytoplasmic aminoacyl-tRNA synthetases, which are autoantigens in idiopathic inflammatory myopathies, activate chemokine receptors on T lymphocytes, monocytes, and immature dendritic cells by recruiting immune cells that could induce innate and adaptive immune responses [13]; <1> the parasite Brugia malayi enzyme, in analogy to the human enzyme, induces human leukocyte chemotaxis and activates G-protein-coupled receptors CXCR1 and CXCR2, but not CXCR3, filarial asparaginyl-tRNA synthetase, AsnRS, is known to be an immunodominant antigen that induces strong human immunoglobulin G3 responses and contributes to the development of chronic inflammatory disease such as lymphatic filariasis, overview [13]) (Reversibility: ?) [13] P ? Substrates and products S ATP + Asp-tRNA + l-Gln <4, 6> (<4,6> in presence of tRNA-dependent amidotransferase AdT, amidation activity [12,14]) (Reversibility: ?) [12, 14] P ? S ATP + Asp-tRNAAsn + l-glutamine <8, 16> (<16> identity elements used by GatCAB to discriminate tRNAAsn from tRNAAsp . GatCAB specifically binds Asp-tRNAAsn . Therefore, modified nucleotides do not play an essential role in GatCAB discrimination of Asp-tRNAAsn from Asp-tRNAAsp [10]; <8> the enzyme transamidates Asp-tRNAAsn and Glu-tRNAGln with similar efficiency [11]) (Reversibility: ?) [10, 11] P ADP + phosphate + Asn-tRNAAsn + l-glutamate S ATP + Asp-tRNAAsn + glutamine <2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18> (<2> dual-specific Asp/Glu-AdT, rates for conversion of Glu to Gln are about twice as fast as the rate of Asp to Asn conversion, enzyme uses glutamine, asparagine or ammonia as amide donors in the presence of ATP, GTP or CTP [4]; <4> no restricted substrate specificity of tRNAdependent AdT: dual-specific Asp/Glu-AdT, enzyme catalyzes transamidation of Asp-tRNAAsn , since Thermus possesses a mischarging Asp-tRNA synthetase, but lacks a mischarging Glu-tRNA synthetase, GatA subunit can discriminate between Asp-tRNAAsn and Glu-tRNAGln and therefore is implicated in binding of the tRNA [2]; <13> single enzyme with both GlntRNA and Asp-tRNA transamidation activities, but functions as Asp-AdT, since Deinococcus do not produce the required mischarged Glu-tRNAGln substrate [1]; <4,13> dual-specific Asp/Glu-AdT is able to amidate both mischarged tRNAGln and tRNAAsn , but functions as Asp-AdT [6,7]; <2, 8,

630

6.3.5.6

P S

P S

P S

P S

P S

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

13, 16> dual tRNA specificity: formation of Asn-tRNA and Gln-tRNA [1,7]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8] ADP + phosphate + Asn-tRNAAsn + glutamate <2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18> (<4> forms Asn-tRNAAsn [2]) [1, 2, 3, 4, 5, 6, 7] ATP + Glu-tRNAGln + l-glutamine <8> (<8> the enzyme transamidates Asp-tRNAAsn and Glu-tRNAGln with similar efficiency. GatCAB uses the amide donor glutamine 129fold more efficiently than asparagine [11]) (Reversibility: ?) [11] ADP + phosphate + Gln-tRNAGln + l-glutamate ATP + Glu-tRNAGln + glutamine <2, 3, 4, 8, 13, 16> (<4,13> dual-specific Asp/Glu-AdT is able to amidate both mischarged tRNAGln and tRNAAsn , but functions as Asp-AdT [1,6]; <2,8,16> dual tRNA specificity: formation of Asn-tRNA and Gln-tRNA [7]; <3> in vitro both Asp-tRNAAsn and GlutRNAGln amidation activities, in vivo only Gln-tRNAGln formation [1,2]; <3> Asp/Glu-AdT carries out Gln-tRNA formation, since Bacillus contains a non-discriminating GluRS, but lacks a mischarging AspRS [1,7]; <2> dual-specific Asp/Glu-AdT, rates for conversion of Glu to Gln are about twice as fast as the rate of Asp to Asn conversion, enzyme uses glutamine, asparagine or ammonia as amide donors in the presence of ATP or GTP [4]; <4,13> in vitro both Asp-tRNAAsn and Glu-tRNAGln amidation activities, in vivo only Asn-tRNAAsn formation [2]) (Reversibility: ?) [1, 2, 4, 6, 7] ADP + Gln-tRNAGln + glutamate <2, 3, 4, 8, 13> [1, 2, 4, 6, 7] ATP + Glu-tRNAGln + glutamine <6> (<6> the enzyme has no GlutRNAGln substrate in its host, but it can transamidate heterologous GlutRNAGln [8]) (Reversibility: ?) [8] ADP + Gln-tRNAGln + glutamate + phosphate ATP + aspartyl-tRNAAsn + l-glutamine <7, 8> (<7> recombinantly produced tRNA substrate [15]; <8> the Halobacter pylori Asp/Glu-Adt can utilize Escherichia coli Asp-tRNAAsn as a substrate, recombinantly produced tRNA substrate [9]) (Reversibility: ?) [9, 15] ADP + phosphate + asparaginyl-tRNAAsn + l-glutamate Asp-tRNAAsn + ? <2, 4, 8, 13, 16> (<4> in vivo only Asn-tRNAAsn formation occurs, since Thermus possesses a mischarging Asp-tRNA synthetase, but lacks a mischarging Glu-tRNA synthetase, and cellular activity of AdTs only depends upon presence or lack of the mischarging amino acyl-tRNA synthetases, explaining why those enzymes have not restricted their specificity [2]; <2> tRNA-dependent transamidation pathway of Asn-tRNA formation, which is required for protein synthesis or under certain metabolic situations for asparagine synthesis [4]; <4,13> AdT is related to the biosynthesis of asparagine rather than to provide charged tRNA for protein biosynthesis [6]; <4,13> tRNA-dependent transamidation pathway of Asn-tRNA formation, only biosynthetic route to asparagine is via Asn-tRNA [5,7]; <16> the mischarging Asp-tRNA synthetase correlates with the absence in the genome of Asn-tRNA synthetase and the presence of Asp-tRNAAsn amidotransferase, employed by the transamidation pathway [3]; <13> gatCAB genes encoded Asp-AdT may be responsi-

631

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

P S

P S

632

6.3.5.6

ble for biosynthesis of asparagine, glutamine as amide source [1]; <2,8> it is likely that the dual specificity amidotransferase serves in Asn-tRNA and Gln-tRNA formation in vivo [4,7]; <13> in vivo only Asn-tRNAAsn formation occurs [2,7]; <4,13,16> enzyme of the two-step pathway for the synthesis of Asn-tRNA that requires misacylated Asp-tRNAAsn as intermediate, which is generated by a Asp-tRNA synthetase [1,2,3,5]; <13> in vivo only Asn-tRNAAsn formation, because the substrate availability of AsptRNAAsn allows only one final product [1]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7] Asn-tRNAAsn + ? <2, 4, 8, 13, 16> [1, 2, 3, 4, 5, 6, 7] Glu-tRNAGln + ? <2, 8> (<2,8> it is likely that the dual specificity amidotransferase serves in Asn-tRNA and Gln-tRNA formation in vivo [4,7]; <2> tRNA-dependent transamidation pathway of Gln-tRNA formation, which is required for protein synthesis or under certain metabolic situations for glutamine synthesis [4]) (Reversibility: ?) [4, 7] Gln-tRNAGln + ? <2, 8> [4, 7] Additional information <1, 2, 4, 5, 6, 7, 8, 11, 12, 13, 15, 16> (<4,13> enzyme specificity depends on the biochemical context, i.e. on the availability of the mischarged aminoacyl-tRNA substrate, which is controlled by the presence of a nondiscriminating GluRS or AspRS [6]; <4> cellular activity of AdTs only depends upon presence or lack of the mischarging amino acyl-tRNA synthetases [2]; <11,12,15,16> archaeal enzyme resembles the bacterial Glu/Asp-AdT, but can only amidate Asp-tRNAAsn and not Glu-tRNAGln [6]; <2> GatA is likely to be the catalytic subunit, GatB may be responsible for tRNA binding and GatC may be involved in a channeling mechanism, in which the misacylated tRNA formed by the non-discriminating AA-tRNA synthetase could be handed off to the AdT [4]; <2,13,16> no substrates: correctly charged Asp-tRNAAsp and GlutRNAGlu [4,7]; <13> A subunit is a tRNA-independent glutaminase, B subunit is a putative tRNA-binding protein [1]; <5> human cytoplasmic aminoacyl-tRNA synthetases, which are autoantigens in idiopathic inflammatory myopathies, activate chemokine receptors on T lymphocytes, monocytes, and immature dendritic cells by recruiting immune cells that could induce innate and adaptive immune responses [13]; <1> the parasite Brugia malayi enzyme, in analogy to the human enzyme, induces human leukocyte chemotaxis and activates G-protein-coupled receptors CXCR1 and CXCR2, but not CXCR3, filarial asparaginyl-tRNA synthetase, AsnRS, is known to be an immunodominant antigen that induces strong human immunoglobulin G3 responses and contributes to the development of chronic inflammatory disease such as lymphatic filariasis, overview [13]; <4,6> a nondiscriminating aspartyl-tRNA synthetase, ND-DRS, first generates a mischarged aspartyl-tRNAAsn that dissociates from the enzyme and binds to a tRNA-dependent amidotransferase, AdT, which then converts the tRNA-bound aspartate into asparagine, the ND-DRS, tRNAAsn , and AdT assemble into a specific ribonucleoprotein complex called transamidosome that remains stable during the overall catalytic process, overview [12,14]; <7,8> in bacteria that lack AsnRS, EC 6.1.1.12, AspRS is

6.3.5.6

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

nondiscriminating, EC 6.1.1.23, and generates both Asp-tRNAAsp and the noncanonical, misacylated Asp-tRNAAsn , this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNAAsn /Glu-tRNAGln amidotransferase, overview [9,15]) (Reversibility: ?) [1, 2, 4, 6, 7, 9, 12, 13, 14, 15] P ? Inhibitors Additional information <2> (<2> not inhibited by N-ethylmaleimide, 5,5’dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate [4]) [4] Cofactors/prosthetic groups ATP <4, 6, 7, 8> [9, 12, 14, 15] Metals, ions Mg2+ <2, 4, 6, 7, 8> (<2> essential for the reaction catalyzed by AdT [4]) [4, 9, 12, 14, 15] Turnover number (min–1) 0.027 <8> (Asn, <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 0.052-2.1 <8> (Gln, <8> 37 C, pH 7.2, cosubstrate: Glu-tRNAGln [11]) [11] 1.3 <8> (Asp-tRNAAsn , <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 3-6 <8> (Gln, <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 3.61 <8> (Asp-tRNAGln , <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 6.1 <8> (ATP, <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 11.8 <8> (Gln, <8> 37 C, pH 7.2, cosubstrate Asp-tRNAAsn + ATP, glutaminase activity [11]) [11] Specific activity (U/mg) 3.5e-005 <2> [4] Km-Value (mM) 0.00095 <8> (Asp-tRNAAsn , <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 0.00118 <8> (Asp-tRNAGln , <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 0.0207 <8> (Gln, <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 0.0224 <8> (Asn, <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] 0.0402 <8> (Gln, <8> 37 C, cosubstrate Asp-tRNAAsn + ATP, glutaminase activity [11]) [11] 0.0509 <8> (Gln, <8> 37 C, cosubstrate Glu-tRNAGln + ATP, glutaminase activity [11]) [11] 0.2068 <8> (ATP, <8> 37 C, pH 7.2, amidotransferase activity [11]) [11] Additional information <4> (<4> steady-state und pre-steady-state kinetics, association of dimeric DRS2, AdT, and tRNAAsn in a ternary complex, AdT binds tRNAAsn with a lower affinity than DRS2, overview [14]) [14]

633

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

6.3.5.6

pH-Optimum 7.2 <4> (<4> assay at [14]) [14] 7.5 <7> (<7> assay at [15]) [15] Temperature optimum ( C) 30 <13> (<13> assay at [1]) [1] 37 <2, 4, 7, 16> (<2,4,7,16> assay at [4,7,14,15]; <4> assay at, suboptimal temperature for the thermophilic enzyme [2]; <16> assay at, as a compromise between optimal enzyme activity and the stability of the reaction product [3]) [2, 3, 4, 7, 14, 15]

4 Enzyme Structure Subunits heterodimer <4> (<4> Asp-AdT is active as a GatAB heterodimer [2]) [2] heterotrimer <2, 3, 4, 11, 12, 13, 15, 16> (<4> possesses the three canonical gatA, gatB and gatC set of genes encoding heterotrimeric AdT: GatABC, GatA and GatB are more tightly bound to each other than to GatC, GatA subunit has conserved Gln-436 [2]; <11,12,15,16> ORFs corresponding to a heterotrimeric amidotransferase resembling the bacterial Glu/Asp-AdT [6]; <2> 1 * 55000, GatA + 1 * 53600, GatB + 1* 11100, GatC, SDS-PAGE [4]; <3,13,16> GatCAB enzyme [3,5,7]) [2, 3, 4, 5, 6, 7] trimer <6> [8] Additional information <4> (<4> association of dimeric DRS2, AdT, and tRNAAsn in a ternary complex: a nondiscriminating aspartyl-tRNA synthetase, ND-DRS, first generates a mischarged aspartyl-tRNAAsn that dissociates from the enzyme and binds to a tRNA-dependent amidotransferase, AdT, which then converts the tRNA-bound aspartate into asparagine, the NDDRS, tRNAAsn , and AdT assemble into a specific ribonucleoprotein complex called transamidosome that remains stable during the overall catalytic process, overview [14]) [14]

