Class 2 Transferases V (Springer Handbook of Enzymes)

Springer Handbook of Enzymes Volume 32 Dietmar Schomburg and Ida Schomburg (Eds.) Springer Handbook of Enzymes Volum...

Author: Dietmar Schomburg | Ida Schomburg | A. Chang

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Springer Handbook of Enzymes Volume 32

Dietmar Schomburg and Ida Schomburg (Eds.)

Springer Handbook of Enzymes Volume 32 Class 2 Transferases V EC 2.4.1.90±2.4.1.232 coedited by Antje Chang

Second Edition

13

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

University to Cologne Institute for Biochemistry Zülpicher Strasse 47 50674 Cologne Germany

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

Library of Congress Control Number: 2006926725 ISBN-10 3-540-32591-3

2nd Edition Springer Berlin Heidelberg New York

ISBN-13 978-3-540-32591-8

2nd Edition Springer Berlin Heidelberg New York

The first edition was published as Volume 12 (ISBN 3-540-60703-X) of the ªEnzyme Handbookº.

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 2006 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 AG, Berlin Printed on acid-free paper 2/3141m-5 4 3 2 1 0

Attention all Users of the ªSpringer Handbook of Enzymesº Information on this handbook can be found on the internet at http://www.springer.com choosing ªChemistryº and then ªReference Worksº. A complete list of all enzyme entries either as an alphabetical Name Index or as the EC-Number Index is available at the above mentioned URL. You can download and print them free of charge. A complete list of all synonyms (> 25,000 entries) used for the enzymes is available in print form (ISBN 3-540-41830-X).

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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) and is now continuing at the University at Cologne, Institute of Biochemistry. The present book ªSpringer Handbook of Enzymesº 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 4,000 ªdifferentº 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. Cologne Autumn 2006

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

List of Deleted and Transferred Enzymes

Since its foundation in 1956 the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) has continually revised and updated the list of enzymes. Entries for new enzymes have been added, others have been deleted completely, or transferred to another EC number in the original class or to different EC classes, catalyzing other types of chemical reactions. The old numbers have not been allotted to new enzymes; instead the place has been left vacant or cross-references given to the changes in nomenclature. Deleted and Transferred Enzymes For EC class 2.4.1.90±2.4.1.232 these changes are: Recommended name

Old EC number Alteration

inulin fructotransferase (depolymerizing, difructofuranose-1,2':2,3'-dianhydrideforming) UDPgalactose-N-acetylglucosamine b-d-galactosyltransferase UDPglucuronate-testosterone glucuronosyltransferase UDPglucuronate-phenol glucuronosyltransferase N-acetyllactosaminea-galactosyltransferase N-acetyllactosaminide a-1,3-galactosyltransferase xyloglucan 6-xylosyltransferase inulin fructotransferase (depolymerizing, difructo furanose-1,2':2',1-dianhydrideforming) zeatin O-b-D-xylosyltransferase

2.4.1.93

transferred to EC 4.2.2.18

2.4.1.98

deleted, included in EC 2.4.1.90

2.4.1.107

2.4.1.151

deleted, included in EC 2.4.1.17 deleted, included in EC 2.4.1.17 deleted, included in EC 2.4.1.87 transferred to EC 2.4.1.87

2.4.1.169 2.4.1.200

transferred to EC 2.4.2.39 transferred to EC 4.2.2.17

2.4.1.204

transferred to EC 2.4.2.40

2.4.1.108 2.4.1.124

XIII

Index of Recommended Enzyme Names

EC-No.

Recommended Name

2.4.1.147

acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase . . . . . . . . . . . . acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase . . . . . . . . . . . . N-acetylgalactosaminyl-proteoglycan 3-b-glucuronosyltransferase N-acetylglucosaminyldiphosphodolichol N-acetylglucosaminyltransferase . . . . . . . . . . . . . . . N-acetylglucosaminyldiphosphoundecaprenol glucosyltransferase. N-acetylglucosaminyldiphosphoundecaprenol N-acetyl-b-D-mannosaminyltransferase . . . . . . . . . . . . N-acetylglucosaminyl-proteoglycan 4-b-glucuronosyltransferase . N-acetyllactosamine synthase . . . . . . . . . . . . . . . . N-acetyllactosaminide a-1,3-galactosyltransferase (transferred to EC 2.4.1.87) . . . . . . . . . . . . . . . . . N-acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase . . N-acetyllactosaminide b-1,6-N-acetylglucosaminyl-transferase . . N-acetylneuraminylgalactosylglucosylceramide b-1,4-N-acetylgalactosaminyltransferase . . . . . . . . . . . . aldose b-D-fructosyltransferase. . . . . . . . . . . . . . . . alizarin 2-b-glucosyltransferase . . . . . . . . . . . . . . . alternansucrase . . . . . . . . . . . . . . . . . . . . . . anthocyanidin 3-O-glucosyltransferase . . . . . . . . . . . . bilirubin-glucuronoside glucuronosyltransferase . . . . . . . . chitobiosyldiphosphodolichol b-mannosyltransferase . . . . . . cinnamate b-D-glucosyltransferase . . . . . . . . . . . . . . cis-p-coumarate glucosyltransferase. . . . . . . . . . . . . . cis-zeatin O-b-D-glucosyltransferase . . . . . . . . . . . . . coniferyl-alcohol glucosyltransferase . . . . . . . . . . . . . 2-coumarate O-b-glucosyltransferase . . . . . . . . . . . . . cyanidin-3-rhamnosylglucoside 5-O-glucosyltransferase . . . . . cytokinin 7-b-glucosyltransferase . . . . . . . . . . . . . . 1,2-diacylglycerol 3-glucosyltransferase . . . . . . . . . . . . diglucosyl diacylglycerol synthase . . . . . . . . . . . . . . 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-D-glucosyltransferase . . . . . . . . . . . . . . . . . . . o-dihydroxycoumarin 7-O-glucosyltransferase . . . . . . . . . dolichyl-diphosphooligosaccharide-protein glycotransferase . . . dolichyl-phosphate a-N-acetylglucosaminyltransferase . . . . . dolichyl-phosphate b-glucosyltransferase . . . . . . . . . . . dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase. . dolichyl-phosphate-mannose-protein mannosyltransferase . . . . flavanone 7-O-b-glucosyltransferase . . . . . . . . . . . . . flavonol 3-O-glucosyltransferase . . . . . . . . . . . . . . . flavonol-3-O-glucoside L-rhamnosyltransferase . . . . . . . . . 2,1-fructan:2,1-fructan 1-fructosyltransferase . . . . . . . . .

2.4.1.148 2.4.1.226 2.4.1.141 2.4.1.188 2.4.1.187 2.4.1.225 2.4.1.90 2.4.1.151 2.4.1.149 2.4.1.150 2.4.1.165 2.4.1.162 2.4.1.103 2.4.1.140 2.4.1.115 2.4.1.95 2.4.1.142 2.4.1.177 2.4.1.209 2.4.1.215 2.4.1.111 2.4.1.114 2.4.1.116 2.4.1.118 2.4.1.157 2.4.1.208 2.4.1.202 2.4.1.104 2.4.1.119 2.4.1.153 2.4.1.117 2.4.1.130 2.4.1.109 2.4.1.185 2.4.1.91 2.4.1.159 2.4.1.100

Page . .

287

. . . .

293 613

. . . .

252 457

. . . . . .

454 610 1

. . . . . .

317 297 307

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

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

368 357 97 248 139 47 256 415 549 576 123 137 142 152 344 545

. . . . . . . . . . .

. . . . . . . . . . .

507 100 155 330 146 205 110 444 21 351 65

XV

Index of Recommended Enzyme Names

2.4.1.222 2.4.1.205 2.4.1.184 2.4.1.135 2.4.1.92 2.4.1.152 2.4.1.163 2.4.1.164 2.4.1.211 2.4.1.146 2.4.1.102 2.4.1.134 2.4.1.136 2.4.1.176 2.4.1.154 2.4.1.97 2.4.1.183 2.4.1.113 2.4.1.112 2.4.1.213 2.4.1.175 2.4.1.224 2.4.1.223 2.4.1.174 2.4.1.96 2.4.1.137 2.4.1.186 2.4.1.131 2.4.1.132 2.4.1.214 2.4.1.122 2.4.1.197 2.4.1.212 2.4.1.218 2.4.1.181 2.4.1.194 2.4.1.126 2.4.1.158 2.4.1.178 2.4.1.121 2.4.1.156 2.4.1.220 2.4.1.232 2.4.1.123

XVI

O-fucosylpeptide 3-b-N-acetylglucosaminyltransferase . . . . . . . galactogen 6b-galactosyltransferase . . . . . . . . . . . . . . . galactolipid galactosyltransferase . . . . . . . . . . . . . . . . galactosylgalactosylxylosylprotein 3-b-glucuronosyltransferase . . . (N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase. . . . . . . . . . . . . . . . 4-galactosyl-N-acetylglucosaminide 3-a-L-fucosyltransferase . . . . b-galactosyl-N-acetylglucosaminylgalactosylglucosyl-ceramide b-1,3-acetylglucosaminyltransferase . . . . . . . . . . . . . . . galactosyl-N-acetylglucosaminylgalactosylglucosyl-ceramide b-1,6-N-acetylglucosaminyltransferase . . . . . . . . . . . . . . 1,3-b-galactosyl-N-acetylhexosamine phosphorylase . . . . . . . . b-1,3-galactosyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase . . . . . . . . . . . . . . b-1,3-galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase . . . . . . . . . . . . . . galactosylxylosylprotein 3-b-galactosyltransferase . . . . . . . . . gallate 1-b-glucosyltransferase . . . . . . . . . . . . . . . . . gibberellin b-D-glucosyltransferase . . . . . . . . . . . . . . . globotriosylceramide b-1,6-N-acetylgalactosaminyl-transferase . . . 1,3-b-D-glucan phosphorylase . . . . . . . . . . . . . . . . . a-1,3-glucan synthase . . . . . . . . . . . . . . . . . . . . . a-1,4-glucan-protein synthase (ADP-forming) . . . . . . . . . . a-1,4-glucan-protein synthase (UDP-forming) . . . . . . . . . . glucosylglycerol-phosphate synthase. . . . . . . . . . . . . . . glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase . . . . . . . . . . . . . . glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-a-N-acetylglucosaminyltransferase . . . . . . . . . . . . . . glucuronyl-galactosyl-proteoglycan 4-a-N-acetylglucosaminyltransferase . . . . . . . . . . . . . . glucuronylgalactosylproteoglycan 4-b-N-acetylgalactosaminyltransferase sn-glycerol-3-phosphate 1-galactosyltransferase . . . . . . . . . . sn-glycerol-3-phosphate 2-a-galactosyltransferase . . . . . . . . . glycogenin glucosyltransferase . . . . . . . . . . . . . . . . . glycolipid 2-a-mannosyltransferase . . . . . . . . . . . . . . . glycolipid 3-a-mannosyltransferase . . . . . . . . . . . . . . . glycoprotein 3-a-L-fucosyltransferase . . . . . . . . . . . . . . glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase . . . . high-mannose-oligosaccharide b-1,4-N-acetylglucosaminyltransferase hyaluronan synthase . . . . . . . . . . . . . . . . . . . . . hydroquinone glucosyltransferase . . . . . . . . . . . . . . . . hydroxyanthraquinone glucosyltransferase . . . . . . . . . . . . 4-hydroxybenzoate 4-O-b-D-glucosyltransferase . . . . . . . . . . hydroxycinnamate 4-b-glucosyltransferase . . . . . . . . . . . . 13-hydroxydocosanoate 13-b-glucosyltransferase . . . . . . . . . hydroxymandelonitrile glucosyltransferase . . . . . . . . . . . . indole-3-acetate b-glucosyltransferase . . . . . . . . . . . . . . indolylacetyl-myo-inositol galactosyltransferase . . . . . . . . . . indoxyl-UDPG glucosyltransferase . . . . . . . . . . . . . . . initiation-specific a-1,6-mannosyltransferase . . . . . . . . . . . inositol 1-a-galactosyltransferase . . . . . . . . . . . . . . . .

599 515 440 231 30 318 362 365 555 282 84 227 236 413 332 52 437 134 129 563 405 604 602 400 49 239 448 210 214 565 174 488 558 584 430 475 192 348 420 170 342 593 640 182

Index of Recommended Enzyme Names

2.4.1.200 2.4.1.93 2.4.1.170 2.4.1.106 2.4.1.230 2.4.1.206 2.4.1.228 2.4.1.179 2.4.1.210 2.4.1.182 2.4.1.180 2.4.1.189 2.4.1.191 2.4.1.190 2.4.1.139 2.4.1.217 2.4.1.143 2.4.1.101 2.4.1.144 2.4.1.145 2.4.1.201 2.4.1.155 2.4.1.199 2.4.1.138 2.4.1.171 2.4.1.127 2.4.1.196 2.4.1.192 2.4.1.161 2.4.1.221 2.4.1.129 2.4.1.198 2.4.1.94 2.4.1.160 2.4.1.166 2.4.1.172 2.4.1.193 2.4.1.128 2.4.1.120 2.4.1.229 2.4.1.173 2.4.1.99 2.4.1.167 2.4.1.125 2.4.1.195 2.4.1.203 2.4.1.216 2.4.1.231 2.4.1.110

inulin fructotransferase (depolymerizing, difructofuranose-1,2':2',1dianhydride-forming) (transferred to EC 4.2.2.17) . . . . . . . . inulin fructotransferase (depolymerizing, difructofuranose-1,2':2,3'dianhydride-forming) (transferred to EC 4.2.2.18) . . . . . . . . isoflavone 7-O-glucosyltransferase . . . . . . . . . . . . . . isovitexin b-glucosyltransferase . . . . . . . . . . . . . . . kojibiose phosphorylase. . . . . . . . . . . . . . . . . . . lactosylceramide 1,3-N-acetyl-b-D-glucosaminyltransferase . . . lactosylceramide 4-a-galactosyltransferase. . . . . . . . . . . lactosylceramide b-1,3-galactosyltransferase . . . . . . . . . . limonoid glucosyltransferase. . . . . . . . . . . . . . . . . lipid-A-disaccharide synthase . . . . . . . . . . . . . . . . lipopolysaccharide N-acetylmannosaminouronosyltransferase . . luteolin 7-O-glucuronosyltransferase . . . . . . . . . . . . . luteolin-7-O-diglucuronide 4'-O-glucuronosyltransferase . . . . luteolin-7-O-glucuronide 7-O-glucuronosyltransferase. . . . . . maltose synthase . . . . . . . . . . . . . . . . . . . . . . mannosyl-3-phosphoglycerate synthase . . . . . . . . . . . . a-1,6-mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase a-1,3-mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase b-1,4-mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase a-1,3-mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase a-1,6-mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase a-1,6-mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase b-mannosylphosphodecaprenol-mannooligosaccharide 6-mannosyltransferase . . . . . . . . . . . . . . . . . . . mannotetraose 2-a-N-acetylglucosaminyltransferase . . . . . . methyl-ONN-azoxymethanol b-D-glucosyltransferase . . . . . . monoterpenol b-glucosyltransferase . . . . . . . . . . . . . nicotinate glucosyltransferase . . . . . . . . . . . . . . . . nuatigenin 3b-glucosyltransferase . . . . . . . . . . . . . . oligosaccharide 4-a-D-glucosyltransferase . . . . . . . . . . . peptide-O-fucosyltransferase. . . . . . . . . . . . . . . . . peptidoglycan glycosyltransferase . . . . . . . . . . . . . . phosphatidylinositol N-acetylglucosaminyltransferase . . . . . . protein N-acetylglucosaminyltransferase. . . . . . . . . . . . pyridoxine 5'-O-b-D-glucosyltransferase . . . . . . . . . . . . raffinose-raffinose a-galactotransferase . . . . . . . . . . . . salicyl-alcohol b-D-glucosyltransferase . . . . . . . . . . . . sarsapogenin 3b-glucosyltransferase . . . . . . . . . . . . . scopoletin glucosyltransferase . . . . . . . . . . . . . . . . sinapate 1-glucosyltransferase . . . . . . . . . . . . . . . . [Skp1-protein]-hydroxyproline N-acetylglucosaminyltransferase . sterol 3b-glucosyltransferase . . . . . . . . . . . . . . . . . sucrose:sucrose fructosyltransferase . . . . . . . . . . . . . sucrose 6F -a-galactotransferase . . . . . . . . . . . . . . . sucrose-1,6-a-glucan 3(6)-a-glucosyltransferase . . . . . . . . thiohydroximate b-D-glucosyltransferase . . . . . . . . . . . trans-zeatin O-b-D-glucosyltransferase . . . . . . . . . . . . trehalose 6-phosphate phosphorylase . . . . . . . . . . . . . a,a-trehalose phosphorylase (configuration-retaining) . . . . . tRNA-queuosine b-mannosyltransferase . . . . . . . . . . . .

. .

500

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

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

38 381 106 631 518 622 423 552 433 428 459 465 462 246 581 259 70 267 278 501 334

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

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

497 242 384 195 485 468 355 596 200 492 39 353 373 386 472 198 165 627 389 56 375 188 479 511 578 634 121

XVII

Index of Recommended Enzyme Names

2.4.1.98 2.4.1.108 2.4.1.107 2.4.1.227 2.4.1.105 2.4.1.219 2.4.1.168 2.4.1.169 2.4.1.207 2.4.1.133 2.4.1.204

XVIII

UDPgalactose-N-acetylglucosamine b-D-galactosyl-transferase (deleted, included in EC 2.4.1.90) . . . . . . . . . . . . UDPglucuronate-phenol glucuronosyltransferase (deleted, included in EC 2.4.1.17) . . . . . . . . . . . . UDPglucuronate-testosterone glucuronosyltransferase (deleted, included in EC 2.4.1.17) . . . . . . . . . . . . undecaprenyldiphospho-muramoylpentapeptide b-N-acetylglucosaminyltransferase . . . . . . . . . . . vitexin b-glucosyltransferase . . . . . . . . . . . . . . vomilenine glucosyltransferase . . . . . . . . . . . . . xyloglucan 4-glucosyltransferase . . . . . . . . . . . . xyloglucan 6-xylosyltransferase (transferred to EC 2.4.2.39) . xyloglucan:xyloglucosyl transferase . . . . . . . . . . . xylosylprotein 4-b-galactosyltransferase . . . . . . . . . zeatin O-b-D-xylosyltransferase (transferred to EC 2.4.2.40) .

. . . .

55

. . . .

109

. . . .

108

. . . . . . . .

616 104 589 377 380 524 221 514

. . . . . . . .

. . . . . . . .

. . . . . . . .

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.

XIX

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,

XX

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.

XXI

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.

XXII

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.

XXIII

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.

XXIV

N-Acetyllactosamine synthase

2.4.1.90

1 Nomenclature EC number 2.4.1.90 Systematic name UDP-galactose:N-acetyl-d-glucosamine 4-b-d-galactosyltransferase Recommended name N-acetyllactosamine synthase Synonyms EC 2.4.1.98 (formerly) Gal-T GalTase N-acetyllactosamine synthetase NAL synthetase UDP-Gal:N-acetylglucosamine b1-4-galactosyltransferase UDP-b-1,4-galactosyltransferase UDP-galactose N-acetylglucosamine b-4-galactosyltransferase UDP-galactose-N-acetylglucosamine b-1,4-galactosyltransferase UDP-galactose-N-acetylglucosamine galactosyltransferase UDP-galactose-acetylglucosamine galactosyltransferase UDP-galactose:N-acetylglucosaminide b1-4-galactosyltransferase UDPgalactose-N-acetylglucosamine b-d-galactosyltransferase UDPgalactose:N-acetylglucosaminyl(b1-4)galactosyltransferase acetyllactosamine synthetase b-1,4-galactosyltransferase b-N-acetylglucosaminide b1-4-galactosyltransferase b1,4-GT b1-4-galactosyltransferase b1-4GalT galactosyltransferase, uridine diphosphogalactose-acetylglucosamine lactosamine synthase lactosamine synthetase lactose synthetase A protein uridine diphosphogalactose-acetylglucosamine galactosyltransferase Additional information (cf. EC 2.4.1.22, not distinguishable from EC 2.4.1.38) CAS registry number 9054-94-8

1

N-Acetyllactosamine synthase

2.4.1.90

2 Source Organism <1> Ovis aries [1] <2> Mus musculus (expression of the enzyme in HeLa cells [48]) [2, 5-7, 9, 15, 34, 48, 51] <3> Rattus norvegicus [3, 4, 18, 19, 21, 22, 23, 24, 30, 36, 39, 42, 43, 44, 60] <4> Bos taurus (calf [26]) [8, 10, 13, 14, 20, 24, 25, 26, 27, 32, 33, 35, 37, 41, 46, 47, 50, 53, 55, 56, 58, 59, 61, 62, 65] <5> Homo sapiens (expressed in Saccharomyces cerevisiae [17]; wild type and recombinant enzyme expressed in Saccharomyces cerevisiae [57]) [11, 12, 16, 17, 28, 29, 38, 42, 45, 49, 52, 54, 56, 57, 63] <6> Sus scrofa [31, 40] <7> Neisseria meningitidis (ATCC 13102 [64]) [64] <8> Neisseria gonorrhoeae (ATCC 31151 [64]) [64]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + N-acetyl-d-glucosamine = UDP + N-acetyllactosamine (not distinguishable from EC 2.4.1.38 which has identical substrate specificities. EC 2.4.1.38/90 is identical with the A protein of EC 2.4.1.22; <2>, the sequences of cDNA isolated from mammary and F9 cell lines are identical, thus indicating that EC 2.4.1.38 and EC 2.4.1.90 are non-distinguishable [5]; <4>, mechanism [32]) Reaction type hexosyl group transfer Natural substrates and products S UDPgalactose + N-acetylglucosaminyl at the non-reducing ends of protein-bound oligosaccharides <2, 3, 4, 5> (<3>, the enzyme may be involved in the synthesis of plasma glycoproteins by the liver during secretion, and may possibly be required for secretion of these proteins [3]; <2>, biosynthesis of carbohydrate moieties of glycoproteins and glycolipids, role in intercellular recognition and adhesion [7]; <4>, biosynthesis of keratan sulfate-like polysaccharides [35]; <4>, enzyme participates in the biosynthesis of the oligosaccharide structures of glycoproteins and glycolipids [14]; <4>, enzyme functions in the coordinate biosynthesis of complex oligosaccharides, proposed to function in intercellular recognition and/or adhesion [25]; <5>, the enzyme may be involved in the synthesis of poly-N-acetyllactosamine, lacto-N-neotetraose and probably lacto-N-neotetraosylceramide in addition to the formation of the Galb14GlcNAc group of glycoprotein sugar chains and lactose [49]; <2>, the enzyme is involved in the biosynthesis of a variety of carbohydrate structures in glycoproteins and glycolipids [51]; <5>, main enzyme responsible for the transfer of galactose residues from UDPgalactose into terminal N-

2

2.4.1.90

N-Acetyllactosamine synthase

acetylglucosamine residues of complex-type oligosaccharides in newly synthesized glycoproteins in the Golgi apparatus. Deficiency of UDP-galactose:N-acetylglucosamine b-1,4-galactosyltransferase I causes the congenital disorder of glycosylation type IId, a severe neurologic disease characterized by a hydrocephalus, myopathy and blood-clotting defects [54]; <3>, the soluble enzyme form from the luminal fluid of the epididymis is suggested to play a role on sperm maturation [60]; <4>, the enzyme facilitates sperm binding to the oocyte zona pellucida [37]) (Reversibility: ? <2, 3, 4, 5> [3, 7, 8, 14, 23, 25, 35, 37, 49, 51, 54, 60]) [3, 7, 8, 14, 23, 25, 35, 37, 49, 51, 54, 60] P ? Substrates and products S UDP-GalNAc + GlcNAc <4> (<4>, transfer of GalNAc is only 1% of galactose transfer in wild type enzyme. Mutant enzyme Y289L exhibits nearly 100% of the galactose transferase activity [55]; <4>, at high concentrations of a-lactalbumin [62]) (Reversibility: ? <4> [55, 62]) [55, 62] P ? S UDPgalactose + 2-acetamido-N-(l-aspart-4-oyl)-1,2-dideoxy-b-glucoside <4> (<4>, 65% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] P ? S UDPgalactose + 3-acetamido-3-deoxy-d-xylose <4> (Reversibility: ? <4> [62]) [62] P ? S UDPgalactose + GlcNAcb-S-p-NP <5> (Reversibility: ? <5> [52]) [52] P UDP + Galb1-4 GlcNAcb-S-p-NP S UDPgalactose + GlcNAcb1-6(GlcNAcb1-2)Mana1-3Manb1O(CH2 )8 COOCH2 -mNP <5> (Reversibility: ? <5> [52]) [52] P UDP + Galb1-4 GlcNAcb1-6(GlcNAcb1-2)Mana1-3Manb1O(CH2 )8 COOCH2 -mNP S UDPgalactose + N-acetamido-3-deoxy-d-glucose <4> (Reversibility: ? <4> [62]) [62] P ? S UDPgalactose + N-acetyl-b-d-glucosaminyl-glycopeptide <4, 6> (<4>, glycoproteins containing terminal nonreducing N-acetylglucosaminyl units [35]; <6>, glycopeptide prepared from porcine IgG immunoglobulin [40]) (Reversibility: ? <4, 6> [35, 40]) [35, 40] P UDP + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminylglycopeptide S UDPgalactose + N-acetylglucosamine <2-6> (<5>, 7% of the activity with GlcNAcb-S-pNP [52]) (Reversibility: ? <2-6> [1-5, 15, 19, 20, 24, 27, 28, 30, 32-35, 40, 41, 42, 44, 47, 51, 52, 55, 56, 57, 61, 62, 63]) [1-5, 15, 19, 20, 24, 27, 28, 30, 32-35, 40, 41, 42, 44, 47, 51, 52, 55, 56, 57, 61, 62, 63] P UDP + N-acetyllactosamine <2-6> [2, 19, 28, 35, 38, 40, 57] S UDPgalactose + N-acetylglucosaminyl-b-1,2-mannosyl-a-1,6-(N-acetylglucosaminyl-b-1,2-mannosyl-a-1,3-)mannosyl-b-1,4-N-acetylglucosami-

3

N-Acetyllactosamine synthase

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 4

2.4.1.90

nyl-b-1,4-(fucosyl-a-1,6-)N-acetylglucosaminyl-asparagine <3> (Reversibility: ? <3> [23]) [23] UDP + galactosyl-b-1,4-N-acetylglucosaminyl-b-1,2-mannosyl-a-1,6-(galactosyl-b-1,4-N-acetylglucosaminyl-b-1,2-mannosyl-a-1,3-)mannosyl-b1,4-N-acetylglucosaminyl-b-1,4-(fucosyl-a-1,6-)N-acetylglucosaminyl-asparagine <3> (<3>, galactose is transferred much faster to the N-acetylglucosaminyl-b-1,2-mannosyl-a-1,3-branch than to the N-acetylglucosaminyl-b-1,2-mannosyl-a-1,6-branch [23]) [23] UDPgalactose + N-acetylglucosaminyl-b-1,3-(N-acetylglucosaminyl-b1,6-)galactose <4> (Reversibility: ? <4> [27]) [27] UDP + galactosyl-b-1,4-N-acetylglucosaminyl-b-1,3-(N-acetylglucosaminyl-b-1,6-)galactose UDPgalactose + N-acetylglucosaminyl-b-1,3-(galactosyl-b-1,4-N-acetylglucosaminyl-b-1,6-)galactose <4> (Reversibility: ? <4> [27]) [27] UDP + galactosyl-b-1,4-N-acetylglucosaminyl-b-1,3-(galactosyl-b-1,4-Nacetylglucosaminyl-b-1,6-)galactose UDPgalactose + N-acetylglucosaminyl-b-1,3-galactose <4> (Reversibility: ? <4> [27]) [27] UDP + galactosyl-b-1,4-N-acetylglucosaminyl-b-1,3-galactose UDPgalactose + N-acetylglucosaminyl-b-1,6-galactose <4> (Reversibility: ? <4> [27]) [27] UDP + galactosyl-b-1,4-N-acetylglucosaminyl-b-1,6-galactose UDPgalactose + UDPglucose <5> (Reversibility: ? <5> [57]) [57] UDP + lactose <5> [57] UDPgalactose + agalacto-ovomucoid <4> (<4>, 65% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] ? UDPgalactose + agalacto-poly-N-acetyllactosamine <5> (Reversibility: ? <5> [49]) [49] ? UDPgalactose + agalactokeratan <4> (<4>, agalactokeratan from bovine cornea and nasal septum, at 5% and 13% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] ? UDPgalactose + a1 -acid glycoprotein <5> (Reversibility: ? <5> [38]) [38] ? UDPgalactose + asialo-agalacto-a1 -acid-glycoprotein <5> (Reversibility: ? <5> [28]) [28] ? UDPgalactose + asialo-agalacto-a1 -glycoprotein <4> (<4>, 42% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] ? UDPgalactose + asialo-agalacto-transferrin <2> (<2>, transfer of galactose to N-acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a b1-4linkage [5]) (Reversibility: ? <2> [5]) [5] ? UDPgalactose + asialogalactofetuin <2> (Reversibility: ? <2> [51]) [51]

2.4.1.90

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

N-Acetyllactosamine synthase

? UDPgalactose + chitobiose <4> (Reversibility: ? <4> [61]) [61] ? UDPgalactose + chitotriose <4> (Reversibility: ? <4> [61]) [61] ? UDPgalactose + degalactosylated fetuin <3, 5, 6> (Reversibility: ? <3, 5, 6> [31, 38, 42, 44]) [31, 38, 42, 44] UDP + fetuin containing b-1,4-galactose linkages <5> [38] UDPgalactose + di-acetylchitobiose <4> (<4>, 54% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] ? UDPgalactose + fetuin <6> (Reversibility: ? <6> [40]) [40] ? UDPgalactose + glucose <2, 3, 4, 5> (<2, 3, 4, 5>, in presence of a-lactalbumin [2, 5, 28, 30, 34, 35, 38, 39, 44, 47, 55, 62, 63]; <5>, enzyme has no lactose synthase activity in presence of a-lactalbumin [52]) (Reversibility: ?<2, 3, 4, 5> [2, 5, 28, 30, 34, 35, 38, 39, 44, 47, 55, 62, 63]) [2, 5, 28, 30, 34, 35, 38, 39, 44, 47, 55, 62, 63] lactose + UDP <2, 3, 4, 5> [2, 5, 28, 30, 34, 35, 38, 39, 44, 47] UDPgalactose + immunoglobulin heavy chain <6> (Reversibility: ? <6> [40]) [40] ? UDPgalactose + lacto-N-triaosylceramide <5> (Reversibility: ? <5> [49]) [49] ? UDPgalactose + lacto-N-triose II <5> (Reversibility: ? <5> [49]) [49] ? UDPgalactose + methyl 2-acetamido-2-deoxy-b-glucoside <4> (<4>, 76% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] ? UDPgalactose + methyl 2-bromo-acetamido-2-deoxy-b-glucoside <4> (<4>, 75% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] ? UDPgalactose + methyl 2-deoxy-2-(p-benzamido)b-glucoside <4> (<4>, 16% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] ? UDPgalactose + ovalbumin <3, 5, 6> (Reversibility: ? <3, 5, 6> [21, 28, 29, 40, 43]) [21, 28, 29, 40, 43] ? UDPgalactose + ovomucoid <3> (Reversibility: ? <3> [18]) [18] UDP + ovomucoid with b-1,4-bound galactose <3> [18] UDPgalactose + p-nitrophenyl 2-acetamido-2-deoxy-b-glucoside <4, 6> (<4>, 67% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4, 6> [35, 40]) [35, 40] 5

N-Acetyllactosamine synthase

2.4.1.90

P ? S UDPgalactose + tri-N-acetylchitotriose <4> (<4>, 64% of the activity with N-acetylglucosamine [35]) (Reversibility: ? <4> [35]) [35] P ? S Additional information <4> (<4>, enzyme also catalyzes transfer of glucose from UDPglucose to N-acetylglucosamine [47]; <4>, regioselectivity towards specific C(4)glucose OH group in the complex protopanaxadiol glycoside ginsenoside Rb1 [50]; <4>, enzyme also catalyzes unusual galactosyl transfer to the 3-OH position of l-sugars [62]) [47, 50, 62] P ? Inhibitors Cu2+ <7, 8> (<7,8>, complete inhibition in presence of Mn2+ [64]) [64] EDTA <2, 3, 6> [5, 30, 31, 39, 44] N-acetylglucosamine <5, 6> (<5>, above 10 mM [38]; <6>, competitively inhibits the transfer of galactose to glycoprotein substrates [40]) [38, 40] N-acetylimidazole <4> (<4>, activity is partially restored by treatment with hydroxylamine [41]) [41] UDP <4, 6> (<4>, treatment with periodate-cleaved UDP and NaCNBH3 results in a loss of 80% of enzyme activity, which is largely prevented by UDPgalactose [13]; <6>, competitively inhibits the transfer of galactose to glycoprotein substrates [40]) [13, 40] UMP <6> (<6>, competitively inhibits the transfer of galactose to glycoprotein substrates [40]) [40] Zn2+ <3, 7, 8> (<7, 8>, complete inhibition in presence of Mn2+ [64]) [21, 64] a-lactalbumin <2, 3, 4, 5> (<2>, partially inhibits reaction with UDPgalactose and asialo-agalacto-transferrin [5]; <3, 4, 5>, inhibits reaction with UDPgalactose and N-acetylglucosamine [24, 35, 57]; <2>, inhibits N-acetyllactosamine synthesis in plasma membrane fraction [34]) [5, 24, 30, 34, 35, 57] a1 -acid glycoprotein <5> (<5>, above 1.4 mM with respect to acceptor sites [38]) [38] p-hydroxymercuribenzoate <5> [45] p-nitrophenyl 2-acetamido-2-deoxy-b-glucoside <6> (<6>, competitively inhibits the transfer of galactose to glycoprotein substrates [40]) [40] phosphatidic acid <3, 4> [20, 22] phosphatidylethanolamine <3> [22] phosphatidylglycerol <3> [22] phosphatidylserine <4> [20] poly(l-Glu) <5> [29] Additional information <4> (<4>, the enzyme is totally inactivated by iodination with lactoperoxidase, EC 1.11.1.7. Substrates protect against inactivation [41]) [41] Activating compounds Triton X-100 <7, 8> (<7,8>, 0.1-5%, 1.5fold stimulation [64]) [64] a-lactalbumin <2, 3, 4> (<4>, stimulates transfer of glucose from UDPglucose to N-acetylglucosamine [47]; <2,3,4,5>, stimulates transfer of galactose 6

2.4.1.90

N-Acetyllactosamine synthase

from UDPgalactose to N-acetylgalactosamine [2, 5, 28, 30, 34, 35, 38, 39, 44, 47, 55, 62, 63]) [2, 5, 28, 30, 34, 35, 38, 39, 44, 47, 55, 62, 63] dimyristoylphosphatidylcholine <3, 4> (<3,4>, activation [20,22]) [20, 22] dioleoylphosphatidylcholine <3> (<3>, activation [22]) [22] dipalmitoylphosphatidylcholine <3> (<3>, activation [22]) [22] distearoylphosphatidylcholine <3> (<3>, activation [22]) [22] histone <4> (<4>, activation [20,29]) [20, 29] lysophosphatidylcholine <4> (<4>, activation [20]) [20] methylphosphatidylic acid <4> (<4>, activation [20]) [20] phosphatidylcholine <3, 4> (<3,4>, activation [20,22]) [20, 22] phosphatidylethanolamine <4> (<4>, activation [20]) [20] phosphatidylglycerol <4> (<4>, activation [20]) [20] poly(l-Arg) <5> (<5>, activation [29]) [29] poly(l-Lys) <5> (<5>, activation [29]) [29] protamine sulfate <5> (<5>, activation [29]) [29] Metals, ions Ca2+ <2, 7, 8> (<2>, can partially replace Mn2+ [2]; <7, 8>, about 50% of the activity with Mn2+ [64]) [2, 64] Co2+ <4> (<4>, activation at 14.9% of the activity with Mn2+ [35]) [35] Fe2+ <7> (<7>, partial activation [64]; <8>, no activation [64]) [64] Mg2+ <2, 7, 8> (<2>, can partially replace Mn2+ [2]; <7,8>, about 50% of the activity with Mn2+ [64]) [2, 64] Mn2+ <1, 2, 3, 4, 5, 6, 7, 8> (<1, 2, 3, 4, 5, 6, 7, 8>, required [1, 2, 5, 21, 29, 30, 31, 32, 33, 35, 38, 39, 42-44, 55, 64]; <2>, optimal concentration is 10 mM [5]; <3>, optimal concentration 3-5 mM [24]; <3,5>, optimal concentration: 12.5 mM [42,44]; <3>, 0.0025 mM required for half-maximal activity [21]; <4>, maximal activity at 4 mM [35]; <3>, maximal activity at 3-5 mM [24]; <5>, Km : 0.03 mM [29]; <6>, optimal concentration is 5-10 mM MnCl2 [31]; <4>, binding of two Mn(II) per mol of enzyme in the ternary enzyme-manganese-UDPgalactose complex. The affinity of the enzyme for manganese is much higher in the presence of UDPgalactose than in its absence [33]; <4>, Km for MnCl2 : 0.34 mM [35]; <5>, Km : 0.4 mM [38]; <5>, 20 mM, stimulates [52]; <7,8>, no activation in presence of Fe2+ , Zn2+ and Cu2+ [64]; <4>, the catalytic domain of the enzyme has two metal binding sites, each with a distinct affinity. Site I binds Mn2+ with high affinity and does not bind Ca2+ . Site II binds a variety of metal ions, including Ca2+ . In the primary metal binding site the Mn2+ ion is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by D254, H347 and M344 [65]) [1, 2, 5, 21, 24, 29, 10, 31, 32, 33, 35, 38, 39, 42, 43, 44, 52, 55, 64, 65] Zn2+ <4> (<4>, activation at 9.2% of the activity with Mn2+ [35]) [35] Turnover number (min±1) Additional information <4> (<4>, turnover number for UDPglucose in absence of a-lactalbumin is 3.48 and 14.8 in presence of a-lactalbumin [47]) [47, 55]