5 Isolation/Preparation/Mutation/Application Source/tissue HEK-293T cell <5> [13] T-lymphocyte <5> [13] leukocyte <5> (<5> primary [13]) [13] Localization cytoplasm <5> [13] Purification <2> (32fold purification of recombinant enzyme, expressed in Escherichia coli) [4] <8> [11]

634

6.3.5.6

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

<13> (partial purification of recombinant enzyme, expressed in Escherichia coli) [1] <16> (purification of recombinant enzyme, expressed in Escherichia coli) [3, 7] Cloning <1> (expression in Escherichia coli) [13] <2> (cloning of the gatC, gatA and gatB genes, situated in an operon-like manner, encoding the GatCAB amidotransferase and overexpression in Escherichia coli BL21-Codon Plus-TM) [4] <3> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <4> (canonical set of amidotransferase genes gatA, gatB and gatC is cloned and sequenced, the holoenzyme is overexpessed in Escherichia coli and exhibits both Asp-tRNAAsn and Glu-tRNAGln transamidation activities, the gatA, gatB and gatC genes are dispersed in the genome and encode polypeptides of 472, 470 and 90 amino acids, removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, but not the Glu-AdT activity) [2] <5> (expression in Escherichia coli) [13] <6> (overexpression in Escherichia coli) [8] <8> (expression in Escherichia coli strain BL21(DE3), co-expression with the cytotoxic nondiscriminating aspartyl-tRNA synthetase, EC 6.1.1.23, which is toxic for the cells, rescues the toxicity, overview) [9] <10> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <11> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <12> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <13> (gatCAB genes encode Asp-AdT) [5] <13> (gatCAB genes encoding Glu/Asp-AdT are cloned and expressed in Escherichia coli) [1] <13> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <14> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <15> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <16> (cloning of the gatCAB genes encoding the Asp/Glu-AdT and overexpression in Escherichia coli) [7] <16> (cloning of the gatCAB operon encoding the Asp-tRNAAsn amidotransferase and expression in Escherichia coli BL21-Codon Plus(DE3)-RIL) [3] <16> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <17> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] <18> (presence of the gatCAB genes encoding Asp/Glu-AdT) [7] Engineering S128T <8> (<8> mutant protein retains significant glutaminase activity and transamidase activity in the presence of Gln [11]) [11] S152A <8> (<8> mutant is glutaminase inactive [11]) [11] S152T <8> (<8> mutant is glutaminase inactive [11]) [11] Additional information <1, 4, 5> (<4> GatDD4 4 mutant AdT possesses a GatA subunit deprived of its natural 44 C-terminal amino acids and shows unaltered Glu-AdT activity, but lacks any detectable Asp-AdT activity, dele-

635

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

6.3.5.6

tion of one base in comparison with the HB27 genome causes a frameshift mutation [2]; <5> the recombinant human enzyme does not induce leukocyte chemotaxis of HEK-293T cells transfected with G-protein-coupled receptors CXCR1 or CXCR2, overview [13]; <1> treatment of HEK-293T cells expressing CXCR2 with the filarial AsnRS, recombinantly expressed in Escherichia coli, parasite AsnRS is chemotactic for human neutrophils and eosinophils, blocks CXCL1-induced calcium flux, and induces mitogen-activated protein kinase, overview [13]) [2, 13] Application pharmacology <2> (<2> enzyme may have potential as a species-specific therapeutic drug target [4]) [4]

6 Stability Temperature stability Additional information <4> (<4> thermophilic enzyme [2]) [2]

References [1] Curnow, A.W.; Tumbula, D.L.; Pelaschier, J.T.; Min, B.; Sçll, D.: GlutamyltRNAGln amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis. Proc. Natl. Acad. Sci. USA, 95, 12838-12843 (1998) [2] Becker, H.D.; Min, B.; Jacobi, C.; Raczniak, G.; Pelaschier, J.; Roy, H.; Klein, S.; Kern, D.; Sçll, D.: The heterotrimeric Thermus thermophilus AsptRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln). FEBS Lett., 476, 140-144 (2000) [3] Tumbula-Hansen, D.; Feng, L.; Toogood, H.; Stetter, K.O.; Sçll, D.: Evolutionary divergence of the archaeal aspartyl-tRNA synthetases into discriminating and nondiscriminating forms. J. Biol. Chem., 277, 37184-37190 (2002) [4] Raczniak, G.; Becker, H.D.; Min, B.; Sçll, D.: A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA in Chlamydia trachomatis. J. Biol. Chem., 276, 45862-45867 (2001) [5] Min, B.; Pelaschier, J.T.; Graham, D.E.; Tumbula-Hansen, D.; Sçll, D.: Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation. Proc. Natl. Acad. Sci. USA, 99, 2678-2683 (2002) [6] Ibba, M.; Sçll, D.: Aminoacyl-tRNA synthesis. Annu. Rev. Biochem., 69, 617-650 (2000) [7] Tumbula, D.L.; Becker, H.D.; Chang, W.Z.; Sçll, D.: Domain-specific recruitment of amide amino acids for protein synthesis. Nature, 407, 106-110 (2000) [8] Akochy, P.M.; Bernard, D.; Roy, P.H.; Lapointe, J.: Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1. J. Bacteriol., 186, 767-776 (2004)

636

6.3.5.6

Asparaginyl-tRNA synthase (glutamine-hydrolysing)

[9] Chuawong, P.; Hendrickson, T.L.: The nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity. Biochemistry, 45, 8079-8087 (2006) [10] Namgoong, S.; Sheppard, K.; Sherrer, R.L.; Soell, D.: Co-evolution of the archaeal tRNA-dependent amidotransferase GatCAB with tRNAAsn . FEBS Lett., 581, 309-314 (2007) [11] Sheppard, K.; Akochy, P.M.; Salazar, J.C.; Soell, D.: The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln . J. Biol. Chem., 282, 11866-11873 (2007) [12] Bernard, D.; Akochy, P.M.; Bernier, S.; Fisette, O.; Brousseau, O.C.; Chenevert, R.; Roy, P.H.; Lapointe, J.: Inhibition by l-aspartol adenylate of a nondiscriminating aspartyl-tRNA synthetase reveals differences between the interactions of its active site with tRNAAsp and tRNAAsn . J. Enzyme Inhib. Med. Chem., 22, 77-82 (2007) [13] Ramirez, B.L.; Howard, O.M.; Dong, H.F.; Edamatsu, T.; Gao, P.; Hartlein, M.; Kron, M.: Brugia malayi asparaginyl-transfer RNA synthetase induces chemotaxis of human leukocytes and activates G-protein-coupled receptors CXCR1 and CXCR2. J. Infect. Dis., 193, 1164-1171 (2006) [14] Bailly, M.; Blaise, M.; Lorber, B.; Becker, H.D.; Kern, D.: The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228-239 (2007) [15] Cardoso, A.M.; Polycarpo, C.; Martins, O.B.; Soell, D.: A non-discriminating aspartyl-tRNA synthetase from Halobacterium salinarum. RNA Biol., 3, 110-114 (2006)

637

Glutaminyl-tRNA synthase (glutaminehydrolysing)

6.3.5.7

1 Nomenclature EC number 6.3.5.7 Systematic name glutamyl-tRNAGln :l-glutamine amido-ligase (ADP-forming) Recommended name glutaminyl-tRNA synthase (glutamine-hydrolysing) Synonyms GatCAB <6, 7> [11, 12] Glu-AdT Glu-tRNAGln amidotransferase Glu-tRNAGln AT GluAdT GatDE <7> [10] amidotransferase B amidotransferase C amidotransferase, glutamyl-transfer ribonucleate (glutamine-specific) glutamyl-tRNA(Gln) amidotransferase glutamyl-tRNAGln amidotransferase CAS registry number 52232-48-1

2 Source Organism <1> <2> <3> <4> <5>

Chlamydomonas reinhardtii (no sequence specified) [6] Bacillus subtilis (no sequence specified) [3, 7, 9] Scenedesmus obliquus (no sequence specified) [1] Geobacillus stearothermophilus (no sequence specified) [5] Streptococcus pyogenes (no sequence specified) (<5> gene R4CL, putative 4-coumarate coenzyme A ligase, i.e. Os02g0177600 protein [2]) [2,4] <6> Helicobacter pylori (no sequence specified) [12] <7> Methanothermobacter thermautotrophicus (no sequence specified) [10,11] <8> Acidithiobacillus ferrooxidans (no sequence specified) [8]

638

6.3.5.7

Glutaminyl-tRNA synthase (glutamine-hydrolysing)

3 Reaction and Specificity Catalyzed reaction ATP + Glu-tRNAGln + l-glutamine = ADP + phosphate + Gln-tRNAGln + lglutamate Natural substrates and products S ATP + Glu-tRNAGln + l-glutamine <2, 3, 5> (<5> organisms lacking GlntRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGln through the transamidation activity of Glu-tRNAGln amidotransferase. The enzyme hydrolyzes Gln to Glu and NH3 , using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis [2]; <3> the enzyme produces Gln-tRNAGln required for plastidal protein biosynthesis [1]; <2> disruption of this operon is lethal. Transamidation is the only pathway to Gln-tRNAGln in Bacillus subtilis. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln , functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, archaea and organelles [3]) (Reversibility: ?) [1, 2, 3] P ADP + phosphate + Gln-tRNAGln + l-glutamate Substrates and products S ATP + Asp-tRNAAsn + l-glutamine <6, 7, 8> (<7> identity elements used by GatCAB to discriminate tRNAAsn from tRNAAsp . GatCAB specifically binds Asp-tRNAAsn . Therefore, modified nucleotides do not play an essential role in GatCAB discrimination of Asp-tRNAAsn from Asp-tRNAAsp [11]; <6> the enzyme transamidates Asp-tRNAAsn and Glu-tRNAGln with similar efficiency [12]) (Reversibility: ?) [8, 11, 12] P ADP + phosphate + Asn-tRNAAsn + l-glutamate <8> [8] S ATP + Glu-tRNAGln + Asn <1, 2> (<2> Asn is much less effective as amide donor than glutamine [3,9]) (Reversibility: ?) [3, 6, 9] P ADP + phosphate + Gln-tRNAGln + Asp S ATP + Glu-tRNAGln + l-asparagine <2> (Reversibility: ?) [7] P ADP + phosphate + Gln-tRNAGln + l-glutamate S ATP + Glu-tRNAGln + l-glutamine <1, 2, 3, 4, 5, 6, 7, 8> (<5> Ser176A is the active-site nucleophile for facilitating Gln hydrolysis by the enzyme [4]; <2> the amidation of Glu-tRNAGln proceeds via a g-phosphorylated intermediate [7]; <5> organisms lacking Gln-tRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGln through the transamidation activity of Glu-tRNAGln amidotransferase. The enzyme hydrolyzes Gln to Glu and NH3 , using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis [2]; <3> the enzyme produces Gln-tRNAGln required for plastidal protein biosynthesis [1]; <2> disruption of this operon is lethal. Transamidation is the only pathway to Gln-tRNAGln in Bacillus subtilis. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated GlutRNAGln , functionally replacing the lack of glutaminyl-tRNA synthetase

639

Glutaminyl-tRNA synthase (glutamine-hydrolysing)

P S P S P S

P

6.3.5.7

activity in Gram-positive eubacteria, cyanobacteria, archaea and organelles [3]; <7> GatDE is a heterodimeric amidotransferase. GatD acts as a glutaminase but only in the presence of both Glu-tRNAGln and the other subunit, GatE. The fact that only Glu-tRNAGln but not tRNA Gln could activate the glutaminase activity of GatD suggests that glutamine hydrolysis is coupled tightly to transamidation. GatE is a Glu-tRNAGln kinase that activates Glu-tRNAGln via g-phosphorylation [10]; <6> the enzyme transamidates Asp-tRNAAsn and Glu-tRNAGln with similar efficiency. GatCAB uses the amide donor glutamine 129fold more efficiently than asparagine [12]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12] ADP + phosphate + Gln-tRNAGln + l-glutamate <1, 2, 4, 5> [2, 3, 4, 5, 6] ATP + Glu-tRNAGln + NH4 Cl <1, 2> (<2> NH4 Cl is much less effective as amide donor than glutamine [3,9]) (Reversibility: ?) [3, 6, 9] ADP + phosphate + Gln-tRNAGln + ? ATP-gS + Glu-tRNAGln + l-glutamine <5> (Reversibility: ?) [2] ? + phosphate + Gln-tRNAGln + l-glutamate <5> [2] Additional information <1, 5> (<5> in absence of the amido acceptor, Glu-tRNAGln , the enzyme has basal glutaminase activity that is unaffected by ATP [2]; <1> the enzyme possesses low glutaminase activity [6]) (Reversibility: ?) [2, 6] ?

Inhibitors 2’-O-(trinitrophenyl)adenosine 5’-triphosphate <5> (<5> IC50: 2.4 mM [2]) [2] 3’-O-(trinitrophenyl)adenosine 5’-triphosphate <5> (<5> IC50: 2.4 mM [2]) [2] 6-diazo-5-oxonorleucine <1> (<1> blocking of glutamine-dependent reaction, no inhibition of ammonia-dependent reaction [6]) [6] ADP <5> (<5> IC50: 0.026 mM [2]) [2] ATP-gS <5> (<5> IC50: 0.19 mM [2]) [2] adenosine 5’-[b,g-methylene]triphosphate <5> (<5> IC50: 2.3 mM [2]) [2] l-methionine-S-sulfoximine <2> (<2> at 1 mM, 3 mM or 5 mM, 20% inhibition [9]) [9] g-Glu boronic acid <5> (<5> IC50: 0.0016 mM [4]) [4] Additional information <2> (<2> no inhibition by 6-diazo-5-oxonorleucine [9]) [9] Activating compounds Additional information <5> (<5> activation of glutaminase activity by ATP or ATP-gS together with Glu-tRNAGln , results either from an allosteric effect due simply to binding of these analogues to the enzyme or from some structural changes that attend ATP or ATP-gS hydrolysis [2]) [2] Metals, ions Mg2+ <1, 2> (<1,2> required [3,6]) [3, 6]

640

6.3.5.7

Glutaminyl-tRNA synthase (glutamine-hydrolysing)

Turnover number (min–1) 0.027 <6> (Asn, <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 0.052-2.1 <6> (Gln, <6> 37 C, pH 7.2, cosubstrate: Glu-tRNAGln [12]) [12] 0.51 <5> (Gln) [2] 0.59 <5> (ATP) [2] 1.3 <6> (Asp-tRNAAsn , <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 3-6 <6> (Gln, <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 3.61 <6> (Asp-tRNAGln , <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 6.1 <6> (ATP, <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 11.8 <6> (Gln, <6> 37 C, pH 7.2, cosubstrate Asp-tRNAAsn + ATP, glutaminase activity [12]) [12] Specific activity (U/mg) 0.009 <2> [3] Km-Value (mM) 0.0002 <5> (tRNA) [2] 0.00095 <6> (Asp-tRNAAsn , <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 0.00118 <6> (Asp-tRNAGln , <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 0.01 <2> (Gln) [9] 0.0159 <5> (Gln) [2] 0.0207 <6> (Gln, <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 0.0224 <6> (Asn, <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] 0.0402 <6> (Gln, <6> 37 C, cosubstrate Asp-tRNAAsn + ATP, glutaminase activity [12]) [12] 0.0509 <6> (Gln, <6> 37 C, cosubstrate Glu-tRNAGln + ATP, glutaminase activity [12]) [12] 0.1-0.2 <2> (Asn) [9] 0.117 <5> (ATP) [2] 0.2068 <6> (ATP, <6> 37 C, pH 7.2, amidotransferase activity [12]) [12] Temperature range ( C) 37-50 <4> [5]

4 Enzyme Structure Molecular weight 120000 <1> (<1> gel filtration, glycerol density gradient sedimentation [6]) [6] Subunits dimer <1> (<1> 2 * 63000, SDS-PAGE [6]) [6] trimer <2> (<2> 1 * 53000 + 1 * 53500 + 1 * 10900, SDS-PAGE [3]) [3]

641

Glutaminyl-tRNA synthase (glutamine-hydrolysing)

6.3.5.7

5 Isolation/Preparation/Mutation/Application Localization chloroplast <3> [1] Purification <1> [6] <2> [3, 7] <4> (overexpressed with pTrcgatCABBST in E. coli) [5] <6> [12] <7> [10] Crystallization <4> [5] Cloning <2> (cloning of the three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the enzyme) [3] <4> [5] <7> [10] <8> (expression in Escherichia coli) [8] Engineering D178E <7> (<7> glutamine hydrolysis is negligible, Gln-tRNAGln formation is undetectable [10]) [10] D178N <7> (<7> glutamine hydrolysis is negligible, Gln-tRNAGln formation is undetectable [10]) [10] K254E <7> (<7> glutamine hydrolysis is negligible, Gln-tRNAGln formation is undetectable [10]) [10] S128T <6> (<6> mutant protein retains significant glutaminase activity and transamidase activity in the presence of Gln [12]) [12] S152A <6> (<6> mutant is glutaminase inactive [12]) [12] S152T <6> (<6> mutant is glutaminase inactive [12]) [12] T101A <7> (<7> glutamine hydrolysis is negligible, Gln-tRNAGln formation is undetectable [10]) [10] T101S <7> (<7> hydrolyzes about 10% of glutamine compared to wild-type enzyme. Compared to wild-type enzyme, the mutant enzyme converts approximately half as much mischarged tRNA substrate to product [10]) [10] T177S <7> (<7> mutant enzyme hydrolyzes the same amount of glutamine as the wild-type enzyme. As the wild-type enzyme, the mutant enzyme transforms most of Glu-tRNAGln to Gln-tRNAGln [10]) [10] T177V <7> (<7> glutamine hydrolysis is negligible. Gln-tRNAGln formation is undetectable [10]) [10]

642

6.3.5.7

Glutaminyl-tRNA synthase (glutamine-hydrolysing)

References [1] Vothknecht, U.C.; Doernemann, D.: Charging of both, plastidial tRNAgln and tRNAglu with glutamate and subsequent amidation of the misacylated tRNAgln by a glutamyl-tRNA amidotransferase in the unicellular green alga Scenedesmus obliquus, mutant C-2A’. Z. Naturforsch. C, 50, 789-795 (1995) [2] Horiuchi, K.Y.; Harpel, M.R.; Shen, L.; Luo, Y.; Rogers, K.C.; Copeland, R.A.: Mechanistic studies of reaction coupling in Glu-tRNAGln amidotransferase. Biochemistry, 40, 6450-6457 (2001) [3] Curnow, A.W.; Hong, K.W.; Yuan, R.; Kim, S.I.; Martins, O.; Winkler, W.; Henkin, T.M.; Soll, D.: Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proc. Natl. Acad. Sci. USA, 94, 11819-11826 (1997) [4] Harpel, M.R.; Horiuchi, K.Y.; Luo, Y.; Shen, L.; Jiang, W.; Nelson, D.J.; Rogers, K.C.; Decicco, C.P.; Copeland, R.A.: Mutagenesis and mechanismbased inhibition of Streptococcus pyogenes Glu-tRNAGln amidotransferase implicate a serine-based glutaminase site. Biochemistry, 41, 6398-6407 (2002) [5] Kwak, J.H.; Shin, K.; Woo, J.S.; Kim, M.K.; Kim, S.I.; Eom, S.H.; Hong, K.W.: Expression, purification, and crystallization of glutamyl-tRNA(Gln) specific amidotransferase from Bacillus stearothermophilus. Mol. Cells, 14, 374-381 (2002) [6] Jahn, D.; Kim, Y.C.; Ishino, Y.; Chen, M.W.; Soll, D.: Purification and functional characterization of the Glu-tRNAGln amidotransferase from Chlamydomonas reinhardtii. J. Biol. Chem., 265, 8059-8064 (1990) [7] Zalkin, H.: Glu-tRNAGln amidotransferase. Methods Enzymol., 113, 303-305 (1985) [8] Salazar, J.C.; Zuniga, R.; Raczniak, G.; Becker, H.; Soll, D.; Orellana, O.: A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans. FEBS Lett., 500, 129-131 (2001) [9] Strauch, M.A.; Zalkin, H.; Aronson, A.I.: Characterization of the glutamyltRNAGln -to-glutaminyl-tRNAGln amidotransferase reaction of Bacillus subtilis. J. Bacteriol., 170, 916-920 (1988) [10] Feng, L.; Sheppard, K.; Tumbula-Hansen, D.; Soell, D.: Gln-tRNAGln formation from Glu-tRNAGln requires cooperation of an asparaginase and a GlutRNAGln kinase. J. Biol. Chem., 280, 8150-8155 (2005) [11] Namgoong, S.; Sheppard, K.; Sherrer, R.L.; Soell, D.: Co-evolution of the archaeal tRNA-dependent amidotransferase GatCAB with tRNAAsn . FEBS Lett., 581, 309-314 (2007) [12] Sheppard, K.; Akochy, P.M.; Salazar, J.C.; Soell, D.: The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln . J. Biol. Chem., 282, 11866-11873 (2007)

643

Aminodeoxychorismate synthase

6.3.5.8

1 Nomenclature EC number 6.3.5.8 (transferred to EC 2.6.1.85. As ATP is not hydrolysed during the reaction, the classification of the enzyme as a ligase was incorrect.) Recommended name aminodeoxychorismate synthase

644

Hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing)

6.3.5.9

1 Nomenclature EC number 6.3.5.9 Systematic name hydrogenobyrinic-acid:l-glutamine amido-ligase (AMP-forming) Recommended name hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing) Synonyms CbiA <1> [7] CobB <1, 2, 4> [1, 4, 5, 6] cobalamin biosynthesis aminotransferase <2> [6] cobyric acid synthase <3> [3] cobyrinic acid a,c-diamide synthase <2, 3, 4> [1, 2, 6] new NaMN:Me2Bza phosphoribosyltransferase enzyme <1> [5] CAS registry number 132053-22-6

2 Source Organism <1> Salmonella typhimurium (no sequence specified) (<1> gene ispS [4,5,7]) [4, 5, 7] <2> Escherichia coli (no sequence specified) [6] <3> Pseudomonas denitrificans (no sequence specified) [2, 3] <4> Pseudomonas denitrificans (UNIPROT accession number: P21632) [1]

3 Reaction and Specificity Catalyzed reaction 2 ATP + hydrogenobyrinic acid + 2 l-glutamine + 2 H2 O = 2 ADP + 2 phosphate + hydrogenobyrinic acid a,c-diamide + 2 l-glutamate Natural substrates and products S 5’-deoxy-5’-adenosyl-cobyrinic acid + ATP + glutamine + H2 O <3> (<3> cobalamin biosynthetic pathway [3]) (Reversibility: ?) [3] P 5’-deoxy-5’-adenosyl-cobyrinic acid ac-diamide + ADP + phosphate + glutamate <3> [3]

645

Hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing)

6.3.5.9

S ATP + cobyrinic acid + l-glutamine + H2 O <3> (<3> cobalamin pathway from cobyrinic acid [2]) (Reversibility: ?) [2] P ADP + phosphate + cobyrinic acid a,c-diamide + l-glutamate <3> [2] S ATP + hydrogenobyrinic acid + l-glutamine + H2 O <2, 3> (<2> vitamin B12 biosynthesis [6]) (Reversibility: ?) [2, 6] P ADP + phosphate + hydrogenobyrinic acid a,c-diamide + l-glutamate <2> [6] S Additional information <1, 4> (<4> cobalamin biosynthetic pathway [1]; <1> required for catabolism of propionate, involved in assembly of the nucleotide loop of cobalamin, alternative activity for the nicotinic acid mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase enzyme CobT [4]) (Reversibility: ?) [1, 4] P ? <1, 4> [1, 4] Substrates and products S 5’-deoxy-5’-adenosyl-cobyrinic acid + ATP + ammonia + H2 O <3> (Reversibility: ?) [3] P 5’-deoxy-5’-adenosyl-cobyrinic acid ac-diamide + ADP + phosphate + ? <3> [3] S 5’-deoxy-5’-adenosyl-cobyrinic acid + ATP + glutamine + H2 O <3> (<3> cobalamin biosynthetic pathway [3]) (Reversibility: ?) [3] P 5’-deoxy-5’-adenosyl-cobyrinic acid ac-diamide + ADP + phosphate + glutamate <3> [3] S ATP + (CN,aq)cobyrinic acid + glutamine + H2 O <3> (Reversibility: ?) [2] P ADP + phosphate + c-monoamide + ? <3> [2] S ATP + (CN,aq)cobyrinic acid -monoamide + glutamine + H2 O <3> (Reversibility: ?) [2] P ADP + phosphate + ? <3> [2] S ATP + (aq)2cobyrinic acid + glutamine + H2 O <3> (Reversibility: ?) [2] P ADP + phosphate + ? <3> [2] S ATP + cobyrinic acid + l-glutamine + H2 O <3, 4> (<3> intermediate formation of cobyrinic acid c-monoamide [2]; <3> cobalamin pathway from cobyrinic acid [2]) (Reversibility: ?) [1, 2] P ADP + phosphate + cobyrinic acid a,c-diamide + l-glutamate <3, 4> [1, 2] S ATP + cobyrinic acid + ammonia + H2 O <3> (Reversibility: ?) [2] P ADP + phosphate + ? <3> [2] S ATP + hydrogenobyrinic acid + l-glutamine + H2 O <1, 2, 3, 4> (<4> amidation of carboxylic groups at positions a and c [1]; <2> vitamin B12 biosynthesis [6]) (Reversibility: ?) [1, 2, 4, 5, 6] P ADP + phosphate + hydrogenobyrinic acid a,c-diamide + l-glutamate <1, 2, 3, 4> [1, 2, 4, 5, 6] S ATP + hydrogenobyrinic acid c-monoamide + l-glutamine + H2 O <3> (Reversibility: ?) [2] P ADP + phosphate + ? <3> [2] S DMB + nicotinic acid mononucleotide <1> (Reversibility: ?) [4] P DMB-ribose-5’-phosphate <1> [4]

646

6.3.5.9

Hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing)