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Specific activity (U/mg) 0.029 <3> [36] 0.035 <6> [40] 0.07 <3> [21] 0.291 <3> [22] 0.535 <5> [38] 4.7 <5> [17] 6.9 <5> (<5>, N-deglycosylated recombinant enzyme [63]) [63] 8.4 <5> (<5>, recombinant enzyme [63]) [63] 10.7 <4> [35] 19.8 <3> [19] Additional information <4, 6> (<4>, determination of b1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection, high sensitivity of product detection [58]) [18, 21, 26, 31, 32, 58] Km-Value (mM) 0.0039 <5> (N-acetylglucosamine) [38] 0.0108 <3> (UDPgalactose) [39] 0.0118 <3> (UDPgalactose, <3>, enzyme from liver microsomal membrane [30]) [30] 0.01205 <3> (UDPgalactose, <3>, enzyme from serum [30]) [30] 0.0125 <3> (UDPgalactose) [44] 0.0127 <2> (UDPgalactose) [34] 0.019 <5> (UDPgalactose) [29] 0.02 <6> (IgG immunoglobulin heavy chain) [40] 0.022 <6> (UDPgalactose) [40] 0.024 <2, 5> (UDPgalactose, <2>, enzyme in pCMGT1-transfected cells [5]) [5, 38] 0.025 <5> (UDPgalactose, <5>, recombinant enzyme [49]) [49] 0.028 <5> (UDPgalactose, <5>, recombinant enzyme [17]) [17] 0.029 <6> (fetuin) [40] 0.0295 <3> (UDPgalactose) [43] 0.03 <3> (UDPgalactose) [21] 0.043 <4> (UDPgalactose) [24] 0.0468 <2> (UDPgalactose) [51] 0.052 <3> (UDPgalactose, <3>, enzyme from serum [24]) [24] 0.054 <6> (ovalbumin) [40] 0.055 <4> (UDPgalactose) [35] 0.056 <2> (asialo-agalacto-transferrin, <2>, enzyme in pCMGT1-transfected cells [5]) [5] 0.0608 <2> (asialogalactofetuin) [51] 0.064 <5> (asialo-agalacto-transferrin, <5>, recombinant enzyme [49]) [49] 0.065 <3> (UDPgalactose, <3>, enzyme from liver [24]) [24] 0.08 <6> (UDPgalactose) [31] 0.082 <5> (UDPgalactose) [17]

8

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0.091 <5> (UDPgalactose, <5>, recombinant enzyme [63]) [63] 0.106 <5> (UDPgalactose) [63] 0.13 <3> (N-acetylglucosaminyl-b-1,2-mannosyl-a-1,6-(N-acetylglucosaminyl-b-1,2-mannosyl-a-1,3-)mannosyl-b-1,4-N-acetylglucosaminyl-b-1,4-(fucosyl-a-1,6-)N-acetylglucosaminyl-asparagine) [23] 0.143 <5> (UDPgalactose, <5>, recombinant N-deglycosylated enzyme [63]) [63] 0.17 <5> (agalacto-poly-N-acetyllactosamine) [49] 0.19 <5> (lacto-N-triose II) [49] 0.2 <5> (UDPgalactose, <5>, reaction with glucose, wild-type enzyme and Ndeglycosylated recombinant enzyme [63]) [63] 0.2 <5> (a1 -acid glycoprotein) [38] 0.21 <5> (UDPgalactose, <5>, reaction with glucose, recombinant enzyme [63]) [63] 0.25 <2> (UDPgalactose) [2] 0.25 <6> (glycopeptide prepared from porcine IgG immunoglobulin) [40] 0.27 <5> (ovalbumin) [29] 0.33 <3> (N-acetylglucosamine, <3>, enzyme from liver [24]) [24] 0.43 <3> (N-acetylglucosaminyl-b-1,2-mannosyl-a-1,6-(galactosyl-b-1,4-Nacetylglucosaminyl-b-1,2-mannosyl-a-1,3-)mannosyl-b-1,4-N-acetylglucosaminyl-b-1,4-(fucosyl-a-1,6-)N-acetylglucosaminyl-asparagine) [23] 0.5 <4> (N-acetylglucosamine) [24] 0.66 <6> (p-nitrophenyl 2-acetamido-2-deoxy-b-glucoside) [40] 0.8 <5> (N-acetylgalactosamine) [28] 0.83 <5> (lacto-N-triaosylceramide) [49] 1 <5> (d-glucose, <5>, recombinant enzyme [63]) [63] 1 <2> (N-acetylglucosamine) [2] 1.1 <5> (d-glucose, <5>, recombinant N-deglycosylated enzyme [63]) [63] 1.49 <3> (N-acetylglucosamine) [24] 1.5 <4> (N-acetylglucosaminyl-b-1,6-galactose, <4>, N-acetylglucosaminylb-1,3-(N-acetylglucosaminyl-b-1,6-)galactose [27]) [27] 1.6 <2> (N-acetylglucosamine) [51] 1.9 <2, 4> (N-acetylglucosamine, <4>, N-acetylglucosaminyl-b-1,3-galactose [27]; <2>, enzyme in pCMGT1-transfected cells [5]) [5, 27] 2 <5> (d-glucose) [63] 2.3 <5> (N-acetylglucosamine) [28] 2.5 <5> (N-acetylglucosamine, <5>, recombinant enzyme [49]) [49] 2.8 <5> (N-acetylglucosamine) [17] 2.8 <4> (agalactokeratan) [35] 3.3 <5> (N-acetylglucosamine) [63] 3.4 <4> (N-acetylglucosaminyl-b-1,3-(galactosyl-b-1,4-N-acetylglucosaminylb-1,6-)galactose) [27] 3.6 <5> (N-acetylglucosamine, <5>, recombinant enzyme [17]) [17] 4 <6> (N-acetylglucosamine) [31] 4.6 <2> (N-acetylglucosamine) [34] 5.8 <6> (N-acetylglucosamine) [40]

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6.28 <3> (galactosyl-b-1,4-N-acetylglucosaminyl-b-1,2-mannosyl-a-1,6-(Nacetylglucosaminyl-b-1,2-mannosyl-a-1,3-)mannosyl-b-1,4-N-acetylglucosaminyl-b-1,4-(fucosyl-a-1,6-)N-acetylglucosaminyl-asparagine) [23] 8.2 <4> (N-acetylglucosamine) [27] 8.3 <5> (N-acetylglucosamine, <5>, recombinant N-deglycosylated enzyme [63]) [63] 10 <5> (N-acetylglucosamine, <5>, recombinant enzyme [63]) [63] 21 <4> (d-glucose) [35] 40 <4> (N-acetylglucosamine) [35] Additional information <5> (<5>, effects of cationic polypeptides [29]; <5>, Km -value for ovalbumin for the N-deglycosylated recombinant enzyme is 6 mg/ml, and 15 mg/ml for the recombinant enzyme [63]) [29, 63] pH-Optimum 5 <2> [51] 6 <3> (<3>, in Tris-maleate buffer [43]) [43] 6.4-7.6 <6> [31] 6.5 <3> [18, 30, 39] 6.5-7 <7, 8> [64] 6.8 <3, 5, 6> [40, 42, 44] 7 <5> [52] 7.5 <5> (<5>, reaction with N-acetylglucosamine and UDPgalactose, reaction with a1 -acid glycoprotein and UDPgalactose [38]) [38] 7.5-10.5 <4> [35] 8.2 <2> [2] pH-Range 5-9.3 <2> (<2>, pH 5.0: about 60% of maximal activity, pH 9.3: about 45% of maximal activity [2]) [2] 5.5-8 <3> (<3>, less than 50% of maximal activity above and below [39]) [39] Temperature optimum ( C) 30 <5> (<5>, reaction with N-acetylglucosamine and UDPgalactose, reaction with a1 -acid glycoprotein and UDPgalactose [38]) [38] 37 <5, 6> (<6>, activity at 37 C is faster than at 31 C or at 27 C [31]) [31, 42] 42 <5> [17] Temperature range ( C) 25-45 <5> (<5>, less than 50% of maximal activity above and below [42]) [42]

4 Enzyme Structure Molecular weight 42960 <2> (<2>, calculation from gene sequence, short form, transmembrane enzyme [7]) [7] 44420 <2> (<2>, long form with NH2 -terminal extension of 13 amino acids, calculation from gene sequence [7]) [7] 10

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44880 <4> (<4>, unglycosylated enzyme, calculation from gene sequence [14]) [14] 57000 <6> (<6>, sucrose density gradient centrifugation [40]) [40] 59000 <4> (<4>, gel filtration [26]) [26] 70000 <6> (<6>, gel filtration [31]) [31] 85000-90000 <5> (<5>, gel filtration [38]) [38] 106000 <5> (<5>, calculation from light-scattering experiments [63]) [63] 440000 <3> (<3>, gel filtration [19]) [19] Additional information <2> (<2>, two related forms of enzyme of 399 and 386 amino acids are synthesized as a consequence of alternative translation initiation. The long enzyme form has a NH2 -terminal extension of 13 amino acids [7]) [7] Subunits ? <3, 4, 5> (<5>, x * 38000, SDS-PAGE [49]; <4>, x * 42200, SDS-PAGE [35]; <3>, x * 43000, SDS-PAGE [44]; <5>, x * 47000, deglycosylated enzyme form, SDS-PAGE [17]; <5>, x * 48000, glycosylated enzyme form [17]; <3>, x * 50000-51000, SDS-PAGE [19]; <4>, x * 51000, SDS-PAGE [26]; <3>, x * 53000, SDS-PAGE [18];<4>, x * 54000, enzyme from milk, SDS-PAGE [32]; <3>, x * 65000-70000, SDS-PAGE [39]; <3>, x * 70000-75000, SDS-PAGE [21]; <5>, x * 70000-80000, SDS-PAGE [38]) [17, 18, 19, 21, 26, 32, 35, 38, 39, 44, 49] dimer <5> (<5>, 2 * 55000, SDS-PAGE [63]) [63] monomer <6> (<6>, 1 * 57000, SDS-PAGE [40]; <6>, 1 * 74000, SDS-PAGE [31]) [31, 40] Posttranslational modification glycoprotein <4, 5> (<5>, structure of mucin-type sugar depends on blood group [12]) [12, 17, 25, 63]

5 Isolation/Preparation/Mutation/Application Source/tissue BALB/3T12-3 cell <2> [34] C127 cell <2> [7] F9 cell <2> [5, 6, 51] HeLa cell <5> (<5>, expression of extremely high levels of mRNA [16]) [11, 16, 17] MDBK cell <4> (<4>, ATCC No. CCL22 [8]) [8] MRK-nu-1 cell <5> [52] amniotic fluid <5> [42] brain <3> [4] colostrum <4> [27, 32, 50] cornea <4> [35] epididymis <3> (<3>, very high enzyme concentration in Golgi apparatus of epididymal duct epithelium from initial segment to intermediate caput, although much lower amounts of enzyme are in efferent ducts, distal caput, 11

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corpus and cauda. Even in the initial segment and caput epididymis, only low levels of soluble enzyme form are detected in the fluid [60]) [60] intestinal mucosa <3> (<3>, ileal-colonic [21]) [21] intestine <3> (<3>, regional distribution [21]) [21] kidney <3> [36] liver <3, 4> (<3>, fetal [42]) [3, 14, 18, 19, 22, 23, 24, 30, 36, 39, 42, 44] lung <3> (<3>, fetal [42]) [42] mammary gland <1, 4> [1, 10] mastocytoma cell <2> [2] mesenteric lymph node <6> [40] milk <4, 5> [12, 13, 20, 14, 32, 33, 35, 41, 45, 47, 57, 6, 63] placenta <3, 5> [11, 42] plasma <5> [38] serum <3, 5> [24, 28, 30, 43, 44] skin fibroblast <5> [29] sperm <4> (<4>, anterior head of sperm head [37]) [37] spermatogonium <2> (<2>, during differentiation from spermatogonia to pachytene spermatocytes the amount of UDP b1,4-galactosyltransferase mRNA is reduced to barley detectable levels [9]) [9] testis <2> (<2>, during differentiation from spermatogonia to pachytene spermatocytes the amount of UDP b1,4-galactosyltransferase mRNA is reduced to barley detectable levels [9]) [9, 51] thymus <4> [26] thyroid gland <6> [31] Localization Golgi apparatus <1, 2, 3, 4, 5, 6> (<4>, short and long enzyme form are resident trans-Golgi proteins, the NH2 -terminal segment contains the cytoplasmic and transmembrane domains [25]) [1, 3, 4, 7, 14, 15, 16, 18, 22, 23, 24, 25, 31, 36, 59] Golgi membrane <2, 3> [4, 15, 22, 36] membrane <2, 3, 4, 6> (<4>, N-terminal hydrophobic segment serves as the membrane anchor, the C-terminal region is oriented within the lumen of the Golgi membranes [14]; <2>, plasma membrane enriched fraction [34]; <3>, the enzyme from kidney appears to be an intrinsic membrane protein [36]) [14, 19, 25, 31, 34, 39, 40, 44, 59, 60] microsome <2, 3, 6> [2, 18, 30, 31, 39] soluble <3-6> (<3>, enzyme exists as a soluble and a membrane-bound form [60]) [13, 18, 30, 35, 38, 40-45, 60] Purification <1> [1] <2> [2, 9, 51] <3> (partial [24]) [18, 19, 21, 22, 24, 30, 36, 39, 44] <4> [26, 27, 35] <5> (expressed in Saccharomyces cerevisiae [17]; recombinant enzyme [49,63]) [17, 30, 38, 49, 63] <6> [31, 40] 12

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<7> (recombinant enzyme [64]) [64] <8> (recombinant enzyme [64]) [64] Crystallization <4> (recombinant enzyme, crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the b1,4-galactosyltransferase-I [46]; crystal structure of enzyme-a-lactalbumin complex with UDP-Glc [47]; crystal structures of the b4-galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose [59]) [46, 47, 55, 59] Cloning <2> (expression in COS-1 cells [5]; cloning and sequencing of the full-length cDNA [6]; HeLa cells expressing the murine enzyme on their surface spread more rapidly on laminin substrates than do control cells [48]; histidinetagged 46000 Da protein produced in Escherichia coli [51]) [5, 6, 48, 51] <2> (isolation and characterization of the genomic locus [15]) [15] <4> (isolation of a cDNA clone that encodes a major portion of galactosyltransferase [8]; expression of short and long enzyme form in CHO-cells [25]; cloning and sequencing of cDNA [10]; expression in Sf9 cells. Sfb4GalT cell, unlike the parental Sf9 cells, can terminally b1,4-galactosylate gp64 during baculovirus infection [53]; expression of wild-type and mutant enzymes in Escherichia coli [61]; expression in Escherichia coli [65]) [8, 10, 25, 53, 61, 65] <5> (recombinant enzyme is N-glycosylated [17]; comparison of sequences of enzyme from placenta and HeLa cells [11]; molecular cloning and nucleotide sequencing [16]; expression in Escherichia coli [49]; enzyme fused to protein A is expressed as a soluble form in COS-7 cells [52]; expression of mutant cDNA from a patient with the congenital disorder of glycosylation type IId leads to the synthesis of a truncated, inactive polypeptide, which is localized to the endoplasmic reticulum [54]; enzymatically active soluble Ndeglycosylated enzyme form [63]) [11, 16, 17, 49, 52, 54, 63] <7> (expression under the control of the T7 promoter in Escherichia coli BL21 [64]) [64] <8> (expression under the control of the T7 promoter in Escherichia coli BL21 [64]) [64] Engineering C134S <4> (<4>, complete loss of activity [61]) [61] C342S <4> (<4>, 33fold increase in the apparent Km -value for UDPgalactose [61]) [61] D254E <4> (<4>, 0.01% of the activity of the wild-type enzyme [65]) [65] D254N <4> (<4>, 0.01% of the activity of the wild-type enzyme [65]) [65] D320A <4> (<4>, when partially activated by Mn2+ binding to the primary site, can be further activated by Co2+ or inhibited by Ca2+ , an effect that is the opposite of what is observed with the wild-type enzyme [65]) [65] D320E <4> (<4>, when partially activated by Mn2+ binding to the primary site, can be further activated by Co2+ or inhibited by Ca2+ , an effect that is the opposite of what is observed with the wild-type enzyme [65]) [65]

13

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D320N <4> (<4>, when partially activated by Mn2+ binding to the primary site, can be further activated by Co2+ or inhibited by Ca2+ , an effect that is the opposite of what is observed with the wild-type enzyme [65]) [65] E317A <4> (<4>, when partially activated by Mn2+ binding to the primary site, can be further activated by Co2+ or inhibited by Ca2+ , an effect that is the opposite of what is observed with the wild-type enzyme [65]) [65] E317D <4> (<4>, when partially activated by Mn2+ binding to the primary site, can be further activated by Co2+ or inhibited by Ca2+ , an effect that is the opposite of what is observed with the wild-type enzyme [65]) [65] E317Q <4> (<4>, when partially activated by Mn2+ binding to the primary site, can be further activated by Co2+ or inhibited by Ca2+ , an effect that is the opposite of what is observed with the wild-type enzyme [65]) [65] H347D <4> (<4>, in presence of Mn2+ retains 0.02% of wild-type enzyme activity, in presence of Co2+ retains 0.085% of wild-type enzyme activity [65]) [65] H347E <4> (<4>, in presence of Mn2+ retains 0.1% of wild-type enzyme activity, in presence of Co2+ retains 0.4% of wild-type enzyme activity [65]) [65] H347N <4> (<4>, in presence of Mn2+ retains 0.07% of wild-type enzyme activity, in presence of Co2+ retains 0.36% of wild-type enzyme activity [65]) [65] H347Q <4> (<4>, in presence of Mn2+ retains 0.28% of wild-type enzyme activity, in presence of Co2+ retains 1.21% of wild-type enzyme activity [65]) [65] M344A <4> (<4>, in presence of Mn2+ retains 54.5% of wild-type enzyme activity, in presence of Co2+ retains 6.15% of wild-type enzyme activity [65]) [65] M344Q <4> (<4>, in presence of Mn2+ retains 15.37% of wild-type enzyme activity, in presence of Co2+ retains 31.08% of wild-type enzyme activity [65]) [65] Y289I <4> (<4>, mutation enhances GalNAc-transferase activity. Km for GlcNAc is increased compared to the wild type [55]) [55] Y289L <4> (<4>, mutation enhances GalNAc-transferase activity. Km for GlcNAc is incereased compared to the wild type [55]) [55] Y289N <4> (<4>, mutation enhances GalNAc-transferase activity. Km for GlcNAc is increased compared to the wild type [55]) [55] Additional information <4> (<4>, N-terminal truncated forms of the enzyme between residues 1-129, do not show any significant difference in the apparent Km -values towards N-acetylglucosamine or linear oligosaccharide acceptors, e.g. for chitobiose and chitotriose, or for the nucleotide donor UDPgalactose. The binding behaviour of N-terminal and C-terminal fragments of the enzyme towards the N-acetylglucosamine-agarose and UDP-agarose columns differ, the former binds preferentially to the N-acetylglucosamine-columns, while the latter binds to UDP-agarose columns via Mn2+ [61]; <4>, mutations of Asp318 and Asp319 abolish enzyme activity [65]) [61, 65]

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Application synthesis <4, 5> (<4>, preparation of a series of specific derivatives of the complex protopanaxadiol glycoside ginsenoside Rb1 [50]; <5>, use of transformed whole yeast cells, expressing the human N-acetylglucosamine b-1,4galactosyltransferase, in synthesis of N-acetyllactosamine [56]) [50, 56]

6 Stability pH-Stability 5-9 <2> [2] Temperature stability 40 <5> (<5>, 4 h, 18% loss of activity [63]) [63] 45 <3> (<3>, stable up to [4]) [4] 50 <5> (<5>, 1 h, complete loss of activity [63]) [63] 56 <5> (<5>, inactivation at [17]) [17] General stability information <3>, ammonium sulfate stabilizes during storage [30] <3>, glycerol stabilizes during storage [30] <3>, more than 50% loss of activity on freezing [44] <4>, Triton X-100 essential for stability during purification [27] Storage stability <1>, 4 C, 0.1% bovine serum albumin, stable for 3 months [1] <3>, -20 C, bovine serum albumin, stable for up to 60 d [30] <3>, -20 C, partially purified enzyme stable for several weeks, purified enzyme stable for 1 week [21] <3>, -20 C, stable for 3 weeks [43] <3>, 4 C, concentrated enzyme [22] <3>, 4 C, more than 50% loss of activity after 1 week [44] <3>, 4 C, purified and concentrated enzyme is stable for 4 weeks [24] <4>, -20 C, 1 mg/ml bovine serum albumin [32] <5>, -20 C, stable fo at least 1 month [17] <5>, -20 C, stable for at least 2 months [42] <6>, -20 C, 0.02 M Tris/HCl buffer, pH 7.5, stable for several months [40]

References [1] Smith, C.A.; Brew, K.: Isolation and characteristics of galactosyltransferase from Golgi membranes of lactating sheep mammary glands. J. Biol. Chem., 252, 7294-7299 (1977) [2] Helting, T.; Erbing, B.: Galactosyltransfer in mouse mastocytoma: purification and properties of N-acetyllactosamine synthetase. Biochim. Biophys. Acta, 293, 94-104 (1973)

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[3] Schachter, H.; Jabbal, I.; Hudgin, R.L.; Pinteric, L.: Intracellular localization of liver sugar nucleotide glycoprotein glycosyltransferases in a Golgi-rich fraction. J. Biol. Chem., 245, 1090-1100 (1970) [4] Deshmukh, D.S.; Bear, W.D.; Soifer, D.: Isolation and characterization of an enriched Golgi fraction from rat brain. Biochim. Biophys. Acta, 542, 284295 (1978) [5] Nakazawa, K.; Furukawa, K.; Kobata, A.; Narimatsu, H.: Characterization of a murine b1-4 galactosyltransferase expressed in COS-1 cells. Eur. J. Biochem., 196, 363-368 (1991) [6] Nakazawa, K.; Ando, T.; Kimura, T.; Narimatsu, H.: Cloning and sequencing of a full-length cDNA of mouse N-acetylglucosamine (b1-4)galactosyltransferase. J. Biochem., 104, 165-168 (1988) [7] Shaper, N.L.; Hollis, G.F.; Douglas, J.G.; Kirsch, I.R.; Shaper, J.H.: Characterization of the full length cDNA for murine b-1,4-galactosyltransferase. Novel features at the 5-end predict two translational start sites at two in-frame AUGs. J. Biol. Chem., 263, 10420-10428 (1988) [8] Shaper, N.L.; Shaper, J.H.; Meuth, J.L.; Fox, J.L.; Chang, H.; Kirsch, I.R.; Hollis, G.F.: Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library. Proc. Natl. Acad. Sci. USA, 83, 1573-1577 (1986) [9] Shaper, N.L.; Wright, W.W.; Shaper, J.H.: Murine b1,4-galactosyltransferase: both the amounts and structure of the mRNA are regulated during spermatogenesis. Proc. Natl. Acad. Sci. USA, 87, 791-795 (1990) [10] Narimatsu, H.; Sinha, S.; Brew, K.; Okayama, H.; Qasba, P.K.: Cloning and sequencing of cDNA of bovine N-acetylglucosamine (b1-4)galactosyltransferase. Proc. Natl. Acad. Sci. USA, 83, 4720-4724 (1986) [11] Watzele, G.; Berger, E.G.: Near identity of HeLa cell galactosyltransferase with the human placental enzyme. Nucleic Acids Res., 18, 7174 (1990) [12] Amano, J.; Straehl, P.; Berger, E.G.; Kochibe, N.; Kobata, A.: Structures of mucin-type sugar chains of the galactosyltransferase purified from human milk. Occurrence of the ABO and Lewis blood group determinants. J. Biol. Chem., 266, 11461-11477 (1991) [13] Yadav, S.; Brew, K.: Identification of a region of UDP-galactose:N-acetylglucosamine b4-galactosyltransferase involved in UDP-galactose binding by differential labeling. J. Biol. Chem., 265, 14163-14169 (1990) [14] D'Agostaro, G.; Bendiak, B.; Tropak, M.: Cloning of cDNA encoding the membrane-bound form of bovine b 1,4-galactosyltransferase. Eur. J. Biochem., 183, 211-217 (1989) [15] Hollis, G.F.; Douglas, J.G.; Shaper, N.L.; Shaper, J.H.; Stafford-Hollis, J.M.; Evans, R.J.; Kirsch, I.R.: Genomic structure of murine b-1,4-galactosyltransferase. Biochem. Biophys. Res. Commun., 162, 1069-1075 (1989) [16] Mengle-Gaw, L.; McCoy-Haman, M.F.; Tiemeier, D.C.: Genomic structure and expression of human b-1,4-galactosyltransferase. Biochem. Biophys. Res. Commun., 176, 1269-1276 (1991) [17] Krezdorn, C.H.; Watzele, G.; Kleene, R.B.; Ivanov, S.X.; Berger, E.G.: Purification and characterization of recombinant human b1-4 galactosyltrans-

16

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[18] [19]

[20] [21]

[22] [23]

[24] [25]

[26]

[27]

[28]

N-Acetyllactosamine synthase

ferase expressed in Saccharomyces cerevisiae. Eur. J. Biochem., 212, 113-120 (1993) Kawano, J.i.; Oinuma, T.; Nakayama, T.; Suganuma, T.: Characterization of b1! 4 galactosyltransferase purified from rat liver microsomes. J. Biochem., 111, 568-572 (1992) Bendiak, B.; Ward, L.D.; Simpson, R.J.: Proteins of the Golgi apparatus. Purification to homogeneity, N-terminal sequence, and unusually large Stokes radius of the membrane-bound form of UDP-galactose:N-acetylglucosamine b1-4galactosyltransferase from rat liver. Eur. J. Biochem., 216, 405417 (1993) Mitranic, M.M.; Moscarello, M.A.: The influence of various lipids on the activity of bovine milk galactosyltransferase. Can. J. Biochem., 58, 809-814 (1980) Weiser, M.M.; Majumdar, S.; Wilson, J.R.; Luther, R.: Distribution, purification and characterization of rat intestinal UDPgalactose:N-acetylglucosaminyl(b1! 4)galactosyltransferase. Biochim. Biophys. Acta, 924, 323-331 (1987) Clark, P.E.; Moscarello, M.A.: The modulating effects of lipids on purified rat liver Golgi galactosyltransferase. Biochim. Biophys. Acta, 859, 143-150 (1986) Paquet, M.R.; Narasimhan, S.; Schachter, H.; Moscarello, M.A.: Branch specificity of purified rat liver Golgi UDP-galactose:N-acetylglucosamine b1,4-galactosyltransferase. Preferential transfer of of galactose on the GlcNAc b1,2-Man a1,3-branch of a complex biantennary Asn-linked oligosaccharide. J. Biol. Chem., 259, 4716-4721 (1984) Paquet, M.R.; Moscarello, M.A.: A kinetic comparison of partially purified rat liver Golgi and rat serum galactosyltransferases. Biochem. J., 218, 745751 (1984) Russo, R.N.; Shaper, N.L.; Taatjes, D.J.; Shaper, J.H.: b1,4-galactosyltransferase: a short NH2 -terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for Golgi retention. J. Biol. Chem., 267, 9241-9247 (1992) Blanken, W.M.; van den Eijnden, D.H.: Biosynthesis of terminal Gala1!3Galb1! 4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide a(1-3)galactosyltransferase from calf thymus. J. Biol. Chem., 260, 12927-12934 (1985) Blanken, W.M.; Hooghwinkel, G.J.M.; van den Eijnden, D.H.: Biosynthesis of blood-group I and i substances. Specificity of bovine colostrum b-Nacetyl-d-glucosaminide b1 leads to 4 galactosyltransferase. Eur. J. Biochem., 127, 547-552 (1982) Berger, E.G.; Kozdrowski, I.; Weiser, M.M.; van den Eijnden, D.H.; Schiphorst, W.E.C.M.: Human serum galactosyltransferase: distinction, separation and product identification of two galactosyltransferase activities. Eur. J. Biochem., 90, 213-222 (1978)

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[29] Rao, G.J.S.; Chyatte, D.; Nadler, H.L.: Enhancement of UDPgalactose: glycoprotein galactosyltransferase in cultured human skin fibroblasts by cationic polypeptides. Biochim. Biophys. Acta, 541, 435-443 (1978) [30] Fraser, I.H.; Wadden, P.; Mookerjea, S.: Purification and stabilization of galactosyltransferase from serum and lysolecithin extracted microsomes. Can. J. Biochem., 58, 878-884 (1980) [31] Bouchilloux, S.: Purification by affinity chromatography and some properties of microsomal galactosyltransferase from pig thyroid. Biochim. Biophys. Acta, 569, 135-144 (1979) [32] Tsopanakis, A.D.; Herries, D.G.: Bovine galactosyl transferase. Substrate.managanese complexes and the role of manganese ions in the mechanism. Eur. J. Biochem., 83, 179-188 (1978) [33] Andree, P.J.; Berliner, L.J.: Metal ion and substrate binding to bovine galactosyltransferase. Biochemistry, 19, 929-934 (1980) [34] Cummings, R.D.; Cebula, T.A.; Roth, S.: Characterization of a galactosyltransferase in plasma membrane-enriched fractions from Balb/c 3T12 cells. J. Biol. Chem., 254, 1233-1240 (1979) [35] Christner, J.E.; Distler, J.J.; Jourdian, G.W.: Biosynthesis of keratan sulfate: purification and properties of a galactosyltransferase from bovine cornea. Arch. Biochem. Biophys., 192, 548-558 (1979) [36] Fleischer, B.; Smigel, M.: Solubilization and properties of galactosyltransferase and sulfotransferase activities of Golgi membranes in Triton X-100. J. Biol. Chem., 253, 1632-1638 (1978) [37] Tengowski, M.W.; Wassler, M.J.; Shur, B.D.; Schatten, G.: Subcellular localization of b1,4-galactosyltransferase on bull sperm and its function during sperm-egg interactions. Mol. Reprod. Dev., 58, 236-244 (2001) [38] Bella, A.; Whitehead, J.S.; Kim, Y.S.: Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction. Biochem. J., 167, 621-628 (1977) [39] Fraser, I.H.; Mookerjea, S.: Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions. Biochem. J., 164, 541-547 (1977) [40] Rao, A.K.; Garver, F.; Mendicino, J.: Biosynthesis of the carbohydrate units of immunoglobulins. 1. Purification and properties of galactosyltransferases from swine mesentary lymph nodes. Biochemistry, 15, 5001-5009 (1976) [41] Chandler, D.K.; Silvia, J.C.; Ebner, K.E.: Inactivation of galactosyltransferase by lactoperoxidase and N-acetylimidazole. Biochim. Biophys. Acta, 616, 179-187 (1980) [42] Nelson, J.D.; Jato-Rodriguez, J.J.; Mookerjea, S.: The occurrence and properties of soluble UDP-galactose:glycoprotein galactosyltransferase in human amniotic fluid. Can. J. Biochem., 52, 42-50 (1974) [43] Wagner, R.R.; Cynkin, M.A.: Glycoprotein metabolism: a UDP-galactoseglycoprotein galactosyltransferase of rat serum. Biochem. Biophys. Res. Commun., 45, 57-62 (1971)

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[44] Fraser, I.H.; Mookerjea, S.: Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum. Biochem. J., 156, 347-355 (1976) [45] Kitchen, B.J.; Andrews, P.: Kinetic studies on the effect of uridine diphosphate galactose and manganous ions on the reaction between lactose synthetase A protein from human milk and p-hydroxymercuribenzoate. Biochem. J., 143, 587-590 (1974) [46] Ramakrishnan, B.; Qasba, P.K.: Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the b1 ,4-galactosyltransferase-I. J. Mol. Biol., 310, 205-218 (2001) [47] Ramakrishnan, B.; Shah, P.S.; Qasba, P.K.: a-Lactalbumin (LA) stimulates milk b-1,4-galactosyltransferase I (b4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine. Crystal structure of b4Gal-T1*LA complex with UDP-Glc. J. Biol. Chem., 276, 37665-37671 (2001) [48] Nguyen, T.T.M.; Hinton, D.A.; Shur, B.D.: Expressing murine b1,4-galactosyltransferase in HeLa cells produces a cell surface galactosyltransferasedependent phenotype. J. Biol. Chem., 269, 28000-28009 (1994) [49] Nakazawa, K.; Furukawa, K.; Narimatsu, H.; Kobata, A.: Kinetic study of human b-1,4-galactosyltransferase expressed in E. coli. J. Biochem., 113, 747-753 (1993) [50] Gebhardt, S.; Bihler, S.; Schubert-Zsilaveez, M.; Riva, S.; Monti, D.; Falcone, L.; Danieli, B.: Biocatalytic generation of molecular diversity: modification of ginsenoside Rb1 by b-1,4-galactosyltransferase and Candida antarctica lipase. Helv. Chim. Acta, 85, 1943-1959 (2002) [51] Uehara, K.; Muramatsu, T.: Molecular cloning and characterization of b1,4-galactosyltransferase expressed in mouse testis. Eur. J. Biochem., 244, 706-712 (1997) [52] Sato, T.; Furukawa, K.; Bakker, H.; Van den Eijnden, D.H.; Van Die, I.: Molecular cloning of a human cDNA encoding b-1,4-galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc b-1,4-galactosyltransferase. Proc. Natl. Acad. Sci. USA, 95, 472-477 (1998) [53] Hollister, J.R.; Shaper, J.H.; Jarvis, D.L.: Stable expression of mammalian b1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells. Glycobiology, 8, 473-480 (1998) [54] Hansske, B.; Thiel, C.; Lubke, T.; Hasilik, M.; Honing, S.; Peters, V.; Heidemann, P.H.; Hoffmann, G.F.; Berger, E.G.; Von Figura, K.; Korner, C.: Deficiency of UDP-galactose:N-acetylglucosamine b-1,4-galactosyltransferase I causes the congenital disorder of glycosylation type IId. J. Clin. Invest., 109, 725-733 (2002) [55] Ramakrishnan, B.; Qasba, P.K.: Structure-based design of b1,4-galactosyltransferase I (b4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity: point mutation broadens b4Gal-T1 donor specificity. J. Biol. Chem., 277, 20833-20839 (2002) [56] Herrmann, G.F.; Elling, L.; Krezdorn, C.H.; Kleene, R.; Berger, E.G.; Wandrey, C.: Use of transformed whole yeast cells expressing b-1,4-galactosyltransferase for the synthesis of N-acetyllactosamine. Bioorg. Med. Chem. Lett., 5, 673-676 (1995) 19

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[57] Elling, L.; Zervosen, A.; Gallego, R.G.; Nieder, V.; Malissard, M.; Berger, E.G.; Vliegenthart, J.F.G.; Kamerling, J.P.: UDP-N-acetyl-a-d-glucosamine as acceptor substrate of b-1,4-galactosyltransferase. Enzymatic synthesis of UDP-N-acetyllactosamine. Glycoconjugate J., 16, 327-336 (1999) [58] Snow, D.M.; Shaper, J.H.; Shaper, N.L.; Hart, G.W.: Determination of b1,4galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection. Anal. Biochem., 271, 36-42 (1999) [59] Gastinel, L.N.; Cambillau, C.; Bourne, Y.: Crystal structures of the bovine b4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose. EMBO J., 18, 3546-3557 (1999) [60] Kawano, J.I.; Ide, S.; Oinuma, T.; Suganuma, T.: Regional distribution of b14 galactosyltransferase in rat epididymis. Acta Histochem. Cytochem., 30, 491-495 (1997) [61] Boeggeman, E.E.; Balajai, P.V.; Qasba, P.K.: Functional domains of bovine b1,4-galactosyltransferase. Glycoconjugate J., 12, 865-878 (1995) [62] Nishida, Y.; Tamakoshi, H.; Kitagawa, Y.; Kobayashi, K.; Thiem, J.: A novel bovine b-1,4-galactosyltransferase reaction to yield b-d-galactopyranosyl(1-3)-linked disaccharides from l-sugars. Angew. Chem., 39, 2000-2003 (2000) [63] Malissard, M.; Borsig, L.; Di Marco, S.; Gruetter, M.G.; Kragl, U.; Wandrey, C.; Berger, E.G.: Recombinant soluble b-1,4-galactosyltransferases expressed in Saccharomyces cerevisiae. Purification, characterization and comparison with human enzyme. Eur. J. Biochem., 239, 340-348 (1996) [64] Park, J.E.; Lee, K.Y.; Do, S.I.; Lee, S.S.: Expression and characterization of b1,4-galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae. J. Biochem. Mol. Biol., 35, 330-336 (2002) [65] Boeggeman, E.; Qasba, P.K.: Studies on the metal binding sites in the catalytic domain of b1,4-galactosyltransferase. Glycobiology, 12, 395-407 (2002)

20

Flavonol 3-O-glucosyltransferase

2.4.1.91

1 Nomenclature EC number 2.4.1.91 Systematic name UDP-glucose:flavonol 3-O-d-glucosyltransferase Recommended name flavonol 3-O-glucosyltransferase Synonyms F3GT GT-I GTI UDP-glucose:flavonol 3-O-glucosyltransferase UDPG:flavonoid-3-O-glucosyltransferase UDPglucose:flavonol O3 -d-glucosyltransferase UFGT glucosyltransferase, uridine diphosphoglucose-flavonol 3-OCAS registry number 50812-18-5