S adenosyl-cobyrinic acid pentaamide + ATP + glutamine + H2 O <3> (Reversibility: ?) [3] P ADP + glutamate + phosphate + ? <3> [3] S adenosyl-cobyrinic acid tetraamide + ATP + glutamine + H2 O <3> (Reversibility: ?) [3] P ADP + glutamate + phosphate + ? <3> [3] S adenosyl-cobyrinic acid triamide + ATP + glutamine + H2 O <3> (Reversibility: ?) [3] P ADP + glutamate + phosphate + ? <3> [3] S a-ribazole + phosphate <1> (Reversibility: ?) [5] P N1 -(5-phospho-a-d-ribosyl)-5,6-dimethylbenzimidazole + H2 O <1> [5] S cobyrinic acid + ATP + NH+4 <1> (<1> model for catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate [7]) (Reversibility: ?) [7] P cobyrinic acid a,c-diamide + ADP + phosphate S cobyrinic acid + ATP + glutamine <1> (<1> model for catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate [7]) (Reversibility: ?) [7] P cobyrinic acid a,c-diamide + ADP + phosphate + glutamate S cobyrinic acid c-monoamide + ATP + NH+4 <1> (Reversibility: ?) [7] P cobyrinic acid a,c-diamide + ADP + phosphate S cobyrinic acid c-monoamide + ATP + glutamine <1> (Reversibility: ?) [7] P cobyrinic acid a,c-diamide + ADP + phosphate + glutamate S Additional information <1, 3, 4> (<3> adenosyl-cobyric acid and hydrogenobyrinic acid a,c-diamide are no substrates [3]; <3> cobyrinic acid a,c-diamide and cobyrinic acid a,c,g-triamide are no substrates [2]; <4> cobalamin biosynthetic pathway [1]; <1> required for catabolism of propionate, involved in assembly of the nucleotide loop of cobalamin, alternative activity for the nicotinic acid mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase enzyme CobT [4]) (Reversibility: ?) [1, 2, 3, 4] P ? <1, 3, 4> [1, 2, 3, 4] Metals, ions Mg2+ <3> (<3> required [2]; <3> amidation reaction is Mg2+ dependent [3]) [2, 3] Turnover number (min–1) 0.008 <1> (cobyrinic acid c-monoamide, <1> mutant enzyme Y46A [7]) [7] 0.04 <1> (cobyrinic acid c-monoamide, <1> mutant enzyme D97N [7]) [7] 0.057 <1> (cobyrinic acid c-monoamide, <1> mutant enzyme L47A [7]) [7] 0.13 <1> (ATP, <1> pH 7.7, wild-type enzyme [7]) [7] 0.16 <1> (glutamine, <1> pH 7.7, wild-type enzyme [7]) [7] 0.16 <1> (cobyrinic acid, <1> pH 7.7, wild-type enzyme [7]) [7] 0.18 <1> (NH+4 , <1> pH 7.7, wild-type enzyme [7]) [7] 0.2 <1> (cobyrinic acid c-monoamide, <1> wild-type enzyme [7]) [7]

647

Hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing)

6.3.5.9

Specific activity (U/mg) 0.068 <3> (<3> adenosyl-cobyrinic acid a,c-diamide as substrate [3]) [3] 0.083 <3> (<3> adenosyl-cobyrinic acid pentaamide as substrate [3]) [3] Km-Value (mM) 0.00021 <3> (hydrogenobyrinic acid c-monoamide, <3> pH 7.6, 30 C [2]) [2] 0.0003 <1> (cobyrinic acid c-monoamide, <1> mutant enzyme D97N [7]) [7] 0.00041 <3> (hydrogenobyrinic acid, <3> pH 7.6, 30 C [2]) [2] 0.00041 <1> (cobyrinic acid c-monoamide, <1> wild-type enzyme [7]) [7] 0.00074 <1> (cobyrinic acid, <1> pH 7.7, wild-type enzyme [7]) [7] 0.00104 <3> (adenosyl-cobyrinic acid a,c-diamide, <3> pH 7.5, 20 C [3]) [3] 0.00121 <3> (adenosyl-cobyrinic acid tetraamide, <3> pH 7.5, 20 C [3]) [3] 0.00122 <3> (adenosyl-cobyrinic acid triamide, <3> pH 7.5, 20 C [3]) [3] 0.00125 <3> (adenosyl-cobyrinic acid pentaamide, <3> pH 7.5, 20 C [3]) [3] 0.0027 <1> (ATP, <1> pH 7.7, wild-type enzyme [7]) [7] 0.0027 <1> (cobyrinic acid c-monoamide, <1> mutant enzyme L47A [7]) [7] 0.011 <3> (ATP, <3> pH 7.5, 20 C, adenosyl-cobyrinic acid a,c-diamide as substrate [3]) [3] 0.011 <1> (cobyrinic acid c-monoamide, <1> mutant enzyme Y46A [7]) [7] 0.012 <3> (ammonia, <3> pH 7.6, 30 C [2]) [2] 0.0203 <3> (glutamine, <3> pH 7.6, 30 C [2]) [2] 0.033 <3> (l-glutamine, <3> pH 7.5, 20 C, adenosyl-cobyrinic acid triamide as substrate [3]) [3] 0.035 <3> (ATP, <3> pH 7.5, 20 C, adenosyl-cobyrinic acid triamide as substrate [3]) [3] 0.045 <3> (l-glutamine, <3> pH 7.5, 20 C, adenosyl-cobyrinic acid a,c-diamide as substrate [3]) [3] 0.049 <3> (l-glutamine, <3> pH 7.5, 20 C, adenosyl-cobyrinic acid pentaamide as substrate [3]) [3] 0.053 <1> (glutamine, <1> pH 7.7, wild-type enzyme [7]) [7] 0.064 <3> (l-glutamine, <3> pH 7.5, 20 C, adenosyl-cobyrinic acid tetraamide as substrate [3]) [3] 0.071 <3> ((CN,aq)cobyrinic acid c-monoamide, <3> pH 7.6, 30 C [2]) [2] 0.088 <3> (ATP, <3> pH 7.5, 20 C, adenosyl-cobyrinic acid pentaamide as substrate [3]) [3] 0.12 <3> (ATP, <3> pH 7.5, 20 C, adenosyl-cobyrinic acid tetraamide as substrate [3]) [3] 0.16 <3> ((CN,aq)cobyrinic acid, <3> pH 7.6, 30 C [2]) [2] 0.25 <3> ((aq)2cobyrinic acid, <3> pH 7.6, 30 C [2]) [2] 20 <3> (ammonia, <3> pH 7.5, 20 C [3]) [3] 26.2 <1> (NH+4 , <1> pH 7.7, wild-type enzyme [7]) [7] pH-Optimum 6.8-8 <1> [7] 7.3 <2, 3> (<3> broad optimum with maximum activity from pH 6.8-8.0 [2]) [2, 6] 7.5 <3> (<3> broad optimum around [3]) [3]

648

6.3.5.9

Hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing)

4 Enzyme Structure Molecular weight 26300 <1> (<1> protein predicted from nucleotide sequence [5]) [5] 28000 <1> (<1> SDS-PAGE [5]) [5] 55000 <1> (<1> gel filtration [7]) [7] 86000 <3> (<3> gel filtration [2]) [2] 97300 <3> (<3> gel filtration [3]) [3] Subunits dimer <2, 3, 4> (<3> 2 * 45000, SDS-PAGE [2]; <2> 2 * 45000 [6]; <4> 2 * 45000, homodimer [1]; <3> 2 * 57000, homodimer, SDS-PAGE [3]) [1, 2, 3, 6] monomer <1> (<1> 1 * 50000, SDS-PAGE [7]) [7]

5 Isolation/Preparation/Mutation/Application Purification <3> [1, 2, 3] Cloning <1> (cloned, sequenced and overexpressed) [5] <1> (expression of wild-type and mutant enzymes in Escherichia coli) [7] <3> [3] <3> (identification of the structural gene) [1] <3> (plasmid pXL 191 is transferre from Escherichia coli MC10 60 to Pseudomonas denitrificans SC510 RifT through conjugation) [2] Engineering D45N <1> (<1> less than 0.2% of amidation activity with glutamine and with NH+4 , compared to wild-type values [7]) [7] D48N <1> (<1> less than 0.3% of amidation activity with glutamine and with NH+4 , compared to wild-type values [7]) [7] D97N <1> (<1> 32.4% of wild-type amidation activity with glutamine, 16.5% of wild-type amidation activity with NH+4 [7]) [7] E90Q <1> (<1> less than 0.02% of amidation activity with glutamine and with NH+4 , compared to wild-type values [7]) [7] L47A <1> (<1> 10.6% of wild-type amidation activity with glutamine, 44% of wild-type amidation activity with NH+4 , mutation specifically decreases the affinity of the enzyme to the cobyrinic acid c-monoamide [7]) [7] Y46A <1> (<1> 3.8% of wild-type amidation activity with glutamine, 2% of wild-type amidation activity with NH+4 , mutation specifically decreases the affinity of the enzyme to the cobyrinic acid c-monoamide [7]) [7]

649

Hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing)

6.3.5.9

6 Stability General stability information <3>, glycerol and DTT are necessary to stabilize the enzyme during purification [3] Storage stability <3>, -20 C, 0.1 M Tris hydrochloride, pH 7.5, 100% activity is lost after 48 h [3] <3>, -20 C, enzyme can be stored in the eluate from purification step v without detectable loss of activity [3] <3>, 0 C, 0.1 M Tris hydrochloride, pH 7.5, 100% activity is lost after 48 h [3]

References [1] Crouzet, J.; Cauchois, L.; Blanche, F.; Debussche, L.; Thibaut, D.; Rouyez, M.C.; Rigault, S.; Mayaux, J.F.; Cameron, B.: Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilobase DNA fragment containing five cob genes and identification of structural genes encoding S-adenosyl-l-methionine: uroporphyrinogen III methyltransferase and cobyrinic acid a,c-diamide synthase. J. Bacteriol., 172, 5968-5979 (1990) [2] Debussche, L.; Thibaut, D.; Cameron, B.; Crouzet, J.; and Blanche, F.: Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans. J. Bacteriol., 172, 6239-6244 (1990) [3] Blanche, F.; Couder, M.; Debussche, L.; Thibaut, D.; Cameron, B.; Cruzet, J.: Biosynthesis of vitamin B12 : stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans. J. Bacteriol., 173, 6046-6051 (1991) [4] Tsang, A.W.; Escalante-Semerena, J.C.: cobB function is required for catabolism of propionate in Salmonella typhimurium LT2: evidence for existence of a substitute function for CobB within the 1,2-propanediol utilization (pdu) operon. J. Bacteriol., 178, 7016-7019 (1996) [5] Tsang, A.W.; Escalante-Semerena, J.C.: CobB, a new member of the SIR2 family of eucaryotic regulatory proteins, is required to compensate for the lack of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase activity in cobT mutants during cobalamin biosynthesis in Salmonella typhimurium LT2. J. Biol. Chem., 273, 31788-31794 (1998) [6] Galperin, M.Y.; Grishin, N.V.: The synthetase domains of cobalamin biosynthesis amidotransferases cobB and cobQ belong to a new family of ATP-dependent amidoligases, related to dethiobiotin synthetase. Proteins, 41, 238247 (2000) [7] Fresquet, V.; Williams, L.; Raushel, F.M.: Mechanism of cobyrinic acid a,cdiamide synthetase from Salmonella typhimurium LT2. Biochemistry, 43, 10619-10627 (2004)

650

Adenosylcobyric acid synthase (glutaminehydrolysing)

6.3.5.10

1 Nomenclature EC number 6.3.5.10 Systematic name adenosylcobyrinic-acid-a,c-diamide:l-glutamine amido-ligase (ADP-forming) Recommended name adenosylcobyric acid synthase (glutamine-hydrolysing) Synonyms 5’-deoxy-5’-adenosylcobyrinic-acid-a,c-diamide:l-glutamine amido-ligase Ado-cobyric acid synthase (glutamine hydrolyzing) CbiP <1, 4, 5> [2, 7, 9] CobQ <2, 3> [2, 5, 6] cobalamin biosynthesis aminotransferase <2> [6] cobyric acid synthase <2> [6] cobyric acid synthetase <1> [9] Additional information <3> (<3> CobQ is a member of the dethiobiotin synthetase superfamily [8]) [8] CAS registry number 137672-90-3

2 Source Organism <1> <2> <3> <4> <5> <6>

Salmonella typhimurium (no sequence specified) [9] Escherichia coli (no sequence specified) [6] Pseudomonas denitrificans (no sequence specified) [2, 3, 4, 5, 8] Salmonella enterica (no sequence specified) [2, 7] Halobacterium sp. (no sequence specified) [7] Pseudomonas denitrificans (UNIPROT accession number: P29932) [1]

651

Adenosylcobyric acid synthase (glutamine-hydrolysing)

6.3.5.10

3 Reaction and Specificity Catalyzed reaction 4 ATP + adenosylcobyrinic acid a,c-diamide + 4 l-glutamine + 4 H2 O = 4 ADP + 4 phosphate + adenosylcobyric acid + 4 l-glutamate (<3> catalytic mechanism [8]) Natural substrates and products S ATP + adenosylcobyrinic acid a,c-diamide + l-glutamine + H2 O <3, 4, 5, 6> (<3> biosynthesis of vitamin B12 , cobalamin biosynthetic pathway [4]; <4> CbiP is involved in the biosynthesis of adenosylcobalamin, i.e. vitamin B12 , anaerobic pathway [2]; <6> cobalamin biosynthetic pathway, involved in coenzyme B12 synthesis [1]; <5> cobinamide biosynthesis [7]; <3> CobQ is involved in the biosynthesis of adenosylcobalamin, i.e. vitamin B12 , aerobic pathway [2]; <3> involved in cobalamin biosynthesis [8]; <4> involved in the de novo cobinamide biosynthesis pathway [7]) (Reversibility: ?) [1, 2, 3, 4, 7, 8] P ADP + phosphate + adenosylcobyric acid + l-glutamate S adenosylcobyrinic acid a,c-diamide + ATP + l-glutamine + H2 O <2> (<2> vitamin B12 biosynthesis [6]) (Reversibility: ?) [6] P adenosylcobyric acid + ADP + phosphate + l-glutamate <2> [6] Substrates and products S ATP + adenosylcobyrinic acid a,c-diamide + l-glutamine + H2 O <3, 4, 5, 6> (<3> biosynthesis of vitamin B12 , cobalamin biosynthetic pathway [4]; <4> CbiP is involved in the biosynthesis of adenosylcobalamin, i.e. vitamin B12 , anaerobic pathway [2]; <6> cobalamin biosynthetic pathway, involved in coenzyme B12 synthesis [1]; <5> cobinamide biosynthesis [7]; <3> CobQ is involved in the biosynthesis of adenosylcobalamin, i.e. vitamin B12 , aerobic pathway [2]; <3> involved in cobalamin biosynthesis [8]; <4> involved in the de novo cobinamide biosynthesis pathway [7]; <4> CbiP catalyzes b, d, e, and g amidation [2]; <3> CobQ amidates the side chains b, d, e, and g generating adenosylcobyric acid, requires glutamine as the amide donor [2]; <6> CobQ catalyzes amidations at positions b, d, e and g [1]; <3> CobQ contains an unusual Triad family, class I, glutamine amidotransferase domain with conserved Cys and His residues, but lacking the Glu residue of the catalytic triad, domain organization, model of substrate binding, catalytic mechanism, CobQ catalyzes amidation of the carboxyls b, d, e, and g in a strict order, one ATP molecule and one glutamine molecule are consumed for each amidation reaction [8]; <3> enzyme catalyzes the stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide, four-step amidation sequence from cobyrinic acid a,c-diamide to cobyric acid via the formation of corbyrinic acid triamide, tetraamide, and pentaamide intermediates, the amidations are carried out in a specific order, enzyme is specific to coenzyme forms of substrates, no amidation of the carboxyl group at position f, l-glutamine is the preferred amide group donor [4]) (Reversibility: ?) [1, 2, 3, 4, 7, 8]