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

Petroselinum hortense [1, 6] Tulipa sp. (cv. Apeldoorn [2]) [2] Vigna mungo [3] Prunus yedoensis (Matsum [4]) [4] Glycine max [5] Hippeastrum sp. [7] Brassica oleracea (cv. Red Danish [8]) [8] Vitis vinifera [9, 13] Euonymus alatus (f. Ciliato-dentatus [10]) [10] Vigna mungo [11] Antirrhinum majus [12] Petunia sp. [14] Paederia scandens (var. Mairei [16]) [16] Petunia hybrida [15]

21

Flavonol 3-O-glucosyltransferase

2.4.1.91

3 Reaction and Specificity Catalyzed reaction UDP-glucose + a flavonol = UDP + a flavonol 3-O-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + a flavonol <1, 7, 8, 12, 14> (<1>, the enzyme is involved in the flavone glycoside pathway in parsley. Enzyme activity is increased by prior illumination of the cell cultures, maximal activity is reached about 24 h after onset of illumination [1]; <7>, enzyme is specific for flavonol glucoside biosynthesis [8]; <8>, the enzyme is critical for anthocyanin biosynthesis in the grape berry [9]; <8>, the principal, if not only, role of the enzyme is to glucosylate anthocyanidins in red fruit during ripening [13]; <12>, in the conversion from colorless leucoanthocyanidin to colored anthocyanidin 3-glucoside, at least two enzymes, anthocyanidin synthase and UDP-glucose:flavonoid 3-O-glucosyltransferase, are postulated to be involved [14]; <14>, the enzyme is responsible for the glycosylation of anthocyanidins to produce stable molecules in the anthocyanin biosynthetic pathway [15]) (Reversibility: ? <1, 7, 8, 12, 14> [1, 8, 9, 14, 15]) [1, 8, 9, 13, 14, 15] P UDP + a flavonol 3-O-d-glucoside Substrates and products S UDPgalactose + isorhamnetin <2> (<2>, isorhamnetin is the best acceptor, 45% of the activity compared to reaction with UDPglucose [2]) (Reversibility: r <2> [2]) [2] P UDP + isorhamnetin 3-O-glucoside S UDPglucose + 3'-O-methylquercetin <1> (<1>, 28% of the activity with quercetin [1]) (Reversibility: ? <1> [1]) [1] P UDP + 3'-O-methylquercetin 3-O-glucoside S UDPglucose + 3,5,7-trihydroxyflavanone <9> (<9>, isoenzyme F3GT1: 22% of the activity with kaempferol, isoenzyme F3GT2: 12% of the activity with kaempferol [10]) (Reversibility: ? <9> [10]) [10] P UDP + 3,5,7-trihydroxyflavanone 3-O-glucoside S UDPglucose + 4,7-dihydroxyflavonol <1> (<1>, 27% of the activity with quercetin [1]) (Reversibility: ? <1> [1]) [1] P UDP + 4,7-dihydroxyflavonol 3-O-glucoside S UDPglucose + 5-deoxyquercetin <1> (<1>, 57% of the activity with quercetin [1]) (Reversibility: ? <1> [1]) [1] P UDP + 5-deoxyquercetin 3-O-glucoside S UDPglucose + 7,4'-dihydroflavonol <9> (<9>, isoenzyme F3GT1: 33% of the activity with kaempferol, isoenzyme F3GT2: 34% of the activity with kaempferol [10]) (Reversibility: ? <9> [10]) [10] P UDP + 7,4'-dihydroflavonol 3-O-glucoside

22

2.4.1.91

Flavonol 3-O-glucosyltransferase

S UDPglucose + apigenin <6> (<6>, at 4.2% the activity with kaempferol [7]) (Reversibility: ? <6> [7]) [7] P UDP + apigenin 3-O-glucoside S UDPglucose + cyanidin <6, 8> (<6>, at 5% the activity with kaempferol [7]) (Reversibility: ? <6, 8> [7, 13]) [7, 13] P UDP + cyanidin 3-O-glucoside S UDPglucose + delphinidin <8> (Reversibility: ? <8> [13]) [13] P UDP + delphinidin 3-O-glucoside S UDPglucose + dihydrokaempferol <6, 9> (<6>, 9.6% of the activity with kaempferol [7]; <9>, isoenzyme F3GT1: 7% of the activity with kaempferol, isoenzyme F3GT2: 7% of the activity with kaempferol [10]) (Reversibility: ? <6, 9> [7, 10]) [7, 10] P UDP + dihydrokaempferol 3-O-glucoside S UDPglucose + dihydroquercetin <6, 9> (<9>, isoenzyme F3GT1: 3% of the activity with kaempferol, isoenzyme F3GT2: 2% of the activity with kaempferol [10]) (Reversibility: ? <6, 9> [7, 10]) [7, 10] P UDP + dihydroquercetin 3-O-glucoside S UDPglucose + fisetin <5, 9> (Reversibility: ? <5, 9> [5, 10]) [5, 10] P UDP + fisetin 3-O-glucoside S UDPglucose + isokuranetin <6> (<6>, at 11.2% the activity with kaempferol [7]) (Reversibility: ? <6> [7]) [7] P UDP + isokuranetin 3-O-glucoside S UDPglucose + isorhamnetin <2, 3, 5, 9, 10, 13> (<2>, isorhamnetin is the best acceptor [2]; <10>, 108% of the activity with kaempferol [11]; <13>, 71% of the activity with kaempferol [16]) (Reversibility: r <2> [2]; ? <3, 5, 9, 10, 13> [3, 5, 10, 11, 16]) [2, 3, 5, 10, 11, 16] P UDP + isorhamnetin 3-O-glucoside S UDPglucose + kaempferol <1, 2, 3, 5, 6, 7, 9, 10, 13> (<1>, 70% of the activity with quercetin [1]; <2>, 45% of the activity with isorhamnetin [2]) (Reversibility: r <1, 2> [2, 6]; ? <1, 3, 5, 6, 7, 9, 10, 13> [1, 3, 5, 7, 8, 10, 11, 16]) [1, 2, 3, 5, 7, 8, 10, 11, 16] P UDP + kaempferol 3-O-glucoside <1, 7> [6, 8] S UDPglucose + kaempferol 3,4'-dimethyl ether <9, 13> (<9>, isoenzyme F3GT1: 4% of the activity with kaempferol, isoenzyme F3GT2: 6% of the activity with kaempferol [10]; <13>, 4% of the activity with kaempferol [16]) (Reversibility: ? <9, 13> [10, 16]) [10, 16] P UDP + kaempferol 3,4'-dimethyl ether 3-O-glucoside S UDPglucose + kaempferol 5,7,4'-trimethyl ether <9, 10, 13> (<9>, isoenzyme F3GT1: 52% of the activity with kaempferol, isoenzyme F3GT2: 11% of the activity with kaempferol [10]; <10>, 9% of the activity with kaempferol [11]; <13>, 4% of the activity with kaempferol [16]) (Reversibility: ? <9, 10, 13> [10, 11, 16]) [10, 11, 16] P UDP + kaempferol 5,7,4'-trimethyl ether 3-O-glucoside S UDPglucose + kaempferol 7-O-glucoside <9, 10> (<9>, isoenzyme F3GT1: 6% of the activity with kaempferol, isoenzyme F3GT2: 5% of the activity with kaempferol [10]; <10>, 10% of the activity with kaempferol [11]) (Reversibility: ? <9, 10> [10, 11]) [10, 11] 23

Flavonol 3-O-glucosyltransferase

2.4.1.91

P UDP + kaempferol 3,7-O-diglucoside S UDPglucose + kaempferol-4'-O-methylether <1, 2, 9, 10> (<1>, 57% of the activity with quercetin [1]; <2>, 13% of the activity with isorhamnetin [2]; <10>, 42% of the activity with kaempferol [11]) (Reversibility: r <2> [2]; ? <1, 9, 10> [1, 10, 11]) [1, 2, 10, 11] P UDP + ? S UDPglucose + malvidin <6, 8> (<6>, at 4% the activity with kaempferol [7]) (Reversibility: ? <6, 8> [7, 13]) [7, 13] P UDP + malvidin 3-O-glucoside S UDPglucose + myricetin <9, 10, 13> (<10>, 20% of the activity with kaempferol [11]; <13>, 43% of the activity with kaempferol [16]) (Reversibility: ? <9, 10, 13> [10, 11, 16]) [10, 11, 16] P UDP + myrecetin 3-O-glucoside S UDPglucose + naringenin <6> (<6>, at 3.2% the activity with kaempferol [7]) (Reversibility: ? <6> [7]) [7] P UDP + naringenin 3-O-glucoside S UDPglucose + pelargonidin <6> (<6>, at 4% the activity with kaempferol [7]) (Reversibility: ? <6> [7]) [7] P UDP + pelargonidin 3-O-glucoside S UDPglucose + quercetin <1-10, 13> (<1>, quercetin is the best acceptor [1]; <2>, 93% of the activity with isorhamnetin [2]; <6>, 61.2% of the activity with kaempferol [7]; <10>, 95% of the activity with quercetin [11]; <13>, 76% of the activity with kaempferol [16]) (Reversibility: r <1, 2> [2, 6]; ? <1, 3-5, 7-10, 13> [1, 3, 4, 5, 8, 10, 11, 13, 16]) [1-8, 10, 11, 13, 16] P UDP + quercetin 3-O-glucoside <1, 7> [1, 6, 8] S UDPglucose + quercetin 5-methyl ether <9, 10> (<9>, isoenzyme F3GT1: 38% of the activity with kaempferol, isoenzyme F3GT2: 25% of the activity with kaempferol [10]; <10>, 7% of the activity with kaempferol [11]) (Reversibility: ? <9, 10> [10, 11]) [10, 11] P UDP + quercetin 5-methyl ether 3-O-glucoside S UDPglucose + quercetin 7-O-glucoside <9, 10> (<9>, isoenzyme F3GT1: 6% of the activity with kaempferol, isoenzyme F3GT2: 6% of the activity with kaempferol [10]; <10>, 12% of the activity with kaempferol [11]) (Reversibility: ? <9, 10> [10, 11]) [10, 11] P UDP + quercetin 3,7-O-diglucoside S UDPglucose + rhamnetin <9, 10, 13> (<9>, isoenzyme F3GT1: 20% of the activity with kaempferol, isoenzyme F3GT2: 82% of the activity with kaempferol [10]; <10>, 98% of the activity with kaempferol [11]; <13>, 70% of the activity with kaempferol [16]) (Reversibility: ? <9, 10, 13> [10, 11, 16]) [10, 11, 16] P UDP + rhamnetin 3-O-glucoside S UDPglucose + rutin <2> (<2>, 4% of the activity with isorhamnetin [2]) (Reversibility: r <2> [2]) [2] P UDP + rutin 3-O-glucoside

24

2.4.1.91

Flavonol 3-O-glucosyltransferase

S Additional information <1, 4, 5, 8> (<1>, no activity with dihydroquercetin [1]; <4,8>, only UDPglucose can serve as glucosyl donor [4,13]; <5>, no activity observed with cinnamic acids or simple phenols [5]) [1, 4, 5, 13] P ? Inhibitors CaCl2 <9, 10> (<9>, 10 mM, 9% inhibition [10]; <10>, 10 mM, 17% inhibition [11]) [10, 11] CoCl2 <9, 10> (<9>, 1 mM, 50% inhibition of isoenzyme F3GT1 and 25% inhibition of isoenzyme F3GT2 [10]; <10>, 1 mM, 35% inhibition [11]) [10, 11] Cu2+ <4, 8, 9, 10, 13> (<4>, 1 mM [4]; <9>, 1 mM CuCl2 , 93% inhibition of isoenzyme F3GT1 and 94% inhibition of isoenzyme F3GT2 [10]; <10>, 1 mM CuCl2 , 96% inhibition [11]) [4, 10, 11, 13, 16] EDTA <9> (<9>, 10 mM, slight inhibition [10]) [10] KCl <9, 10> (<9>, 10 mM, 9-10% inhibition [10]; <10>, 10 mM, 17% inhibition [11]) [10, 11] Mg2+ <8, 10> (<10>, 1 mM MgCl2 , 18% inhibition [11]) [11, 13] Mn2+ <9, 10> (<9>, 10 mM MnCl2 , 52% inhibition of isoenzyme F3GT1 and 28% inhibition of isoenzyme F3GT2 [10]; <10>, 10 mM MnCl2 , 35% inhibition [11]) [10, 11] NEM <9, 10, 13> [10, 11, 16] PCMB <2, 9, 10, 13> [2, 10, 11, 16] Zn2+ <4, 8, 9, 10, 13> (<4>, 1 mM [4]; <9>, 1 mM ZnCl2 , 93% inhibition of isoenzyme F3GT1 and 94% inhibition of isoenzyme F3GT2 [10]; <10>, 1 mM ZnCl2 , 96% inhibition [11]) [4, 10, 11, 13, 16] dithioerythritol <9, 13> [10, 16] iodoacetamide <9, 13> [10, 16] iodoacetate <9, 10, 13> [10, 11, 16] phenylmercuriacetate <9, 10> [10, 11] Additional information <1> (<1>, enzyme is inhibited in the crude extract [1]) [1] Activating compounds 2-mercaptoethanol <2, 3, 9, 10, 13> (<9>, 14 mM, activation of isoenzyme F3GT1 to 160%, activation of isoenzyme F3GT2 to 136% [10]; <10>, 14 mM, activation to 157% [11]; <13>, 14 mM, stimulates to 134% [16]; <2>, 1 mM, activation to 120% [2]; <3>, 14 mM, stimulates [3]) [2, 3, 10, 11, 16] EDTA <9, 10> (<9>, slight activation [10]; <10>, 1 mM, activation to 173% [11]) [10, 11] dithioerythritol <2, 10> (<2>, 1 mM, activation to 124% [2]; <10>, 10 mM, activation to 215% [11]) [2, 11] glutathione <2> (<2>, 1 mM, activation to 118% [2]) [2] glycine-HCl <1> (<1>, glycine-HCl buffer stimulates about 2fold at pH 9 in comparison with Tris-HCl buffer [1]) [1] sucrose <2> (<2>, 5%, activation to 123% [2]) [2]

25

Flavonol 3-O-glucosyltransferase

2.4.1.91

Metals, ions Mg2+ <4, 9> (<4>, 1 mM, stimulates [4]; <9>, 10 mM, slight stimulatory effect on isoenzyme F3GT1 and F3GT2 [10]) [4, 10] Specific activity (U/mg) Additional information <6, 7, 13> [7, 8, 16] Km-Value (mM) 0.00067 <10> (kaempferol) [11] 0.001 <1> (quercetin, <1>, less than 0.001 mM [1]) [1] 0.00114 <13> (kaempferol) [16] 0.00121 <4> (quercetin) [4] 0.007 <7> (quercetin) [8] 0.0098 <4> (UDPglucose) [4] 0.012 <7> (kaempferol) [8] 0.0125 <6> (kaempferol) [7] 0.0133 <13> (UDPglucose) [16] 0.015 <8> (quercetin, <8>, recombinant enzyme [13]) [13] 0.016 <8> (delphinidin, <8>, recombinant enzyme [13]) [13] 0.03 <8> (cyanidin, <8>, recombinant enzyme [13]) [13] 0.0357 <8> (malvidin, <8>, recombinant enzyme [13]) [13] 0.07 <6> (cyanidin, <6>, about [7]) [7] 0.07 <6> (quercetin, <6>, about [7]) [7] 0.126 <5> (quercetin) [5] 0.172 <5> (kaempferol) [5] 0.18 <7> (UDPglucose) [8] 0.2 <5> (isorhamnetin) [5] 0.27 <5> (fisetin) [5] 0.3 <5> (UDPglucose) [5] 0.5 <1> (UDPglucose) [1] 0.69 <3> (kaempferol) [3] 1 <6> (UDPglucose) [7] 1.67 <3, 10> (UDPglucose) [3, 11] 1.9 <8> (UDPglucose, <8>, recombinant enzyme [13]) [13] pH-Optimum 5 <6> [7] 5.8-6.2 <7> [8] 7.5 <4, 9, 10, 13> (<10>, in histidine-HCl buffer [11]; <13>, Tris-HCl buffer [16]) [4, 10, 11, 16] 8 <1, 8, 10, 13> (<1>, broad pH-optimum around pH 8 [1]; <10>, in imidazole-HCl buffer [11]; <8>, reaction with quercetin or cyanidin [13]; <13>, imidazole-HCl buffer [16]) [1, 11, 13, 16] 8.5 <5> [5] 8.5-9 <2> [2]

26

2.4.1.91

Flavonol 3-O-glucosyltransferase

pH-Range 4-9 <6> (<6>, pH 4.0: about 60% of maximal activity, pH 9.0: about 70% of maximal activity [7]) [7] 4.5-8 <7> (<7>, pH 4.5: about 80% of maximal activity, pH 8.0: about 55% of maximal activity [8]) [8]

4 Enzyme Structure Molecular weight 40000 <2> (<2>, gel filtration [2]) [2] 43000 <3, 10, 13> (<3,10,13>, gel filtration [3,11,16]) [3, 11, 16] 48000 <9> (<9>, gel filtration [10]) [10] 49000 <6> (<6>, gel filtration [7]) [7] 51000 <4> (<4>, gel filtration [4]) [4] 59000 <7> (<7>, gel filtration [8]) [8] Subunits dimer <6, 7> (<6>, 2 * 24500, SDS-PAGE [7]; <7>, 2 * 59000, SDS-PAGE [8]) [7, 8]

5 Isolation/Preparation/Mutation/Application Source/tissue anther <2> [2] cell suspension culture <1, 5> (<5>, the specific activity increases with age of the culture, reaching a maximum late in the growth cycle [5]) [1, 5] exocarp <8> (<8>, gene expression in the red-skin sports seems to be the result of a mutation in this gene or a mutation in a regulatory gene controlling its expression [9]) [9] flower <14> (<14>, mRNA expression increases in the early developmental stages, reaching the maximum at the stage before flower opening [15]) [15] fruit <8> [13] leaf <4, 8, 9, 13> (<9,13>, young [10,16]) [4, 10, 13, 16] petal <6> [7] seedling <3, 7, 10> [3, 8, 11] Purification <2> (partial [2]) [2] <3> [3] <4> [4] <6> [7] <7> [8] <8> (recombinant enzyme [13]) [13] <9> (isoenzyme F3GT1 and F3GT2 [10]) [10] <10> (partial [11]) [11] <13> (partial [16]) [16] 27

Flavonol 3-O-glucosyltransferase

2.4.1.91

Cloning <8> [13] <11> (a binary vector containing an UDPglucose:flavonoid-3-O-glucosyltransferase cDNA under the control of the cauliflower mosaic virus 35S promoter is used to transform Eustoma grandiflorum Grise. Of four independent transgenic lines recovered, one produces high levels of the UDPglucose:flavonoid-3-O-glucosyltransferase transcript and synthesizes 3-O-glucosylated anthocyanins novel to Eusoma grandiflorum, as well as enhanced levels of 3-Oglucosylated flavonols [12]) [12] <12> (expression in Escherichia coli [14]) [14] <14> [15]

6 Stability General stability information <8>, at least three freeze-thaw cycles can be tolerated without apparent loss of activity [13] Storage stability <8>, -20 C or 4 C, the recombinant enzyme is stable for at least 120 h [13] <8>, -80 C, stable for many months [13] <10>, -20 C, 20 mM imidazole-HCl, pH 8.0, 14 mM 2-mercaptoethanol, 10% glycerol, 35% loss of activity after 2 days, 61% loss of activity after 1 week [11] <13>, -20 C, 20 mM Tris-HCl, pH 7.5, 14 mM 2-mercaptoethanol, 10% glycerol, 15% loss of activity after 2 days [16]

References [1] Sutter, A.; Grisebach, H.: UDP-glucose:flavonol 3-0-glucosyltransferase from cell suspension cultures of parsley. Biochim. Biophys. Acta, 309, 289295 (1973) [2] Kleinehollenhorst, G.; Behrens, H.; Pegels, G.; Srunk, N.; Wiermann, R.: Formation of flavonol 3-O-diglycosides and flavonol 3-O-triglycosides by enzyme extracts from anthers of Tulipa cv apeldoorn - characterization and activity of 3 different O-glycosyltransferases during anther development. Z. Naturforsch. C, 37, 587-599 (1982) [3] Ishikura, N.; Mato, M.: Partial purification and some properties of flavonol 3-O-glycosyltransferases from seedlings of Vigna mungo, with special reference to the formation of kaempferol 3-O-galactoside and 3-O-glucoside. Plant Cell Physiol., 34, 329-335 (1993) [4] Ishikura, N.; Kazumi, Y.: Detection and characterization of UDP-glucose:flavonoid O-glucosyltransferases in the leaves of Prunus yedoensis Matsum. Plant Cell Physiol., 31, 1109-1115 (1990)

28

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Flavonol 3-O-glucosyltransferase

[5] Poulton, J.E.; Kauer, M.: Identification of an UDP-glucose:flavonol 3-Oglucosyl-transferase from cell suspension cultures of soybean (Glycine max L.). Planta, 136, 53-59 (1977) [6] Sutter, A.; Grisebach, H.: Free reversibility of the UDP-glucose:flavonol 3-Oglucosyltransferase reaction. Arch. Biochem. Biophys., 167, 444-447 (1975) [7] Hrazdina, G.: Purification and properies of a UDPglucose: flavonoid 3-Oglucosyltransferase from Hippeastrum petals. Biochim. Biophys. Acta, 955, 301-309 (1988) [8] Sun, Y.; Hrazdina, G.: Isolation and characterization of a UDPglucose:flavonol O3 -glucosyltransferase from illuminated red cabbage (Brassica oleracea cv. Red Danish) seedlings. Plant Physiol., 95, 570-576 (1991) [9] Larson, R.L.: Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin. Phytochemistry, 10, 3073-3076 (1971) [10] Larson, R.L.; Lonergan, C.M.: Multiple forms of flavonol O-glucosyltransferases in young leaves of Euonymus alatus f. ciliato-denatus. Planta, 103, 361-364 (1972) [11] Ishikura, N.; Mato, M.: Partial purification and some properties of flavonol 3-O-glycosyltransferases from seedlings of Vigna mungo, with special reference to the formation of kaempferol 3-O-galactoside and 3-O-glucoside. Plant Cell Physiol., 34, 329-335 (1993) [12] Schwinn, K.E.; Davies, K.M.; Deroles, S.C.; Markham, K.R.; Miller, R.M.; Bradley, J.M.; Manson, D.G.; Given, N.K.: Expression of an Antirrhinum majus UDP-glucose:flavonoid-3-O-glucosyltransferase transgene alters flavonoid glycosylation and acylation in lisianthus (Eustoma grandiflorum Grise.). Plant Sci., 125, 53-61 (1997) [13] Ford, C.M.; Boss, P.K.; Hoj, P.B.: Cloning and characterization of Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, a homolog of the enzyme encoded by the maize bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo. J. Biol. Chem., 273, 9224-9233 (1998) [14] Nakajima, J.I.; Tanaka, Y.; Yamazaki, M.; Saito, K.: Reaction mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis. J. Biol. Chem., 276, 25797-25803 (2001) [15] Yamazaki, M.; Yamagishi, E.; Gong, Z.; Fukuchi-Mizutani, M.; Fukui, Y.; Tanaka, Y.; Kusumi, T.; Yamaguchi, M.; Saito, K.: Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmentally regulated expression. Plant Mol. Biol., 48, 401-411 (2002) [16] Ishikura, N.; Yang, Z.Q.; Teramoto, S.: UDP-d-glucose:flavonol 3-O- and 7O-glucosyl transferases from young leaves of Paederia seandens var. mairei. Z. Naturforsch. C, 48, 563-569 (1993)

29

(N-Acetylneuraminyl)galactosylglucosylceramide N-acetylgalactosaminyltransferase

2.4.1.92

1 Nomenclature EC number 2.4.1.92 Systematic name UDP-N-acetyl-d-galactosamine:(N-acetylneuraminyl)-d-galactosyl-d-glucosylceramide N-acetyl-d-galactosaminyltransferase Recommended name (N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase Synonyms GM2 synthase GM2/GD2-synthase GalNAcT UDP acetylgalactosamine-(N-acetylneuraminyl)-d-galactosyl-d-glucosylceramide UDP-N-acetylgalactosamine GM3 N-acetylgalactosaminyltransferase UDP-N-acetylgalactosaminyltransferase I acetylgalactosaminyltransferase acetylgalactosaminyltransferase acetylgalactosaminyltransferase, uridine diphosphoacetylgalactosamineganglioside GM3 ganglioside GM2 synthase ganglioside GM3 acetylgalactosaminyltransferase uridine diphosphoacetylgalactosamine-acetylneuraminylgalactosylglucosylceramide uridine diphosphoacetylgalactosamine-ganglioside GM3 acetylgalactosaminyltransferase uridine diphosphoacetylgalactosamine-hematoside acetylgalactosaminyltransferase CAS registry number 67338-98-1

2 Source Organism <1> Rattus norvegicus [1-4, 7-10] <2> Homo sapiens [3, 5, 18]

30

2.4.1.92

<3> <4> <5> <6>

(N-Acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase

Mus musculus (embryo [11]) [6, 11] Cricetulus griseus [12] Homo sapiens [13-17, 19] Mus musculus [17]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-galactosamine + (N-acetylneuraminyl)-d-galactosyl-d-glucosylceramide = UDP + N-acetyl-d-galactosaminyl-(N-acetylneuraminyl)-dgalactosyl-d-glucosylceramide (<1> mechanism [7]) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-galactosamine + (N-acetylneuraminyl)-d-galactosyl-dglucosylceramide <1, 5> (<1> involved in biosynthesis of gangliosides in brain [2]; <1> model for ganglioside biosynthesis [7]; <5> relative glycosyltransferase levels involved in determining the ganglioside composition of cells [16]) (Reversibility: ? <1, 5> [2, 7, 16]) [2, 7, 16] P UDP + N-acetyl-d-galactosaminyl-(N-acetylneuraminyl)-d-galactosyl-dglucosylceramide Substrates and products S GM3(O-acetyl-(N-glycolyl)neuraminic acid) + UDP-N-acetyl-d-galactosamine <1> (<1> less effective substrate [2]) (Reversibility: ? <1> [1]) [2] P UDP + N-acetyl-d-galactosaminyl-GM3(O-acetyl-(N-glycolyl)neuraminic acid) S UDP-N-acetyl-d-galactosamine + (N-acetylneuraminyl)-d-galactosyl-dglucosylceramide <1-3, 5> (<1,5> i.e. GM3(N-acetylneuraminic acid), best substrate [2,17]; <1,2> from human spleen or rat liver [3]; <1,3> 73%-78% as effective as GM3(N-glycolylneuraminic acid) [6,10]; <1> less effective: GM3(O-acetyl-(N-glycolyl)neuraminic acid) [2];) (Reversibility: ? <1-3, 5> [2-4, 6-10, 17]) [2-4, 6-10, 17] P UDP + N-acetyl-d-galactosaminyl-(N-acetylneuraminyl)-d-galactosyl-dglucosylceramide <1, 3> (i.e. GM2(N-acetylneuraminic acid)-ganglioside) [2-4, 6-10] S UDP-N-acetyl-d-galactosamine + GD3-ganglioside <1, 3, 5> (<3> GD3(N-glycolylneuraminic acid): 66% as effective as GM3(N-glycolylneuraminic acid), GD3(N-acetylneuraminic acid): 76% as effective as GM3(N-glycolylneuraminic acid) [6]) (Reversibility: ? <1, 3, 5> [6, 7, 17]) [6, 7, 17] P UDP + GD2-ganglioside <1> [7] S UDP-N-acetyl-d-galactosamine + GM3(N-glycolylneuraminic acid) <1, 3> (Reversibility: ? <1, 3> [6, 10]) [6, 10] P UDP + N-acetyl-d-galactosaminyl-GM3(N-glycolylneuraminic acid)

31

(N-Acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase

2.4.1.92

S UDP-N-acetyl-d-galactosamine + GM3(N-glycolylneuraminic acid)-ganglioside <1, 3> (<1,3> best substrate [6, 10]; <1> not as effective as GM3(N-acetylneuraminic acid) [2]) (Reversibility: ? <1, 3> [2, 6, 10]) [2, 6, 10] P UDP + GM2(N-glycolylneuraminic acid)-ganglioside <1> [10] S UDP-N-acetyl-d-galactosamine + N-acetylneuraminelactose <1> (<1> i.e. sialyllactose [10]) (Reversibility: ? <1> [10]) [10] P UDP + N-acetyl-d-galactosaminyl-N-acetylneuraminelactose S UDP-N-acetyl-d-galactosamine + SM3-ganglioside <1> (<1> best substrate, no substrates are SM4-ganglioside, Gb3- and Gb4-ceramide [9]) (Reversibility: ? <1> [9]) [9] P UDP + SM2-ganglioside <1> [7] S UDP-N-acetyl-d-galactosamine + lactosylceramide <1> (Reversibility: ? <1> [7]) [7] P UDP + GA2-ganglioside <1> [7] S Additional information <1, 3, 5> (<3> poor substrate: sialyllactose [6]; <1> poor substrate: GM2-, GM1-, GD1b- and GT1-gangliosides and various ceramides [2]; <1> acceptor specificity [4]; <1> no substrate: N-acetylglucosamine [2]; <3> no substrate: lactosylceramide, sialylparagloboside, GM1b(N-glycolylneuraminic acid), GD1a(N-acetylneuraminic acid), GT1b(N-acetylneuraminic acid), GQ1b(N-acetylneuraminic acid), GM1a(N-acetylneuraminic acid), GD1b(N-acetylneuraminic acid), globoside [6]; <1> no substrate: blood type H glycoprotein, deglucosylated bovine submaxillary mucin, various ceramides [10]; <1> no substrates are SM4-ganglioside, Gb3- and Gb4-ceramide [9]; <5> overview on substrate specificity [17]) [2, 4, 6, 9, 10, 17] P ? Inhibitors CMP <1> [3] Chelex-100 <1> [10] EDTA <1> [10] GD3 <1> (<1> GM3 as substrate [7]) [7] GM3 <1> (<1> GD3 as substrate [7]) [7] IMP <1> [3] UDPgalactose <1> [10] adenosine-5'-[a,b-methylene]triphosphate <1> [3] ganglioside GD1a <1> [8] ganglioside GM1 <1> [8] ganglioside GM2 <1> [8] ganglioside GQ1b <1> (<1> not in detergent-solubilized enzyme assays, kinetics [8]) [8] ganglioside GT1b <1> [8] Activating compounds CF-54 <1> [2] Triton X-100 <1, 3> [3, 4, 6-8] Tween 80 <1> [2] 32

2.4.1.92

(N-Acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase

cardiolipin <1> [3] detergents <1, 3> (<1,3> requirement, solubilized enzyme, e.g. Triton X-100, [3,4,6-8]; <1> CF-54, Tween 80 [2]) [2-4, 6-8] endogen lipid factor <3> [6] heptylthioglucoside <1> (<1> activation [9]) [9] octylglucoside <1> (<1> activation [3,8]; <1> optimal: 0.8% [3]) [3, 8] phosphatidylglycerol <1> (<1> activation, detergent-free assay [4,8]) [4, 8] Metals, ions Ca2+ <3> (<3> activation, 27% as effective as Mn2+ [6]; <1> not [2,3,9,10]) [6] Cd2+ <1> (<1> activation, can replace Mn2+ to some extent [3]) [3] Co2+ <1, 3> (<1> activation, can replace Mn2+ to some extent [3]; <3> 62% as effective as Mn2+ [6]; <1> not [10]) [3, 6] Cu2+ <1> (<1> activation, can replace Ni2+ to some extent [10]) [10] Fe2+ <1, 3> (<1> activation, can replace Mn2+ [3]; <3> activation, can replace Ni2+ to some extent, 17% as effective as Mn2+ [6]) [3, 6, 10] Mg2+ <3> (<3> activation, 22% as effective as Mn2+ [6]; <1> not [2,3,9,10]) [6] Mn2+ <1, 3> (<3> requirement, 2.5-10 mM [6]; <1> can replace Ni2+ to some extent [10]) [2, 3, 6, 9, 10] Ni2+ <1, 3> (<1> requirement, most active, 10-20 mM [10]; <1> can replace Mn2+ to some extent [3]; <3> 5% as effective as Mn2+ [6]; <1> not [2]) [3, 6, 10] Additional information <1> (<1> no activation by K+ , Al3+ , [2]; <1> no activation by Zn2+ [9,10]; <1> no activation by Ba2+ [3]) [2, 3, 9, 10] Specific activity (U/mg) 0.000149 <1> [10] 0.0004-0.0005 <1> [3] 3.6 <3> [6] Km-Value (mM) 0.007 <3> (UDP-N-acetyl-d-galactosamine) [6] 0.0166 <1> (GM3) [2] 0.017 <1> (UDP-N-acetyl-d-galactosamine) [10] 0.026 <1> (UDP-N-acetyl-d-galactosamine, <1> plus GM3 [9]) [9] 0.027 <3> (GD3(N-glycolylneuraminic acid)) [6] 0.035 <1> (UDP-N-acetyl-d-galactosamine) [3] 0.057 <1> (UDP-N-acetyl-d-galactosamine) [2] 0.082 <1> (UDP-N-acetyl-d-galactosamine, <1> plus SM3 [9]) [9] 0.1 <1> (GM3) [3] 0.16 <3> (GM3(N-glycolylneuraminic acid)) [6] 0.19 <1> (GM3) [9] 0.35 <3> (GD3(N-acetylneuraminic acid)) [6] 2.1 <1> (GM3(N-acetylneuraminic acid)) [9] Additional information <1> (<1> kinetic study [7]) [7]

33

(N-Acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase

2.4.1.92

pH-Optimum 6.7-6.9 <1> [10] 6.8-7.2 <1> [2] 7-7.5 <1> [9] 7.2 <1> [3] 7.5-7.9 <3> [6] pH-Range 6.2-8.5 <1> (<1> about half-maximal activity at pH 6.2 and about 85% of maximal activity at pH 8.5) [9] Temperature optimum ( C) 37 <1, 3> (<1,3> assay at [2,4,6-8]) [2, 4, 6-8]

4 Enzyme Structure Molecular weight 120000 <1> (<1> gel filtration [10]) [10] Subunits dimer <1, 3> (<1> 2 * 64000, SDS-PAGE [10]; <3> 2 * 65000, SDS-PAGE [6]) [6, 10] Additional information <3, 4, 5> (<3> probably enzyme complex with sialyltransferase II [11]; <4> enzyme is homodimer with antiparallel orientation of catalytic domains and intersubunit disulfide bonds [12]; <5> dimer formation occurs in ER, strategy to test for dimerization of Golgi membrane proteins [14]; <2> enzyme is functionally coupled to GD3 synthase [18]) [11, 12, 14, 18] Posttranslational modification glycoprotein <5> (<5> 3 N-glycosylation sites at 79,179, 274 [17]; <5> all 3 glycosylation sites occupied by glycans, elimination of one or several sites leads to decrease in activity up to 90% [14]) [14, 17]

5 Isolation/Preparation/Mutation/Application Source/tissue NG 108-15 cell <1> (<1> cell suspension culture [1]) [1] brain <1> [1, 2, 9] liver <1, 3> [3, 4, 6-8, 10] melanoma cell line <2> [5] neuroblastoma cell line <2> [5] ovary <4> [12] Localization Golgi apparatus <1, 3, 5> (<5> dimerization occurs in the ER [14]; <2> functionally coupled to GD3 synthase [18]) [3, 4, 6-8, 13, 14, 17, 18]

34

2.4.1.92

(N-Acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase

microsome <1> [2] mitochondrion <1> [2] Purification <1> (Triton X-100 solubilized, affinity chromatography on GM3-acid Sepharose [10]) [10] <3> [6] Cloning <2> (human cDNA clones by transfecting with polyoma T antigen, host recipient: mouse melanoma cell line B16 [1]) [5] Engineering D356A/D356N <5> (<5> variation of DXD motif, activity almost completely lost [19]) [19] D356E <5> (<5> variation of DXD motif, weak activity [19]) [19] D356N/D357N/D358N <5> (<5> variation of DXD motif, activity almost completely lost, no alteration in nucleotide binding [19]) [19] D357N <5> (<5> variation of DXD motif, weak activity [19]) [19] D358A/D358N <5> (<5> variation of DXD motif, activity almost completely lost [19]) [19] V352A <5> (<5> 167% of wild type activity [19]) [19] W354A <5> (<5> 24% of wild type activity [19]) [19] Additional information <3-6> (<3> fusion protein with green, red or yellow fluorescent protein, localization to ER and Golgi, probably forms complex with sialyl-transferase II [11]; <4> site directed mutations in several Cys residues reveal that enzyme is homodimer with antiparallel orientation of catalytic domains and intersubunit disulfide bonds [12]; <5> 3 different epitopetagged forms of enzyme, enzyme is homodimer and has disulfide bonds, is localized to Golgi [13]; <5> elimination of one or several glycosylation sites leads to decrease in activity up to 90% [14]; <6> overexpression of gene leads to shift of ganglioside components from b-series to a-series [17]) [11-13, 15, 17]

6 Stability General stability information <1>, detergents stabilize [10] <3>, glycerol, 20% v/v, stabilizes [6] Storage stability <1>, -80 C, several months, crude Triton X-100 extract [9] <1>, -80 C, stable in 20% v/v glycerol, 1% v/v Triton X-100 [10] <3>, -70 C, 3 months, crude [6] <3>, 4 C, purified enzyme, at least 6 days in 20% v/v glycerol, 1% w/v heptylthioglucoside, 1 M sucrose, 0.2 M NaCl and 1 mM EDTA in 0.03 M cacodylate buffer, pH 6.9 [6]

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(N-Acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase

2.4.1.92

References [1] Scheideler, M.A.; Dawson, G.: Direct demonstration of the activation of UDP-N-acetylgalactosamine: [GM3]N-acetylgalactosaminyltransferase by cyclic AMP. J. Neurochem., 46, 1639-1643 (1986) [2] Dicesare, J.L.; Dain, J.A.: The enzymic synthesis of ganglioside. IV. UDP-Nacetylgalactosamine:(N-acetylneuraminyl)-galactosylglucosyl ceramide Nacetylgalactosaminyltransferase in rat brain. Biochim. Biophys. Acta, 231, 385-393 (1971) [3] Senn, H.J.; Cooper, C.; Warnke, P.C.; Wagner, M.; Decker, K.: Ganglioside biosynthesis in rat liver. Characterization of UDP-N-acetylgalactosamine GM3 acetylgalactosaminyltransferase. Eur. J. Biochem., 120, 59-67 (1981) [4] Klein, D.; Pohlentz, G.; Schwarzmann, G.; Sandhoff, K.: Substrate specificity of GM2 and GD3 synthase of Golgi vesicles derived from rat liver. Eur. J. Biochem., 167, 417-424 (1987) [5] Nagata, Y.; Yamashiro, S.; Yodoi, J.; Lloyd, K.O.; Shiku, H.; Furukawa, K.: Expression cloning of b1,4 N-acetylgalactosaminyltransferase cDNAs that determine the expression of GM2 and GD2 gangliosides [published erratum appears in J Biol Chem 1994 Mar 4;269(9):7045]. J. Biol. Chem., 267, 1208212089 (1992) [6] Hashimoto, Y.; Sekine, M.; Iwasaki, K.; Suzuki, A.: Purification and characterization of UDP-N-acetylgalactosamine GM3/GD3 N-acetylgalactosaminyltransferase from mouse liver. J. Biol. Chem., 268, 25857-25864 (1993) [7] Pohlentz, G.; Klein, D.; Schwarzmann, G.; Schmitz, D.; Sandhoff, K.: Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver. Proc. Natl. Acad. Sci. USA, 85, 7044-7048 (1988) [8] Yusuf, H.K.M.; Schwarzmann, G.; Pohlentz, G.; Sandhoff, K.: Oligosialogangliosides inhibit GM2- and GD3-synthesis in isolated Golgi vesicles from rat liver. Biol. Chem. Hoppe-Seyler, 368, 455-462 (1987) [9] Nagai, K.I.; Ishizuka, I.: Biosynthesis of monosulfogangliotriaosylceramide and GM2 by N-acetylgalactosaminyltransferase from rat brain. J. Biochem., 101, 1115-1127 (1987) [10] Yanagisawa, K.; Taniguchi, N.; Makita, A.: Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 b-N-acetylgalactosaminyltransferase from rat liver. Biochim. Biophys. Acta, 919, 213-220 (1987) [11] Bieberich, E.; MacKinnon, S.; Silva, J.; Li, D.D.; Tencomnao, T.; Irwin, L.; Kapitonov, D.; Yu, R.K.: Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases. Biochemistry, 41, 11479-11487 (2002) [12] Li, J.; Yen, T.Y.; Allende, M.L.; Joshi, R.K.; Cai, J.; Pierce, W.M.; Jaskiewicz, E.; Darling, D.S.; Macher, B.A.; Young, W.W., Jr.: Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains. J. Biol. Chem., 275, 41476-41486 (2000) [13] Jaskiewicz, E.; Zhu, G.; Taatjes, D.J.; Darling, D.S.; Zwanzig, G.E., Jr.; Young, W.W., Jr.: Cloned b1,4N-acetylgalactosaminyltransferase: subcellular locali-

36

2.4.1.92

[14]

[15]

[16]

[17]

[18] [19]

(N-Acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase

zation and formation of disulfide bonded species. Glycoconjugate J., 13, 213-223 (1996) Zhu, G.; Jaskiewicz, E.; Bassi, R.; Darling, D.S.; Young, W.W., Jr.: b1 ,4 NAcetylgalactosaminyltransferase (GM2/GD2/GA2 synthase) forms homodimers in the endoplasmic reticulum: a strategy to test for dimerization of Golgi membrane proteins. Glycobiology, 7, 987-996 (1997) Haraguchi, M.; Yamashiro, S.; Furukawa, K.; Takamiya, K.; Shiku, H.; Furukawa, K.: The effects of the site-directed removal of N-glycosylation sites from b-1,4-N-acetylgalactosaminyltransferase on its function. Biochem. J., 312, 273-280 (1995) Ruan, S.; Raj, B.K.M.; Furukawa, K.; Lloyd, K.O.: Analysis of melanoma cells stably transfected with b1,4GalNAc transferase (GM2/GD2 synthase) cDNA: relative glycosyltransferase levels play a dominant role in determining ganglioside expression. Arch. Biochem. Biophys., 323, 11-18 (1995) Furukawa, K.; Takamiya, K.; Furukawa, K.: b1,4-N-acetylgalactosaminyltransferase-GM2/GD2 synthase: a key enzyme to control the synthesis of brain-enriched complex gangliosides. Biochim. Biophys. Acta, 1573, 356362 (2002) Daniotti, J.L.; Martina, J.A.; Giraudo, C.G.; Zurita, A.R.; Maccioni, H.J.F.: GM3 a2,8-sialyltransferase (GD3 synthase): protein characterization and sub-Golgi location in CHO-K1 cells. J. Neurochem., 74, 1711-1720 (2000) Li, J.; Rancour, D.M.; Allende, M.L.; Worth, C.A.; Darling, D.S.; Gilbert, J.B.; Menon, A.K.; Young, W.W., Jr.: The DXD motif is required for GM2 synthase activity but is not critical for nucleotide binding. Glycobiology, 11, 217-229 (2001)

37

Inulin fructotransferase (depolymerizing, difructofuranose-1,2':2,3'-dianhydrideforming)

2.4.1.93

1 Nomenclature EC number 2.4.1.93 (transferred to EC 4.2.2.18) Recommended name inulin fructotransferase (depolymerizing, difructofuranose-1,2':2,3'-dianhydride-forming)

38

Protein N-acetylglucosaminyltransferase

2.4.1.94

1 Nomenclature EC number 2.4.1.94 Systematic name UDP-N-acetyl-d-glucosamine:protein b-N-acetyl-d-glucosaminyltransferase Recommended name protein N-acetylglucosaminyltransferase Synonyms O-GlcNAc transferase OGT <1, 5-14> [7-12] acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-protein uridine diphospho-N-acetylglucosamine:polypeptide b-N-acetylglucosaminyltransferase uridine diphosphoacetylglucosamine-protein acetylglucosaminyltransferase CAS registry number 72319-34-7

2 Source Organism <-2> no activity in Saccharomyces cerevisiae (<-2> DNA sequence of OGT [8]) [8] <-1> no activity in Escherichia coli (<-1> DNA sequence of OGT [8]) [8] <1> Rattus norvegicus (Sprague-Dawley [3]) [1-3, 7, 11-13] <2> Oryctolagus cuniculus [2, 4-6] <3> Saccharomyces cerevisiae (mutant 2180-1A-5mnn2 [4]) [4] <4> Cercopithecus aethiops (COS-1 cell line [6]) [6] <5> Rattus norvegicus (gene Ogt encoding the 110 kDa a-subunit [7]) [7] <6> Mesocricetus auratus (chinese hamster [7]) [7] <7> Homo sapiens [8, 10] <8> Arabidopsis thaliana [8] <9> Rhodobacter sp. [8] <10> Caenorhabditis elegans [8] <11> Oryza sativa [8] <12> Hordeum vulgare [8] <13> petunia [8] <14> Mus musculus (Ogt gene, located on the X chromosome [9]) [9]

39

Protein N-acetylglucosaminyltransferase

2.4.1.94

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + protein = UDP + 4-N-(N-acetyl-d-glucosaminyl)-protein (<1> the enzyme achieves both high specificity and a remarkable diversity of substrates by complexing with a variety of targeting proteins via its TPR protein-protein interaction domains [13]; <2, 3> sequence Asn-X-Thr(Ser) at glycosylation site Asn-34 is necessary [4,5]) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + protein <1, 4, 5, 7-9, 14> (<1> involved in regulation of transcription and of granular secretion, important component of the glucose sensing mechanism in the pancreas [11]; <1> important role in hexosamine pathway [11]; <7> enzyme modulates insulin action, modifies kinases involved in the signaling cascade [10]; <14> role in control of gene transcription, neurofilament assembly, and the emergence of diabetes and neurologic disease, essential for embryonic stem cell viability and for ontogeny [9]; <7,8> possible role in ectopic protein interactions, transcriptional activation, protein degradation, and glucose sensing [8]; <8> enzyme serves to attenuate the gibberillin pathway [8]; <1, 5, 7-9> enzyme contains multiple tandem repeats of the tetratricopeptide repeat motif at the N-terminus, i.e. TPR: regulation by protein-protein interaction independent of the enzymic catalytic site at the C-terminus [7, 8, 10, 11, 13]; <5> regulation model [7]; <7-9> TPR structure and function [8]) (Reversibility: ? <1, 4, 5, 7-9, 14> [6-11, 13]) [6-11] P UDP + 4-N-(N-acetyl-d-glucosaminyl)-protein <1, 4, 5, 7-9, 14> [6-11, 13] S UDP-N-acetylglucosamine + nuclear pore protein p62 <4> (<4> recombinant rat protein substrate expressed in COS-1 cells [6]) (Reversibility: ? <4> [6]) [6] P UDP + nuclear pore protein with O-linked N-acetylglucosamine <4> [6] Substrates and products S UDP-N-acetyl-d-glucosamine + protein <1-5, 7-14> (<1> substrate: transcription factors [11]; <7> enzyme performs also autoglycosylation [10]; <7-9> substrates are many proteins, e.g. transcription factors, kinases, cytoskeletal proteins, and nuclear pore proteins [8]) (Reversibility: ? <15, 7-14> [1-13]) [1-13] P UDP + 4-N-(N-acetyl-d-glucosaminyl)-protein <1-5, 7-14> (<1, 7, 14> Nacetyl-d-glucosamine is attached to Ser or Thr [9-11]) [1-13] S UDP-N-acetylglucosamine + OIP106 <1> (<1> multiple tandem repeats of the tetratricopeptide repeat motif at the N-terminus, i.e. TPR, function as substrate docking sites [13]; <1> OIP106 interacts with TPRs 2-6 of the enzyme [13]; <1> OIP106 is a member of the family of OGT-interacting proteins, i.e. OIPs [13]) (Reversibility: ? <1> [13]) [13]

40

2.4.1.94

Protein N-acetylglucosaminyltransferase

P UDP + OIP106 with O-linked N-acetylglucosamine <1> [13] S UDP-N-acetylglucosamine + RNA polymerase II <1> (Reversibility: ? <1> [11]) [11] P UDP + RNA polymerase II with O-linked N-acetylglucosamine <1> [11] S UDP-N-acetylglucosamine + Tyr-Ser-Asp-Ser-Pro-Ser-Thr-Ser-Thr <1> (<1> extremely high affinity for UDP-N-acetylglucosamine [1]) (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + N-acetylglucosamine with O-linkage to peptide <1> (<1> no epimerization to N-acetylgalactosamine of the attached monosaccharide [2]) [2] S UDP-N-acetylglucosamine + Tyr-Ser-Gly-Ser-Pro-Ser-Thr-Ser-Thr <1> (Reversibility: ? <1> [2]) [2] P UDP + N-acetylglucosamine with O-linkage to peptide <1> [2] S UDP-N-acetylglucosamine + YSDSGSTST <7> (Reversibility: ? <7> [10]) [10] P UDP + N-acetylglucosamine with O-linkage to peptide <7> [10] S UDP-N-acetylglucosamine + YSDSPSTST <7> (Reversibility: ? <7> [10]) [10] P UDP + N-acetylglucosamine with O-linkage to peptide <7> [10] S UDP-N-acetylglucosamine + casein kinase II <1, 7> (<1> CK II peptide [13]; <7> protein from human glioblastoma [10]) (Reversibility: ? <1, 7> [10, 13]) [10, 13] P UDP + casein kinase II with O-linked N-acetylglucosamine <1, 7> [10, 13] S UDP-N-acetylglucosamine + glycogen synthase kinase-3 <7> (<7> protein from rabbit skeletal muscle [10]) (Reversibility: ? <7> [10]) [10] P UDP + glycogen synthase kinase-3 with O-linked N-acetylglucosamine <7> [10] S UDP-N-acetylglucosamine + nuclear pore protein p62 <1, 2, 4, 7> (<4> recombinant rat protein substrate [6]) (Reversibility: ? <1, 2, 4, 7> [6, 10, 11, 13]) [6, 10, 11, 13] P UDP + nuclear pore protein with O-linked N-acetylglucosamine <1, 2, 4, 7> [6, 10, 11, 13] S UDP-N-acetylglucosamine + pancreatic ribonuclease A <1-3> (Reversibility: ? <1-3> [3-5]) [3-5] P UDP + pancreatic ribonuclease A with N-acetylglucosamine linked to Asp-34 <1-3> [3-5] S Additional information <1, 7> (<1> nuclear pore protein p62 and OIP106 are competing for the enzymes substrate docking domain [13]; <7> E. coli proteins are no substrates [10]; <1> no substrates: Tyr-Ser-Asp-SerGly-Ser-Thr-Ser-Thr and Tyr-Ser-Asp-Ser-Pro [2]) [2, 10] P ? Inhibitors 4-thiouridine diphosphate <1> (<1> irreversible inactivation by covalent attachment to the active site, photoinactivation [1]) [1] CDP <1> (<1> weak [1]) [1]

41

Protein N-acetylglucosaminyltransferase

2.4.1.94

EDTA <1> (<1> 5 mM, slight [2]) [2] Tunicamycin <3> (<1> not inhibitory [2,3]) [4] UDP <1> (<1> strong [2]) [1, 2] UDP-N-acetylgalactosamine <1> [1] UDP-N-acetylglucosamine <1> [1] UMP <1> [1] UTP <1> [1] free tetratricopeptide repeat motif peptides <1> (<1> i.e. TPR peptides [13]; <1> inhibition by the different tetratricopeptide repeat motif peptides of the enzyme is specific for single substrates [13]; <1> from the enzyme, recombinantly expressed in Escherichia coli, purified [13]) [13] Additional information <1> (<1> no inhibition by N-acetylglucosamine [2]) [2] Activating compounds Triton X-100 <2> (<2> 1.7fold at 0.04% [5]) [5] Metals, ions Ca2+ <1> (<1> activation [3]) [3] Co2+ <1, 2> (<1,2> activation [3,5]) [3, 5] Mn2+ <1-3> (<3> 2fold activation at 25 mM, slight activation at 20 mM and above 50 mM [4]; <1,2> activation [1,3,5]) [1, 3-5] Ni2+ <2> (<2> slight activation [5]) [5] Additional information <1, 2> (<2> Mg2+ is ineffective [5]; <1> no requirement for divalent cations [2]) [2, 5] Specific activity (U/mg) 0.00111 <1> [1] Additional information <1> [3] Km-Value (mM) 0.0005 <7> (UDP-N-acetylglucosamine) [10] 0.000545 <1> (UDP-N-acetylglucosamine) [1] 0.0012 <1, 7> (nuclear pore protein p62) [10, 13] 0.00335 <1> (OIP106) [13] 0.0046 <1> (UDP-N-acetylglucosamine) [3] 9.87 <1> (Tyr-Ser-Asp-Ser-Pro-Ser-Thr-Ser-Thr) [1] pH-Optimum 6 <1> [1] 6.4 <1> [3] 7.2 <3> (<3> assay at [4]) [4] 7.4 <1, 7> (<1,7> assay at [10,13]) [10, 13] pH-Range 6-7.5 <1> (<1> rapid decrease of activity below pH 6.0 [1]) [1] Temperature optimum ( C) 20 <1> (<1> assay at [1]) [1] 37 <1, 3, 7> (<1,3,7> assay at [4,10,11]) [4, 10, 11]

42

2.4.1.94

Protein N-acetylglucosaminyltransferase

4 Enzyme Structure Molecular weight 340000 <1> (<1> gel filtration [1]) [1] Additional information <1> (<1> amino acid sequence determination [12]) [12] Subunits ? <7> (<7> x * 103000, SDS-PAGE [10]) [10] trimer <1, 5> (<1,5> 1 * 78000 + 2 * 110000, a2 b, SDS-PAGE [1,7,11]) [1, 7, 11] Additional information <1, 5, 7-9> (<7-9> three-dimensional structure of the protein domains I and II, conserved amino acid sequences [8]; <5> amino acid sequence of 110 kDa a-subunit, alignment [7]; <1> a-subunits contain the catalytic site [1]) [1, 7, 8] Posttranslational modification glycoprotein <1, 5, 7> (<1> O-glycosylated [12]; <7> enzyme performs autoglycosylation [10]; <1,5,7> a- and b-subunits contain N-acetylglucosamine [7,10,11]) [7, 10-12]

5 Isolation/Preparation/Mutation/Application Source/tissue COS-1 cell <4> [6] Langerhans cell <1> (<1> A cells [11]) [11] bone marrow <14> [9] brain <1, 5, 14> [7, 9, 12] embryonic stem cell <14> [9] exocrine acinar cell <1> (<1> euchromatin [11]) [11] heart <1, 5, 14> [7, 9, 12] kidney <1, 5, 14> (<1> not [12]) [7, 9] liver <1, 2, 5, 14> [1-5, 7, 9, 11] lung <1, 5, 14> [7, 9, 12] lymph node <14> [9] ovary <5> [7] ovary cell line <6> [7] pancreas <1, 14> (<1> glucagon-secreting cells [11]; <1> polypeptide secreting cells [11]; <14> very low RNA amount [9]) [9, 11, 12] pancreatic b cell <1> [11] reticulocyte <2> (<2> commercial product [2,6]) [2, 6] skeletal muscle <1, 5, 14> [7, 9] small intestine <14> [9] spinal cord <14> [9] spleen <1, 5, 14> [7, 9, 12] stomach <14> [9]

43

Protein N-acetylglucosaminyltransferase

2.4.1.94

testis <14> [9] thymus <5, 14> (<1,5> highest activity [7]) [7, 9] uterus <5> [7] Additional information <5> (<5> enzyme activity does not in all tissues correlate with the amount of expressed protein [7]; <5> several Ogt-derived proteins present in most tissues [7]) [7] Localization cytosol <1, 5-14> (<1> 85% of overall activity [2]) [1, 2, 7-11, 13] membrane <1-3> (<1> 15% of overall activity [2]) [2-4] microsome <1> (<1> located on the cytosolic side [2]) [2, 3] nucleus <1, 5-14> (<1> restricted to euchromatin [11]) [7-11, 13] rough endoplasmic reticulum <2> [5] secretory granule <1> (<1> A cells [11]) [11] zymogen granule <1> [11] Purification <1> (His-tagged TPRs of the enzyme recombinant from Escherichia coli [13]; from brain [12]; partial [3]) [1, 3, 12, 13] <5> [7] <7> (recombinant S-tagged fusion protein from Escherichia coli [10]) [10] Cloning <1> (overexpression of His-tagged TPRs of the enzyme in Escherichia coli [13]; expression via baculovirus system, recombinant wild-type and deletion mutants [13]) [8, 13] <5> (overexpression of the Ogt gene encoding the 110 kDa a-subunit in Escherichia coli and transiently in HEK293 cells, DNA sequence analysis: gene Ogt is no member of a multigene family, the sequence contains multiple tandem repeats of the tetratricopeptide repeat motif [7]) [7] <7> (expression in Escherichia coli as S-tagged fusion protein [10]) [8, 10] <10> [8] <11> [8] <12> [8] <13> [8] Engineering Additional information <7, 8, 14> (<7> deletion of the first 3 TPR of the Nterminal sequence greatly reduces enzyme activity with macromolecular substrates, but not with synthetic peptides, deletion of the first 6 TPR increases the autoglycosylation activity [10]; <14> gene-targeting conditional mutagenesis of the single copy gene in male embryonic stem cells and germ line, deletion is lethal in ontogeny [9]; <8> deletional or disrupting mutation of the Spindly SPY locus affects the enzyme and the plant signaling, e.g. increased gibberillin signaling, altered phenotypes [8]) [8, 9]

44

2.4.1.94

Protein N-acetylglucosaminyltransferase

6 Stability pH-Stability 6.8 <1> (<1> irreversible inactivation during purification [1]) [1] General stability information <1>, inhibitor 4-thiouridine diphosphate stabilizes [1] Storage stability <1>, -20 C, 20 mM Tris/HCl buffer, pH 7.5, 40% ethylene glycol plus cytochrome c or bovine serum albumin, several months [1]

References [1] Haltiwanger, R.S.; Blomberg, M.A.; Hart, G.W.: Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide b-N-acetylglucosaminyltransferase. J. Biol. Chem., 267, 9005-9013 (1992) [2] Haltiwanger, R.S.; Holt, G.D.; Hart, G.W.: Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphosphoN-acetylglucosamine:peptide b-N-acetylglucosaminyltransferase. J. Biol. Chem., 265, 2563-2568 (1990) [3] Arakawa, H.; Mookerjea, S.: Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A. Eur. J. Biochem., 140, 297-302 (1984) [4] Khalkhali, Z.; Marshall, R.D.; Reuvers, F.; Habets-Willems, C.; Boer, P.: Glycosylation in vitro of an asparagine sequon catalysed by preparations of yeast cell membranes. Biochem. J., 160, 37-41 (1976) [5] Khalkhali, Z.; Marshall, R.D.: Glycosylation of ribonuclease A catalysed by rabbit liver extracts. Biochem. J., 146, 299-307 (1975) [6] Starr, C.M.; Hanover, J.A.: Glycosylation of nuclear pore protein p62. Reticulocyte lysate catalyzes O-linked N-acetylglucosamine addition in vitro. J. Biol. Chem., 265, 6868-6873 (1990) [7] Kreppel, L.K.; Blomberg, M.A.; Hart, G.W.: Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique OGlcNAc transferase with multiple tetratricopeptide repeats. J. Biol. Chem., 272, 9308-9315 (1997) [8] Roos, M.D.; Hanover, J.A.: Structure of O-linked GlcNAc transferase: Mediator of glycan-dependent signaling. Biochem. Biophys. Res. Commun., 271, 275-280 (2000) [9] Shafi, R.; Iyer, S.P.N.; Ellies, L.G.; O'Donnell, N.; Marek, K.W.; Chui, D.; Hart, G.W.; Marth, J.D.: The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny. Proc. Natl. Acad. Sci. USA, 97, 5735-5739 (2000)

45

Protein N-acetylglucosaminyltransferase

2.4.1.94

[10] Lubas, W.A.; Hanover, J.A.: Functional expression of O-linked GlcNAc transferase: domain structure and substrate specificity. J. Biol. Chem., 275, 10983-10988 (2000) [11] Akimoto, Y.; Hart, G.W.; Hirano, H.: Distribution of O-GlcNAc transferase in the rat pancreas. Acta Histochem. Cytochem., 33, 163-167 (2000) [12] Konrad, R.J.; Tolar, J.F.; Hale, J.E.; Knierman, M.D.; Becker, G.W.; Kudlow, J.E.: Purification of the O-glycosylated protein p135 and identification as OGlcNAc transferase. Biochem. Biophys. Res. Commun., 288, 1136-1140 (2001) [13] Iyer, S.P.; Hart, G.W.: Roles of the tetratricopeptide repeat domain in OGlcNAc transferase targeting and protein substrate specificity. J. Biol. Chem., 30, 30 (2003)

46

Bilirubin-glucuronoside glucuronosyltransferase

2.4.1.95

1 Nomenclature EC number 2.4.1.95 Systematic name bilirubin-glucuronoside:bilirubin-glucuronoside d-glucuronosyltransferase Recommended name bilirubin-glucuronoside glucuronosyltransferase Synonyms bilirubin glucuronoside glucuronosyltransferase bilirubin monoglucuronide transglucuronidase glucuronosyltransferase, bilirubin glucuronoside CAS registry number 71822-22-5

2 Source Organism <1> Rattus norvegicus (Wistar) [1-3]

3 Reaction and Specificity Catalyzed reaction 2 bilirubin-glucuronoside = bilirubin + bilirubin-bisglucuronoside Reaction type glucuronyl group transfer hexosyl group transfer Substrates and products S bilirubin monoglucuronide + bilirubin monoglucuronide <1> (Reversibility: ? <1> [1-3]) [1-3] P bilirubin diglucuronide + bilirubin <1> [1] Specific activity (U/mg) Additional information <1> [2, 3] Km-Value (mM) 0.033 <1> (bilirubin monoglucuronide) [2] 0.035 <1> (bilirubin monoglucuronide) [3] 47

Bilirubin-glucuronoside glucuronosyltransferase

2.4.1.95

pH-Optimum 6.6 <1> [1]

4 Enzyme Structure Molecular weight 160000 <1> (<1>, gel filtration [2,3]) [2, 3] Subunits ? <1> (<1>, x * 28000, SDS-PAGE [2,3]) [2, 3]

5 Isolation/Preparation/Mutation/Application Source/tissue liver <1> [1-3] Localization microsome <1> [2] plasma membrane <1> [1-3] Purification <1> [2, 3]

6 Stability General stability information <1>, following incubation of solubilized plasma membrane with pronase at 37 C for 180 min, 80% of enzyme activity is lost [3]

References [1] Jansen, P.L.M.; Chowdhury, J.R.; Fischberg, E.B.; Arias, I.M.: Enzymatic conversion of bilirubin monoglucuronide to diglucuronide by rat liver plasma membranes. J. Biol. Chem., 252, 2710-2716 (1977) [2] Chowdhury, J.R.; Arias, I.M.: Dismutation of bilirubin monoglucuronide. Methods Enzymol., 77, 192-197 (1981) [3] Chowdhury, J.R.; Chowdhury, N.R.; Bhargava, M.M.; Arias, I.M.: Purification and partial characterization of rat liver bilirubin glucuronoside glucuronosyltransferase. J. Biol. Chem., 254, 8336-8339 (1979)

48

sn-Glycerol-3-phosphate 1galactosyltransferase

2.4.1.96

1 Nomenclature EC number 2.4.1.96 Systematic name UDP-galactose:sn-glycerol-3-phosphate 1-a-d-galactosyltransferase Recommended name sn-glycerol-3-phosphate 1-galactosyltransferase Synonyms JFP-synthase UDP-Gal:sn-glycero-3-phosphoric acid 1-a-galactosyl-transferase UDPgalactose:sn-glycerol-3-phosphate a-d-galactosyltransferase galactosyltransferase, glycerol 3-phosphate 1agalactosyltransferse, uridine diphosphogalactose-glycerol phosphate isofloridoside-phosphate synthase Additional information (cf. EC 2.4.1.137) CAS registry number 9076-70-4

2 Source Organism <1> Ochromonas malhamensis [1, 2, 5] <2> Poterioochromonas malhamensis (Peterfi [4]) [3, 4]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + sn-glycerol 3-phosphate = UDP + a-d-galactosyl-(1,1')-snglycerol 3-phosphate Reaction type hexosyl group transfer Natural substrates and products S UDPgalactose + sn-glycerol 3-phosphate <1> (<1>, enzyme is involved in regulation of osmotic balance in Ochromonas [1,2]) (Reversibility: ? <1> [1]) [1, 2] P UDP + a-d-galactosyl sn-glycerol 3-phosphate <1> [1] 49

sn-Glycerol-3-phosphate 1-galactosyltransferase

2.4.1.96

Substrates and products S UDPgalactose + sn-glycerol 3-phosphate <1, 2> (Reversibility: ? <1, 2> [1-5]) [1-5] P UDP + a-d-galactosyl sn-glycerol 3-phosphate <1, 2> (<1>, i.e. isofloridoside phosphate [1]) [1-5] Activating compounds Additional information <1, 2> (<2>, proteolytic activation mechanism might be the first step in the activation of IFP-synthase after cell shrinkage due to osmotic water loss. About 1 h later when proteolytic activation is no longer evident and the isofloridoside pool is almost filled up a second maximum of active IFP-synthase is evident. This type of enzyme exhibits different molecular properties and might by involved in the turnover of the isofloridoside pool [4]; <1>, enzyme appears to exist as an inactive proenzyme which can be activated by incubation of crude cell extracts with endogenous or exogenous proteases [5]) [4, 5] Specific activity (U/mg) 198 <2> [3] Km-Value (mM) 0.01 <2> (UDPgalactose) [3] 0.02 <2> (sn-glycerol 3-phosphate) [3] pH-Optimum 7-8 <2> [3] pH-Range 6-9 <2> (<2>, about 50% of activity maximum at pH 6 and pH 9 [3]) [3]

4 Enzyme Structure Molecular weight 68000 <2> (<2>, gel filtration [3]) [3] Subunits monomer <2> (<2>, 1 * 70000, SDS-PAGE [3]) [3] Posttranslational modification proteolytic modification <1, 2> (<2>, proteolytic activation mechanism might be the first step in the activation of IFP-synthase after cell shrinkage due to osmotic water loss. About 1 h later when proteolytic activation is no longer evident and the isofloridose pool is almost filled up a second maximum of active IFP-synthase is evident. This type of enzyme exhibits different molecular properties and might by involved in the turnover of the isofloridoside pool [4]; <1>, enzyme appears to exist as an inactive proenzyme which can be activated by incubation of crude cell extracts with endogenous or exogenous proteases [5]) [4, 5]

50

2.4.1.96

sn-Glycerol-3-phosphate 1-galactosyltransferase

5 Isolation/Preparation/Mutation/Application Purification <2> [3]

6 Stability Temperature stability 0 <1> (<1>, 30 min, enzyme in crude extract, 50% loss of activity [1]) [1] 30 <1> (<1>, almost complete inactivation after some min [1]) [1] General stability information <1>, a combination of dithioerythritol and bovine serum g-globulin stabilizes the enzyme during cell destruction [1] <1>, the presumable proenzyme is fully stable even over 20 h in cell extract at pH 7.8 containing bovine serum albumin at 0 C, activation of the proenzyme is followed by rapid inactivation [5] <2>, cetyltrimethylammonium bromide, optimal concentration for stabilization, 0.075% w/v at pH 8.5, 0.1% w/v at pH 7.2, 0.2 w/v at pH 6.2 [3] <2>, glycerol, 30% v/v, stabilizes the enzyme at pH 7.8 but not at lower or higher pH values [3] <2>, unstable to repeated freezing and thawing [3]

References [1] Kauss, H.; Schobert, B.: First demonstration of UDP-Gal:sn-glycero-3-phosphoric acid 1-a-galactosyl-transferase and its possible role in osmoregulation. FEBS Lett., 19, 131-135 (1971) [2] Kauss, H.; Quader, H.: In vitro activation of a galactosyl transferase involved in the osmotic regulation of Ochromonas. Plant Physiol., 58, 295-298 (1976) [3] Thomson, K.S.: Purification of UDPgalactose:sn-glycerol-3-phosphate a-dgalactosyltransferase from Poterioochromonas malhamensis. Biochim. Biophys. Acta, 759, 154-159 (1983) [4] Kauss, H.; Thomson, K.S.; Thomson, M.; Jeblick, W.: Osmotic regulation. Physiological significance of proteolytic and nonproteolytic activation of isofloridoside-phosphate synthase. Plant Physiol., 63, 455-459 (1979) [5] Kauss, H.; Thomson, K.S.; Tetour, M.; Jeblick, W.: Proteolytic activation of a galactosyl tranferase involved in osmotic regulation. Plant Physiol., 61, 35-37 (1978)

51

1,3-b-D-Glucan phosphorylase

2.4.1.97

1 Nomenclature EC number 2.4.1.97 Systematic name 1,3-b-d-glucan:phosphate a-d-glucosyltransferase Recommended name 1,3-b-d-glucan phosphorylase Synonyms 1,3-b-d-glucan:orthophosphate glucosyltransferase b-(1-3)glucan phosphorylase laminarin phosphorylase laminarin phosphoryltransferase phosphorylase, 1,3-b-glucan Additional information (different from EC 2.4.1.30 and EC 2.4.1.31) CAS registry number 37340-31-1

2 Source Organism <1> Ochromonas malhamensis [1] <2> Acacia verek [2]

3 Reaction and Specificity Catalyzed reaction (1,3-b-d-glucosyl)n + phosphate = (1,3-b-d-glucosyl)n-1 + a-d-glucose 1phosphate Reaction type hexosyl group transfer Natural substrates and products S (1,3-b-d-glucosyl)n + phosphate <2> (<2>, enzyme may be a key factor in the regulation of cell-wall extension and build up by switching hydrolase and synthase activity in a balance dependent manner [2]) (Reversibility: ? <2> [2]) [2] P (1,3-b-d-glucosyl)n-1 + a-d-glucose 1-phosphate 52

2.4.1.97

1,3-b-D-Glucan phosphorylase

Substrates and products S (1,3-b-d-glucosyl)n + phosphate <1, 2> (<2>, in 50 mM citrate-phosphate buffer, pH 6.5, the phosphorolytic cleavage of laminarin and laminaribiose can be observed. In 0.02 M imidazole buffer, pH 6.5, the phosphorylase equilibrium lies in the direction of the synthesis reaction, a-d-glucose-1-phosphate as glucosyl donor is incorporated into laminaribiose [2]) (Reversibility: r <2> [2]; ? <1> [1]) [1, 2] P (1,3-b-d-glucosyl)n-1 + a-d-glucose 1-phosphate S laminaribiose + glucose 1-phosphate <2> (<2>, in 50 mM citrate-phosphate buffer, pH 6.5, the phosphorolytic cleavage of laminarin and laminaribiose can be observed. In 0.02 M imidazole buffer, pH 6.5, the phosphorylase equilibrium lies in the direction of the synthesis reaction, a-dglucose-1-phosphate as glucosyl donor is incorporated into laminaribiose [2]) (Reversibility: r <2> [2]) [2] P phosphate + laminarin Inhibitors AMP <1> (<1>, non-competitive inhibition at high concentration [1]) [1] Cofactors/prosthetic groups AMP <1> (<1>, low concentration: stimulation, Km : 0.04 mM [1]) [1] Specific activity (U/mg) 10.92 <1> [1] Km-Value (mM) 0.25 <2> (glucose) [2] 0.34 <2> (laminaribiose, <2>, phosphorolytic reaction [2]) [2] 0.47 <2> (laminarin, <2>, phosphorolytic reaction [2]) [2] 1 <1> (laminarin) [1] 1.45 <2> (laminaribiose, <2>, reaction with glucose 1-phosphate [2]) [2] 1.7 <2> (laminarin, <2>, reaction with glucose 1-phosphate [2]) [2] 12 <1> (glucose 1-phosphate) [1] 25 <1> (phosphate) [1] Ki-Value (mM) 20 <1> (AMP) [1] pH-Optimum 5.5 <1> [1] pH-Range 5-5.5 <1> (<1>, pH 5.0: 50% of activity maximum, pH 5.5: activity maximum [1]) [1] Temperature optimum ( C) 22.5 <1> [1] Temperature range ( C) 16-27 <1> (<1>, about 50% of activity maximum at 16 C and 27 C [1]) [1]

53

1,3-b-D-Glucan phosphorylase

2.4.1.97

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <2> [2] Localization cell wall <2> (<2>, bound [2]) [2] Purification <1> [1] Crystallization <1> [1]

6 Stability General stability information <1>, freezing and thawing, loss of activity [1] Storage stability <1>, 0-4 C, stable for several weeks [1]

References [1] Albrecht, G.J.; Kauss, H.: Purification, crystallization and properties of a b(1!3)-glucan phosphorylase from Ochromonas malhamensis. Phytochemistry, 10, 1293-1298 (1971) [2] Lienart, Y.; Comtat, J.; Barnoud, F.: Wall-bound 1,3-b-d-glucan:orthophosphate glucosyltransferase activity from Acacia cultured cells. Plant Sci., 58, 165-170 (1988)

54

UDPgalactose-N-acetylglucosamine b-D-galactosyl-transferase

2.4.1.98

1 Nomenclature EC number 2.4.1.98 (deleted, included in EC 2.4.1.90) Recommended name UDPgalactose-N-acetylglucosamine b-d-galactosyl-transferase

55

Sucrose:sucrose fructosyltransferase

2.4.1.99

1 Nomenclature EC number 2.4.1.99 Systematic name sucrose:sucrose 1'-b-d-fructosyltransferase Recommended name sucrose:sucrose fructosyltransferase Synonyms 1-SST SST fructosyltransferase, sucrose 1Fsucrose-sucrose 1-fructosyltransferase sucrose:sucrose 1-fructosyltransferase Additional information (the enzymes EC 2.4.1.9 and EC 2.4.1.99 have overlapping substrate specificities, each being able to use 2 sucrose molecules. When the predominant product is 1-kestose the enzyme is classified as EC 2.4.1.99, when the predominant products are higher fructooligosaccharides the enzyme is classified as EC 2.4.1.9) CAS registry number 73379-56-3

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

56

Hordeum vulgare [1, 6, 10, 15] Pennisetum americanum [2] Asparagus officinalis [3, 6, 25] Helianthus tuberosus [4, 11, 15, 20, 25] Allium cepa [5, 6, 25] Dactylis glomerata [6] Triticum aestivum [6, 15, 17] Cichorium intybus (L. var. foliosum cv. [8]; L. var. foliosum cv. Flash [18,19]) [7, 8, 15, 18, 19, 25] Festuca arundinacea [9] Taraxacum officinale [12] Aspergillus foetidus [13] Cynara scolymus [14]

2.4.1.99

<13> <14> <15> <16> <17> <18> <19> <20> <21> <22> <23> <24> <25> <26> <27> <28> <29> <30>

Sucrose:sucrose fructosyltransferase

Bromus pictus [15] Polymnia sonchifolia [16] Cichorium intybus [21] Helianthus tuberosus [22] Agropyron sp. (Agropyron cristatum Gaertn. * Agropyron desertorum Schult [23]) [23] Agave vera cruz [24, 25] Aspergillus sp. [25] Aureobasidium pullulans [25] Aspergillus niger [25] Fusarium sp. [25] Agave americana [25] Crinum longifolium [25] Beta vulgaris [25] Lactuca sativa [25] Lycoris radiata [25] Taraxacum officinale [25] Aureobasidium sp. [25] Aspergillus phoenicis [25]