652

6.3.5.10

Adenosylcobyric acid synthase (glutamine-hydrolysing)

P ADP + phosphate + adenosylcobyric acid + l-glutamate S ATP + adenosylcobyrinic acid a,c-diamide + ammonia + H2 O <3> (<3> ammonia can replace l-glutamine as amide group donor, but l-glutamine is preferred [4]) (Reversibility: ?) [4] P ADP + phosphate + adenosylcobyric acid + ? S ATP + adenosylcobyrinic acid diamide + l-glutamine + H2 O <1> (<1> four carboxylates attached to the corrinoid ring are amidated in a dissociative fashion. The four carboxylates are amidated in a sequence beginning with carboxylate e and followed in turn by carboxylates d, b, and g [9]) (Reversibility: ?) [9] P ADP + phosphate + adenosylcobyric acid + l-glutamate S ATP + adenosylcobyrinic acid pentaamide + l-glutamine + H2 O <1, 3> (<1> four carboxylates attached to the corrinoid ring are amidated in a dissociative fashion. The four carboxylates are amidated in a sequence beginning with carboxylate e and followed in turn by carboxylates d, b, and g [9]) (Reversibility: ?) [4, 9] P ADP + phosphate + adenosylcobyric acid + l-glutamate S ATP + adenosylcobyrinic acid tetraamide + l-glutamine + H2 O <1, 3> (<3> two-step amidation [4]; <1> four carboxylates attached to the corrinoid ring are amidated in a dissociative fashion. The four carboxylates are amidated in a sequence beginning with carboxylate e and followed in turn by carboxylates d, b, and g [9]) (Reversibility: ?) [4, 9] P ADP + phosphate + adenosylcobyric acid + l-glutamate S ATP + adenosylcobyrinic acid triamide + l-glutamine + H2 O <1, 3> (<3> three-step amidation [4]; <1> four carboxylates attached to the corrinoid ring are amidated in a dissociative fashion. The four carboxylates are amidated in a sequence beginning with carboxylate e and followed in turn by carboxylates d, b, and g [9]) (Reversibility: ?) [4, 9] P ADP + phosphate + adenosylcobyric acid + l-glutamate S adenosylcobyrinic acid a,c-diamide + ATP + l-glutamine + H2 O <2, 3> (<2> vitamin B12 biosynthesis [6]) (Reversibility: ?) [5, 6] P adenosylcobyric acid + ADP + phosphate + l-glutamate <2, 3> [5, 6] S cobinic acid a,c-diamide + ATP + l-glutamine + H2 O <3> (Reversibility: ?) [5] P cobinamide + ADP + phosphate + l-glutamate <3> [5] S Additional information <3> (<3> no amidation of adenosylcobyric acid, hydrogenobyrinic acid a,c-diamide, monocyano or diaqua form of cobyrinic acid a,c-diamide [4]) (Reversibility: ?) [4] P ? Inhibitors cobyric acid <1> [9] Cofactors/prosthetic groups ATP <3> (<3> requirement [2]; <3> ATP-dependent [4,8]) [2,4,8] Metals, ions Mg2+ <3> (<3> Mg2+ -dependent [4]; <3> bound [8]) [4, 8]

653

Adenosylcobyric acid synthase (glutamine-hydrolysing)

Turnover number (min–1) 2.4 <1> (adenosylcobyrinic 3.6 <1> (adenosylcobyrinic 3.6 <1> (adenosylcobyrinic 8.4 <1> (adenosylcobyrinic

6.3.5.10

acid triamide, <1> 30 C, pH 7.5 [9]) [9] acid diamide, <1> 30 C, pH 7.5 [9]) [9] acid tetraamide, <1> 30 C, pH 7.5 [9]) [9] acid pentaamide, <1> 30 C, pH 7.5 [9]) [9]

Specific activity (U/mg) 0.083 <3> (<3> pH 7.5, adenosylcobyrinic acid a,c-diamide as substrate [4]) [4] Km-Value (mM) 0.0007 <1> (adenosylcobyrinic acid pentaamide, <1> 30 C, pH 7.5 [9]) [9] 0.0008 <1> (adenosylcobyrinic acid tetraamide, <1> 30 C, pH 7.5 [9]) [9] 0.001 <1> (adenosylcobyrinic acid triamide, <1> 30 C, pH 7.5 [9]) [9] 0.00104 <3> (adenosylcobyrinic acid a,c-diamide, <3> pH 7.5 [4]) [4] 0.00121 <3> (adenosylcobyrinic acid tetraamide, <3> pH 7.5 [4]) [4] 0.00122 <3> (adenosylcobyrinic acid triamide, <3> pH 7.5 [4]) [4] 0.00125 <3> (adenosylcobyrinic acid pentaamide, <3> pH 7.5 [4]) [4] 0.0027 <1> (adenosylcobyrinic acid diamide, <1> 30 C, pH 7.5 [9]) [9] 0.011 <3> (ATP, <3> pH 7.5, adenosylcobyrinic acid a,c-diamide as substrate [4]) [4] 0.033 <3> (l-glutamine, <3> pH 7.5, adenosylcobyrinic acid triamide as substrate [4]) [4] 0.035 <3> (ATP, <3> pH 7.5, adenosylcobyrinic acid triamide as substrate [4]) [4] 0.045 <3> (l-glutamine, <3> pH 7.5, adenosylcobyrinic acid a,c-diamide as substrate [4]) [4] 0.049 <3> (l-glutamine, <3> pH 7.5, adenosylcobyrinic acid pentaamide as substrate [4]) [4] 0.064 <3> (l-glutamine, <3> pH 7.5, adenosylcobyrinic acid tetraamide as substrate [4]) [4] 0.088 <3> (ATP, <3> pH 7.5, adenosylcobyrinic acid pentaamide as substrate [4]) [4] 0.12 <3> (ATP, <3> pH 7.5, adenosylcobyrinic acid tetraamide as substrate [4]) [4] 20.4 <3> (ammonia, <3> pH 7.5, adenosylcobyrinic acid a,c-diamide as substrate [4]) [4] Additional information <3> [4, 8] Ki-Value (mM) 0.0059 <1> (cobyric acid) [9] pH-Optimum 7.5 <2, 3> (<3> broad pH-optimum around [4]) [4, 6, 8]

654

6.3.5.10

Adenosylcobyric acid synthase (glutamine-hydrolysing)

4 Enzyme Structure Molecular weight 52000 <3> (<3> predicted from DNA sequence of the cobQ gene [5]) [5] 97300 <3> (<3> gel filtration [4]) [4] Subunits dimer <2, 3> (<3> 2 * 57000, SDS-PAGE [5]; <2,3> 2 * 57000 [6,8]) [5, 6, 8] homodimer <3, 6> (<3> 2 * 57000, SDS-PAGE [4]; <3> 2 * 57000 [2]; <6> 2 * 51982, CobQ sequence calculation, 2 * 57000 [1]) [1, 2, 4]

5 Isolation/Preparation/Mutation/Application Purification <3> [1] <3> (45fold) [4] Cloning <3> (cobQ gene) [5] <3> (cobQ gene, sequencing, genomic organization) [1] <4> (cbiP gene) [7] <5> (cbiP gene) [7] Engineering Additional information <4, 5> (<4> cbiP mutants, nutritional studies [7]; <5> mutant strain JE6738 with in-frame deletion of cbiP, deficient in cobinamide biosynthesis, auxotrophic for adenosylcorbyric acid [7]) [7]

6 Stability Oxidation stability <3>, sensitive to oxidation [4] General stability information <3>, 20% w/v and 1 mM dithiothreitol are necessary to stabilize enzyme during purification [4] Storage stability <3>, -20 C or 0 C, purified enzyme, 0.1 M Tris hydrochloride, pH 7.5, 48 h, 100% loss of activity [4] <3>, -20 C, purified enzyme, 50 mM Tris hydrochloride, pH 7.5, 0.1 M sodium chloride, 1 mM dithiothreitol, 20% glycerol, several weeks, stable [4]

655

Adenosylcobyric acid synthase (glutamine-hydrolysing)

6.3.5.10

References [1] Crouzet, J.; Levy-Schil, S.; Cameron, B.; Cauchois, L.; Rigault, S.; Rouyez, M.C.; Blanche, F.; Debussche, L.; Thibaut, D.: Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing five cob genes and identification of structural genes encoding Cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase. J. Bacteriol., 173, 6074-6087 (1991) [2] Warren, M.J.; Raux, E.; Schubert, H.L.; Escalante-Semerena, J.C.: The biosynthesis of adenosylcobalamin (vitamin B12 ). Nat. Prod. Rep., 19, 390-412 (2002) [3] Blanche F.; Maton, L.; Debussche, L.; Thibaut, D.: Purification and characterization of cob(II)yrinic acid a,c-diamide reductase from Pseudomonas denitrificans. J. Bacteriol., 174, 7452-7454 (1992) [4] Blanche, F.; Couder, M.; Debussche, L.; Thibaut, D.; Cameron, B.; Cruzet, J.: Biosynthesis of vitamin B12 : stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans. J. Bacteriol., 173, 6046-6051 (1991) [5] Blanche, F.; Couder, M.; Debussche, L.; Thibaut, D.; Cameron, B.; Cruzet, J.: Biosynthesis of vitamin B12 : stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans. J.Bacteriol., 173, 6046-6051 (1991) [6] Galperin, M.Y.; Grishin, N.V.: The synthetase domains of cobalamin biosynthesis amidotransferases cobB and cobQ belong to a new family of ATP-dependent amidoligases, related to dethiobiotin synthetase. Proteins, 41, 238247 (2000) [7] Woodson, J.D.; Zayas, C.L.; Escalante-Semerena, J.C.: A new pathway for salvaging the coenzyme B12 precursor cobinamide in archaea requires cobinamide-phosphate synthase (CbiB) enzyme activity. J. Bacteriol., 185, 71937201 (2003) [8] Galperin, M.Y.; Grishin, N.V.: The synthetase domains of cobalamin biosynthesis amidotransferases CobB and CobQ belong to a new family of ATPdependent amidoligases, related to dethiobiotin synthetase. Proteins Struct. Funct. Genet., 41, 238-247 (2000) [9] Williams, L.; Fresquet, V.; Santander, P.J.; Raushel, F.M.: The multiple amidation reactions catalyzed by cobyric acid synthetase from Salmonella typhimurium are sequential and dissociative. J. Am. Chem. Soc., 129, 294-295 (2007)

656

Acetone carboxylase

6.4.1.6

1 Nomenclature EC number 6.4.1.6 Systematic name acetone:carbon-dioxide ligase (AMP-forming) Recommended name acetone carboxylase Synonyms carboxylase, acetone CAS registry number 189258-15-9

2 Source Organism <1> <2> <3> <4> <5> <6>

Rhodococcus rhodochrous (no sequence specified) [2] Rhodobacter capsulatus (no sequence specified) [1, 2, 5, 7] Rhodomicrobium vannielii (no sequence specified) [1] Xanthobacter autotrophicus (no sequence specified) [4, 6] Thiosphaera pantotropha (no sequence specified) [1] Xanthobacter sp. Py2 (no sequence specified) [2,3]

3 Reaction and Specificity Catalyzed reaction acetone + CO2 + ATP + 2 H2 O = acetoacetate + AMP + 2 phosphate (<2> biotin-independent, activity is strongly induced by growth on acetone [1]; <1> activity is induced in suspensions of acetone- and isopropanol-grown cells [2]) Reaction type carboxylation Natural substrates and products S acetone + CO2 + ATP + H2 O <2> (<2> initial step in acetone metabolism [1]) (Reversibility: ?) [1] P acetoacetate + AMP + phosphate <2> [1]

657

Acetone carboxylase

6.4.1.6

S Additional information <4> (<4> not present at detectable levels in cells grown with carbon sources other than acetone [6]) (Reversibility: ?) [6] P ? Substrates and products S acetone + CO2 + ATP + H2 O <1, 2, 3, 4, 5, 6> (<1> low activity with ATP [2]; <2> phosphate buffer, CoA and magnesium ions needed [1]; <2> initial step in acetone metabolism [1]; <2> it is proposed that a-abstraction from acetone occurs in concert with transfer of the g-phosphoryl group of ATP to the carbonyl oxygen, generating phosphoenol acetone as the activated nucleophile for attack on CO2 [5]; <4> no detectable activity observed with CTP, ITP, GTP, XTP or pyrophosphate [6]) (Reversibility: ?) [1, 2, 3, 5, 6, 7] P acetoacetate + AMP + phosphate <1, 2, 3, 5, 6> [1, 2, 3] S acetone + CO2 + GTP + H2 O <1> (<1> better activity than with ATP [2]) (Reversibility: ?) [2] P acetoacetate + GMP + phosphate <1> [2] S butanone + CO2 + ATP + H2 O <1, 2, 6> (<2> 47% of the activity with acetone [5]) (Reversibility: ?) [1, 2, 3, 5] P 3-oxopentanoate + AMP + phosphate <1, 2, 6> [1, 2, 3] S Additional information <1, 4, 6> (<1,6> enzyme expressed by acetone and isopropanol grown cells, not by propylene or glucose grown cells [2,3]; <6> 2-pentanone, 3-pentanone, 2-hexanone and chloroacetone are substrates [3]; <1> 2-pentanone, 3-pentanone and 2-hexanone as well as short-chain aliphatic alkanes and alkenes, including propane, are substrates. Several other nucleoside triphosphates are used [2]; <4> not present at detectable levels in cells grown with carbon sources other than acetone [6]) (Reversibility: ?) [2, 3, 6] P ? Inhibitors ATP <2> (<2> in high concentrations [1]) [1] Additional information <1, 6> (<1> no inhibition by avidin [2]) [2, 3] Cofactors/prosthetic groups ATP <1, 2, 6> (<1> low activity [2]; <6> obligate requirement [3]) [1, 2, 3] GTP <1> [2] ITP <1> [2] Additional information <1> (<1> nucleoside triphosphate required, ATP does not support acetone carboxylation [2]) [2] Activating compounds acetyl-CoA <2, 5> (<2> dependent on, gives more reproducible and larger degree of stimulation than CoA [1]) [1] CoA <2> (<2> stimulates acetone-dependent CO2 fixation [1]) [1] NAD(P)H <2> [1]