3 Reaction and Specificity Catalyzed reaction 2 sucrose = d-glucose + b-d-fructofuranosyl-(2!1)-b-d-fructofuranosyl ad-glucopyranoside Reaction type fructosyl group transfer hexosyl group transfer Natural substrates and products S sucrose + sucrose <1, 4, 7, 8, 10, 12, 13> (<7>, first enzyme in the biosynthetic pathway of most fructans [6]; <8>, key enzyme in fructan biosynthesis. After 1 month of growth plantelets do not contain the enzyme. The onset of fructan synthesis coincides with the increase in 1-SST activity in roots. Expression of the 1-SST gene can be observed in roots and leaves of stressed plants [8]; <1>, the inducible enzyme is the key enzyme diverting sucrose into the biosynthetic pathways of the fructans by formation of 1kestose, can act in concert with sucrose-sucrose fructosyl-6-transferase to initiate fructan accumulation in barley leaves [10]; <4>, the enzyme is involved in fructan biosynthesis [11]; <10>, key enzyme initiating fructan synthesis [12]; <12>, the enzyme is the entry point of fructan synthesis, giving rise to the accumulation of 1-kestose and nystose on expression in plant storage organs [14]; <1,4,7,8,13>, the enzyme initiates fructan biosynthesis [15]; <7>, the enzyme is important in fructan accumulation during cold hardening of winter wheat [17]; <8>, EC 2.1.4.99, EC 2.4.1.100 and EC 3.2.1.26 simultaneously control fructan in young chicory 57

Sucrose:sucrose fructosyltransferase

2.4.1.99

roots [19]) (Reversibility: ? <1, 4, 7, 8, 10, 12, 13> [6, 8, 10, 11, 12, 14, 15, 19]) [6, 8, 10, 11, 12, 14, 15, 17, 19] P d-glucose + 1F-b-d-fructosylsucrose Substrates and products S 1,1-nystose + 1,1-nystose <4> (<4>, self-transfructosylation at a low rate [11]) (Reversibility: ? <4> [11]) [11] P 1,1,1-fructosylnystose + 1-kestose <4> [11] S 1-kestose + 1-kestose <4> (<4>, self-transfructosylation at a low rate [11]) (Reversibility: ? <4> [11]) [11] P 1,1-nystose + sucrose <4> [11] S sucrose + 6G(1-b-fructofuranosyl)n -sucrose <3> (Reversibility: ? <3> [3]) [3] P d-glucose + 1F-b-fructofuranosyl-6G(1-b-fructofuranosyl)2 -sucrose <3> [3] S sucrose + neokestose <3> (Reversibility: ? <3> [3]) [3] P d-glucose + 1F,6G-di-b-fructofuranosylsucrose <3> [3] S sucrose + sucrose <1-30> (Reversibility: r <3, 18> [3, 24]; ? <1-17, 19-30> [1-25]) [1-25] P d-glucose + 1F-b-d-fructosylsucrose <3, 4, 5, 8, 9, 18> (<9>, and moderate production of nystose [9]; <1>, production of 1-kestose exclusively [10]; <12>, 1-kestose is the only fructan product [14]; <4>, formation of isokestose and upon prolonged incubation some nystose [20]) [3, 4, 5, 7, 9, 11, 20, 24] S Additional information <4, 8, 18> (<4>, the enzyme also catalyzes the removal of the terminal fructosyl unit from both 1-kestose and 1,1-nystose, which results in the release of sucrose and 1-kestose, respectively and free fructose. The enzyme has no hydrolytic activity against sucrose [11]; <8>, de-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes: sucrose:sucrose 1-fructosyl transferase and fructan:fructan 1-fructosyl transferase [18]; <4>, in combination EC 2.4.1.99 and EC 2.4.1.100 can synthesize long-chain inulins in vitro from sucrose [20]; <18>, free fructose, glucose and raffinose also serves as acceptor for fructose units [24]) [11, 18, 20, 24] P ? Inhibitors Ag+ <3, 4, 5, 12, 18, 23> (<3>, 1 mM, 71% inhibition [3]; <4>, 1 mM AgNO3, 92% inhibition [4]; <5>, 0.25 mM AgNO3, 32.2% inhibition [5]; <4>, 1 mM, 15% inhibition [11]) [3, 4, 5, 11, 14, 24, 25] Al3+ <23> [25] Ca2+ <5, 7, 17> (<5>, 0.25 mM CaCl2 , 10.6% inhibition [5]; <7>, 10 mM, 62% inhibition [6]; <17>, CaCl2 [23]) [5, 6, 23] Co2+ <5> (<5>, 0.25 mM CoCl2 , 12.7% inhibition [5]) [5] CsCl2 <17> [23] Cu2+ <4, 5, 7, 12, 29> (<5>, 0.25 mM CuSO4, 11.1% inhibition [5]; <7>, 10 mM, 89% inhibition [6]; <4>, 1 mM CuSO4, 47% inhibition [11]) [5, 6, 11, 14, 25] 58

2.4.1.99

Sucrose:sucrose fructosyltransferase

Hg2+ <3, 4, 5, 18, 23, 29> (<3>, 0.1 mM, 81% inhibition [3]; <4>, HgCl2 , 97% inhibition [4]; <5>, 0.025 mM HgCl2 , 53.8% inhibition [5]) [3, 4, 5, 23, 25] K+ <1, 6, 7> (<7>, 10 mM KCl, 20% inhibition, inhibition by ionic stength may be responsible for approximately 50% of the KCl inhibition [6]; <3,5>, no inhibition by KCl [6]) [6] Mn2+ <3, 4, 5, 7> (<3>, 1.0 mM, 75% inhibition [3]; <5>, 0.25 mM MnSO4, 45.7% inhibition [5]; <7>, 10 mM, 44% inhibition [6]; <4>, 5 mM 20% inhibition [11]) [3, 5, 6, 11] NEM <4> (<4>, 1 mM, 10% inhibition [11]) [11] PCMB <3, 4, 5, 18> (<3>, 0.12 mM, 22% inhibition [3]; <4>, 0.1 mM, 93% inhibition [4]; <5>, 0.03 mM, 63% inhibition [5]) [3, 4, 5, 23, 24] Pb2+ <23, 29> [25] Sn2+ <23> [25] UDP <18> [24] Zn2+ <7, 12> (<7>, 10 mM, 91% inhibition [6]) [6, 14] choline chloride <7> [6] iodoacetamide <4> (<4>, 1 mM, 10% inhibition [11]) [11] Activating compounds cysteine <18> (<18>, activates [24]) [24] pyridoxal-HCl <4> (<4>, 20 mM, stimulation to 147% of the control [11]) [11] pyridoxine <4> (<4>, 20 mM, stimulation to 120% of the control [11]) [11] Metals, ions Ca2+ <23> (<23>, activates [25]) [25] Co2+ <23> (<23>, activates [25]) [25] Cu2+ <18> (<18>, activates [24]) [24] Li+ <23> (<23>, activates [25]) [25] Mg2+ <18, 23, 29> (<18,23,29>, activates [24,25]) [24, 25] Specific activity (U/mg) 0.126 <4> [4] 2.97 <3> [3] Additional information <1, 4, 7, 8, 13> (<1,4,7,8,13>, direct and rapid colorimetric procedure for the assay of plant extracts [15]) [11, 15, 20] Km-Value (mM) 0.083 <5> (sucrose) [5] 0.11 <3> (sucrose) [3] 1.8 <18> (sucrose) [24] 74 <2> (sucrose) [2] 200 <1> (sucrose) [10] 266 <7> (sucrose) [6] Ki-Value (mM) 122 <7> (K+ ) [6]

59

Sucrose:sucrose fructosyltransferase

2.4.1.99

pH-Optimum 3.5-5 <4> [11] 5 <2, 3, 17> [2, 3, 23] 5.4 <4, 5> [4, 5] 5.5 <7> [6] 5.7 <18> [24] pH-Range 2.7-6.5 <4> (<4>, about 50% of maximal activity at pH 2.7 and at pH 6.5 [11]) [11] Temperature optimum ( C) 20-25 <4> (<4>, synthesis of 1-kestose [11]) [11] 30 <1, 17> [10, 23] 34 <4> [4] 37 <18> [24] Temperature range ( C) 5-37 <4> (<4>, 50% of maximal activity at 5 C and at 37 C, synthesis of 1kestose [11]) [11]

4 Enzyme Structure Molecular weight 62000 <18> (<18>, gel filtration [24]) [24] 65000 <3> (<3>, gel filtration [3]) [3] 67000 <4> (<4>, gel filtration [4,11]) [4, 11] 68000 <5> (<5>, gel filtration [5]) [5] 70500 <2> [2] 90000 <4> (<4>, non-denaturing PAGE [11]) [11] 180000 <11> (<11>, gel filtration [13]) [13] Subunits ? <10> (<10>, x * 61500, calculation from nucleotide sequence [12]) [12] dimer <4, 11> (<4>, 1 * 26000 + 1 * 59000, SDS-PAGE [20]; <11>, 2 * 90000, SDS-PAGE [13]) [13, 20] Additional information <4> (<4>, electrophoresis or gel filtration under denaturing conditions yields a 27000 Da and a 55000 Da fragment [11]) [11] Posttranslational modification glycoprotein <10, 11> (<10>, the enzyme contains six potential N-glycosylation sites [12]; <11>, the enzyme is extensively glycosylated [13]) [12, 13]

5 Isolation/Preparation/Mutation/Application Source/tissue bulb <5> [6, 25] disc <12> [14] 60

2.4.1.99

Sucrose:sucrose fructosyltransferase

grain <2> [2] leaf <1, 6, 7, 8, 25> (<1>, growth zones [1]; <8>, of the seedling [8]; <8>, expression of the 1-SST gene can be observed in roots and leaves of stressed plants [8]; <7>, higher levels of activity are found in leaf tissue of snow mold-resistant cultivar, which accumulates more fructan than other cultivars [17]) [1, 6, 8, 10, 17, 25] plantlet <8> (<8>, after 1 month of growth the activity is twice as high as in N-rich plants [19]) [19] primary root <12> [14] receptacle <10> (<10>, at the flowering stage [12]) [12] rhizophore <14> (<14>, activity increase up to 8 months of cultivation, decreasing to initial values at the end of the growth period [16]) [16] root <3, 7, 8, 10, 15> (<8>, of the seedling [8]; <8>, the onset of fructan synthesis coincides with the increase in 1-SST activity in roots. Expression of the 1-SST gene can be observed in roots and leaves of stressed plants [8]; <10>, at the flowering stage. At the pre-flowering stage the 1-SST mRNA concentration and the 1-SST activity are higher in root phloem than in xylem [12]; <7>, the level of the enzyme transcript increases from autumn to early winter in the crown-tissue of all field-grown wheat cultivars examined [17]) [3, 7, 8, 12, 15, 17, 21, 25] seed <5> [5] seedling <8> [8] shoot <1, 7, 13> [15] stem <1, 3, 7, 10> (<10>, at the flowering stage [12]) [6, 12] tuber <4> [4, 11, 15, 20] tuberous root <14> (<14>, higher activitiy values are found at the beginning of the tuberization, 3-months-old plants, and at the flowering phase, 7 months-old plants [16]) [16] Purification <1> (partial [1]) [1] <2> (partial [2]) [2] <3> [3] <4> [4, 10, 20] <5> [5] <8> [7] <9> [9] <11> [13] <18> [24] Cloning <7> (expression in Pichia pastoris [17]) [17] <9> (expression in tobacco protoplasts and in Pichia pastoris [9]) [9] <10> [12] <11> (expression in Saccharomyces cerevisiae [13]) [13]

61

Sucrose:sucrose fructosyltransferase

2.4.1.99

<12> (subcloned into a pUC18 derivative and expressed in tobacco protoplasts under the control of the cauliflower mosaic virus 35S RNA promotor [14]) [14] <15> [21] <16> [22] Application nutrition <21, 29> (<21,29>, production of fructooligosaccharides from sucrose as alternative sweeteners with low calories, no cariogenicity, safety for diabetic and bifidus-stimulating functionality. The production yield of fructooligosaccharides using enzymes originated from plants is low and mass production of the enzyme is quite limited by seasonal conditions. Therefore industrial production depends chiefly on fungal enzymes [25]) [25]

6 Stability pH-Stability 4-8 <3> (<3>, 30 C, stable [3]) [3] Temperature stability 20-37 <5> (<5>, 10 min, stable [5]) [5] 30 <3> (<3>, pH 4.0-8.0, 30 min, stable [3]) [3] 45 <3, 5> (<3>, pH 5.0-6.5, 50% loss of activity after 30 min, pH 4.0 and pH 8.0, more than 90% loss of activity after 30 min [3]; <5>, 10 min, residual activity is 52% at pH 4.6 and at pH 5.4, 37% at pH 4.0, 35% at pH 6.0 and 5% at pH 7.0 [5]) [3, 5] 50-60 <5> (<5>, 10 min, inactivation [5]) [5] 60 <3> (<3>, 10 min, 82% loss of activity [3]) [3] General stability information <20>, operational stability in semibatch culture 20-60 days, operational stability in continous culture is 100 days [25] <29>, operational stability in continous culture is 30 days [25]

References [1] Luscher, M.; Hochstrasser, U.; Boller, T.; Wiemken, A.: Isolation of sucrose:sucrose 1-fructosyltransferase (1-SST) from barley (Hordeum vulgare). New Phytol., 145, 225-232 (2000) [2] Asthir, B.; Singh, R.; Gupta, A.K.: Sucrose-sucrose fructosyltransferase in relation to fructans in developing grains of pearl millet, Pennisetum americanum. Indian J. Exp. Biol., 33, 233-235 (1995) [3] Shiomi, N.; Izawa, M.: Purification and characterization of sucrose:sucrose 1-fructosyltransferase from the roots of Asparagus (Asparagus officinalis L.). Agric. Biol. Chem., 44, 603-614 (1980)

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Sucrose:sucrose fructosyltransferase

[4] Praznik, W.; Beck, R.H.F.; Spies, T.: Isolation and characterization of sucrose:sucrose 1F-b-d-fructosyltransferase from tubers of Helianthus tuberosus. Agric. Biol. Chem., 54, 2429-2431 (1990) [5] Shiomi, N.; Kido, H.; Kiriyama, S.: Purification and properties of sucrose:sucrose 1F-b-d-fructosyltransferase in onion seeds. Phytochemistry, 24, 695-698 (1985) [6] Chevalier, P.M.; Rupp, R.A.: Inhibition of sucrose:sucrose fructosyl transferase by cationic and ionic strength. Plant Physiol., 101, 589-594 (1993) [7] Singh, R.; Bhatia, I.S.: Substrate specificity of fructosyl transferase from chicory roots. Phytochemistry, 10, 2037-2039 (1971) [8] De Roover, J.; Vandenbranden, K.; Van Laere, A.; Van den Ende, W.: Drought induces fructan synthesis and 1-SST (sucrose:sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.). Planta, 210, 808-814 (2000) [9] Luscher, M.; Hochstrasser, U.; Vogel, G.; Aeschbacher, R.; Galati, V.; Nelson, C.J.; Boller, T.; Wiemken, A.: Cloning and functional analysis of sucrose:sucrose 1-fructosyltransferase from tall fescue. Plant Physiol., 124, 1217-1227 (2000) [10] Simmen, U.; Obenland, D.; Boller, T.; Wiemken, A.: Fructan synthesis in excised barley leaves. Identification of two sucrose-sucrose fructosyltransferases induced by light and their separation from constitutive invertases. Plant Physiol., 101, 459-468 (1993) [11] Koops, A.J.; Jonker, H.H.: Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus Colombia. II. Purification of sucrose:sucrose 1-fructosyltransferase and reconstitution of fructan synthesis in vitro with purified sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1-fructosyltransferase. Plant Physiol., 110, 11671175 (1996) [12] Van den Ende, W.; Van Laere, A.: Purification and properties of an invertase with sucrose:sucrose fructosyltransferase (SST) activity from the roots of Cichorium intybus L. New Phytol., 123, 31-37 (1993) [13] Rehm, J.; Willmitzer, L.; Heyer, A.G.: Production of 1-kestose in transgenic yeast expressing a fructosyltransferase from Aspergillus foetidus. J. Bacteriol., 180, 1305-1310 (1998) [14] Hellwege, E.M.; Gritscher, D.; Willmitzer, L.; Heyer, A.G.: Transgenic potato tubers accumulate high levels of 1-kestose and nystose: functional identification of a sucrose sucrose 1-fructosyltransferase of artichoke (Cynara scolymus) blossom disks. Plant J., 12, 1057-1065 (1997) [15] Puebla, A.F.; Battaglia, M.E.; Salerno, G.L.; Pontis, H.G.: Sucrose-sucrose fructosyl transferase activity: A direct and rapid colorimetric procedure for the assay of plant extracts. Plant Physiol. Biochem., 37, 699-702 (1999) [16] Itaya, N.M.; Machado de Carvalho, M.A.; Figueiredo-Ribeiro, R.D.C.L.: Fructosyl transferase and hydrolase activities in rhizophores and tuberous roots upon growth of Polymnia sonchifolia (Asteraceae). Physiol. Plant., 116, 451-459 (2002) [17] Kawakami, A.; Yoshida, M.: Molecular characterization of sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase associated 63

Sucrose:sucrose fructosyltransferase

[18]

[19] [20] [21] [22] [23] [24] [25]

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with fructan accumulation in winter wheat during cold hardening. Biosci. Biotechnol. Biochem., 66, 2297-2305 (2002) Van Den Ende, W.; Van Laere, A.: De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose:sucrose 1-fructosyl transferase and fructan:fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus). Planta, 200, 335-342 (1996) Van den Ende, W.; De Roover, J.; Van Laere, A.: Effect of nitrogen concentration on fructan and fructan-metabolizing enzymes in young chicory plants (Cichorium intybus). Physiol. Plant., 105, 2-8 (1999) Luescher, M.; Erdin, C.; Sprenger, N.; Hochstrasser, U.; Boller, T.; Wiemken, A.: Inulin synthesis by a combination of purified fructosyltransferases from tubers of Helianthus tuberosus. FEBS Lett., 385, 39-42 (1996) De Halleux, S.; van Cutsem, P.: Cloning and sequencing of the 1-SST cDNA from chicory root. Plant Physiol., 113, 1003 (1997) Van der Meer, I.M.; Koops, A.J.; Hakkert, J.C.: Cloning of the fructan biosynthesis pathway of Jerusalem artichoke. Plant J., 15, 489-500 (1998) Chatterton, N.J.; Harrison, P.A.; Thornley, W.R.; Bennett, J.H.: Characterization of sucrose:sucrose fructosyltransferase from crested wheatgrass. New Phytol., 109, 29-33 (1988) Satyanarayana, M.N.: Biosynthesis of oligosaccharides and fructans in Agave vera cruz: I. Properties of a partially purified transfructosylase. Indian J. Biochem. Biophys., 13, 261-266 (1976) Yun, J.W.: Fructooligosaccharides - occurence, preparation, and application. Enzyme Microb. Technol., 19, 107-117 (1996)

2,1-Fructan:2,1-fructan 1-fructosyltransferase

2.4.1.100

1 Nomenclature EC number 2.4.1.100 Systematic name 2,1-b-d-fructan:2,1-b-d-fructan 1-b-d-fructosyltransferase Recommended name 2,1-fructan:2,1-fructan 1-fructosyltransferase Synonyms 1-FFT FFT fructan:fructan 1-fructosyl transferase fructan:fructan fructosyl transferase fructosyltransferase, 1,2-b-d-fructan 1FCAS registry number 73379-55-2

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

Allium cepa [1] Helianthus tuberosus (Colombia [7]) [2, 7, 13] Taraxacum officinale (Weber [3]) [3] Triticum aestivum [4] Lolium rigidum (gaudin [5]) [5, 6] Cichorium intybus (L. var. foliosum cv. Flash [8,10,11]) [8, 10, 11, 12] Polymnia sonchifolia [9]

3 Reaction and Specificity Catalyzed reaction [b-d-fructosyl-(2!1)-]m + [b-d-fructosyl-(2!1)-]n = [b-d-fructosyl(2!1)-]m-1 + [b-d-fructosyl-(2!1)-]n+1 Reaction type hexosyl group transfer

65

2,1-Fructan:2,1-fructan 1-fructosyltransferase

2.4.1.100

Natural substrates and products S (1,2-b-d-fructosyl)m + (1,2-b-d-fructosyl)n <2, 4, 6, 7> (<2>, the enzyme is involved in the synthesis of b-2,1-linked fructose polymers [7]; <4>, role of enzyme in synthesis of fructan [4]; <6>, in combination with the purified chicory root sucrose:sucrose 1-fructosyl transferase, i.e. EC 2.4.1.99, the enzyme synthesizes a range of naturally occuring chicory fructans from sucrose as the sole substrate [8]; <7>, the enzyme can be involved in chain length distribution of the fructan molecules found in rhizophores and in tuberous roots [9]; <6>, EC 2.4.1.100, EC 2.4.1.99 and EC 3.2.1.26 simultaneously control fructan in young chicory roots [11]) (Reversibility: ? <2, 4, 6, 7> [4, 7, 8, 9, 11]) [4, 7, 8, 9, 11] P ? Substrates and products S (1,2-b-d-fructosyl)m + (1,2-b-d-fructosyl)n <1-7> (<3>, active on different oligofructans of the inulin series [3]; <2,3>, 1-kestose-dependent nystose production [2,3]; <2>, transfers fructosyl groups from oligofructans (degree of polymerization: 3-8) of the inulin series [2]; <2>, 1-kestose is an efficient donor of fructosyl units to sucrose [2]; <4>, enzyme is specific for fructosyl transfer from b-2,1-linked 1-kestose or fructan to sucrose and b-2,1-fructosyl transfer to other fructans [4]) (Reversibility: ? <1-7> [1-13]) [1-13] P (1,2-b-d-fructosyl)m-1 + (1,2-b-d-fructosyl)n+1 <1-7> (<1>, the enzyme produces tetrasaccharides and higher polymers from trisaccharide [1]) [1-13] S 1,1-nystose + 1,1-nystose <6> (Reversibility: ? <6> [10]) [10] P sucrose + 1-kestose + oligofructan DP5 + oligofructan DP6 <6> [10] S 1-kestose + 1-kestose <4, 5, 6> (Reversibility: ? <4, 5, 6> [4, 5, 6, 10]) [4, 5, 6, 10] P 1,1-nystose + 1,1,1-logose <4> (<4>, inulin-type tetra- and pentasaccharides [4]; <6>, 1,1-nystose + sucrose + oligofructans DP5 and DP6 [10]) [4, 10] S 1-kestose + 6-kestose <4> (Reversibility: ? <4> [4]) [4] P 1,6-nystose + 6,1-nystose <4> [4] S 6-kestose + 1-kestose + sucrose <1> (Reversibility: ? <1> [1]) [1] P mixture of tetrasacharides of b-2,6- and b-2,1-linked fructans <1> [1] S 6G-kestose + 6G-kestose <5> (Reversibility: ? <5> [5]) [5] P ? S 6G-kestose + sucrose <5> (Reversibility: ? <5> [5]) [5] P tetrasaccharide <5> (<5>, some synthesis of 1-kestose and resynthesis of 6G-kestose also occurs [5]) [5] S fructose + inulin <6> (Reversibility: ? <6> [12]) [12] P oligosaccharide <6> (<6>, reducing fructofuranosyl-only oligosaccharides [12]) [12] S sucrose + 1,1-nystose <6> (Reversibility: ? <6> [10]) [10] P ? S sucrose + 1-kestose <2, 4, 6> (Reversibility: ? <2, 4, 6> [2, 4, 10]) [2, 4, 10]

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P 1,1-nystose <4> [4] S sucrose + 1-kestose <5> (Reversibility: ? <5> [5]) [5] P 1-kestose + 6G-kestose + tetrasaccharide <5> (<5>, transfer of fructosyl residue from 1-kestose to sucrose results in resynthesis of 1-kestose. Tetrasaccharides and 6G-kestose are also synthesized [5]) [5] S sucrose + inulin <6> (Reversibility: ? <6> [10, 12]) [10, 12] P oligosaccharide <6> (<6>, as a series of non-reducing b-(2, 1)-glucofructo-oligosaccharides [12]) [12] S sucrose + oligofructan <6> (Reversibility: ? <6> [10]) [10] P ? S Additional information <2, 6> (<6>, fructofuranosyl-only oligosaccharides can be synthesized in vitro from fructose and inulin [12]; <2>, enzyme is inactive when incubated individually with sucrose [13]; <2>, in combination EC 2.4.1.99 and EC 2.4.1.100 can synthesize long-chain inulins in vitro from sucrose [13]) [12, 13] P ? Inhibitors Ag+ <6> [10] Cu2+ <6> [10] Hg2+ <6> [10] Zn2+ <6> [10] sucrose <6> (<6>, competitive inhibitor of donor substrates at 8-50 mM [10]; <6>, competitive inhibitor at high concentrations [12]) [10, 12] Specific activity (U/mg) 14.5 <6> [10] Additional information <1> [1] Km-Value (mM) 0.2 <2> (sucrose) [2] 119 <6> (1-kestose) [10] 152 <6> (1,1-nystose) [10] 199 <6> (fructose) [12] Ki-Value (mM) 15 <6> (sucrose) [10] pH-Optimum 5.5-6.5 <6> [10] 5.5-7 <2> [7] 6.5 <2-4> [2-4] pH-Range 5-8 <4> (<4>, pH 5.0: about 70% of maximal activity, pH 8.0: about 65% of maximal activity [4]) [4] Temperature optimum ( C) 25-35 <2> [7] 30 <4> [4]

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2.4.1.100

Temperature range ( C) 5-40 <4> (<4>, 5 C: about 70% of maximal activity, 40 C: about 75% of maximal activity [4]) [4]

4 Enzyme Structure Molecular weight 50000 <5> (<5>, gel filtration [5]) [5] 69000 <6> (<6>, gel filtration [10]) [10] 70000 <2> [7] 72800 <2> (<2>, non-denaturing gel electrophoresis [2]) [2] Subunits ? <3> (<3>, x * 49000, SDS-PAGE [3]) [3] dimer <6> (<6>, 1 * 17000 + 1 * 52000, SDS-PAGE [10]) [10] monomer <2> (<2>, 1 * 70000, SDS-PAGE [7]) [7] Posttranslational modification glycoprotein <3, 6> [3, 10]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <5> [5] leaf base <1> (<1>, inner leaf base [1]) [1] leaf blade <4> [4] plantlet <6> (<6>, effect of nitrogen concentration [11]) [11] rhizophore <7> [9] root <6> [8, 10, 12] tuber <2> [2, 7, 13] tuberous root <7> [9] Purification <1> [1] <2> [7, 13] <3> [3] <4> [4] <5> [5, 6] <6> [8, 12]

6 Stability Temperature stability 30 <3> (<3>, 1 h, stable [3]) [3] 40 <2> (<2>, 1 h, up to 20% loss of activity below [2]) [2]

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References [1] Henry, R.J.; Darbyshire, B.: Sucrose:sucrose fructosyltransferase and fructan:fructan fructosyltransferase from Allium cepa. Phytochemistry, 19, 1017-1020 (1980) [2] Luescher, M.; Frehner, M.; Noesberger, J.: Purification and characterization of fructan:fructan fructosyltransferase from Jerusalem artichoke (Helianthus tuberosus K.). New Phytol., 123, 717-724 (1993) [3] Luescher, M.; Frehner, M.; Noesberger, J.: Purification and some properties of fructan:fructan fructosyltransferase from dandelion (Taraxacum officinale Weber). New Phytol., 123, 437-442 (1993) [4] Jeong, B.R.; Housley, T.L.: Purification and characterization of wheat b(2!1) fructan:fructan fructosyl transferase activity. Plant Physiol., 100, 199-204 (1992) [5] St. John, J.A.; Bonnett, G.D.; Simpson, R.J.; Tanner, G.J.: A fructan: fructan fructosyltransferase activity from Lolium rigidum. New Phytol., 135, 235247 (1997) [6] St. John, J.A.; Sims, I.M.; Bonnett, G.D.; Simpson, R.J.: Identification of products formed by a fructan:fructan fructosyltransferase activity from Lolium rigidum. New Phytol., 135, 249-257 (1997) [7] Koops, A.J.; Jonker, H.H.: Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus Colombia. I. Fructan:fructan fructosyl transferase. J. Exp. Bot., 45, 1623-1631 (1994) [8] Van Den Ende, W.; Van Laere, A.: De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose:sucrose 1-fructosyl transferase and fructan:fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus). Planta, 200, 335-342 (1996) [9] Itaya, N.M.; Machado de Carvalho, M.A.; Figueiredo-Ribeiro, R.D.C.L.: Fructosyl transferase and hydrolase activities in rhizophores and tuberous roots upon growth of Polymnia sonchifolia (Asteraceae). Physiol. Plant., 116, 451-459 (2002) [10] Van den Ende, W.; Van Wonterghem, D.; Verhaert, P.; Dewil, E.; Van Laere, A.: Purification and characterization of fructan: fructan fructosyl transferase from chicory roots. Planta, 199, 493-502 (1996) [11] Van den Ende, W.; De Roover, J.; Van Laere, A.: Effect of nitrogen concentration on fructan and fructan-metabolizing enzymes in young chicory plants (Cichorium intybus). Physiol. Plant., 105, 2-8 (1999) [12] Van den Ende, W.; De Roover, J.; Van Laere, A.: In vitro synthesis of fructofuranosyl-only oligosaccharides from inulin and fructose by purified chicory root fructan:fructan fructosyl transferase. Physiol. Plant., 97, 346-352 (1996) [13] Luescher, M.; Erdin, C.; Sprenger, N.; Hochstrasser, U.; Boller, T.; Wiemken, A.: Inulin synthesis by a combination of purified fructosyltransferases from tubers of Helianthus tuberosus. FEBS Lett., 385, 39-42 (1996)

69

a-1,3-Mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase

2.4.1.101

1 Nomenclature EC number 2.4.1.101 Systematic name UDP-N-acetyl-d-glucosamine:3-(a-d-mannosyl)-b-d-mannosyl-glycoprotein 2-b-N-acetyl-d-glucosaminyltransferase Recommended name a-1,3-mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase Synonyms GlcNAc-T I <1, 7, 12> [1, 13, 22] GnT-I <1, 2, 5, 9-11, 13> [14, 16-21, 23] N-acetylglucosaminyltransferase I N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase I NT <5> [11] UDP-N-GlcNAc:a3-d-mannoside b-1,2-N-acetylglucosaminyltransferase I <13> [23] UDP-N-acetyl-d-glucosamine:glycoprotein (N-acetyl-d-glucosamine to a-dmannosyl-1,3-(R1)-b-d-mannosyl-R2) b-1,2-N-acetyl-d-glucosaminyltransferase UDP-N-acetylglucosaminyl:a-1,3-d-mannoside-b-1,2-N-acetylglucosaminyltransferase I UDP-N-acetylglucosaminyl:a-3-d-mannoside b-1,2-N-acetylglucosaminyltransferase I acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-a-1,3mannosylglycoprotein b-1,2-Na-1,3-mannosyl-glycoprotein b-1,2-N-acetylglucosaminyltransferase uridine diphosphoacetylglucosamine-a-1,3-mannosylglycoprotein b-1,2-Nacetylglucosaminyltransferase Additional information (cf. EC 2.4.1.143, EC 2.4.1.144, EC 2.4.1.145 and EC 2.4.1.155) CAS registry number 102576-81-8

2 Source Organism <-3> no activity in Saccharomyces cerevisiae [17] <-2> no activity in Autographa californica nuclear polyhedrosis baculovirus [16] 70

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<-1> no activity in Spodoptera frugiperda [16] <1> Rattus norvegicus (transferases A and B [5]; male Donryu [5]) [1, 5, 7, 14, 15, 17] <2> Oryctolagus cuniculus [2, 10, 12, 18] <3> Sus scrofa [3, 6, 12] <4> Bos taurus [4, 8, 12] <5> Homo sapiens (recombinant enzyme as fusion protein with maltose-binding protein [21]) [11, 16, 19, 21] <6> Gallus gallus [12] <7> Mesocricetus auratus (wild-type and mutant cell lines [13]) [12, 13, 22] <8> Acer pseudoplatanus (sycamore [9]) [9] <9> Solanum tuberosum (constitutive single copy gene [20]) [20] <10> Nicotiana tabacum (constitutive single copy gene [20]) [20] <11> Arabidopsis thaliana (constitutive single copy gene [20]) [20] <12> Cricetulus griseus (1 difference to published sequence U65791: C1340 is A1340 [22]) [22] <13> Xenopus laevis (2 isozymes A and B [23]) [23] <14> Spodoptera frugiperda [23]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + 3-(a-d-mannosyl)-b-d-mannosyl-R = UDP + 3-(2-[N-acetyl-b-d-glucosaminyl]-a-d-mannosyl)-b-d-mannosyl-R (R represents the reminder of the N-linked oligosaccharide in the glycoprotein acceptor. Note that this enzyme acts before N-acetylglucosaminyltransferase II, III, IV, V and VI.; <2> SGC domain of SGC superfamily [18]; <2,12> active site [18,22]; <12> R303 is important for enzyme activity, conserved throughout all species [22]; <12> D212 is the central residue of the DxD motif, conserved throughout all species [22]; <2> DxD motif [18]; <2> ordered sequential bi bi mechanism [18]; <2> mechanism [10]) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + a-d-mannosyl-1,6-(a-d-mannosyl-1,3)a-d-mannosyl-1,6-(a-d-mannosyl-1,3)-b-d-mannosyl-1,4-N-acetyl-dglucosaminyl-1,4-N-acetyl-d-glucosaminyl-Asn-peptide <1, 2, 4, 5, 13> (<1, 2, 5, 13> key enzyme of biosynthesis of complex and hybrid N-glycans [1,10,21,23]; <4> stimulates galactosyltransferase of bovine milk [8]) (Reversibility: ? <1, 2, 4, 5, 13> [1, 8, 10, 21, 23]) [1, 8, 10, 21, 23] P UDP + a-d-mannosyl-1,6-(a-d-mannosyl-1,3)-a-d-mannosyl-1,6-(Nacetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-1,4-Nacetyl-d-glucosaminyl-R S UDP-N-acetyl-d-glucosamine + ovalbumin glycopeptide V <4> (<4> physiological substrate [4]) (Reversibility: ? <4> [4]) [4] 71

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2.4.1.101

P UDP + ovalbumin glycopeptide IIIA S Additional information <7> (<7> enzyme initiates the biosynthesis of heparan sulfate [13]) [13] P ? Substrates and products S UDP-N-acetyl-d-glucosamine + GlcUAcb1-3Galb1-O-naphthalenemethanol <7> (<7> synthetic substrate [13]) (Reversibility: ? <7> [13]) [13] P ? S UDP-N-acetyl-d-glucosamine + Man5 GlcNAc2 <5, 12> (<5> best substrate [21]) (Reversibility: ? <5, 12> [21, 22]) [21, 22] P ? S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-b-d-mannosyl-1,4-N-acetylglucosamine1,4-(fucose-1,6)-N-acetyl-glucosamine-Asn-peptide <4> (<4> poor substrate [4]; <2,3> no activity [2,6]) (Reversibility: ? <4> [4]) [4] P UDP + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6)-b-d-mannosyl-1,4-N-acetylglucosamine-1,4-(fucose-1,6)-N-acetyl-glucosamine-Asn-peptide <4> [4] S UDP-N-acetyl-d-glucosamine + a-d-mannosyl-1,3-(a-d-mannosyl-1,6)b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R <1-4, 6-8, 13> (<13> recombinant isozyme A and B mutant T223A [23]; <14> no activity with pyridylaminated substrate [23]; <2-4> R: H [2,4,6,10]; <1,8,13> pyridylamine [9,14,23]; <2-4,6,7> 1,4-N-acetyl-d-glucosaminyl-R [12]; <2> 1,4-Nacetyl-d-glucosaminyl-Asn [10]; <2> synthetic-b-d-mannosyl-(1,6-anhydro)-derivative acts as substrate, too [10]) (Reversibility: ? <1-4, 6-8, 13> [2, 4, 6, 9, 10, 12, 14, 23]) [2, 4, 6, 9, 10, 12, 14, 23] P UDP + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(a-d-mannosyl1,6)-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R <1-4, 6-8> (<2> R: H [2,10]; <1,8> R: pyridylamine [9,14]) [2, 9, 10, 12, 14] S UDP-N-acetyl-d-glucosamine + a-d-mannosyl-1,3-b-d-mannosyl-1,4-Nacetyl-d-glucosamine <2, 3> (Reversibility: ? <2, 3> [2, 6]) [2, 6] P UDP + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3-b-d-mannosyl1,4-N-acetyl-d-glucosamine <2> [2] S UDP-N-acetyl-d-glucosamine + a-d-mannosyl-1,6-(a-d-mannosyl-1,3)a-d-mannosyl-1,6-(a-d-mannosyl-1,3)-b-d-mannosyl-1,4-N-acetyl-dglucosaminyl-R <1-8, 13> (<2-4> R: H [2,4,6]; <1,2,4> R: 1,4-N-acetyl-dglucosaminyl-Asn [1,4,5,8,10,15]; <4> R: (fucose-1,6)-1,4-N-acetyl-d-glucosaminyl-Asn [4]; <4> R: 4-N-acetyl-d-glucosaminyl-Asn-peptide [4]; <1> pyridylaminated oligosaccharide [14]; <1,8> R: pyridinylamine [9,14]; <4> can act on a-mannosyl-1,6-b-mannosyl-terminus if a-mannosyl-1,3-b-mannosyl-terminus is not available, appreciable higher Km -value [4]; <3> absolute specificity for terminal branched mannosyl residues [3]; <1,2> essential for activity: unsubstituted equatorial hydroxyl on C-4 of b-linked mannosyl-residue [1,10]; <1-3,7> substrate specificity [13,10,12]; <4> does not act on a-mannosyl-1,2-a-mannosyl-, a-mannosyl-1,3-a-mannosyl-, a-mannosyl-1,6-a-mannosyl-termini [4]; <1> an

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P

S

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

P S P S P S P

a-1,3-Mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase

axial 2-hydroxyl group on the b-linked mannose of the substrate are essential for the enzyme [1]) (Reversibility: ? <1-8, 13> [1-16, 21, 23]) [1-16, 21, 23] UDP + a-d-mannosyl-1,6-(a-d-mannosyl-1,3)-a-d-mannosyl-1,6-(Nacetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R <1-8> (<1,3,5> transfers an N-acetylglucosamine in b-1,2-linkage to mannosyl-1,3-b-mannosyl-terminus [1,3,16]; <2-4> R: H [2,4,6]; <1> R: 1,4-N-acetyl-d-glucosaminyl-1,4-N-acetyl-d-glucosaminylAsn [15]; <1,2,4> R: 1,4-N-acetyl-d-glucosaminyl-Asn [1,4,5,8,10]; <4> R: (fucose-1,6)-1,4-N-acetyl-d-glucosaminyl-Asn [4]; <8> pyridinylamine [9]) [1, 2-6, 8-10, 12-14, 16, 21] UDP-N-acetyl-d-glucosamine + asialofetuin <1> (<1> b-galactosidase-, b-N-acetylhexosamidase- and a-mannosidase-treated asialofetuin, b-galactosidase- and b-N-acetylhexosamidase-treated asialofetuin [5]) (Reversibility: ? <1> [5]) [5] ? UDP-N-acetyl-d-glucosamine + b-galactosidase- and b-N-acetylhexosamidase-treated asialotransferrin <1> (Reversibility: ? <1> [5]) [5] ? UDP-N-acetyl-d-glucosamine + dehexoso-orosomucoid <2> (Reversibility: ? <2> [2]) [2] ? UDP-N-acetyl-d-glucosamine + desialo-degalacto-dehexosamine-orosomucoid <4> (Reversibility: ? <4> [8]) [8] ? UDP-N-acetyl-d-glucosamine + fowl plague virus hemagglutinin <5> (<5> recombinant coexpression of enzyme and substrate in SF9 insect cells [16]) (Reversibility: ? <5> [16]) [16] UDP + fowl plague virus hemagglutinin with terminal N-acetyl-d-glucosaminyl residues <5> [16] UDP-N-acetyl-d-glucosamine + glycopeptides prepared from porcine IgC <3> (Reversibility: ? <3> [3]) [3] ? UDP-N-acetyl-d-glucosamine + ovalbumin <1, 3, 5> (<5> best substrate, recombinant enzyme expressed in SF9 cells, unmodified and as tagged fusion protein [16]; <3> substrate: glycopeptides prepared from ovalbumin [3]) (Reversibility: ? <1, 3, 5> [3, 5, 16, 19]) [3, 5, 16, 19] ? UDP-N-acetyl-d-glucosamine + ovalbumin glycopeptide III A-C <4> (Reversibility: ? <4> [4]) [4] ? UDP-N-acetyl-d-glucosamine + ovalbumin glycopeptide IV <4> (Reversibility: ? <4> [4]) [4] ? UDP-N-acetyl-d-glucosamine + ovalbumin glycopeptide V <1, 4> (<1,4> best substrate [4,5]) (Reversibility: ? <1, 4> [4, 5]) [4, 5] UDP + ovalbumin glycopeptide IIIA <4> [4] 73

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2.4.1.101

S Additional information <1, 2, 4> (<1> specificity for several synthetic acceptor substrate analogues, overview [15]; <1> substrates: dolichol phosphate, asialo-bovine submaxillary mucin or galactosylated asialo-bovine submaxillary mucin, different specificities of transferase A and B, overview [5]; <2> acts on synthetic substrate analogues [10]; <1> substrates: free and protein-matrix-bound glycans [7]; <2> no acceptor substrates are N-acetyl-d-glucosamine, N-acetyl-d-glucosaminyl-1,2-a-dmannosyl-1,3-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6)-b-dmannosyl-1,4-N-acetyl-d-glucosaminyl-1,4-N-acetyl-d-glucosaminyl-Asn [10]) [2, 4, 5, 7, 8, 10, 15] P ? Inhibitors 2'-O-methyl-UDP <2> (<2> weak [10]) [10] 2'-dUDP <2> (<2> weak [10]) [10] 5-bromo-UTP <2> [10] 5-mercury-UDP <2> [10] Ca2+ <5> [21] EDTA <2, 4> (<2,4> 3 mM [2,4]) [2, 4] TDP <2> [10] UDP <2> [2, 10] UDP-N-acetylgalactosamine <2> (<2> weak [10]) [10] UDP-N-acetylglucosamine <2> (<2> competitively to UDP [10]) [10] UDP-glucose <2> (<2> weak [10]) [10] UMP <2> (<2> weak [10]) [10] UTP <2> (<2> weak [10]) [10] Zn2+ <5> [21] a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-4-O-methyl-b-d-mannosyl-1,4-Nacetyl-d-glucosamine <1> (<1> weak [1]) [1] a-d-mannosyl-1,6-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-dmannosyl-1,4-N-acetyl-d-glucosaminyl-R <2> [10] antibodies to pig liver N-acetylglucosaminyltransferase I <3> [6] Additional information <1, 2> (<2> high ionic strength, e.g. 0.1 M NaCl [2]; <1> several inhibitory synthetic acceptor substrate analogues, overview [15]; <1> strategies for inhibitor design based on substrate specificities of the enzyme [15]; <2> no inhibitors are CDP, UDPhexanolamine, AMP [10]) [2, 10, 15] Activating compounds CHAPS <7> (<7> slight activation [13]) [13] Triton X-100 <1, 2, 7> (<2,7> slight activation [12,13]) [5, 10, 12, 13] Zwittergent 3-12 <7> (<7> slight activation [13]) [13] Additional information <2> (<2> no activation by 2-mercaptoethanol [10,12]) [10, 12] Metals, ions Ba2+ <2> (<2> slight activation [10,12]; <2> no activation [2]) [10, 12] Ca2+ <2, 7> (<7> can substitute Mn2+ [13]; <2> slight activation [2,10,12]) [2, 10, 12, 13]

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Cd2+ <2> (<2> activation [2,10,12]; <2> can substitute for Mn2+ with 20% [10]; <2> 78% efficiency [2]) [2, 10, 12] Co2+ <2, 5> (<5> requirement [21]; <2> activation [2,10,12]; <2> can substitute for Mn2+ with 80% [10]; <2> 91% efficiency [2]) [2, 10, 12, 21] Fe2+ <2> (<2> activation [2,10,12]; <2> can substitute for Mn2+ with 65% efficiency [10]; <2> slight [2]) [2, 10, 12] Mg2+ <2> (<2> activation [2,10,12]; <2> can substitute for Mn2+ with 40% [10]; <2> 61% efficiency [2]) [2, 10, 12] Mn2+ <1-8, 13> (<2> binds not independently of UDP-N-acetyl-d-glucose [18]; <7> can also use Ca2+ [13]; <1-8> requirement [2-10,12-14,21]; <1> 20-80 mM [14]; <4> 18-26 mM [4]; <1> 2 mM [5]; <2> 25-100 mM [12]) [210, 12-14, 18, 21, 23] Ni2+ <2> (<2> activation [2,10,12]; <2> can substitute for Mn2+ with 20% [10]; <2> 34% efficiency [2]) [2, 10, 12] Sn2+ <2> (<2> slight activation [10,12]) [10, 12] Zn2+ <2> (<2> activation [2,10,12]; <2> can substitute for Mn2+ with 8% [10]; <2> 19% efficiency [2]) [2, 10, 12] Additional information <2> (<2> no activation by Hg2+ [2]; <2> no activation by Sr2+, Pb2+ [10,12]; <2> no activation by Cu2+ [2,10,12]) [2, 10, 12] Specific activity (U/mg) 0.0000002 <7> (<7> wild-type S49 cell [13]) [13] 0.00000084 <7> (<7> wild-type CHO cell [13]) [13] 0.000015 <1> (<1> transferase A, liver [5]) [5] 0.00065 <1> (<1> transferase B, hepatoma [5]) [5] 0.079 <4> (<4> a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-b-d-mannosyl-1,4N-acetyl-d-glucosaminyl-R immunoglobulin G glycopeptide [4]) [4] 0.106 <4> (<4> a1 -acid glycoprotein treated with mild acid, b-galactosidase and b-N-acetylglucosamidase [4]) [4] 0.6 <13> (<13> recombinant isozyme A [23]) [23] 2.51 <2> (<2> purified enzyme [2]) [2] 3.37 <3> (<3> purified enzyme [3]) [3] 4.8 <3> (<3> liver [6]) [6] 19.8 <2> (<2> low-molecular weight form [10]) [10] 20 <1> (<1> purified enzyme [15]) [15] Additional information <5, 12> (<12> wild-type and mutant cell lines [22]; <5> in vivo assay of recombinant enzyme expressed in Sf9 cells with coexpressed fowl plague virus HA as endogenous substrate [16]) [16, 22] Km-Value (mM) 0.036 <7> (UDP-N-acetyl-d-glucosamine) [13] 0.0384 <2> (UDP-N-acetyl-d-glucosamine) [10] 0.0384 <2> (a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-b-d-mannosyl-1,4-Nacetyl-d-glucosaminyl-R) [10] 0.046 <1> (UDP-N-acetyl-d-glucosamine, <1> recombinant catalytic fragment [14]) [14]

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0.078 <2> (UDP-N-acetyl-d-glucosamine, <2> with a-d-mannosyl-1,3-(a-dmannosyl-1,6)-a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-b-d-mannosyl-1,4-Nacetyl-d-glucosamine [12]) [12] 0.078 <2> (a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-a-d-mannosyl-1,3-(a-dmannosyl-1,6)-b-d-mannosyl-1,4-N-acetyl-d-glucosamine) [12] 0.1 <4> (UDP-N-acetyl-d-glucosamine) [4] 0.12 <4> (ovalbumin glycopeptide V) [4] 0.17 <12> (UDP-N-acetyl-d-glucosamine, <12> wild-type CHO [22]) [22] 0.2 <4> (a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-b-d-mannosyl-1,4-N-acetyld-glucosaminyl-R, <4> immunoglobulin G glycopeptide [4]) [4] 0.25 <2> (a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-a-d-mannosyl-1,3-(a-dmannosyl-1,6)-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-1,4-N-acetylglucosamine) [12] 0.26 <12> (Man5 GlcNAc2 , <12> wild-type CHO [22]) [22] 0.33 <1> (ovalbumin glycopeptide V, <1> transferase A [5]) [5] 0.39 <3> (a-d-mannosyl-1,6-(a-d-mannosyl-1,3)-a-d-mannosyl-1,6-(a-dmannosyl-1,3)-b-d-mannosyl-1,4-N-acetyl-d-glucosamine) [6] 0.43 <8> (N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(a-d-mannosyl1,6)-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-pyridinylamine) [9] 0.44 <1> (ovalbumin, <1> transferase A [5]) [5] 0.45 <2> (a-d-mannosyl-1,6-(a-d-mannosyl-1,3)-a-d-mannosyl-1,6-(a-dmannosyl-1,3)-b-d-mannosyl-1,4-N-acetyl-d-glucosamine) [2] 0.47 <1> (a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-a-d-mannosyl-1,3-(a-dmannosyl-1,6)-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-1,4-N-acetylglucosamine, <1> recombinant catalytic fragment [14]) [14] 0.483 <5> (Man5 GlcNAc2 , <5> recombinant fusion protein [21]) [21] 1.6 <13> (pyridylaminated Man3 GlcNAc2 , <13> recombinant isozyme A [23]) [23] 2.03 <2> (a-d-mannosyl-1,3-(a-d-mannosyl-1,6)-b-d-mannosyl-1,4-N-acetyl-d-glucosamine) [10] 4.5 <1> (ovalbumin, <1> transferase B [5]) [5] 5.43 <12> (Man5 GlcNAc2 , <12> mutant Lec1A [22]) [22] 7.4 <4> (a-mannosyl-1,3-b-mannosyl-1,4-N-acetylglucosamine) [4] 7.56 <12> (UDP-N-acetyl-d-glucosamine, <12> mutant Lec1A [22]) [22] 10 <4> (N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(a-d-mannosyl1,6)-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R, <4> immunoglobulin G glycopeptide [4]) [4] Additional information <1, 2, 4> (<1> Km -values for several synthetic acceptor substrate analogues, overview [15]; <4> interaction with bovine milk galactosyltransferase influences the Km -values [8]; <1> elimination of 4-hydroxyl of the a-1,3-linked mannose of the substrate increases the Km -value 20fold [1]; <1> kinetic data of free and protein-matrix-bound acceptor substrates [7]; <2> kinetic study [10]) [1, 7, 8, 10, 15]

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Ki-Value (mM) 0.034 <2> (UDP-N-acetylglucosamine) [10] 1.8 <2> (a-d-mannosyl-1,6-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl1,3)-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R) [10] Additional information <1> (<1> several inhibitory synthetic acceptor substrate analogues, overview [15]) [15] pH-Optimum 5-8 <7> (<7> broad optimum [13]) [13] 5.6 <2> [10, 12] 6 <4, 8> [4, 9] 6.3 <2, 13> (<13> assay at [23]) [2, 23] 6.5 <5> (<5> recombinant fusion protein [21]) [21] 7 <1> (<1> recombinant catalytic fragment [14]) [14] 7-7.3 <1> [5] pH-Range 5.1-6.9 <2> (<2> about half-maximal activity at pH 5.1 and about 70% of maximal activity at pH 6.9 [10]) [10] 5.8-6.7 <2> [2] 6.5-9 <5> (<5> recombinant fusion protein [21]) [21] Temperature optimum ( C) 37 <1-4, 7, 8, 13> (<1-4,7,8,13> assay at [2,4-10,12,14,23]) [2, 4-10, 12, 14, 23] 40 <5> (<5> recombinant fusion protein [21]) [21] Temperature range ( C) 0-70 <5> (<5> recombinant fusion protein [21]) [21]

4 Enzyme Structure Molecular weight 52000 <3> (<3> SDS-PAGE [6]) [6] 59000 <3> (<3> SDS-PAGE [6]) [6] Additional information <2, 3, 5, 9-11, 13> (<13> type II membrane proteins: short N-terminal cytoplasmic tail of 4 amino acid residues, transmembrane domain of 22 residues, stem region of 81 residue for isozyme A and 77 residues for isozyme B, amino acid sequences alignment [23]; <9-11> shorter hydrophobic membrane anchor domain compared to animal enzymes, amino acid sequences alignment [20]; <5> enzyme exists as high molecular weight complex, which do not require the transmembrane domain nor the cytoplasmic tail of the enzyme for complex formation [19]; <2> 2 domain protein, determination of acceptor substrate-, nucleotide sugar- and metal-binding sites [18]; <3> two MW-species of pig liver enzyme: major MW 52000 and minor MW 59000, SDS-PAGE [6]; <2> two MW-species [2,10,12]; <2> separable by gel filtration [10,12]) [2, 6, 10, 12, 18, 19, 20, 23]

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Subunits ? <1, 2, 5, 13> (<13> x * 65000, recombinant isozyme B, SDS-PAGE [23]; <1> x * 51000, recombinant, tagged protein, SDS-PAGE [17]; <5> x * 50000, recombinant enzyme, SDS-PAGE [16]; <1> x * 46000, recombinant catalytic fragment, SDS-PAGE [14]; <2> x * 58000 + x * 46000, SDS-PAGE [2]; <2> x * 45000 + x * 50000 + x * 54000, low molecular weight form, SDS-PAGE, major band: MW 45000 [10,12]) [2, 10, 12, 14, 16, 17, 23] Posttranslational modification glycoprotein <5, 9-11> (<9-11> one glycosylation site [20]; <5> O-glycosylated [19]) [19, 20] no glycoprotein <1> (<1> recombinant catalytic fragment, no glycosylation sites [14]) [14]

5 Isolation/Preparation/Mutation/Application Source/tissue CHO cell <7, 12> (<12> wild-type, Lec1 cells are deficient in enzyme activity, mutant Lec1A cells show weak enzyme activity, 2% compared to wild-type, and produce complex N-glycans [22]; <7> wild-type and mutant F6 and F8 [13]) [13, 22] S49 cell <7> (<7> wild-type and mutants Thy 1-a and Thy 1-b [13]) [13] SF-21 cell <14> [23] cell suspension culture <8> [9] colostrum <4> [4, 8, 12] flower <11> [20] heart <13> (<13> isozyme B, low content of isozyme A [23]) [23] hepatoma cell <1> (<1> diethylnitrosamine- or dimethylaminoazobenzeneinduced hepatoma, Morris 5123D hepatoma or AH-109A, solid or ascitic [5]) [5] kidney <13> (<13> isozyme B, low content of isozyme A [23]) [23] leaf <11> [20] liver <1-3, 13> (<13> isozymes A and B [23]) [1, 2, 5-7, 10, 12, 14, 15, 23] muscle <13> (<13> isozymes A and B [23]) [23] ovary <13> (<13> isozymes A and B [23]) [23] oviduct <6> [12] root <11> [20] stem <11> [20] tracheal mucosa <3, 6> [3, 6, 12] Localization Golgi apparatus <1, 5, 8, 13> (<13> medial enzyme [23]; <5> medial Golgi enzyme, exists as high molecular weight complex, which do not require the transmembrane domain nor the cytoplasmic tail of the enzyme for complex formation [19]; <1> in recombinant Saccharomyces cerevisisae [17]; <5> resident type II transmembrane protein [11,16]) [7, 9, 11, 16, 17, 19, 23] cytosol <1, 3, 4> (<1> transferase B, hepatoma [5]) [3-5, 8] 78

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a-1,3-Mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase

endoplasmic reticulum <1> (<1> in recombinant Saccharomyces cerevisisae [17]) [17] membrane <1, 2, 5, 8> (<5> membrane-bound recombinant chimeras [19]; <1> transferase A [5]; <4> not [4,8]) [5, 7, 9-12, 19] microsome <3, 7> [3, 6, 13] vacuole <1> (<1> in recombinant Saccharomyces cerevisisae [17]) [17] Additional information <1> (<1> mammalian Golgi retention signal does not function in Saccharomyces cerevisiae cells [17]) [17] Purification <1> (recombinant N-terminally His-tagged protein from Escherichia coli [14]; partial [5]; two forms: transferase A and B, the latter only in hepatoma [5]) [1, 5, 14, 15] <2> (recombinant catalytic fragment from Sf9 insect cells [18]; two molecular weight forms separable by gel filtration, purification of low-molecular weight form [10]) [2, 10, 12, 18] <3> (affinity chromatography [3]; liver [6]) [3, 6] <4> (partial [4]) [4] <5> (large scale, recombinant maltose-binding fusion protein [21]) [21] <13> (recombinant glutathione S-transferase fusion isozymes and mutants from insect Sf21 cells [23]) [23] Crystallization <2> (catalytic enzyme fragment in presence and absence of UDP-N-acetyl-dglucosamine and Mn2+ , hanging drop vapour diffusion method, protein 10 mg/ml + 10 mM MES, pH 5.5, + 270 mM KCl + 2 mM MnCl2 , 10 mM UDPN-acetyl-d-glucosamine, well-solution: 100 mM Tris-HCl, pH 7.9, 15-20% polyethylene glycol , X-ray structure determination and analysis [18]) [18] Cloning <1> (expression in Saccharomyces cerevisiae, tagged with c-Myc epitope [17]; expression of the enzymes catalytic domain in Escherichia coli strain BL21, unmodified catalytic domain and His-tagged at the C-terminal and at the Nterminal end [14]) [14, 17] <2> (expression of catalytic fragment in Spodoptera frugiperda Sf9 cells via baculovirus infection [18]) [18] <5> (functional expression of c-Myc-tagged full length clone and chimera TfR/GnT1myc and exchange mutants of the latter in CHO Lec 1 cells, lacking GnT-1 activity [19]; functional expression of unmodified enzyme and as tagged fusion protein in Spodoptera frugiperda Sf9 cells via baculovirus infection, coexpression of fowl plague virus hemagglutinin HA as endogenous substrate for in vivo activity assay [16]; cDNA clone, constructed type-II-surface membrane-protein/human transferase chimeras are transfected into Madin-Darby canine kidney cells [11]) [11, 16, 19, 21] <9> (cloning and DNA sequence determination and analysis [20]) [20] <10> (cloning and DNA sequence determination and analysis [20]) [20] <11> (cloning and DNA sequence determination and analysis [20]) [20]

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<13> (functional expression of isozymes A and B in Spodoptera frugiperda Sf21 cells as glutathione-S-transferase fusion proteins via baculovirus infection, isozyme B is folded less efficiently [23]; cloning of isozymes A and B, 2 separate genes, DNA sequence determination and analysis [23]) [23] Engineering C907T <12> (<12> point mutation in mutant cell line Lec1A.3E and Lec1A.5J, altering interactions important for stabilization of the structure, correction of the mutation by site-directed mutagenesis restores enzyme activity [22]) [22] D77N <5> (<5> exchange mutation in chimera TfR/GnT1myc, no effect on Golgi localization and inclusion into high molecular weight complexes, no catalytic activity [19]) [19] G634A <12> (<12> point mutation in mutant cell line Lec1A.2C, disrution of DxD motif responsible for interaction with UDP-GlcNAc and Mn2+ , correction of the mutation by site-directed mutagenesis restores enzyme activity [22]) [22] R83S <5> (<5> exchange mutation in chimera TfR/GnT1myc, no effect on Golgi localization and inclusion into high molecular weight complexes, no catalytic activity [19]) [19] R85S <5> (<5> exchange mutation in chimera TfR/GnT1myc, no effect on Golgi localization and inclusion into high molecular weight complexes, no catalytic activity [19]) [19] T223A <13> (<13> isozyme B, improvement of properties of isozyme B to the level of isozyme A [23]) [23] Additional information <5, 9-12> (<12> 1 difference to published sequence with accession number U65791: C1340 is A1340 [22]; <9,10> construction of antisense transgenic plants via Agrobacterium tumefaciens transfection of the own gene in antisense orientation, regulation analysis [20]; <9,10> transient transfection of isolated protoplasts of Arabidopsis thaliana defective mutant with cDNA in sense and antisense orientation [20]; <11> natural point mutation contains mRNA but no enzyme activity [20]; <5> a truncated, soluble enzyme form, consisting of stem region and catalytic domain, is accumulated in the Golgi apparatus prior to secretion [19]; <5> construction of chimeric proteins with different portions of the N-terminal ectodomain of the enzyme, modification to ectodomain of type II surface membrane protein, chimeras are retained in the Golgi apparatus [11]) [11, 19, 20, 22] Application biotechnology <1, 5> (<5> immobilization of the enzyme as maltose-binding fusion protein on an amylose resin for production of high-mannose type oligosaccharides [21]; <1> production of glycoprotein therapeutics in recombinant Saccharomyces cerevisiae adapted to production of hybrid- and complex-type carbohydrates [17]) [17, 21]

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6 Stability General stability information <3>, Triton X-100, 0.1%, and albumin, 0.01%, stabilize solubilized enzyme [3] <3>, freeze-thawing, solubilized enzyme, unstable to [3] <2, 3>, repeated freeze-thawing, stable to [2, 6] Storage stability <2>, -20 C, 20% glycerol v/v, up to a year [2] <2>, 4 C, in 20% glycerol, 0.1% Triton X-100, 25 mM MES-buffer, pH 6.5, 10 mM MnCl2 , 1 mM PMSF, 0.1 mM 6-aminocaproic acid and 0.02% NaN3 , several months [12] <3>, -20 C, at least 6 months [6] <3>, 3 C, at least a month [3] <4>, 4 C, in dilute solution, t1=2 : 1 day [4] <4>, 4 C, in the presence of albumin, at least a month [4]

References [1] Möller, G.; Reck, F.; Paulsen, H.; Kaur, K.J.; Sarkar, M.; Schachter, H.; Brockhausen, I.: Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc: Man-a-3R-b-2-N-acetylglucosaminyltransferase I using synthetic substrate analogs. Glycoconjugate J., 9, 180-190 (1992) [2] Oppenheimer, C.L.; Hill, R.L.: Purification and characterization of a rabbit liver a1 ! 3 mannoside b1 ! 2 N-acetylglucosaminyltransferase. J. Biol. Chem., 256, 799-804 (1981) [3] Mendicino, J.; Chandrasekaran, E.V.; Rao Anumula, K.; Davila, M.: Isolation and properties of a-d-mannose:b-1,2-N-acetylglucosaminyltransferase from trachea mucosa. Biochemistry, 20, 967-976 (1981) [4] Harpaz, N.; Schachter, H.: Control of glycoprotein synthesis. Bovine colostrum UDP-N-acetylglucosamine:a-d-mannoside b2-N-acetylglucosaminyltransferase I. Separation from UDP-N-acetylglucosamine:a-d-mannoside b 2-N-acetylglucosaminyltransferase II, partial purification, and substrate specificity. J. Biol. Chem., 255, 4885-4893 (1980) [5] Miyagi, T.; Tsuiki, S.: Studies on UDP-N-acetylglucosamine: a-mannoside b-N-acetylglucosaminyltransferase of rat liver and hepatomas. Biochim. Biophys. Acta, 661, 148-157 (1981) [6] Oppenheimer, C.L.; Eckhardt, A.E.; Hill, R.L.: The nonidentity of porcine N-acetylglucosaminyltransferases I and II. J. Biol. Chem., 256, 11477-11482 (1981) [7] Shao, M.C.; Wold, F.: The effect of the protein matrix on glycan processing in glycoproteins. Kinetic analysis of three rat liver Golgi enzymes. J. Biol. Chem., 263, 5771-5774 (1988)

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[8] Moscarello, M.A.; Mitranic, M.M.; Vella, G.: Stimulation of bovine milk galactosyltransferase activity by bovine colostrum N-acetylglucosaminyltransferase I. Biochim. Biophys. Acta, 831, 192-200 (1985) [9] Tezuka, K.; Hayashi, M.; Ishihara, H.; Akazawa, T.; Takahashi, N.: Studies on synthetic pathway of xylose-containing N-linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.). Eur. J. Biochem., 203, 401-413 (1992) [10] Nishikawa, Y.; Pegg, W.; Paulsen, H.; Schachter, H.: Control of glycoprotein synthesis. Purification and characterization of rabbit liver UDP-N-acetylglucosamine:a-3-d-mannoside b-1,2-N-acetylglucosaminyltransferase I. J. Biol. Chem., 263, 8270-8281 (1988) [11] Tang, B.L.; Wong, S.H.; Low, S.H.; Hong, W.: The transmembrane domain of N-glucosaminyltransferase I contains a Golgi retention signal. J. Biol. Chem., 267, 10122-10126 (1992) [12] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and O-glycan synthesis. Methods Enzymol., 179, 351-397 (1989) [13] Fritz, T.A.; Gabb, M.M.; Wei, G.; Esko, J.D.: Two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate. J. Biol. Chem., 269, 28809-28814 (1994) [14] Nishiu, J.; Kioka, N.; Fukada, T.; Sakai, H.; Komano, T.: Characterization of rat N-acetylglucosaminyltransferase I expressed in Escherichia coli. Biosci. Biotechnol. Biochem., 59, 1750-1752 (1995) [15] Reck, F.; Springer, M.; Meinjohanns, E.; Paulsen, H.; Brockhausen, I.; Schachter, H.: Synthetic substrate analogs for UDP-GlcNAc:mana1-3R b1-2-N-acetylglucosaminyltransferase I. Substrate specificity and inhibitors for the enzyme. Glycoconjugate J., 12, 747-754 (1995) [16] Wagner, R.; Liedtke, S.; Kretzschmar, E.; Geyer, H.G.; Geyer, R.; Klenk, H.D.: Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human b1,2-N-acetylglucosaminyltransferase I. Glycobiology, 6, 165-175 (1996) [17] Yoshida, S.; Suzuki, M.; Yamano, S.; Takeuchi, M.; Ikenaga, H.; Kioka, N.; Sakai, H.; Komano, T.: Expression and characterization of rat UDP-N-acetylglucosamine: a-3-d-mannoside b-1,2-N-acetylglucosaminyltransferase I in Saccharomyces cerevisiae. Glycobiology, 9, 53-58 (1999) [18] Unligil, U.M.; Zhou, S.; Yuwaraj, S.; Sarkar, M.; Schachter, H.; Rini, J.M.: Xray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. EMBO J., 19, 5269-5280 (2000) [19] Opat, A.S.; Houghton, F.; Gleeson, P.A.: Medial Golgi but not late Golgi glycosyltransferases exist as high molecular weight complexes. Role of luminal domain in complex formation and localization. J. Biol. Chem., 275, 1183611845 (2000) [20] Wenderoth, I.; Von Schaewen, A.: Isolation and characterization of plant Nacetyl glucosaminyltransferase I (GntI) cDNA sequences. Functional analyses in the Arabidopsis cgl mutant and in antisense plants. Plant Physiol., 123, 1097-1108 (2000)

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[21] Fujiyama, K.; Ido, Y.; Misaki, R.; Moran, D.G.; Yanagihara, I.; Honda, T.; Nishimura, S.I.; Yoshida, T.; Seki, T.: Human N-acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides. J. Biosci. Bioeng., 92, 569-574 (2001) [22] Chen, W.; Unligil, U.M.; Rini, J.M.; Stanley, P.: Independent Lec1A CHO glycosylation mutants arise from point mutations in N-acetylglucosaminyltransferase I that reduce affinity for both substrates. Molecular consequences based on the crystal structure of GlcNAc-TI. Biochemistry, 40, 8765-8772 (2001) [23] Mucha, J.; Svoboda, B.; Frohwein, U.; Strasser, R.; Mischinger, M.; Schwihla, H.; Altmann, F.; Hane, W.; Schachter, H.; Glossl, J.; Mach, L.: Tissues of the clawed frog Xenopus laevis contain two closely related forms of UDPGlcNAc:a3-d-mannoside b-1,2-N-acetylglucosaminyltransferase I. Glycobiology, 11, 769-778 (2001)

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b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

2.4.1.102

1 Nomenclature EC number 2.4.1.102 Systematic name UDP-N-acetyl-d-glucosamine:O-glycosyl-glycoprotein (N-acetyl-d-glucosamine to N-acetyl-d-galactosamine of b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-R) b-1,6-N-acetyl-d-glucosaminyltransferase Recommended name b-1,3-galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase Synonyms C2GnT <4> [14] O-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase I acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-mucin b-(1-6)acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-mucin b-(1-6)-, A b6-N-acetylglucosaminyltransferase core 2 GlcNAc-T <3, 9, 11> [8, 17] core 2 N-acetylglucosaminyltransferase <9> [9] core 2 acetylglucosaminyltransferase core 2 b-1,6-N-acetylglucosaminyltransferase <9> [19] core 6-b-GlcNAc-T <4> [7] core 6-b-GlcNAc-transferase A uridine diphosphoacetylglucosamine-mucin b-(1-6)-acetylglucosaminyltransferase uridine diphosphoacetylglucosamine-mucin b-(1-6)-acetylglucosaminyltransferase A Additional information <4, 15> (cf. EC 2.4.1.146-148; <4,15> core 2 b6-GalNAc-transferase, EC 2.4.1.102, exists in 2 types, type L and type M, the latter shows an additional core 4 b6-GalNAc transferase activity [18,20]) [18, 20] CAS registry number 87927-97-7 95978-15-7

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b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

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

Canis familiaris [1-5] Sus scrofa [1-3, 7, 10, 16] Rattus norvegicus [1-3, 7, 10, 17] Homo sapiens [1, 6, 7, 10, 12-14, 20-22] Oryctolagus cuniculus [1] Bos taurus [1] monkey [1] Ovis aries [1] Mus musculus (inducible by cytokines IL-4, and IL-12/STAT4 [19]) [7, 9, 11, 17, 19] Gallus gallus [7] Cricetulus griseus [8] Homo sapiens (isozyme 3 [15]) [15] Homo sapiens (isozyme 1 [15]) [15] Homo sapiens (isozyme 2 [15]) [15] Bovine herpesvirus type 4 (i.e. BHV-4 [18]) [18]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-R = UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-R Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-R <1-9> (<9> involved in the development of cardiomyopathy [17]; <9> involved in regulation of downstream intracellular signal transduction pathways [17,19]; <1-9> involved in biosynthesis of O-glycans core class 2 [1,7,17]; <1-8> involved in mucin oligosaccharide biosynthesis [1,2]; <9> increased activity during E- and P-selectin ligand biosynthesis [19]) [1, 2, 7, 17, 19] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-R S Additional information <1-9> (<3,9> enzyme is developmentally regulated, increased activity in hearts of diabetic organisms, regulation by hyperglycemia and insulin [17]; <4> enzyme activity is increased in patients with chronic myelogenous leukemia CML or acute myeloid leukemia AML [7,10]; <4> patients with Wiskott-Aldrich-Syndrome show an altered enzyme activity and inversed enzyme stimulation in T cells, enzyme activity

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2.4.1.102

in platelets is 3fold higher compared to normal organisms [6]; <1-8> overview: structure of O-glycans [1]) [1, 6, 7, 10, 17] P ? Substrates and products S UDP-N-acetyl-d-glucosamine + 3-deoxy-b-d-galactosyl-1,3-N-acetyl-dgalactosaminyl-benzyl <2-4, 9> (Reversibility: ? <2-4,9> [7]) [7] P UDP + 3-deoxy-b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-Nacetyl-d-galactosaminyl-benzyl S UDP-N-acetyl-d-glucosamine + 4-deoxy-b-d-galactosyl-1,3-N-acetyl-dgalactosaminyl-benzyl <2-4, 9> (Reversibility: ? <2-4,9> [7]) [7] P UDP + 4-deoxy-b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-Nacetyl-d-galactosaminyl-benzyl S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-glucosaminyl-1,3-N-acetyla-d-galactosaminyl-R <15> (<15> i.e. core 3 [18]; <15> enzyme shows both core 2 b6-GalNAc-transferase activity, EC 2.4.1.102, and core 4 b6GalNAc-transferase activity, EC 2.4.1.148, and is termed a type M core 2 b6-GalNAc-transferase [18]) (Reversibility: ? <15> [18]) [18] P UDP + N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,3)N-acetyl-a-d-galactosaminyl-R <15> (<15> i.e. core 4 [18]) [18] S UDP-N-acetyl-d-glucosamine + N-acetylgalactosaminyl-Ser(Thr)-mucin <1-8> (Reversibility: ? <1-8> [1]) [1] P ? S UDP-N-acetyl-d-glucosamine + antifreeze glycoprotein polypeptide <1> (Reversibility: ? <1> [5]) [5] P ? S UDP-N-acetyl-d-glucosamine + asialo-a1 acid glycoprotein <1> (<12> no activity [15]) (Reversibility: ? <1> [4,5]) [4, 5] P ? S UDP-N-acetyl-d-glucosamine + asialo-blood group A negative PSM <1> (<1> acceptor contains no fucose [4]) (Reversibility: ? <1> [4]) [4] P ? S UDP-N-acetyl-d-glucosamine + asialo-submaxillary mucin <1, 12> (<1> ovine asialo-submaxillary mucin [5]; <12> bovine asialo-submaxillary mucin [15]; <12> low activity [15]) (Reversibility: ? <1,12> [15]) [5, 15] P ? S UDP-N-acetyl-d-glucosamine + asialofetuin <1, 9, 12> (<1> low activity [5]) (Reversibility: ? <1,9,12> [5,11,15]) [5, 11, 15] P ? S UDP-N-acetyl-d-glucosamine + asialoglycophorin A <9, 12> (<9,12> best substrate [11,15]) (Reversibility: ? <9,12> [11,15]) [11, 15] P ? S UDP-N-acetyl-d-glucosamine + benzyl 2-acetamido-2-deoxy-a-d-galactopyranoside <2, 3> (Reversibility: ? <2,3> [2]) [2] P ?