658

6.4.1.6

Acetone carboxylase

Metals, ions Fe <4> (<4> contains 0.7 mol Fe per mol of enzyme [6]) [6] K+ <2, 4, 6> (<6> required as stimulatory but not essential cofactor [2]; <2> monovalent cation required: K+ , Rb+ or NH+4 [5]; <4> monovalent ion, K+ or NH+4 , at concentration of 20-80 mM, required for optimal acetone carboxylase activity [6]) [2, 5, 6] Mg2+ <1, 2, 6> (<2> required [5]; <2> required for assay, stimulating [1]; <6> required as stimulatory but not essential cofactor [2]; <1> stimulatory effect on enzyme activity [2]) [1, 2, 3, 5] Mn2+ <4> (<4> contains 1.3 mol Mn per mol of enzyme [6]) [6] Mn2+ <2> (<2> enzyme contains 1.9 Mn2+ per a2 b2 g2 multimer, tightly bound and not removed upon dialysis against various metal ion chelators. Presence of a mononuclear Mn2+ center with possible spin coupling of two mononuclear sites. Manganese is essential for acetone carboxylation [7]; <2> tightly bound to the enzyme and not removed upon dialysis against various metal chelators. Presence of a mononuclear Mn2+ center, with possible spin coupling of two mononuclear sites. Mn2+ is essential for acetone carboxylation [7]) [7] NH+4 <1, 2, 4> (<1> stimulatory effect on enzyme activity [2]; <2> monovalent cation required: K+ , Rb+ or NH+4 [5]; <4> monovalent ion, K+ or NH+4 , at concentration of 20-80 mM, required for optimal acetone carboxylase activity [6]) [2, 5, 6] Rb+ <2> (<2> monovalent cation required: K+ , Rb+ or NH+4 [5]) [5] Zn <4> (<4> contains 1.0 mol Zn per mol of enzyme [6]) [6] Additional information <1, 6> (<1> dependent on the presence of a divalent metal [2]; <6> addition of Fe2+ , Mn2+ , Zn2+ , Ca2+ , Co2+ , Cu2+ or Ni2+ do not stimulate the enzyme activity above the maximal levels obtained in the presence of Mg2+ alone [3]) [2, 3] Turnover number (min–1) 1.33 <4> (acetone) [6] Specific activity (U/mg) 0.0015 <1> (<1> cell extracts [2]) [2] 0.014 <1> (<1> with ITP as nucleoside triphosphate [2]) [2] 0.016 <1> (<1> with GTP as nucleoside triphosphate [2]) [2] 0.07 <1> (<1> cell suspension [2]) [2] 0.094 <6> (<6> usage of butanone [3]) [3] 0.225 <6> (<6> usage of acetone at 30 C and pH 7.6 [3]) [2, 3] 0.24 <4> [6] Additional information <1, 2> (<2> a few hundred pmol per minute and mg protein [1]; <1> GTP shows best specific activity of tested nucleoside triphosphates: ATP, ITP, XTP, CTP and UTP [2]) [1, 2] Km-Value (mM) 0.0000042 <4> (CO2 ) [6] 0.0078 <4, 6> (acetone) [3, 6] 0.03 <2> (acetone) [1]

659

Acetone carboxylase

6.4.1.6

0.122 <4, 6> (ATP) [3, 6] 4.17 <6> (CO2, <6> cosubstrate bicarbonate [3]) [3] pH-Optimum 7.2 <2> [1] 7.6 <6> [3] 8 <1> (<1> assay at [2]) [2] Temperature optimum ( C) 30 <1, 2, 6> (<2> assay at [1,2]) [1, 2, 3]

4 Enzyme Structure Molecular weight 353000 <4, 6> (<4> gel filtration [6]; <6> native molecular weight of enzyme complex [3]) [3, 6] Subunits ? <2> (<2> x * 70000 + x * 85000, also 60000 Da and small amounts of 35000 Da subunits exist, SDS-PAGE [1]) [1] hexamer <4> (<4> a2 b2 g2 , 2 * 79000 + 2 * 68000 + 2 * 23000, SDS-PAGE [6]; <4> a2 b2 g2 , 2 * 85300 + 2 * 78300 + 2 * 19600, MALDI- TOF [6]) [6] trimer <1, 6> (<1> 1 * 85000, 1 * 74000, 1 * 16000, SDS-PAGE [2]; <6> 1 * 68400, 1 * 78800, 1 * 23000 by SDS-PAGE and 1 * 78300, 1 * 85300, 1 * 19600 by mass spectrometry. Subunits have an a2 b2 g2 quaternary structure [2,3]) [2, 3]

5 Isolation/Preparation/Mutation/Application Source/tissue culture condition:acetylacetone-grown cell <4> [6] Additional information <4> (<4> not present at detectable levels in cells grown with carbon sources other than acetone [6]) [6] Purification <1> (DEAE-Sepharose chromatography, but hydrophobic interaction, gel filtration and anion-exchange chromatography by Q-Sepharose resulted in large loss of activity) [2] <2> [5, 7] <2> (ion-exchange chromatography) [1] <4> [6] <6> (DEAE-Sepharose chromatography, gel filtration with Sephacryl S-300 column, HiLoad Q-Sepharose column) [3] Crystallization <4> (hanging drop vapor diffusion method. The enzyme crystallizes in a primitive orthorhombic point group P222, with unit-cell parameters a =

660

6.4.1.6

Acetone carboxylase

76.2 , b = 122.0 , c = 264.2 . One abg half of the large protein complex is located in the asymmetric unit in this crystal form, 3.2 resolution) [4]

6 Stability Oxidation stability <4>, sensitive to oxygen [6] General stability information <4>, 4 C, enzyme in cell extract, stable for several days [6] Storage stability <2>, -20 C [1] <6>, 4 C, pH 6.5-8.0, activity stable for several days [3]

References [1] Birks, S.J.; Kelly, D.J.: Assay and properties of acetone carboxylase, a novel enzyme involved in acetone-dependent growth and CO2 fixation in Rhodobacter capsulatus and other photosynthetic and denitrifying bacteria. Microbiology, 143, 755-766 (1997) [2] Clark, D.D.; Ensign, S.A.: Evidence for an inducible nucleotide-dependent acetone carboxylase in Rhodococcus rhodochrous B276. J. Bacteriol., 181, 2752-2758 (1999) [3] Sluis, M.K.; Ensign, S.A.: Purification and characterization of acetone carboxylase from Xanthobacter strain Py2. Proc. Natl. Acad. Sci. USA, 94, 8456-8461 (1997) [4] Nocek, B.; Boyd, J.; Ensign, S.A.; Peters, J.W.: Crystallization and preliminary X-ray analysis of an acetone carboxylase from Xanthobacter autotrophicus strain Py2. Acta Crystallogr. Sect. D, 60, 385-387 (2004) [5] Boyd, J.M.; Ensign, S.A.: ATP-dependent enolization of acetone by acetone carboxylase from Rhodobacter capsulatus. Biochemistry, 44, 8543-8553 (2005) [6] Sluis, M.K.; Larsen, R.A.; Krum, J.G.; Anderson, R.; Metcalf, W.W.; Ensign, S.A.: Biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10. J. Bacteriol., 184, 2969-2977 (2002) [7] Boyd, J.M.; Ellsworth, H.; Ensign, S.A.: Bacterial acetone carboxylase is a manganese-dependent metalloenzyme. J. Biol. Chem., 279, 46644-46651 (2004)

661

2-Oxoglutarate carboxylase

6.4.1.7

1 Nomenclature EC number 6.4.1.7 Recommended name 2-oxoglutarate carboxylase Synonyms CFI <2> [2] OGC <2> [2] carboxylating factor for ICDH <2> (<2> incorrect [2]) [2] oxalosuccinate synthetase <2> [2]

2 Source Organism <1> Rattus norvegicus (no sequence specified) [1] <2> Hydrogenobacter thermophilus (no sequence specified) [2, 3]

3 Reaction and Specificity Catalyzed reaction ATP + 2-oxoglutarate + HCO3- = ADP + phosphate + oxalosuccinate Natural substrates and products S ATP + 2-oxoglutarate + HCO3- <2> (<2> first step of the reductive carboxylation from 2-oxoglutarate to isocitrate. Oxalosuccinate, the product of the reaction is unstable and is quickly converted into isocitrate by the action of EC 1.1.1.41 [3]) (Reversibility: ?) [2, 3] P ADP + phosphate + oxalosuccinate Substrates and products S ATP + 2-oxoglutarate + HCO-3 <1, 2> (<2> first step of the reductive carboxylation from 2-oxoglutarate to isocitrate. Oxalosuccinate, the product of the reaction is unstable and is quickly converted into isocitrate by the action of EC 1.1.1.41 [3]) (Reversibility: ?) [1, 2, 3] P ADP + phosphate + oxalosuccinate Inhibitors avidin <2> [2]

662

6.4.1.7

2-Oxoglutarate carboxylase

Cofactors/prosthetic groups biotin <2> (<2> enzyme contains biotin [2,3]) [2,3] Metals, ions Mg2+ <2> (<2> required [2]) [2] Turnover number (min–1) 30.6 <2> (2-oxoglutarate, <2> pH 8.5, 20 C [3]) [3] 31.7 <2> (ATP, <2> pH 8.5, 20 C [3]) [3] Km-Value (mM) 0.788 <2> (ATP, <2> pH 8.5, 20 C [3]) [3] 1.03 <2> (2-oxoglutarate, <2> pH 8.5, 20 C [3]) [3] pH-Optimum 8 <2> [3] Temperature optimum ( C) 70-80 <2> [3]

4 Enzyme Structure Molecular weight 998000 <2> (<2> gel filtration [2]) [2] Subunits ? <2> (<2> x * 72000 + x * 49000, likely subunit structure a8 b8 , SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue liver <1> [1] Localization cytosol <1> [1] Purification <2> [2] <2> (recombinant) [3] Cloning <2> (a fragment containing a and b subunit genes is engineered for heterologous expression in Escherichia coli) [3] <2> (expression in Escherichia coli) [2]

663

2-Oxoglutarate carboxylase

6.4.1.7

6 Stability Temperature stability 90 <2> (<2> inactivation above [3]) [3] Storage stability <2>, 4 C, stable for at least 1 month [3]

References [1] Keech, D.B.; Mattoo, A.K.; Carabott, M.J.J.; Wallace, J.C.: The ATP-dependent reductive carboxylation of 2-oxoglutarate using cytosol from rat liver. Biochem. Biophys. Res. Commun., 71, 712-718 (1976) [2] Aoshima, M.; Ishii, M.; Igarashi, Y.: A novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK-6. Mol. Microbiol., 51, 791-798 (2004) [3] Aoshima, M.; Igarashi, Y.: A novel oxalosuccinate-forming enzyme involved in the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6. Mol. Microbiol., 62, 748-759 (2006)

664

Magnesium chelatase

6.6.1.1

1 Nomenclature EC number 6.6.1.1 Systematic name Mg-protoporphyrin IX magnesium-lyase Recommended name magnesium chelatase Synonyms CHLI <7> [33] CHLI protein <4> [34] Mg chelatase <4> [34] Mg-chelatase Mg-protoporphyrin IX magnesio-lyase Oil Yellow1 <13> (<13> Oil yellow1 (Oy1) gene encodes the I subunit of magnesium chelatase [35]) [35] Oy1 <13> (<13> Oil yellow1 (Oy1) gene encodes the I subunit of magnesium chelatase [35]) [35] ZmChlI <13> [35] magnesium-chelatase magnesium-protoporphyrin IX chelatase magnesium-protoporphyrin chelatase protoporphyrin IX Mg-chelatase protoporphyrin IX magnesium-chelatase CAS registry number 9074-88-8

2 Source Organism <1> Chlamydomonas reinhardtii (no sequence specified) [28] <2> Hordeum vulgare (no sequence specified) (<2> gene VPE3 [9]) [2, 9, 11, 24, 29] <3> Pisum sativum (no sequence specified) (<3> hoxH, b-subunit [1]) [1, 2, 20] <4> Zea mays (no sequence specified) [34] <5> Nicotiana tabacum (no sequence specified) (<5> ST6GALNAC4, SIAT3C, SIAT7D [7,8,19,21,22]) [7,8,19,21,22,36]