86

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b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-dgalactosaminyl-benzyl <2-4, 9> (<4, 9, 10> at high concentration inhibitory [7]) (Reversibility: ? <2-4,9> [7,10]) [7, 10] P ? S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosamine <1-8> (Reversibility: ? <1-8> [1,5]) [1, 5] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosamine S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-(CH2 )8 CO2 Me <9> (<9> synthetic disaccharide [9]) (Reversibility: ? <9> [9]) [9] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-(CH2 )8 CO2 Me S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-R <1-9, 11, 12, 15> (<4> large scale preparation [21]; <9> highly specific for both substrates [11]; <2> highly specific for UDP-N-acetyl-dglucosamine [16]; <1-9, 12> i.e. core class 1, R: polypeptide [1, 11, 15]; <1, 4, 12> substrate specificity [5, 7, 15]) (Reversibility: ? <1-9, 11, 12, 15> [1-8, 11-22]) [1-8, 11-22] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-R <1-9, 11, 12> (<1-9, 11, 12> i.e. core class 2 [1, 2, 4, 5, 7, 8, 11-15, 21]) [1, 2, 4, 5, 7, 8, 11-16, 21, 22] S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl <1-10> (<2-4, 9, 10> requirement for the 4- and 6-hydroxyls of N-acetyl-galactosamine and the hydroxyl of galactose in the acceptor substrate [7]) (Reversibility: ? <1-10> [1, 2, 4, 5, 7, 14, 21, 22]) [1, 2, 4, 5, 7, 14, 21, 22] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-benzyl <4> [21] S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-methyl <1-8> (Reversibility: ? <1-8> [1,5]) [1, 5] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-methyl S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-methylumbelliferyl <12> (Reversibility: ? <12> [15]) [15] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-methylumbelliferyl S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-o-nitrophenyl <1-8> (Reversibility: ? <1-8> [1,2,5,7]) [1, 2, 5, 7] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-o-nitrophenyl S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-p-nitrophenyl <1-8, 12> (<4> inhibitory under UV-radiation [10]) (Reversibility: ? <1-8,12> [1,5,7,10,15,21]) [1, 5, 7, 10, 15, 21] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-p-nitrophenyl <4, 12> [15, 21]

87

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2.4.1.102

S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-phenyl <1-8> (Reversibility: ? <1-8> [1,4,7]) [1, 4, 7] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-phenyl S UDP-N-acetyl-d-glucosamine + fetuin <1> (<12> no activity [15]) (Reversibility: ? <1> [5]) [5] P ? S UDP-N-acetyl-d-glucosamine + fucosyl-a-1,2-galactosyl-b-1,3-N-acetyld-galactosaminyl-R <1-8> (<1-8> low activity [1]) (Reversibility: ? <18> [1]) [1] P UDP + fucosyl-a-1,2-galactosyl-b-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-Nacetyl-d-galactosaminyl-R S UDP-N-acetyl-d-glucosamine + galactosyl-b-1,3-N-acetyl-d-galactosaminitol, d-fucosyl-b-1,3-N-acetyl-a-d-galactosaminyl-benzyl <1> (Reversibility: ? <1> [5]) [5] P ? S UDP-N-acetyl-d-glucosamine + galactosyl-b-1,3-N-acetyl-b-d-glucosaminyl-methyl <1> (Reversibility: ? <1> [5]) [5] P UDP + galactosyl-b-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-b-dglucosaminyl-methyl S UDP-N-acetyl-d-glucosamine + galactosyl-b-1,3-N-acetyl-b-d-glucosaminyl-p-nitrophenyl <1, 11> (Reversibility: ? <1,11> [5,8]) [5, 8] P UDP + galactosyl-b-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-b-dglucosaminyl-p-nitrophenyl S UDP-N-acetyl-d-glucosamine + p-nitrophenyl 2-acetamido-2-deoxy-3-O(b-d-galactopyranosyl)-a-d-galactopyranoside <9> (Reversibility: ? <9> [19]) [19] P ? S UDP-N-acetyl-d-glucosamine + phenyl 2-acetamido-2-deoxy-a-d-galactopyranoside <2, 3> (Reversibility: ? <2,3> [2]) [2] P ? S UDP-N-acetyl-d-glucosamine + submaxillary mucin polypeptide <1-8> (<1> porcine or ovine origin of acceptor substrate [4]; <1-8> porcine acceptor substrate [1,5]) (Reversibility: ? <1-8> [1,4,5]) [1, 4, 5] P ? S UDP-N-acetyl-d-glucosamine + trachea mucin <2> (<2> porcine or human trachea mucin [16]; <2> acceptor substrate is deglycosylated [16]) (Reversibility: ? <2> [16]) [16] P ? S Additional information <1-8, 12> (<12> no activity human transferrin and IgG, N-acetylgalactosamine, core 3 substrates [15]; <2,4> attachment of UDPGlcNAc to serine or threonine residue [14, 16]; <1> no substrates: lactose, Galb1-3GlcNAc, l-Fuca1-2Galb1-3GalNAc-OH, a- and b-methylglycosides of galactose [5]; <1-8> no substrate: galactosyl-b-1,3-N-acetyld-galactosamine [1,5]) [1, 5, 14-16] P ?

88

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b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

Inhibitors 12-O-tetradecanoylphorbol-13-acetate <4> (<4> i.e. TPA [13]; <4> about 10fold decreased activity, down-regulation of native enzyme in KM3 leukemia cell line, but not of the recombinant enzyme [13]) [13] Ca2+ <1> (<1> slight inhibition [4]) [4] Cu2+ <2-4, 9> [7] EDTA <1> (<1> slight inhibition [4]) [4] Mn2+ <1-9> (<9> slight inhibition [11]; <2> concentration above 7.5 mM [16];<1> slight stimulation at 5 mM, inhibition at higher concentrations [4]) [1, 4, 11, 16] Triton X-100 <2> (<2> activation at 0.1% v/v, inhibition at higher concentrations, pig stomach [1]) [1] Zn2+ <1, 9> (<1> strong inhibition [4]) [4, 11] b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl <4, 9> (<4, 9> high concentration [7]) [7] b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-p-nitrophenyl <4> (<4> and other nitrophenyl-sugar-derivatives [10]; <4> strong inhibition under UVradiation at 350 nm, specific for substrate binding site, preincubation with b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl partially protects [10]) [10] substrate analogues lacking the 6-hydroxyl of the N-acetylgalactosamine <4> (<4> weak [10]) [10] tunicamycin <4> (<4> prevents secretion of recombinant enyzme from Sf9 cells [12]; <4> inactivation [12]) [12] Additional information <11> (<11> no effect of dimethyl sulfoxide, retinoic acid, phorbol ester, cholera toxin [8]) [8] Activating compounds Triton X-100 <1-8> (<1-8> activation, 0.1% v/v [1]; <1> 4fold stimulation at 0.125% v/v [4]; <2> inactivates at higher concentrations, pig stomach [1]) [1, 4] cytokines <9> (<9> induction by IL-4 and IL-12/STAT4 [19]) [19] empigen BB <1> (<1> activation, detergent [4]) [4] sodium butyrate <11> (<11> induction after treatment of cells for 24 h, 16fold increase in activity, blocked by actinomycin D and cycloheximide [8]) [8] Additional information <9, 11> (<9> no induction by Ag+ [19]; <11> no effect of dimethyl sulfoxide, retinoic acid, phorbol ester, cholera toxin [8]) [8, 19] Metals, ions Mg2+ <1, 9> (<9> activation [11]; <1> slight stimulation, 5 mM [4]) [4, 11] Mn2+ <1-8> (<4> not required [21]; <2> required, 5-7.5 mM [16]; <1-8> slight stimulation, 5 mM, inhibits at higher concentration [1,4]) [1, 4, 16] Additional information <2-4, 9> (<2-4,9> stimulation by divalent cations [7]) [7]

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Specific activity (U/mg) 0.0000048 <4> (<4> normal granulocytes [21]) [21] 0.0000056 <4> (<4> untreated recombinant KM3 leukemia cell line [13]) [13] 0.000013 <4> (<4> unstimulated normal T-lymphocytes [6]) [6] 0.00002 <4> (<4> CML granulocytes [21]) [21] 0.00004 <4> (<4> EBV B-cell line from healthy individuals [6]) [6] 0.000087 <4> (<4> AML cells [21]) [21] 0.00015 <4> (<4> undifferentiated CaCo-2, colonic adenocarcinoma, cells [22]) [22] 0.00034 <4> (<4> CaCo-2, colonic adenocarcinoma, cells differentiated to enterocytes [22]) [22] 0.00067 <4> (<4> recombinant enzyme in Panc1-MUC1 cells [14]) [14] Additional information <2-4, 9, 10> (<2-4,9,10> overview: enzyme activities with several substrates [7]) [7, 12] Km-Value (mM) 0.0063 <2> (UDP-N-acetyl-d-glucosamine) [16] 0.36 <4> (b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl, <4> undifferentiated CaCo-2, colonic adenocarcinoma, cells [22]) [22] 0.43 <4> (b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl, <4> CaCo-2, colonic adenocarcinoma, cells differentiated to enterocytes [22]) [22] 0.5 <11> (UDP-N-acetyl-d-glucosamine) [8] 0.52 <1> (galactosyl-b-1,3-N-acetyl-d-galactosaminyl-a-p-nitrophenyl) [1, 5] 0.6 <4> (b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-p-nitrophenyl, <4> AML cell line [7]) [7] 0.77 <1> (galactosyl-b-1,3-N-acetyl-a-d-galactosaminyl-benzyl) [1, 5] 0.8 <4> (b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-benzyl, <4> AML cell line [7]) [7] 0.86 <1> (galactosyl-b-1,3-N-acetyl-d-galactosaminyl-a-o-nitrophenyl) [1, 5] 0.92 <1> (galactosyl-b-1,3-N-acetyl-b-d-galactosaminyl-p-nitrophenyl) [1, 5] 1 <1-8> (UDP-N-acetyl-d-glucosamine) [1, 4] 1.1 <2, 10> (b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-benzyl) [7] 1.2 <3> (b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-benzyl) [7] 1.2 <1> (galactosyl-b-1,3-N-acetyl-d-galactosamine) [5] 1.2 <1> (galactosyl-b-1,3-N-acetyl-a-d-galactosaminyl-phenyl) [1] 1.4 <2> (b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-benzyl, <4> colon [7]) [7] 2 <9> (3-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl) [7] 2 <4> (b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-o-nitrophenyl, <4> AML cell line [7]) [7] 2 <9> (b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-benzyl) [7] 3.1 <9> (4-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl) [7] 3.3 <4> (3-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl, <4> AML cell line [7]) [7] 4.2 <3> (4-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl) [7] 4.2 <1> (galactosyl-b-1,3-N-acetyl-a-d-galactosaminyl-methyl) [1, 5] 4.3 <3> (3-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl) [7]

90

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b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

4.5 <4> (3-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl, <4> colon [7]) [7] 4.54 <11> (UDP-N-acetyl-d-glucosamine, <11> butyrate treated cells [8]) [8] 5 <2> (4-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl) [7] 5.2 <1> (asialo-blood group A negative PSM) [4] 5.2 <1-8> (porcine submaxillary mucin polypeptide) [1, 5] 5.5 <2> (3-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl) [7] 6.7 <4> (4-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl, <4> AML cell line [7]) [7] 10 <4> (4-deoxy-b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-benzyl, <4> colon [7]) [7] 10 <4> (b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-phenyl, <4> AML cell line [7]) [7] 12 <9> (b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl) [7] 21 <4> (b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl, <4> colon [7]) [7] 23 <1> (antifreeze glycoprotein polypeptide) [5] 29 <4> (b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl, <4> AML cells [7]) [7] 37 <4> (b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl, <4> CML cells [7]) [7] Additional information <2, 4> [7, 16] Ki-Value (mM) 0.95 <4> (b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl, <4> AML cells [7]) [7] 1.5 <4> (b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl, <4> colon [7]) [7] 2 <9> (b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl) [7] 2.5 <4> (b-d-galactosyl-1,3-(6-deoxy)-N-acetyl-d-galactosaminyl-benzyl, <4> CML cells [7]) [7] pH-Optimum 7 <1-8> (<4> assay at [21]) [1, 4, 12] 7.2 <2> [16] 7.4 <11> (<11> assay at [8]) [8] pH-Range 6.7-7.8 <2> (<2> broad optimum [16]) [16] Temperature optimum ( C) 25 <1> (<1> assay at [4]) [4] 30 <1-8> (<1-8> assay at [1]) [1] 37 <4, 11> (<4,11> assay at [7,8,12,21]) [7, 8, 12, 21]

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4 Enzyme Structure Molecular weight 70000 <2> (<2> gel filtration [16]) [16] Additional information <12> (<12-14> amino acid sequence [15]) [15] Subunits monomer <2> (<2> 1 * 70000, SDS-PAGE [16]) [16] Posttranslational modification glycoprotein <4> (<4> binds concanavalin A [12]; <4> N-glycans at both glycosylation sites [12]) [12]

5 Isolation/Preparation/Mutation/Application Source/tissue B-cell <4> [6] CACO-2 cell <4> [22] CHO cell <11> [8] CML cell <4> (<4> chronic myelogenous leukemia granulocyte cell line [7,21]; <4> 4fold increased level compared to normal granulocytes [21]) [7, 21] HT-29 cell <4> (<4> colonic adenocarcinoma cell line [20]) [20] KG-1 cell <4> (<4> erythroleukemia cell line [20]) [20] KM-3 cell <4> (<4> pre-B lymphocytic leukemia cell line [13]) [13] LS-180 cell <4> (<4> colonic adenocarcinoma cell line [20]) [20] SW-1116 cell <4> (<4> colonic grade II tumour cell line [20]) [20] SW-403 cell <4> (<4> colonic grade III tumour cell line [20]) [20] SW-48 cell <4> (<4> colonic large ulcerating grade IV tumour cell line, low activity [20]) [20] T-lymphocyte <4, 9> (<9> wild-type and transgenic CD4+ T cell lines, induction by cytokines IL-4, and IL-12/STAT4 [19]) [6, 19] acute myeloid leukemia cell <4> (<4> acute myeloid leukemia blast cell line [7,10,21]; <4> 18fold increased level compared to normal granulocytes [21]) [7, 10, 13, 21] blood platelet <4> [6] colon <2-4, 7, 12> (<12> low content [15]) [1, 3, 10, 15, 20] colonic adenocarcinoma cell <4> [22] colonic mucosa <3> [2, 7] gastric mucosa <2> [2, 7, 10] granulocyte <4> [21] heart <9> [17] intestinal cancer cell line <4> (<4> NCI498, ileo-caecal carcinoma cell line [20]) [20] intestine <5> [1] kidney <9> (<9> commercial acetone powder [9]) [7, 9]

92

2.4.1.102

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

leukocyte <12> (<12> peripheral blood [15]) [15] lymphocytic leukemia cell <4> (<4> YC2C2 lymphocytic leukemia cell line [20]) [20] ovary <11, 12> (<12> low content [15]) [8, 15] oviduct <10> [7] peripheral blood <12> [15] small intestine <3, 12> [1, 15] spleen <12> (<12> low content [15]) [15] stomach <2, 3, 7, 8> [1, 3] submaxillary gland <1, 3> (<2> not [1,3]) [1-5] testis <12> (<12> low content [15]) [15] thymus <12> (<12> high content [15]) [15] trachea <2, 6> [1, 16] tracheal epithelium <2> [16] Additional information <2-4, 15> (<4> not in breast cancer cell lines [20]; <15> expression during virus replication cycle, MDBK cells [18]; <3> not in rat liver [1]) [1, 18, 20] Localization extracellular <4, 12> (<4,12> secretion into medium of the recombinant enzyme from Sf9 cells [12,15]) [12, 15] microsome <1-8, 10> [1, 3-5, 7, 10, 16] Purification <2> [16] <3> (recombinant as GST fusion protein from a bacterial expression system [17]) [17] <4> (partially, recombinant from Sf9 insect cells [12]) [12] <9> (single-step, affinity chromatography on UDP-hexanolamine Sepharose [9]; pilot scale, recombinant protein A-tagged enzyme, fluidized bed system [11]) [9, 11] Cloning <4> (expression in human pancreatic cancer cell line Panc1-MUC1, which lacks enzyme C2GnT activity, alters the expression of MUC1-epitope in the same cell line [14]; overexpression in KM3 leukemia cell line [13]; expression in Spodoptera frugiperda Sf9 insect cells via baculovirus infection, truncated enzyme form [12]) [12, 13] <9> (expression in PC12 cells [17]; overexpression in CHO cells as protein Atagged fusion protein [11]) [11, 17] <12> (DNA sequence determination and analysis [15]; coexpression of isozyme 3 with leukosialin as substrate in CHO cells for in vivo activity assay of the enzyme [15]; cloning and expression of isozyme 3 in Spodoptera frugiperda Sf9 insect cells via baculovirus infection, secretion of the enzyme [15]) [15] <15> (functional expression in CHO cells, direction of enzyme activity onto the cell surface, a soluble chimeric enzyme form shows also core 4 6b-GalNAc transferase activity, DNA and amino acid sequence determination [18]) [18]

93

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

2.4.1.102

Engineering T60A <4> (<4> site-directed mutagenesis, highly reduced activity [12]) [12] T60A/T97A <4> (<4> site-directed mutagenesis, no activity, no secretion of Sf9 cells [12]) [12] T97A <4> (<4> site-directed mutagenesis, 50% reduced activity [12]) [12] Additional information <9> (<9> construction of transgenic mice by microinjection of rat cDNA fused to a rat cardiac-specific promotor into pronuclei of fertilized FVB mouse eggs, overexpression and increase of enzyme activity in the heart leading to cardiac hypertrophy, elevated content of sialylated Oglycans, induction of c-fos expression, and phenotypic changes [17]) [17] Application molecular biology <4> (<4> b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-pnitrophenyl and other nitrophenyl-sugar-derivatives are useful as specific inhibitors and as affinity label [10]) [10] synthesis <9> (<9> chemical-enzymatic synthesis of sialyl-Lex-containing hexasaccharides found on O-linked glycoproteins, process involves several enzymes of the pathway [9]) [9]

6 Stability Temperature stability 25 <1> (<1> 25 C, 2 h [4]) [4] 37 <1-8> (<1> unstable [4]; <1-8> decrease of activity, in the presence of Triton X-100 [1]) [1, 4] General stability information <1>, freeze-thawing, after 2 cycles 13% loss of activity [4] <4>, freeze-drying and resuspension in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 95% remaining activity [12] Storage stability <1>, frozen in liquid nitrogen, several months [4] <4>, -70 C, crude enzyme extract, stable up to 1 year [12] <4>, 4 C, stable in solution or after lyophilization for several months [12] <1-8>, -70 C, microsomal preparation, detergent-free 0.25 M sucrose suspension, several years [1]

References [1] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and O-glycan synthesis. Methods Enzymol., 179, 351-397 (1989) [2] Brockhausen, I.; Rachaman, E.S.; Matta, K.L.; Schachter, H.: The separation by liquid chromatography (under elevated pressure) of phenyl, benzyl, and O-nitrophenyl glycosides of oligosaccharides. Analysis of substrates and

94

2.4.1.102

[3]

[4] [5]

[6]

[7]

[8] [9]

[10]

[11]

[12] [13]

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

products for four N-acetyl-d-glucosaminyl-transferases involved in mucin synthesis. Carbohydr. Res., 120, 3-16 (1983) Brockhausen, I.; Matta, K.L.; Orr, J.; Schachter, H.: Mucin synthesis. UDPb3-N-acetylglucosaminyltransferase and UDPGlcNAc:GalNAc-R GlcNAc:GlcNAc b 1-3GalNAc-R (GlcNAc to GalNAc) b6-N-acetylglucosaminyltransferase from pig and rat colon mucosa. Biochemistry, 24, 1866-1874 (1985) Williams, D.; Schachter, H.: Mucin synthesis. I. Detection in canine submaxillary glands of an N-acetylglucosaminyltransferase which acts on mucin substrates. J. Biol. Chem., 255, 11247-11252 (1980) Williams, D.; Longmore, G.; Matta, K.L.; Schachter, H.: Mucin synthesis. II. Substrate specificity and product identification studies on canine submaxillary gland UDP-GlcNAc:Gal b1-3GalNAc(GlcNAc leads to GalNAc) b6-Nacetylglucosaminyltransferase. J. Biol. Chem., 255, 11253-11261 (1980) Higgins, E.A.; Siminovitch, K.A.; Zhuang, D.; Brockhausen, I.; Dennis, J.W.: Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome. J. Biol. Chem., 266, 6280-6290 (1991) Kuhns, W.; Rutz, V.; Paulsen, H.; Matta, K.L.; Baker, M.A.; Barner, M.; Granovsky, M.; Brockhausen, I.: Processing O-glycan core 1, Galb1-3GalNAca-R. Specificities of core 2, UDP-GlcNAc: Galb1-3GalNAc-R(GlcNAc to GalNAc) b6-N-acetylglucosaminyltransferase and CMP-sialic acid: Galb1-3GalNAc-R a3-sialyltransferase. Glycoconjugate J., 10, 381-394 (1993) Datti, A.; Dennis, J.W.: Regulation of UDP-GlcNAc:Galb1-3GalNAc-R b1-6N-acetylglucosaminyltransferase (GlcNAc to GalNAc) in chinese hamster ovary cells. J. Biol. Chem., 268, 5409-5416 (1993) Oehrlein, R.; Hindsgaul, O.; Palcic, M.M.: Use of the core-2-N-acetylglucosaminyltransferase in the chemical-enzymic synthesis of a sialyl-LeX-containing hexasaccharide found on O-linked glycoproteins. Carbohydr. Res., 244, 149-159 (1993) Toki, D.; Granovsky, M.A.; Reck, F.; Kuhns, W.; Baker, M.A.; Matta, K.L.; Brockhausen, I.: Inhibition of UDP-GlcNAc:Gal b1-3GalNAc-R (GlcNAc to GalNAc) b6-N-acetylglucosaminyltransferase from acute myeloid leukaemia cells by photoreactive nitrophenyl substrate derivatives. Biochem. Biophys. Res. Commun., 198, 417-423 (1994) Zeng, S.; Dinter, A.; Eisenkratzer, D.; Biselli, M.; Wandrey, C.; Berger, E.G.: Pilot scale expression and purification of soluble protein A tagged b1,6Nacetylglucosaminyltransferase in CHO cells. Biochem. Biophys. Res. Commun., 237, 653-658 (1997) Toki, D.; Sarkar, M.; Yip, B.; Reck, F.; Joziasse, D.; Fukuda, M.; Schachter, H.; Brockhausen, I.: Expression of stable human O-glycan core 2 b-1,6-N-acetylglycosaminyltransferase in Sf9 insect cells. Biochem. J., 325, 63-69 (1997) Nakamura, M.; Kudo, T.; Narimatsu, H.; Furukawa, Y.; Kikuchi, J.; Asakura, S.; Yang, W.; Iwase, S.; Hatake, K.; Miura, Y.: Single glycosyltransferase, core 2 b1-6-N-acetylglucosaminyltransferase, regulates cell surface sialyl-Lex expression level in human pre-B lymphocytic leukemia cell line KM3 treated with phorbolester. J. Biol. Chem., 273, 26779-26789 (1998) 95

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

2.4.1.102

[14] Beum, P.V.; Singh, J.; Burdick, M.; Hollingsworth, M.A.; Cheng, P.W.: Expression of core 2 b-1,6-N-acetylglucosaminyltransferase in a human pancreatic cancer cell line results in altered expression of MUC1 tumor-associated epitopes. J. Biol. Chem., 274, 24641-24648 (1999) [15] Schwientek, T.; Yeh, J.-C.; Levery, S.B.; Keck, B.; Merkx, G.; Van Kessel, A.G.; Fukuda, M.; Clausen, H.: Control of O-glycan branch formation: molecular cloning and characterization of a novel thymus-associated core 2 b1,6-Nacetylglucosaminyltransferase. J. Biol. Chem., 275, 11106-11113 (2000) [16] Mendicino, J.; Sangadala, S.: Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from swine trachea epithelium. Mol. Cell. Biochem., 185, 135-145 (1998) [17] Koya, D.; Dennis, J.W.; Warren, C.E.; Takahara, N.; Schoen, F.J.; Nishio, Y.; Nakajima, T.; Lipes, M.A.; King, G.L.: Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice. FASEB J., 13, 2329-2337 (1999) [18] Vanderplasschen, A.; Markine-Goriaynoff, N.; Lomonte, P.; Suzuki, M.; Hiraoka, N.; Yeh, J.-C.; Bureau, F.; Willems, L.; Thiry, E.; Fukuda, M.; Pastoret, P.-P.: A multipotential b-1,6-N-acetylglucosaminyltransferase is encoded by bovine herpesvirus type 4. Proc. Natl. Acad. Sci. USA, 97, 5756-5761 (2000) [19] Lim, Y.-C.; Xie, H.; Come, C.E.; Alexander, S.I.; Grusby, M.J.; Lichtman, A.H.; Luscinskas, F.W.: IL-12, STAT4-dependent up-regulation of CD4+ T cell core 2 b-1,6-n-acetylglucosaminyltransferase, an enzyme essential for biosynthesis of P-selectin ligands. J. Immunol., 167, 4476-4484 (2001) [20] Vavasseur, F.; Yang, J.-M.; Dole, K.; Paulsen, H.; Brockhausen, I.: Synthesis of O-glycan core 3: characterization of UDP-GlcNAc:GalNAc-R b3-N-acetyl-glucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines. Glycobiology, 5, 351-357 (1995) [21] Brockhausen, I.; Kuhns, W.; Schachter, H.; Matta K.L.; Sutherland, D.R.; Baker, M.A.: Biosynthesis of O-glycans in leukocytes from normal donors and from patients with leukemia: increase in O-glycan core 2 UDP-GlcNAc:Galb3GalNAca-R (GlcNAc to GalNAc) b(1,6)-N-acetylglucosaminyltransferase in leikemic cells. Cancer Res., 51, 1257-1263 (1991) [22] Brockhausen, I.; Romero, P.A.; Herscovics: : Glycosyltransferase changes upon differentation of CaCo-2 human colonic adenocarcinoma cells. Cancer Res., 51, 3136-3142 (1991)

96

Alizarin 2-b-glucosyltransferase

2.4.1.103

1 Nomenclature EC number 2.4.1.103 Systematic name UDP-glucose:1,2-dihydroxy-9,10-anthraquinone 2-O-b-d-glucosyl transferase Recommended name alizarin 2-b-glucosyltransferase Synonyms glucosyltransferase, uridine diphosphoglucose-alizarin CAS registry number 74506-41-5

2 Source Organism <1> Streptomyces aureofaciens (mutant strain B96 [1,2]; strain Bg, 84-25, NMG2 and UV61 [2]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + alizarin = UDP + 1-hydroxy-2-(b-d-glucosyloxy)-9,10-anthraquinone (<1>, mechanism [1]) Reaction type hexosyl group transfer Substrates and products S UDP-glucose + 1,4-dihydroxy-9,10-anthraquinone <1> (<1>, 7% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-hydroxy-4-(b-d-glucosyloxy)-9,10-anthraquinone S UDP-glucose + 1,5-dihydroxy-9,10-anthraquinone <1> (<1>, 2% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-hydroxy-5-(b-d-glucosyloxy)-9,10-anthraquinone

97

Alizarin 2-b-glucosyltransferase

2.4.1.103

S UDP-glucose + alizarin <1> (<1>, i.e. 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1,2]) [1, 2] P UDP + 1-hydroxy-2-(b-d-glucosyloxy)-9,10-anthraquinone <1> [1, 2] S UDPglucose + 1,3-dihydroxy-9,10-anthraquinone <1> (<1>, 48% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-hydroxy-3-(b-d-glucosyloxy)-9,10-anthraquinone S UDPglucose + 1,8-dihydroxy-9,10-anthraquinone <1> (<1>, 9% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-hydroxy-8-(b-d-glucosyloxy)-9,10-anthraquinone S UDPglucose + 1-hydroxy-3-methoxy9,10-anthraquinone <1> (<1>, 2% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 3-methoxy-1-(b-d-glucosyloxy)-9,10-anthraquinone S UDPglucose + 1-hydroxy-9,10-anthraquinone <1> (<1>, 3% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-(b-d-glucosyloxy)-9,10-anthraquinone S UDPglucose + 2,6-dihydroxy-9,10-anthraquinone <1> (<1>, 51% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-hydroxy-6-(b-d-glucosyloxy)-9,10-anthraquinone S UDPglucose + 2-hydroxy-9,10-anthraquinone <1> (<1>, 32% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 2-(b-d-glucosyloxy)-9,10-anthraquinone S UDPglucose + 3-hydroxy-1-methoxy-9,10-anthraquinone <1> (<1>, 52% of the activity with 1,2-dihydroxy-9,10-anthraquinone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-methoxy-3-(b-d-glucosyloxy)-9,10-anthraquinone Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 0.0108 <1> (UDPglucose, <1> pH 7.4, 30 C [1]) [1] 0.11 <1> (alizarin, <1> pH 7.4, 30 C [1]) [1] pH-Optimum 7.1 <1> (<1>, glucosylation of 1,2-dihydroxy-9,10-anthraquinone [1]) [1] pH-Range 6.8-7.6 <1> (<1>, about half-maximal activity at pH 6.7 and 7.6 [1]) [1] Temperature optimum ( C) 30 <1> [1]

98

2.4.1.103

Alizarin 2-b-glucosyltransferase

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

6 Stability Storage stability <1>, -20 C, 2 months [1]

References [1] Mateju, J.; Cudlin, J.; Steinerova, N.; Blumauerova, M.; Vanek, Z.: Partial purification and properties of glucosyltransferase from Streptomyces aureofaciens. Folia Microbiol., 24, 205-210 (1979) [2] Mateju, J.; Nohynek, M.: Alizarin glucosyl transferase activity in Streptomyces aureofaciens mutans. Folia Microbiol., 36, 314-316 (1991)

99

o-Dihydroxycoumarin 7-O-glucosyltransferase

2.4.1.104

1 Nomenclature EC number 2.4.1.104 Systematic name UDP-glucose:7,8-dihydroxycoumarin 7-O-b-d-glucosyltransferase Recommended name o-dihydroxycoumarin 7-O-glucosyltransferase Synonyms CGTase UDP-glucose:o-dihydroxycoumarin glucosyltransferase UDPglucose:7,8-dihydroxycoumarin 7-O-b-d-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-o-dihydroxycoumarin 7-OCAS registry number 74114-37-7

2 Source Organism <1> Nicotiana tabacum (L. cv. Wisconsin No. 38 [1]) [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + 7,8-dihydroxycoumarin = UDP + daphnin Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + 7,8-dihydroxycoumarin <1> (<1>, i.e. daphnetin, involved in the glycosylation of daphnetin, esculetin and possibly scopoletin to their corresponding 7-O-glucosides daphnin, chicoriin and scopolin [1]) (Reversibility: ? <1> [1]) [1] P UDP + daphnin <1> [1] S UDPglucose + esculetin <1> (<1>, i.e. daphnetin, involved in the glycosylation of daphnetin, esculetin and possibly scopoletin to their corre-

100

2.4.1.104

o-Dihydroxycoumarin 7-O-glucosyltransferase

sponding 7-O-glucosides daphnin, chicoriin and scopolin [1]) (Reversibility: ? <1> [1]) [1] P UDP + cichoriin <1> [1] S UDPglucose + scopoletin <1> (<1>, i.e. daphnetin, involved in the glycosylation of daphnetin, esculetin and possibly scopoletin to their corresponding 7-O-glucosides daphnin, chicoriin and scopolin [1]) (Reversibility: ? <1> [1]) [1] P UDP + scopolin <1> [1] Substrates and products S UDPglucose + 4-coumaric acid <1> (Reversibility: ? <1> [3]) [3] P UDP + 4-O-glucosylcoumaric acid S UDPglucose + 4-methylumbelliferone <1> (Reversibility: ? <1> [3]) [3] P UDP + 7-O-glucosyl-4-methylumbelliferone S UDPglucose + 7,8-dihydroxycoumarin <1> (<1>, i.e. daphnetin [1]) (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + daphnin <1> [1, 2] S UDPglucose + baicalein <1> (Reversibility: ? <1> [3]) [3] P UDP + 7-O-glucosylbaicalein S UDPglucose + caffeic acid <1> (<1>, 56% of the activity with 7,8-dihydroxycoumarin [1]; <1>, 50% of the activity with 7,8-dihydroxycoumarin [2]) (Reversibility: ? <1> [1, 2, 3]) [1, 2, 3] P UDP + ? S UDPglucose + esculetin <1> (<1>, 95% of the activity with 7,8-dihydroxycoumarin [1,2]; <1>, sequential mechanism [3]) (Reversibility: ? <1> [1, 2, 3]) [1, 2, 3] P UDP + cichoriin <1> [1, 2] S UDPglucose + ferulic acid <1> (<1>, low activity [3]) (Reversibility: ? <1> [3]) [3] P UDP + 4-O-glucosylferulic acid S UDPglucose + formononetin <1> (Reversibility: ? <1> [3]) [3] P UDP + 7-O-glucosylformononetin S UDPglucose + hydrangetin <1> (<1>, 30% of the activity with 7,8-dihydroxycoumarin [1]; <1>, 30% of the activity with 7,8-dihydroxycoumarin [2]) (Reversibility: ? <1> [1]) [1, 2] P UDP + hydrangin <1> [1] S UDPglucose + kaempferol <1> (Reversibility: ? <1> [3]) [3] P UDP + 7-O-glucosylkaempferol S UDPglucose + protocatechuic acid <1> (<1>, 12% of the activity with 7,8-dihydroxycoumarin [1]) (Reversibility: ? <1> [1]) [1] P UDP + ? S UDPglucose + quercetin <1> (Reversibility: ? <1> [3]) [3] P UDP + 7-O-glucosylquercetin S UDPglucose + scopoletin <1> (<1>, 25% of the activity with 7,8-dihydroxycoumarin [1]; <1>, 25% of the activity with 7,8-dihydroxycoumarin [2]; <1>, sequential mechanism [3]) (Reversibility: ? <1> [1, 2, 3]) [1, 2, 3]

101

o-Dihydroxycoumarin 7-O-glucosyltransferase

2.4.1.104

P UDP + scopolin <1> [1] S UDPglucose + syringic acid <1> (<1>, 10% of the activity with 7,8-dihydroxycoumarin [1]) (Reversibility: ? <1> [1]) [1] P UDP + 4-O-glucosyl syringic acid S UDPglucose + umbelliferone <1> (<1>, 52% of the activity with 7,8-dihydroxycoumarin [1]; <1>, 25% of the activity with 7,8-dihydroxycoumarin [2]) (Reversibility: ? <1> [1, 2]) [1, 2, 3] P UDP + skimmin <1> [1] S UDPglucose + vanillic acid <1> (<1>, 14% of the activity with 7,8-dihydroxycoumarin [1]) (Reversibility: ? <1> [1]) [1] P UDP + 4-O-glucosylvanillic acid Inhibitors Co2+ <1> (<1>, 1 mM, 87% inhibition [3]) [3] Cu2+ <1> (<1>, 1 mM, 55% inhibition [3]) [3] Zn2+ <1> (<1>, 1 mM, 96% inhibition [3]) [3] Activating compounds 2-mercaptoethanol <1> (<1>, 10 mM, activation to 141% of control [3]) [3] Hg2+ <1> [3] PCMB <1> [3] Metals, ions Co2+ <1> (<1>, 1 mM, activation to 114% of control [3]) [3] Cu2+ <1> (<1>, 1 mM, activation to 112% of control [3]) [3] Mn2+ <1> (<1>, 1 mM, activation to 133% of control [3]) [3] Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 0.025 <1> (esculetin) [3] 0.043 <1> (UDPglucose) [3] 0.05 <1> (UDPglucose, <1>, reaction with esculetin [1]) [1] 0.095 <1> (daphnetin) [1, 2] 0.11 <1> (esculetin) [1, 2] 0.15 <1> (scopoletin) [3] 1.43 <1> (scopoletin) [1] pH-Optimum 7.5 <1> [1, 3] Temperature optimum ( C) 45 <1> [3]

4 Enzyme Structure Molecular weight 47000-50000 <1> (<1>, gel filtration [3]) [3]

102

2.4.1.104

o-Dihydroxycoumarin 7-O-glucosyltransferase

Subunits monomer <1> (<1>, 1 * 49000, SDS-PAGE [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture <1> [1, 2, 3] Purification <1> (partial [1,2]) [1, 2, 3]

References [1] Ibrahim, R.K.; Boulay, B.: Purification and some properties of UDP-glucose:o-dihydroxycoumarin 7-O-glucosyltransferase from tobacco cell cultures. Plant Sci. Lett., 18, 177-184 (1980) [2] Ibrahim, R.K.: Position specificity of an o-dihydroxycoumarin glucosyltransferase from tobacco cell suspension culture. Phytochemistry, 19, 2459-2460 (1980) [3] Taguchi, G.; Imura, H.; Maeda, Y.; Kodaira, R.; Hayashida, N.; Shimosaka, M.; Okazaki, M.: Purification and characterization of UDP-glucose:hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells. Plant Sci., 157, 105-112 (2000)

103

Vitexin b-glucosyltransferase

2.4.1.105

1 Nomenclature EC number 2.4.1.105 Systematic name UDP-glucose:vitexin 2''-O-b-d-glucosyltransferase Recommended name vitexin b-glucosyltransferase Synonyms glucosyltransferase, uridine diphosphoglucose-vitexin 2''CAS registry number 76828-68-7

2 Source Organism <1> Silene alba (Armenia population [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + vitexin = UDP + vitexin 2''-O-b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + vitexin <1> (<1>, pathway in flavone glycoside metabolism [1]) [1] P UDP + vitexin 2''-O-b-d-glucoside Substrates and products S UDPglucose + vitexin <1> (Reversibility: ? <1> [1]) [1] P UDP + vitexin 2''-O-b-d-glucoside <1> [1] Metals, ions Ca2+ <1> (<1>, 2.5fold stimulation at 2 mM [1]) [1] Co2+ <1> (<1>, 2fold stimulates at 2 mM [1]) [1] Mg2+ <1> (<1>, stimulation [1]) [1] Mn2+ <1> (<1>, 2.5fold stimulates at 2 mM [1]) [1] 104

2.4.1.105

Vitexin b-glucosyltransferase

Km-Value (mM) 0.01 <1> (vitexin) [1] 0.2 <1> (UDPglucose) [1] pH-Optimum 7.5 <1> [1]

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

References [1] Heinsbroek, R.; van Brederode, J.; van Nigtevecht, G.; Maas, J.; Kamsteeg, J.; Besson, E.; Chopin, J.: The 2''-O-glucosylation of vitexin and isovitexin in petals of Silene alba is catalysed by two different enzymes. Phytochemistry, 19, 1935-1937 (1980)

105

Isovitexin b-glucosyltransferase

2.4.1.106

1 Nomenclature EC number 2.4.1.106 Systematic name UDP-glucose:isovitexin 2''-O-b-d-glucosyltransferase Recommended name isovitexin b-glucosyltransferase Synonyms glucosyltransferase, uridine diphosphoglucose-isovitexin 2''uridine diphosphoglucose-isovitexin 2''-glucosyltransferase CAS registry number 72102-99-9

2 Source Organism <1> Silene alba [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + isovitexin = UDP + isovitexin 2''-O-b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + isovitexin <1> (<1>, pathway in flavone glycoside metabolism [1]) (Reversibility: ? <1> [1]) [1] P UDP + isovitexin 2''-O-b-d-glucoside <1> [1] Substrates and products S UDPglucose + isoorientin <1> (Reversibility: ? <1> [2]) [2] P UDP + isoorientin 2''-O-b-d-glucoside <1> [2] S UDPglucose + isovitexin <1> (<1>, ionisation grade of substrates influences their binding to the enzyme [2]) (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + isovitexin 2''-O-b-d-glucoside <1> [1, 2]