665

Magnesium chelatase

6.6.1.1

<6> Glycine max (no sequence specified) (<6> alkB4 gene from Rhodococcus NRRL B-16531 [4,17]) [4,17] <7> Arabidopsis thaliana (no sequence specified) [2,6,18,33] <8> Rhodobacter capsulatus (no sequence specified) [2,3,5,10,15,19,27,30] <9> Cucumis sativus (no sequence specified) [1,2,16] <10> Rhodobacter sphaeroides (no sequence specified) [2, 5, 13, 23, 31] <11> Synechocystis sp. (no sequence specified) [2, 6, 13, 14, 25, 26, 32] <12> Chlorobium vibrioforme (no sequence specified) [12] <13> Zea mays (UNIPROT accession number: Q4FE78) [35]

3 Reaction and Specificity Catalyzed reaction ATP + protoporphyrin IX + Mg2+ + H2 O = ADP + phosphate + Mg-protoporphyrin IX + 2 H+ (<5,7,8,10,12> subunits H, I and D from bchH, bchI and bchD genes combine to form the enzyme complex, the complex is active only when the three proteins are present [2,12,15,18,22]; <3,5,6,7,8,9,10,11> complex three-subunit enzyme [1,2,3,4,8,10,13,19,21]; <2,3,5,6,7,8,9,10,11,12> this is the first committed step of chlorophyll biosynthesis and is a branchpoint of two major routes in the tetrapyrrole pathway [1, 2, 4, 5, 6, 9, 11, 12, 13, 14, 15, 16, 19, 20, 23]; <3> linear reaction for at least 60 min under standard incubation [1]) Reaction type Ligation Natural substrates and products S ATP + deuteroporphyrin IX + Mg2+ + H2 O <11> (<11> magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis [32]) (Reversibility: ?) [32] P ADP + phosphate + Mg-deuteroporphyrin IX + H+ S ATP + protoporphyrin IX + Mg2+ + H2 O <2, 3, 5, 6, 7, 8, 9, 11> (<11> magnesium chelatase H subunit markedly enhances magnesium protoporphyrin methyltransferase catalysis by accelerating the formation and breakdown of the catalytic intermediate, providing a kinetic link between the first two reactions of chlorophyll biosynthesis with the signalling molecule magnesium protoporphyrin as the common factor [25]; <2> the enzyme catalyzes the insertion of magnesium into protoporphyrin IX, the first unique step of the chlorophyll biosynthetic pathway [29]; <7> insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway [33]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 17, 18, 20, 21, 22, 25, 29, 33] P ADP + phosphate + Mg-protoporphyrin IX + H+ <2, 3, 5, 6, 7, 8, 9, 11> [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 17, 18, 20, 21, 22] S Additional information <1, 8, 10> (<8> BchH is the rate-limiting component of Mg chelatase in cell extracts, and its slective inactivation during adaption to aerobic growth may account for the rapid inactivation of Mg

666

6.6.1.1

Magnesium chelatase

chelatase in vivo when anaerobically growing cells are exposed to O2 in the light [10]; <1> the pattern of changes in RNA transcript levels of the magnesium chelatase genes, chlH, chlD and chlI of Chlamydomonas reinhardtii grown under synchronous culture conditions in light/dark cycles are similar. Light is involved in regulation [28]; <10> mutant with a nonfunctional magnesium chelatase subunit D assembles a Zn-BChl photosystem [31]) (Reversibility: ?) [10, 28, 31] P ? Substrates and products S ATP + deuteroporphyrin IX + Mg2+ + H2 O <8, 11> (<11> magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis [32]; <8> ATP utilization by magnesium chelatase is solely connected to the I-subunit [30]; <11> MgATP2- binding occurs after the rate-determining step, nucleotide binding acts to clamp the chelatase in a product complex [32]) (Reversibility: ?) [26, 30, 32] P ADP + phosphate + Mg-deuteroporphyrin IX + H+ S ATP + protoporphyrin IX + Mg2+ + H2 O <2, 3, 4, 5, 6, 7, 8, 9, 10, 11> (<3> omission of any of the substrates results in complete loss of activity. Optimum concentration of Mg2+ is lower for intact than broken and reconstituted chloroplasts [1]; <8,10> optimum activity at 11.5 mM Mg2+ [5]; <11> magnesium chelatase H subunit markedly enhances magnesium protoporphyrin methyltransferase catalysis by accelerating the formation and breakdown of the catalytic intermediate, providing a kinetic link between the first two reactions of chlorophyll biosynthesis with the signalling molecule magnesium protoporphyrin as the common factor [25]; <2> the enzyme catalyzes the insertion of magnesium into protoporphyrin IX, the first unique step of the chlorophyll biosynthetic pathway [29]; <7> insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway [33]; <4> Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. The Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions [34]) (Reversibility: ?) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 17, 18, 20, 21, 22, 25, 27, 29, 33, 34] P ADP + phosphate + Mg-protoporphyrin IX + H+ <2, 3, 5, 6, 7, 8, 9, 10, 11> [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 17, 18, 20, 21, 22] S Additional information <1, 8, 10> (<8> BchH is the rate-limiting component of Mg chelatase in cell extracts, and its slective inactivation during adaption to aerobic growth may account for the rapid inactivation of Mg chelatase in vivo when anaerobically growing cells are exposed to O2 in the light [10]; <1> the pattern of changes in RNA transcript levels of the magnesium chelatase genes, chlH, chlD and chlI of Chlamydomonas reinhardtii grown under synchronous culture conditions in light/dark cycles are similar. Light is involved in regulation [28]; <10> mutant with a nonfunctional magnesium chelatase subunit D assembles a Zn-BChl photosystem [31]) (Reversibility: ?) [10, 28, 31] P ?

667

Magnesium chelatase

6.6.1.1

Inhibitors Mg2+ <9> (<9> marked inhibition above 10 mM [16]) [16] N-ethylmaleimide <3, 11> (<3> potent, IC50 of 0.02 mM [1]; <11> binds to ChlI subunit and inhibits its ATPase activity. The ChlI- ChlD-ATP complex forms but cannot catalyse magnesium chelation. Prior incubation with MgATP2- affords protection. Full protection can also be obtained with 5 mM ATP or 5 mM ADP alone [14]) [1, 14] urea <8> (<8> 20% inhibition with 100 mM, 50% inhibition with 250 mM, 90% inhibition with 800 mM [15]) [15] thiomerosal <8, 10> (<8,10> 60% inhibition with 0.022 mM, in the absence of DTT [5]) [5] thioredoxin <7> (<7> ATPase activity of recombinant CHLI1 is fully inactivated by oxidation and easily recovered by thioredoxin-assisted reduction [33]) [33] Activating compounds 5-aminolevulinic acid <2> (<2> 0.01 mM, increases activity [11]) [11] ATP <3, 5, 8, 10> (<3> required for activation and chelation steps [2]) [2, 8, 22] Metals, ions Mg2+ <3, 5, 9, 11> (<3,5,9> required [1,8]; <11> the magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction. Magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP2- [26]) [1, 8, 26] Turnover number (min–1) 0.013 <11> (deuteroporphyrin IX) [26] Specific activity (U/mg) 0.0000024 <3> (<3> pellet of broken fractionated chloroplasts [1]) [1] 0.0000026 <3> (<3> supernatant of broken fractionated chloroplasts [1]) [1] 0.00002 <3> (<3> broken unfractionated chloroplasts [1]) [1] 0.0001 <3> (<3> intact chloroplasts [1]) [1] Additional information <3> (<3> 3fold to 4fold higher values than in cucumber chloroplasts [1]) [1] Km-Value (mM) 0.00089 <8, 10> (protoporphyrin IX, <8,10> pH 8.2, 20-22 C, 192000 * g supernatant [5]) [5] 0.00123 <8> (protoporphyrin IX, <8> pH 8.0, 30 C [15]) [15] 0.00283 <8, 10> (protoporphyrin IX, <8,10> pH 8.2, 20-22 C, 20000 * g supernatant [5]) [5] 0.0032 <11> (deuteroporphyrin IX) [26] 0.0057 <11> (deuteroporphyrin, <11> pH 7.7, 37 C, 100 nM ChlH subunit [13]) [13] 0.008 <11> (deuteroporphyrin, <11> pH 7.7, 37 C, 200 nM ChlH subunit [13]) [13]

668

6.6.1.1

Magnesium chelatase

0.026 <2> (ATP, <2> pH 8.5, 32 C [9]) [9] 0.45 <11> (ATP) [26] 0.46 <7> (ATP) [33] pH-Optimum 8 <8> [15] Temperature optimum ( C) 20-22 <8, 10> (<8,10> assay at [5]) [5] 28 <5> (<5> assay at [7]) [7] 30 <8> (<8> assay at [10]) [10] 32 <2> (<2> assay at [9]) [9]

4 Enzyme Structure Subunits ? <2, 5, 6, 7, 8, 10, 11, 12> (<7> x * 44000, SDS-PAGE, ChlI subunit [18]; <6> x * 153491, calculation from sequence of cDNA, ChlH subunit [17]; <12> x * 38000, SDS-PAGE, a construct expressing I subunit [12]; <12> x * 42000, SDS-PAGE, a construct expressing I subunit [12]; <5> x * 82900, calculation from sequence of cDNA, D subunit [22]; <8> x * 73000, calculation from sequence of cDNA, D subunit [2]; <2> x * 90000-130000, gel filtration, BchI subunit [9]; <6,7> x * 46000, calculation from sequence of cDNA, I subunit [2,4]; <10> x * 87000, calculation from sequence of cDNA, D subunit [2]; <11> x * 60000, calculation from sequence of cDNA, D subunit [2]; <6> x * 40000, SDS-PAGE, I subunit [4]; <2> x * 40000 + x * 70000 + x * 140000 [24]) [2, 4, 9, 12, 17, 18, 22, 24] dimer <10> (<10> 2 * 40000, BchI subunit [23]) [23] octamer <10> (<10> 8 * 70000, D subunit, the molecular mass of the polymeric protein is approximately 550000 Da [23]) [23] trimer <8> (<8> 1 * 40000 (I- subunit) + 1 * 70000 (D-subunit) + 1 * 140000 (H-subunit) [30]) [30] Additional information <2, 3, 5, 6, 8, 10, 11, 12> (<11> 1 * 148000, gel filtration, ChlH subunit [13]; <10> 1 * 110000, gel filtration, BchH subunit [13]; <10> 1 * 140000, BchH subunit [23]; <8,10,12> the enzyme has three subunits, BchI, BchH and BchD [2,3,12,23]; <5> M1 and M2 domains of ChlD subunit participate in homodimerization and interaction between ChlI and ChlD [8]; <11> ChlI subunit forms high-molecular-mass aggregates [6]; <8> I subunit is hexameric [3]; <11> ChlH forms high-molecular aggregates when preincubated with ATP and Mg2+ [13]; <3> H subunit is a monomer and I subunit is a dimer, determined by dynamic-light-scattering studies [2]; <2,3,5,6> the enzyme has three subunits, ChlI, ChlH and ChlD [2,4,22]; <2> the 40000 Da subunit functions as a chaperon that is esential for the survival of the 70000 Da subunit. The ATPase activity of the 40000 Da subunit is essential for this function. Binding between the two subunits is not sufficient to maintain the 70000 Da subunit in the cell [24]) [2, 3, 4, 6, 8, 12, 13, 22, 23, 24]

669

Magnesium chelatase

6.6.1.1

5 Isolation/Preparation/Mutation/Application Source/tissue cotyledon <6, 9> [1, 4] grain <2> [9] leaf <2, 5, 6> (<5> 4-week-old [7]; <2> 6-day-old [11]; <5> generation of transgenic tobacco lines with RNAi silenced expression of the glutamate 1semialdehyde aminotransferase (GSA) gene does not cause a decrease in the transcript levels after inactivation of HEMA and GSA-expression. Enzyme activity for Mg chelatase is lower in parallel to the loss of chlorophyll and heme content [36]) [7, 11, 17, 21, 24, 36] plant <5> (<5> 5-week-old [19]) [19] seed <3, 6, 9> [4, 16, 20] Localization chloroplast <2, 3, 5, 6, 9> [1, 4, 11, 16, 21] chloroplast stroma <6> [17] membrane <6, 9> [2, 17] Additional information <3> (<3> multicomponent enzyme, requires at least two proteins, one membrane-associated and one soluble [2]) [2] Purification <7> (recombinant enzyme) [33] <8> (cation exchange chromatography) [15] Crystallization <8> (multiple wavelength anomalous dispersion method) [3] Cloning <2> (cloning and sequencing of xantha-f mutants (140000 Da subunit)) [29] <3> (expression in Escherichia coli) [20] <4> (expression of mutant maize ChlI alleles in Nicotiana benthamiana results in the formation of chlorotic lesions within 4 d of inoculation. Transient expression provides a convenient, high-throughput, qualitative assay for functional variation in the CHLI protein) [34] <5> [22] <5> (expression of central CHLD region in yeast) [8] <7> (expression in Escherichia coli) [33] <8> (BchD, BchI and BchH proteins are expressed in Escherichia coli from the respective cloned Rhodobacter capsulatus genes) [10] <8> (Rhodobacter capsulatus H-subunit produced in Escherichia coli) [30] <8> (expression in Escherichia coli) [15] <8> (expression of bchD gene in Escherichia coli) [2, 10] <10> (expression in Escherichia coli) [13, 23] <10> (expression of bchD gene in Escherichia coli) [2] <11> (expression in Escherichia coli) [13, 14] <12> (expression in Escherichia coli) [12]

670

6.6.1.1

Magnesium chelatase

Engineering D207N <2> (<2> altered restriction enzyme site, no measurable magnesium chelatase activity, ATPase activity not affected [9]) [9] L111F <2> (<2> altered restriction enzyme site, no measurable magnesium chelatase activity, ATPase activity not affected [9]) [9] R298K <2> (<2> altered restriction enzyme site, no measurable magnesium chelatase activity, ATPase activity not affected [9]) [9]

6 Stability pH-Stability Additional information <8> (<8> inactive below pH 6.0 and above pH 10.5 [15]) [15] General stability information <3>, 26% loss of activity in chloroplasts subjected to hypotonic lysis and freeze-thaw cycles [1] <9>, high concentrations of protoporphyrin, ATP and Mg2+ during gentle lysis, stabilise [2]

References [1] Walker, C.J.; Weinstein, J.D.: In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions. Proc. Natl. Acad. Sci. USA, 88, 5789-5793 (1991) [2] Walker, C.J.; Willows, R.D.: Mechanism and regulation of Mg-chelatase. Biochem. J., 327, 321-333 (1997) [3] Fodje, M.N.; Hansson, A.; Hansson, M.; Olsen, J.G.; Gough, S.; Willows, R.D.; Al-Karadaghi, S.: Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase. J. Mol. Biol., 311, 111-122 (2001) [4] Nakayama, M.; Masuda, T.; Sato, N.; Yamagata, H.; Bowler, C.; Ohta, H.; Shioi, Y.; Takamiya, K.: Cloning, subcellular localization and expression of CHL1, a subunit of magnesium-chelatase in soybean. Biochem. Biophys. Res. Commun., 215, 422-428 (1995) [5] Gorchein, A.: Cell-free activity of magnesium chelatase in Rhodobacter spheroides and Rhodobacter capsulatus. Biochem. Soc. Trans., 25, 82S (1997) [6] Reid, J.D.; Hunter, C.N.: Current understanding of the function of magnesium chelatase. Biochem. Soc. Trans., 30, 643-645 (2002) [7] Papenbrock, J.; Mock, H.-P.; Kruse, E.; Grimm, B.: Expression studies in tetrapyrrole biosynthesis: inverse maxima of magnesium chelatase and ferrochelatase activity during cyclic photoperiods. Planta, 208, 264-273 (1999)

671

Magnesium chelatase

6.6.1.1

[8] Grafe, S.; Saluz, H.-P.; Grimm, B.; Hanel, F.: Mg-chelatase of tobacco: the role of the subunit CHL D in the chelation step of protoporphyrin IX. Proc. Natl. Acad. Sci. USA, 96, 1941-1946 (1999) [9] Hansson, A.; Willows, R.D.; Roberts, T.H.; Hansson, M.: Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA+ hexamers. Proc. Natl. Acad. Sci. USA, 99, 13944-13949 (2002) [10] Willows, R.D.; Lake, V.; Roberts, T.H.; Beale, S.I.: Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus. J. Bacteriol., 185, 3249-3258 (2003) [11] Yaronskaya, E.B.; Rassadina, V.V.; Averina, N.G.: Regulation of magnesium chelatase activity during excessive accumulation of porphyrins in green barley leaves. Russ. J. Plant Physiol., 49, 771-775 (2002) [12] Petersen, B.L.; Jensen, P.E.; Gibson, L.C.D.; Stummann, B.M.; Hunter, C.N.; Henningsen, K.W.: Reconstitution of an active magnesium chelatase enzyme complex from the bchI, -D, and -H gene products of the green sulfur bacterium Chlorobium vibrioforme expressed in Escherichia coli. J. Bacteriol., 180, 699-704 (1998) [13] Karger, G.A.; Reid, J.D.; Hunter, C.N.: Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex. Biochemistry, 40, 9291-9299 (2001) [14] Jensen, P.E.; Reid, J.D.; Hunter, C.N.: Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation. Biochem. J., 352, 435-441 (2000) [15] Willows, R.D.; Beale, S.I.: Heterologous expression of the Rhodobacter capsulatus BchI, -D, and -H genes that encode magnesium chelatase subunits and characterization of the reconstituted enzyme. J. Biol. Chem., 273, 34206-34213 (1998) [16] Fuesler, T.P.; Wright, L.A., Jr.; Castelfranco, P.A.: Properties of magnesium chelatase in greening etioplasts. Metal ion specificity and effect of substrate concentrations. Plant Physiol., 67, 246-249 (1981) [17] Nakayama, M.; Masuda, T.; Bando, T.; Yamagata, H.; Ohta, H.; Takamiya, K.: Cloning and expression of the soybean chlH gene encoding a subunit of Mg-chelatase and localization of the Mg2+ concentration-dependent ChlH protein within the chloroplast. Plant Cell Physiol., 39, 275-284 (1998) [18] Rissler, H.M.; Collakova, E.; DellaPenna, D.; Whelan, J.; Pogson, B.J.: Chlorophyll biosynthesis. Expression of a second Chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis. Plant Physiol., 128, 770-779 (2002) [19] Papenbrock, J.; Mock, H.-P.; Tanaka, R.; Kruse, E.; Grimm, B.: Role of magnesium chelatase activity in the early steps of the tetrapyrrole biosynthetic pathway. Plant Physiol., 122, 1161-1169 (2000) [20] Luo, M.; Weinstein, J.D.; Walker, C.J.: Magnesium chelatase subunit D from pea: characterization of the cDNA, heterologous expression of an enzymatically active protein and immunoassay of the native protein. Plant Mol. Biol., 41, 721-731 (1999)

672

6.6.1.1

Magnesium chelatase

[21] Papenbrock, J.; Pfundel, E.; Mock, H.-P.; Grimm, B.: Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants. Plant J., 22, 155-164 (2000) [22] Papenbrock, J.; Grafe, S.; Kruse, E.; Hanel, F.; Grimm, B.: Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I. Plant J., 12, 981-990 (1997) [23] Willows, R.D.; Gibson, L.C.; Kanangara, C.G.; Hunter, C.N.; von Wettstein, D.: Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides. Eur. J. Biochem., 235, 438-443 (1996) [24] Lake, V.; Olsson, U.; Willows, R.D.; Hansson, M.: ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo. Eur. J. Biochem., 271, 2182-2188 (2004) [25] Shepherd, M.; McLean, S.; Hunter, C.N.: Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis. FEBS J., 272, 4532-4539 (2005) [26] Reid, J.D.; Hunter, C.N.: Magnesium-dependent ATPase activity and cooperativity of magnesium chelatase from Synechocystis sp. PCC6803. J. Biol. Chem., 279, 26893-26899 (2004) [27] Willows, R.D.; Hansson, A.; Birch, D.; Al-Karadaghi, S.; Hansson, M.: EM single particle analysis of the ATP-dependent BchI complex of magnesium chelatase: an AAA+ hexamer. J. Struct. Biol., 146, 227-233 (2004) [28] Lake, V.; Willows, R.D.: Rapid extraction of RNA and analysis of transcript levels in Chlamydomonas reinhardtii using real-time RT-PCR: Magnesium chelatase chlH, chlD and chlI gene expression. Photosynth. Res., 77, 69-76 (2003) [29] Olsson, U.; Sirijovski, N.; Hansson, M.: Characterization of eight barley xantha-f mutants deficient in magnesium chelatase. Plant Physiol. Biochem., 42, 557-564 (2004) [30] Sirijovski, N.; Olsson, U.; Lundqvist, J.; Al-Karadaghi, S.; Willows, R.D.; Hansson, M.: ATPase activity associated with the magnesium chelatase Hsubunit of the chlorophyll biosynthetic pathway is an artefact. Biochem. J., 400, 477-484 (2006) [31] Jaschke, P.R.; Beatty, J.T.: The photosystem of Rhodobacter sphaeroides assembles with zinc bacteriochlorophyll in a bchD (magnesium chelatase) mutant. Biochemistry, 46, 12491-12500 (2007) [32] Viney, J.; Davison, P.A.; Hunter, C.N.; Reid, J.D.: Direct measurement of metal-ion chelation in the active site of the AAA(+) ATPase magnesium chelatase. Biochemistry, 46, 12788-12794 (2007) [33] Ikegami, A.; Yoshimura, N.; Motohashi, K.; Takahashi, S.; Romano, P.G.; Hisabori, T.; Takamiya, K.; Masuda, T.: The CHLI1 subunit of Arabidopsis thaliana magnesium chelatase is a target protein of the chloroplast thioredoxin. J. Biol. Chem., 282, 19282-19291 (2007) [34] Sawers, R.J.; Farmer, P.R.; Moffett, P.; Brutnell, T.P.: In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis. Plant Methods, 2, 15 (2006)

673

Magnesium chelatase

6.6.1.1

[35] Sawers, R.J.; Viney, J.; Farmer, P.R.; Bussey, R.R.; Olsefski, G.; Anufrikova, K.; Hunter, C.N.; Brutnell, T.P.: The maize Oil yellow1 (Oy1) gene encodes the I subunit of magnesium chelatase. Plant Mol. Biol., 60, 95-106 (2006) [36] Hedtke, B.; Alawady, A.; Chen, S.; Boernke, F.; Grimm, B.: HEMA RNAi silencing reveals a control mechanism of ALA biosynthesis on Mg chelatase and Fe chelatase. Plant Mol. Biol., 64, 733-742 (2007)

674

Cobaltochelatase

6.6.1.2

1 Nomenclature EC number 6.6.1.2 Systematic name hydrogenobyrinic-acid-a,c-diamide:cobalt cobalt-ligase (ADP-forming) Recommended name cobaltochelatase Synonyms CobN-CobST CobN-CobST <1> [2] CobNST <1> [2] cobalt chelatase <1> [2] cobaltochelatase <1> [2] gene cobN/gene cobS cobaltochelatase <1> [2] holocobalamin synthase <1> [2] hydrogenobyrinic acid a,c-diamide cobaltochelatase <1> [2] CAS registry number 81295-49-0

2 Source Organism <1> Pseudomonas denitrificans (no sequence specified) [1, 2]

3 Reaction and Specificity Catalyzed reaction ATP + hydrogenobyrinic acid a,c-diamide + Co2+ + H2 O = ADP + phosphate + cob(II)yrinic acid a,c-diamide + H+ Natural substrates and products S ATP + hydrogenobyrinic acid a,c-diamide + Co2+ <1> (Reversibility: ?) [1] P ADP + phosphate + cob(II)yrinic acid a,c-diamide + H+ S hydrogenobyrinic acid a,c-diamide + ATP + Co2+ <1> (Reversibility: ?) [2] P ADP + phosphate + cob(II)yrinic acid a,c-diamide + H+ <1> [2]

675

Cobaltochelatase

6.6.1.2

Substrates and products S ATP + hydrogenobyrinic acid a,c-diamide + Co2+ <1> (Reversibility: ?) [1, 2] P ADP + phosphate + cob(II)yrinic acid a,c-diamide + H+ <1> [2] S CTP + hydrogenobyrinic acid a,c-diamide + Co2+ <1> (<1> 45% of activity with respect to ATP [2]) (Reversibility: ?) [2] P CDP + phosphate + cob(II)yrinic acid a,c-diamide + H+ <1> [2] S ITP + hydrogenobyrinic acid a,c-diamide + Co2+ <1> (<1> 5% of activity with respect to ATP [2]) (Reversibility: ?) [2] P IDP + phosphate + cob(II)yrinic acid a,c-diamide + H+ <1> [2] S dATP + hydrogenobyrinic acid a,c-diamide + Co2+ <1> (<1> 65% of activity with respect to ATP [2]) (Reversibility: ?) [2] P dADP + phosphate + cob(II)yrinic acid a,c-diamide + H+ <1> [2] S hydrogenobyrinic acid a,c-diamide + ATP + Co2+ <1> (Reversibility: ?) [2] P ADP + phosphate + cob(II)yrinic acid a,c-diamide + H+ <1> [2] Inhibitors ADP <1> (<1> 50% inhibition at 8 mM [2]) [2] AMP <1> (<1> 15% inhibition at 10 mM [2]) [2] Cu2+ <1> (<1> 50% inhibition at 0.002 mM [2]) [2] diphosphate <1> (<1> 50% inhibition at 5 mM [2]) [2] Fe2+ <1> (<1> 50% inhibition at 0.05 mM [2]) [2] N-ethylmaleimide <1> (<1> 50% inhibition at 0.005 mM [2]) [2] Ni2+ <1> (<1> 50% inhibition at 0.003 mM [2]) [2] Zn2+ <1> (<1> 50% inhibition at 0.0005 mM [2]) [2] adenylyl (b,g-methylene)-diphosphonate <1> (<1> 50% inhibition at 5 mM [2]) [2] adenylyl-imidodiphosphate <1> (<1> 50% inhibition at 2 mM [2]) [2] Metals, ions Mg2+ <1> (<1> ATP binding cation [2]) [2] Specific activity (U/mg) Additional information <1> (<1> 0.0057 units for CobST subunit, 1 unit of CobN is defined as the amount of CobST necessary to reconstruct 1 unit of the entire complex. 0.006 for CobN subunit, 1 unit of CobN is defined as the amount of CobN necessary to reconstruct 1 unit of the entire complex [2]) [2] Km-Value (mM) 0.000085 <1> (hydrogenobyrinic acid a,c-diamide, <1> pH 8.0, 30 C [2]) [2] 0.0042 <1> (Co2+ , <1> pH 8.0, 30 C [2]) [2] 0.22 <1> (ATP, <1> pH 8.0, 30 C [2]) [2]

676

6.6.1.2

Cobaltochelatase

4 Enzyme Structure Molecular weight 258000 <1> (<1> gel filtration [2]) [2] Subunits trimer <1> (<1> 1 * 140000, CobN + 1 * 38000, namely CobS + 1 * 80000, namely CobT, gel filtration, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Purification <1> (purification includes: Mono Q HR 10/10, Penyl-Superose and Mono Q HR 5/5 chromatographies for CobN subunit, Mono Q HR 10/10, PhenylSuperose and AGATP for CobST subunit) [2]

References [1] Debussche, L.; Thibaut, D.; Cameron, B.; Crouzet, J.; Blanche, F.: Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans. J. Bacteriol., 175, 7430-7440 (1993) [2] Debussche, L.; Couder, M.; Thibaut, D.; Cameron, B.; Crouzet, J.; Blanche, F.: Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzime B12 biosynthesis in Pseudomonas denitrificans. J.BACTERIOL., 174, 7445-7451 (1992)

677