106

2.4.1.106

Isovitexin b-glucosyltransferase

Inhibitors Mn2+ <1> (<1>, above 25 mM [1]) [1] Metals, ions Ca2+ <1> (<1>, stimulation [1]) [1] Co2+ <1> (<1>, stimulation [1]) [1] Mg2+ <1> (<1>, stimulation [1]) [1] Mn2+ <1> (<1>, stimulation [1]) [1] Km-Value (mM) 0.09 <1> (isovitexin) [1, 2] 0.3 <1> (UDPglucose, <1>, reaction with isovitexin [1,2]) [1, 2] 0.45 <1> (isoorientin) [2] 0.75 <1> (UDPglucose, <1>, reaction with isoorientin [2]) [2] pH-Optimum 7.5 <1> (<1>, reaction with isoorientin [2]) [2] 8.5 <1> (<1>, reaction with isovitexin [1,2]) [1, 2] pH-Range 6.5-8.5 <1> (<1>, pH 6.5: about 40% of maximal activity, pH 8.5: about 30% of maximal activity, reaction with isoorientin [2]) [2] 7.5-9.5 <1> (<1>, about 50% of maximal activity at pH 7.5 and at pH 9.5, reaction with isovitexin [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [2] petal <1> [1, 2]

References [1] Heinsbroek, R.; van Brederode, J.; van Nigtevecht, G.; Maas, J.; Kamsteeg, J.; Besson, E.; Chopin, J.: The 2''-O-glucosylation of vitexin and isovitexin in petals of Silene alba is catalysed by two different enzymes. Phytochemistry, 19, 1935-1937 (1980) [2] Van Brederode, J.; Chopin, J.; Kamsteeg, J.; van Nigtevecht, G.; Heinsbroek, R.: The pH dependent substrate specificity of UDP-fglucose:isovitexin 2''-Oglucosyltransferase. Phytochemistry, 18, 655-656 (1979)

107

UDPglucuronate-testosterone glucuronosyltransferase

1 Nomenclature EC number 2.4.1.107 (deleted, included in EC 2.4.1.17) Recommended name UDPglucuronate-testosterone glucuronosyltransferase

108

2.4.1.107

UDPglucuronate-phenol glucuronosyltransferase

2.4.1.108

1 Nomenclature EC number 2.4.1.108 (deleted, included in EC 2.4.1.17) Recommended name UDPglucuronate-phenol glucuronosyltransferase

109

Dolichyl-phosphate-mannose-protein mannosyltransferase

2.4.1.109

1 Nomenclature EC number 2.4.1.109 Systematic name dolichyl-phosphate-d-mannose:protein O-d-mannosyltransferase Recommended name dolichyl-phosphate-mannose-protein mannosyltransferase Synonyms PMT <1, 3-9> [7-13, 15, 16] dolichol phosphomannose-protein mannosyltransferase mannosyltransferase, dolichol phosphomannose-protein protein O-d-mannosyltransferase protein O-mannosyltransferase <1> [9] protein mannosyltransferase <1> [8] Additional information <1, 8, 9> (<8,9> PMT3 behaves as a dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase, EC 2.4.1.130 [8]; <1> PMT3: no clearly determined dolichyl-phosphate-mannose-protein mannosyltransferase activity [13]) [8, 13] CAS registry number 74315-99-4

2 Source Organism <1> Saccharomyces cerevisiae (strain BY4743 [16]; genes PMT1-6 [13]; enzyme form other than PMT2 [11]; enzyme form other than PMT1 [10]; gene PMT2 [8,9,15]; gene PMT1 [7-11,15]) [1, 2, 4, 5, 7-11, 13, 15, 16] <2> Candida albicans (strain ATCC 26555 [6]; 2005 E [3]) [3, 6] <3> Saccharomyces cerevisiae (<3> PMT2 [11,12]) [11, 12] <4> Saccharomyces cerevisiae (gene PMT1 [12]) [12] <5> Saccharomyces cerevisiae (gene PMT5 [12]) [12] <6> Saccharomyces cerevisiae (gene PMT6 [12]) [12] <7> Saccharomyces cerevisiae (gene PMT7 [12]) [12] <8> Saccharomyces cerevisiae (gene PMT3 [8,12]) [8, 12] <9> Saccharomyces cerevisiae (gene PMT4 [8,12]) [8, 12] <10> Rattus norvegicus (C-mannosylating enzyme [14]) [14]

110

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3 Reaction and Specificity Catalyzed reaction dolichyl phosphate d-mannose + protein = dolichyl phosphate + O-d-mannosylprotein (<1> structure-function analysis [15]) Reaction type hexosyl group transfer Natural substrates and products S dolichyl phosphate d-mannose + protein <1-9> (<1> acceptor substrate specificities of PMT1-4,6 [13]; <3-9> acceptor substrates: secretory proteins [12]; <1-9> production of cell-wall mannoproteins [1-5,8,12]) [1-13, 15] P dolichyl phosphate + O-d-mannosylprotein S dolichyl phosphate d-mannose + protein Aga2 <1> (<1> i.e. small-agglutinin [13]; <1> protein is located at the cell surface [13]; <1> activity is not affected by disruption mutations of PMT1-4 [13]) [13] P dolichyl phosphate + O-d-mannosylprotein Aga2 S dolichyl phosphate d-mannose + protein Bar1 <1> (<1> protein is located in the medium [13]; <1> PMT1 and PMT2, not PMT3 and PMT4 [13]) [13] P dolichyl phosphate + O-d-mannosylprotein Bar1 S dolichyl phosphate d-mannose + protein Ggp1/Gas1 <1> (<1> protein is located at the cell surface [13]; <1> PMT4 and PMT6, not PMT1-3 [13]) [13] P dolichyl phosphate + O-d-mannosylprotein Ggp1/Gas1 S dolichyl phosphate d-mannose + protein Kex2 <1> (<1> protein is located in the Golgi apparatus [13]; <1> PMT4, not PMT1-3 [13]) [13] P dolichyl phosphate + O-d-mannosylprotein Kex2 S dolichyl phosphate d-mannose + protein Kre9 <1> (<1> protein is located in the Golgi apparatus [13]; <1> mainly PMT1 and PMT2, not PMT3 and PMT4 [13]) [13] P dolichyl phosphate + O-d-mannosylprotein Kre9 S dolichyl phosphate d-mannose + protein Pir2/hsp150 <1> (<1> PMT1 [15]; <1> protein is located in the medium [13]; <1> PMT1, PMT2, and to some extent PMT4, not PMT3 [13]) [13, 15] P dolichyl phosphate + O-d-mannosylprotein Pir2/hsp150 S dolichyl phosphate d-mannose + protein chitinase 1 <1> (<1> PMT1 [15]; <1> protein is located in the cell wall and medium [13]; <1> PMT1, PMT4, PMT6 and especially PMT2, not PMT3 [13]) [13, 15] P dolichyl phosphate + O-d-mannosylprotein chitinase 1 S dolichyl phosphate d-mannose + ribonuclease 2 <10> (<10> C-mannosylation activity [14]; <10> biosynthetic pathway [14]) [14] P dolichyl phosphate + ribonuclease 2-d-mannose S Additional information <1> (<1> defective mutants are used to investigate the substrate specificities of PMT1-4,6 in vivo [13]) [13] P ? 111

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2.4.1.109

Substrates and products S dolichyl phosphate d-mannose + Ac-Ala-Thr-Ala-NH2 <2> (Reversibility: ? <2> [3]) [3] P dolichyl phosphate + O-d-mannosyl-Ac-Ala-Thr-Ala-NH2 <2> [3] S dolichyl phosphate d-mannose + Ac-Tyr-Ala-Thr-Ala-Val-NH2 <1, 2> (<1> PMT1 [15]; <1> PMT1 transfers preferably to the threonine and valine residues, the additional enzyme form prefers the serine residue, both depending on the sequence of the acceptor substrate peptide [10]; <1> recombinant yeast overproducing PMT1 and PMT2 [9]) (Reversibility: ir <1> [9]; ? <1,2> [3,10,15]) [3, 9, 10, 15] P dolichyl phosphate + O-d-mannosyl-Ac-Tyr-Ala-Thr-Ala-Val-NH2 <1, 2> [3, 9, 10, 15] S dolichyl phosphate d-mannose + Ac-Tyr-Asn-Pro-Thr-Ser-Val-NH2 <13> (Reversibility: ir <1> [9]; ? <1-3> [3,11]) [3, 9, 11] P dolichyl phosphate + O-d-mannosyl-Ac-Tyr-Asn-Pro-Thr-Ser-Val-NH2 <1-3> [3, 9, 11] S dolichyl phosphate d-mannose + AcSSSSSNH2 <1> (<1> PMT1-3, PMT5, PMT6, not PMT4 [13]) (Reversibility: ir <1> [9]; ? <1> [13]) [9, 13] P dolichyl phosphate + O-d-mannosyl-AcSSSSSNH2 <1> [9, 13] S dolichyl phosphate d-mannose + Asn-Ala-Thr-Val-dinitrophenyl <1> (Reversibility: ? <1> [2]) [2] P dolichyl phosphate + O-d-mannosyl-Asn-Ala-Thr-Val-dinitrophenyl <1> [2] S dolichyl phosphate d-mannose + Lys-Pro-Ser-Gly-Tyr <1> (Reversibility: ? <1> [4]) [4] P dolichyl phosphate + O-d-mannosyl-Lys-Pro-Ser-Gly-Tyr <1> [4] S dolichyl phosphate d-mannose + Lys-Pro-Thr-Gly-Tyr <1> (Reversibility: ? <1> [4]) [4] P dolichyl phosphate + O-d-mannosyl-Lys-Pro-Thr-Gly-Tyr <1> [4] S dolichyl phosphate d-mannose + Lys-Pro-Thr-Pro-Tyr <1> (Reversibility: ? <1> [4]) [4] P dolichyl phosphate + O-d-mannosyl-Lys-Pro-Thr-Pro-Tyr <1> [4] S dolichyl phosphate d-mannose + Pro-Thr-Val <1> (Reversibility: ? <1> [4]) [4] P dolichyl phosphate + O-d-mannosyl-Pro-Thr-Val <1> [4] S dolichyl phosphate d-mannose + Pro-Tyr-Thr-Val <1> (Reversibility: ? <1> [4]) [4] P dolichyl phosphate + O-d-mannosyl-Pro-Tyr-Thr-Val <1> [4] S dolichyl phosphate d-mannose + RSPSPSTQ <1> (Reversibility: ? <1> [10]) [10] P dolichyl phosphate + O-d-mannosyl-RSPSPSTQ <1> [10] S dolichyl phosphate d-mannose + Tyr-Ala-Thr-Ala-Val <2-9> (<3-9> wild-type and mutants [12]) (Reversibility: ? <2-9> [3,12]) [3, 12] P dolichyl phosphate + O-d-mannosyl-Tyr-Ala-Thr-Ala-Val <2-9> [3, 12] S dolichyl phosphate d-mannose + Tyr-Asn-Leu-Thr-Ser-Val <1> (Reversibility: ? <1> [4]) [4] P dolichyl phosphate + O-d-mannosyl-Tyr-Asn-Leu-Thr-Ser-Val <1> [4] 112

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Dolichyl-phosphate-mannose-protein mannosyltransferase

S dolichyl phosphate d-mannose + Tyr-Asn-Pro-Thr-Ser-Val <1, 2> (Reversibility: ? <1,2> [3,4,16]) [3, 4, 16] P dolichyl phosphate + O-d-mannosyl-Tyr-Asn-Pro-Thr-Ser-Val <1, 2> [3, 4, 16] S dolichyl phosphate d-mannose + Tyr-Asn-Pro-Thr-Ser-Val-NH2 <1> (Reversibility: ? <1> [7]) [7] P dolichyl phosphate + O-a-d-mannosyl-Tyr-Asn-Pro-Thr-Ser-Val-NH2 <1> [7] S dolichyl phosphate d-mannose + Tyr-Leu-Thr-Ala-Val <2> (Reversibility: ? <2> [3]) [3] P dolichyl phosphate + O-d-mannosyl-Tyr-Leu-Thr-Ala-Val <2> [3] S dolichyl phosphate d-mannose + Tyr-Pro-Thr-Ala-Val <2> (Reversibility: ? <2> [3]) [3] P dolichyl phosphate + O-d-mannosyl-Tyr-Pro-Thr-Ala-Val <2> [3] S dolichyl phosphate d-mannose + biotin-Tyr-Ala-Thr-Ala-Val-NH2 <1, 2> (Reversibility: ? <1,2> [3,7]) [3, 7] P dolichyl phosphate + O-d-mannosyl-N-biotinyl-Tyr-Ala-Thr-Ala-Val-NH2 <1, 2> [3, 7] S dolichyl phosphate d-mannose + biotin-Tyr-Pro-Thr-Ala-Val-NH2 <2> (Reversibility: ? <2> [3]) [3] P dolichyl phosphate + O-d-mannosyl-N-biotinyl-Tyr-Pro-Thr-Ala-Val-NH2 <2> [3] S dolichyl phosphate d-mannose + biotin-Tyr-Thr-Ala-Val-NH2 <2> (Reversibility: ? <2> [3]) [3] P dolichyl phosphate + O-d-mannosyl-N-biotinyl-Tyr-Thr-Ala-Val-NH2 <2> [3] S dolichyl phosphate d-mannose + human ribonuclease 2 <10> (<10> recombinant wild-type RNase from E. coli is no substrate [14]; <10> whole protein or N-terminal dodecapeptide containing Trp7 [14]; <10> C-mannosylation activity [14]; <10> no C-mannosylation activity of Trp7 when Trp10 is exchanged for Ala in the acceptor substrate [14]) (Reversibility: ? <10> [14]) [14] P dolichyl phosphate + human ribonuclease 2-d-mannose <10> (<10> dmannose is bound at Trp7 forming a C-C linkage [14]) [14] S dolichyl phosphate d-mannose + protein <1-9> (<1> acceptor substrate specificities of PMT1-4,6 [13]; <3-9> acceptor substrates: secretory proteins [12]) (Reversibility: ? <1-9> [1-13,15]) [1-13, 15] P dolichyl phosphate + O-d-mannosylprotein <1-9> [1-13, 15] S dolichyl phosphate d-mannose + protein <1-9> (<1> strictly stereospecific for the anomeric configuration of phosphoryl-linkage of the donor substrate, a saturated a-isoprene unit in the dolichyl moiety is required [7]; <1> acceptor substrate specificity study [5]; <1> acceptor substrate: HCl-treated cell wall mannoprotein from Saccharomyces cerevisiae [1]; <1> acceptor substrate: human-granulocyte-macrophage colony-stimulating-factor-derived peptide(4-11) [5]; <1> preferred chain lengthin decreasing order: C100, C80, C55, C35 [2,7]; <1> acidic amino acids strongly inhibit acceptor activity, as do glycine and proline residues as 113

Dolichyl-phosphate-mannose-protein mannosyltransferase

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

P

2.4.1.109

amino-terminal and carboxy-terminal neighbours [4]; <1-9> the enzyme transfers mannosyl residues to the hydroxyl of serine or threonine residues [1-8,10-13]) (Reversibility: ir <1> [1,9]; ? <1-9> [2-8,10-13,15]) [113, 15] dolichyl phosphate + O-d-mannosylprotein <1-9> [1-13, 15] dolichyl phosphate d-mannose + protein Aga2 <1> (<1> i.e. small-agglutinin [13]; <1> activity is not affected by disruption mutations of PMT1-4 [13]) (Reversibility: ? <1> [13]) [13] dolichyl phosphate + O-d-mannosylprotein Aga2 <1> [13] dolichyl phosphate d-mannose + protein Bar1 <1> (<1> PMT1 and PMT2, not PMT3 and PMT4 [13]) (Reversibility: ? <1> [13]) [13] dolichyl phosphate + O-d-mannosylprotein Bar1 <1> [13] dolichyl phosphate d-mannose + protein Ggp1/Gas1 <1> (<1> PMT4 and PMT6, not PMT1-3 [13]) (Reversibility: ? <1> [13]) [13] dolichyl phosphate + O-d-mannosylprotein Ggp1/Gas1 <1> [13] dolichyl phosphate d-mannose + protein Kex2 <1> (<1> PMT4, not PMT1-3 [13]) (Reversibility: ? <1> [13]) [13] dolichyl phosphate + O-d-mannosylprotein Kex2 <1> [13] dolichyl phosphate d-mannose + protein Kre9 <1> (<1> mainly PMT1 and PMT2, not PMT3 and PMT4 [13]) (Reversibility: ? <1> [13]) [13] dolichyl phosphate + O-d-mannosylprotein Kre9 <1> [13] dolichyl phosphate d-mannose + protein Pir2/hsp150 <1> (<1> PMT1 [15]; <1> PMT1, PMT2, and to some extent PMT4, not PMT3 [13]) (Reversibility: ? <1> [13,15]) [13, 15] dolichyl phosphate + O-d-mannosylprotein Pir2/hsp150 <1> [13, 15] dolichyl phosphate d-mannose + protein chitinase 1 <1> (<1> PMT1 [15]; <1> PMT1, PMT4, PMT6 and especially PMT2, not PMT3 [13]) (Reversibility: ? <1> [13,15]) [13, 15] dolichyl phosphate + O-d-mannosylprotein chitinase 1 <1> [13, 15] dolichyl phosphate d-mannose + ribonuclease 2 <10> (<10> C-mannosylation activity [14]) (Reversibility: ? <10> [14]) [14] dolichyl phosphate + ribonuclease 2-d-mannose <10> (<10> d-mannose is bound at Trp7 forming a C-C linkage [14]) [14] Additional information <1, 8, 9> (<8,9> PMT3 behaves as a dolichylphosphate-mannose-glycolipid a-mannosyltransferase, EC 2.4.1.130 [8]; <1> PMT3: no clearly determined dolichyl-phosphate-mannose-protein mannosyltransferase activity [13]; <1> PMT1 and PMT2 function as a complex [9,13]; <1> PMT4 and PMT6 probably function as an active dimer [13]) [8, 9, 13] ?

Inhibitors CuSO4 <1> (<1> 10 mM, complete inhibition [16]) [16] Mg2+ <1, 2> (<2> above 5 mM [3]) [3, 10] chymotrypsin <10> (<10> inactivation [14]) [14] glycerol <1> (<1> at 50% inhibitory [16]; <1> at 20% inhibitory [7]) [7, 16]

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phosphatidylinositol <2> [3] Additional information <1> (<1> no inhibition by EDTA [4,16]; <1> not affected by Triton X-100 up to 0.2% [1]) [1, 4, 16] Activating compounds deoxycholate <1> (<1> 0.025%, stimulates slightly [1]) [1] phosphatidylcholine <2> (<2> stimulates [3]) [3] Additional information <1> (<1> not affected by Triton X-100 up to 0.2% [1]) [1] Metals, ions Mg2+ <1, 2, 10> (<1> required, 10 mM [16]; <1> 2fold activation [4]; <1> optimal stimulation at 7 mM [1]; <1> Mg2+ or Mn2+ stimulates [1]; <2> not required [3]) [1, 3, 4, 11, 14, 16] Mn2+ <1> (<1> Mg2+ or Mn2+ stimulates [1]) [1] Specific activity (U/mg) 0.0000012 <2> (<2> substrate YNPTSV [3]) [3] 0.0000677 <1> (<1> partially purified enzyme [4]) [4] 0.00057 <1> (<1> partially purified enzyme [1]) [1] 0.0035 <1> (<1> partially purified enzyme [7]) [7] Additional information <1, 10> (<1> assay development on microtiter plates [16]; <1> wild-type and deletion mutants [15]; <1> specific activity of the additional enzyme form is 7fold higher than that of PMT1, substrate AcYNPTSV-NH2 [10]) [2, 4, 9, 10, 11, 14-16] Km-Value (mM) 0.075 <2> (biotin-Tyr-Thr-Ala-Val-NH2 ) [3] 0.1 <2> (biotin-Tyr-Leu-Ala-Val-NH2 ) [3] 0.25 <2> (Ac-Tyr-Ala-Thr-Ala-Val-NH2 ) [3] 0.4 <1> (dolichyl phosphate d-mannose) [4] 0.85 <2> (biotin-Tyr-Pro-Thr-Ala-Val-NH2 ) [3] 1 <1> (Tyr-Asn-Pro-Thr-Ser-Val) [16] 2 <1> (Ac-Tyr-Ala-Thr-Ala-Val-NH2 , <1> PMT1 [10]) [10] 2.2 <2> (Tyr-Ala-Thr-Ala-Val) [3] 2.5 <2> (Tyr-Leu-Thr-Ala-Val) [3] 3.3 <1> (Tyr-Asn-Pro-Thr-Ser-Val) [4] 4.3 <2> (Ac-Tyr-Asn-Pro-Thr-Ser-Val-NH2 ) [3] 6.7 <2> (Ac-Ala-Thr-Ala-NH2 ) [3] 7.3 <2> (Tyr-Pro-Thr-Ala-Val) [3] 10 <1> (RSPSPSTQ, <1> additional enzyme form [10]) [10] 15 <1> (Ac-Tyr-Ala-Thr-Ala-Val-NH2 , <1> additional enzyme form [10]) [10] 20 <1> (RSPSPSTQ, <1> PMT1 [10]) [10] Additional information <1> (<1> Km value depends on chain length and asaturation of the acceptor substrate [2]) [2]

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pH-Optimum 5.7 <1> [1] 6.5 <1> (<1> assay at [13]; <1> PMT1 null mutant, optimum of the additional enzyme form [10]) [10, 13] 7 <1> (<1> assay at [7]) [7] 7.2 <10> (<10> assay at [14]) [14] 7.5 <1> (<1> assay at [5,16]; <1> PMT1 wild-type [10]) [4, 5, 10, 16] 7.5-8 <2> (<2> the best buffers are bicine pH 7.7, tricine pH 8.0 and HEPES pH 7.5 [3]) [3] Temperature optimum ( C) 20 <1> (<1> assay at [16]) [16] 22 <1> (<1> additional enzyme form [10]) [10] 25 <2> (<2> assay at [3]) [3] 26 <10> (<10> about [14]) [14] 30 <1> (<1> PMT1 [10]; <1> assay at [7]) [7, 10] 37 <10> (<10> assay at [14]) [14] Temperature range ( C) 28-37 <2> (<2> in vivo [6]) [6] Additional information <1> (<1> 40% remaining activity at 0 C [16]) [16]

4 Enzyme Structure Subunits ? <1> (<1> x * 92000, SDS-PAGE [4]; <1> x * 92000, PMT1, SDS-PAGE [4,9]; <1> x * 78000, PMT2, SDS-PAGE [9]) [4, 9] Additional information <1> (<1> topology model of PMT1 [15]; <1> central hydrophilic loop is essential for catalytic activity, not complex formation, and is conserved throughout the PMT-family [15]; <1> Arg138 is crucial for complex formation between PMT1 and PMT2, N-terminal third of the protein is essential for complex formation [15]; <1> PMT1 and PMT2 function as a complex [9,13,15]; <1> PMT4 and PMT6 probably function as an active dimer [13]) [9, 13, 15] Posttranslational modification glycoprotein <1, 3> (<1> construction of diverse deletion mutants with different numbers of N-glycosylation sites [15]; <1> PMT1: 3 N-glycosylation sites [15]; <1,3> PMT1 and PMT2 contain both 3 putative N-glycosylation sites [11]; <1> may contain 4 carbohydrate chains [4]) [4, 11, 15]

5 Isolation/Preparation/Mutation/Application Source/tissue liver <10> [14]

116

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Localization endoplasmic reticulum <1> (<1> integral membrane protein [15]) [4, 12, 15] membrane <1-3, 10> (<1,3> integral, multiple transmembranal domains [11,15]; <1> bound [1]) [1-11, 14-16] microsome <1, 2, 10> [3, 7, 14] spheroplast <1> [16] Purification <1> (immunoaffinity chromatography, co-purification of PMT1 and PMT2 as a complex, no immuno-cross reactivity [9]; solubilization with 0.5% deoxycholate and 1.2% CHAPS [7]; <1> partial [1,4,7]) [1, 4, 7, 9] Cloning <1> (chromosome mapping of PMT1 [11]; cloning and overexpression of PMT2 in yeast strain GFUII-4B, showing no alteration of enzyme activity, and co-overexpression with PMT1 in yeast strain TF1.8, leading to 3fold increase in enzyme activity in vitro, thus PMT1 and 2 function as a complex [9]) [9, 11] <3> (DNA sequence determination, chromosome mapping, [11]) [11] <8> (DNA sequence determination [8]) [8, 12] <9> (DNA sequence determination [9]) [8, 12] Engineering D96A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, reduced activity [15]) [15] E78A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, highly reduced activity [15]) [15] H346A/H348A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, reduced activity [15]) [15] H411A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, slightly reduced activity [15]) [15] H472A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, reduced activity [15]) [15] K234A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, slightly reduced activity [15]) [15] L399A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, reduced activity [15]) [15] L408A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, highly reduced activity [15]) [15] L408A/H411A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, highly reduced activity [15]) [15] N370A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, slightly reduced activity [15]) [15] Q359A/Q360A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, reduced activity [15]) [15] Q493A/E495A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, reduced activity [15]) [15]

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R138A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, highly reduced activity [15]) [15] R398A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, reduced activity [15]) [15] R469A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, slightly reduced activity [15]) [15] R64 A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, highly reduced activity [15]) [15] W253A <1> (<1> site-directed mutagenesis, exchange of conserved residue in the central loop, slightly reduced activity [15]) [15] Additional information <1, 3, 4, 8, 9> (<1> construction of diverse deletion mutants of PMT1, effect on activity and complex formation with PMT2, overview [15]; <1> construction of defective mutants of PMT1-4,6, determination of substrate specificities [13]; <8,9> PMT3 and 4 gene disruption does not alter enzyme activity [8]; <3,4,8,9> construction of all single, double and triple mutants of the genes PMT1-4 by gene disruption and crosses, characterization concerning growth, morphology, and their sensitivity to killer toxin K1, sorbitol dependence, caffeine and calcofluor white, overview [12]; <3> PMT2 haploid mutants grow slightly slower than the wild-type, the enzyme is required but not essential for normal vegetative growth of the cells [11]; <1,3> PMT1 and PMT2 double disruption mutant shows severe growth defect but retain residual activity due to an additional enzyme [11]; <3> PMT2 disruption mutant shows reduced in vitro and in vivo activity [11]; <1> PMT1 null mutant shows increased heat lability, a-d-mannose transfer only to serine residue of the substrate peptide Ac-YNPTSV-NH2 , not to threonine and valine like with the wild-type [10]; <1> homozygous diploid double disruption mutants of PMT1 and PMT2 lead to reduced enzyme activity and a severe defect in sporulation, lethal phenotype when combined with a disruption mutation in dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase, EC 2.4.1.130, genes [8]) [8, 10-13]

6 Stability pH-Stability 4.9 <1> (<1> inactivation below pH 4.9 [7]) [7] Temperature stability 26 <10> (<10> 22 h, stable [14]) [14] 37 <10> (<10> 60 min, stable [14]) [14] 95 <10> (<10> inactivation [14]) [14] General stability information <1>, stable in solution with deoxycholate and CHAPS [7] Storage stability <1>, -80 C, partially purified enzyme, indefinitely stable [7] <1>, 23 C, partially purified enzyme, 2% glycerol, at least 12 h stable [7]

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2.4.1.109

Dolichyl-phosphate-mannose-protein mannosyltransferase

<1>, 4-23 C, partially purified enzyme, stable for at least 1 week [7] <1>, 4 C, solubilized with 0.5% Triton X-100, loss of 80% activity within 5 days [7]

References [1] Babczinski, P.; Haselbeck, A.; Tanner, W.: Yeast mannosyl transferases requiring dolichyl phosphate and dolichyl phosphate mannose as substrate. Partial purification and characterization of the solubilized enzyme. Eur. J. Biochem., 105, 509-515 (1980) [2] Palamarczyk, G.; Lehle, L.; Mankowski, T.; Chojnacki, T.; Tanner, W.: Specificity of solubilized yeast glycosyl transferases for polyprenyl derivatives. Eur. J. Biochem., 105, 517-523 (1980) [3] Weston, A.; Nassau, P.M.; Henley, C.; Marriott, M.S.: Protein O-mannosylation in Candida albicans. Determination of the amino acid sequences of peptide acceptors for protein O-mannosyltransferase. Eur. J. Biochem., 215, 845-849 (1993) [4] Strahl-Bolsinger, S.; Tanner, W.: Protein O-glycosylation in Saccharomyces cerevisiae. Purification and characterization of the dolichyl-phosphate-dmannose-protein O-d-mannosyltransferase. Eur. J. Biochem., 196, 185-190 (1991) [5] Lorenz, C.; Strahl-Bolsinger, S.; Ernst, J.F.: Specific in vitro O-glycosylation of human granulocyte-macrophage colony-stimulating-factor-derived peptides by O-glycosyltransferases of yeast and rat liver cells. Eur. J. Biochem., 205, 1163-1167 (1992) [6] Arroyo-Flores, B.L.; Calvo-Mendez, C.; Flores-Carreon, A.; Lopez-Romero, E.: Biosynthesis of glycoproteins in Candida albicans: activity of dolichol phosphate mannose synthase and protein mannosylation in a mixed membrane fraction. Microbiology, 141, 2289-2294 (1995) [7] Dotson, S.B.; Rush, J.S.; Ricketts, A.D.; Waechter, C.J.: Mannosylphosphoryldolichol-mediated O-mannosylation of yeast glycoproteins: stereospecificity and recognition of the a-isoprene unit by a purified mannosyltransferase. Arch. Biochem. Biophys., 316, 773-779 (1995) [8] Immervoll, T.; Gentzsch, M.; Tanner, W.: PMT3 and PMT4, two new members of the protein-O-mannosyltransferase gene family of Saccharomyces cerevisiae. Yeast, 11, 1345-1351 (1995) [9] Gentzsch, M.; Immervoll, T.; Tanner, W.: Protein O-glycosylation in Saccharomyces cerevisiae: the protein O-mannosyltransferases Pmt1p and Pmt2p function as heterodimer. FEBS Lett., 377, 128-130 (1995) [10] Gentzsch, M.; Strahl-Bolsinger, S.; Tanner, W.: A new Dol-P-Man:protein Od-mannosyltransferase activity from Saccharomyces cerevisiae. Glycobiology, 5, 77-82 (1995) [11] Lussier, M.; Gentzsch, M.; Sdicu, A.-M.; Bussey, H.; Tanner, W.: Protein Oglycosylation in yeast. The PMT2 gene specifies a second protein O-mannosyltransferase that functions in addition to the PMT-1 encoded activity. J. Biol. Chem., 270, 2770-2775 (1995) 119

Dolichyl-phosphate-mannose-protein mannosyltransferase

2.4.1.109

[12] Gentzsch, M.; Tanner, W.: The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital. EMBO J., 15, 5752-5759 (1996) [13] Gentzsch, M.; Tanner, W.: Protein-O-glycosylation in yeast: protein-specific mannosyltransferases. Glycobiology, 7, 481-486 (1997) [14] Doucey, M.A.; Hess, D.; Cacan, R.; Hofsteenge, J.: Protein C-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor. Mol. Biol. Cell, 9, 291-300 (1998) [15] Girrbach, V.; Zeller, T.; Priesmeier, M.; Strahl-Bolsinger, S.: Structure-function analysis of the dolichyl phosphate-mannose:protein O-mannosyltransferase ScPmt1p. J. Biol. Chem., 275, 19288-19296 (2000) [16] Hendershot, L.L.; Aeed, P.A.; Kezdy, F.J.; Elhammer, A.P.: An efficient assay for dolichyl phosphate-mannose: protein O-mannosyltransferase. Anal. Biochem., 307, 273-279 (2002)

120

tRNA-queuosine b-mannosyltransferase

2.4.1.110

1 Nomenclature EC number 2.4.1.110 Systematic name GDP-mannose:tRNAAsp -queuosine O-5''-b-d-mannosyltransferase Recommended name tRNA-queuosine b-mannosyltransferase CAS registry number 9055-06-5

2 Source Organism <1> Rattus norvegicus (male Donryu [1]) [1]

3 Reaction and Specificity Catalyzed reaction GDP-mannose + tRNAAsp -queuosine = GDP + tRNAAsp -O-5''-b-d-mannosylqueuosine Reaction type hexosyl group transfer Natural substrates and products S GDPmannose + tRNAAsp -queuosine <1> (Reversibility: ? <1> [1]) [1] P GDP + tRNAAsp -O-5''-b-d-mannosylqueuosine <1> [1] Substrates and products S GDPmannose + tRNAAsp -queuosine <1> (<1>, strict specificity for E. coli tRNAAsp , transfer with concomitant inversion of anomeric configuration at sugar C-1-carbon, no substrates are tRNATyr , tRNAHis or tRNAAsn [1]) (Reversibility: ? <1> [1]) [1] P GDP + tRNAAsp -O-5''-b-d-mannosylqueuosine <1> [1] Inhibitors EDTA <1> [1]

121

tRNA-queuosine b-mannosyltransferase

2.4.1.110

Metals, ions Mg2+ <1> (<1>, requirement [1]) [1] NaCl <1> (<1>, 0.1 M NaCl stimulates more than 2fold [1]) [1] pH-Optimum 7.5 <1> [1]

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

References [1] Okada, N.; Nishimura, S.: Enzymatic synthesis of Q nucleoside containing mannose in the anticodon of tRNA: isolation of a novel mannosyltransferase from a cell-free extract of rat liver. Nucleic Acids Res., 4, 2931-2937 (1977)

122

Coniferyl-alcohol glucosyltransferase

2.4.1.111

1 Nomenclature EC number 2.4.1.111 Systematic name UDP-glucose:coniferyl-alcohol 4'-b-d-glucosyltransferase Recommended name coniferyl-alcohol glucosyltransferase Synonyms CAGT UDP-glucose coniferyl alcohol glucosyltransferase glucosyltransferase, uridine diphosphoglucose-coniferyl alcohol uridine diphosphoglucose-coniferyl alcohol glucosyltransferase CAS registry number 61116-23-2

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

Rosa sp. [1] Picea abies [2, 3] Forsythia ovata [4] Linum usitatissimum [1] Glycine max (v. Kanrich [1]) [1] Petroselinum hortense (<1>, weak activity [1]) [1] Parthenocissus tricuspidata (<1>, weak activity [1]) [1] Nicotiana tabacum (<1>, weak activity [1]) [1] Cicer arietinum (<1>, weak activity [1]) [1] Pinus strobus [5] Fagus grandifolia (Ehrh Bark [6]) [6] Pinus banksiana (Lamb [7]) [7, 8]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + coniferyl alcohol = UDP + coniferin (<2>, mechanism [3])

123

Coniferyl-alcohol glucosyltransferase

2.4.1.111

Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + coniferyl alcohol <1, 2, 4-9> (<1,4-9>, the role of this enzyme is believed to catalyze the transfer of glucose from UDPglucose to coniferyl or sinapyl alcohol as storage intermediates in lignin biosynthesis [1]; <2>, participates in lignification of spruce [2,3]) (Reversibility: ? <1, 2, 4-9> [1-3]) [1-3] P ? Substrates and products S UDPglucose + caffeic acid <1> (<1>, glucosylated at 18% of the activity with coniferyl alcohol [1]) (Reversibility: ? <1> [1]) [1] P UDP + ? S UDPglucose + coniferyl alcohol <1-12> (<2>, forward reaction is about six times faster than the reverse reaction [3]; <11>, Z-coniferyl alcohol is readily converted into Z-coniferin. E-coniferyl alcohol is only poorly converted into E-coniferin [6]) (Reversibility: r <2> [3]; ? <1-12> [1, 2, 4-8]) [1-8] P UDP + coniferin <1, 3-10> (i.e. coniferyl alcohol 7-O-b-d-glucopyranoside; <11>, Z-coniferyl alcohol is readily converted into Z-coniferin. Econiferyl alcohol is only poorly converted into E-coniferin [6]) [1-6] S UDPglucose + coniferyl aldehyde <1, 2, 10> (<1>, glucosylated at 46% of the activity with coniferyl alcohol [1]; <2>, glucosylated at 63% the rate of coniferyl alcohol [3]) (Reversibility: ? <1, 2, 10> [1, 3, 5]) [1, 3, 5] P UDP + coniferaldehyde-4-O-b-d-glucopyranoside <10> [5] S UDPglucose + ferulic acid <1, 3> (<1>, glucosylated at 20% of the activitry with coniferyl alcohol [1]) (Reversibility: ? <1, 3> [1, 4]) [1, 4] P UDP + ferulic acid-4-O-b-d-glucopyranoside S UDPglucose + isorhamnetin <1, 3> (<1>, glucosylated at 22% of the activity with coniferyl alcohol [1]; <3>, glucosylated at 18% of the activity with coniferyl alcohol [4]) (Reversibility: ? <1, 3> [1, 4]) [1, 4] P UDP + isorhamnetin 7-O-b-d-glucopyranoside S UDPglucose + quercetin <1> (<1>, glucosylated at 22% of the activity with coniferyl alcohol [1]) (Reversibility: ? <1> [1]) [1] P UDP + quercetin 7-O-b-d-glucopyranoside S UDPglucose + scopoletin <1, 3> (Reversibility: ? <1, 3> [1, 4]) [1, 4] P UDP + scopoletin 7-O-b-d-glucopyranoside S UDPglucose + scopoletin <3> (<3>, glucosylated at 10% of the activity with coniferyl alcohol [4]) (Reversibility: ? <3> [4]) [4] P UDP + scopoletin-7-O-b-d-glucopyranoside S UDPglucose + sinapaldehyde <2, 3, 10> (<2>, glucosylated at 48% the rate of coniferyl alcohol [3]; <3>, glucosylated at 82% the rate of coniferyl alcohol [4]) (Reversibility: ? <2, 3, 10> [3, 4, 5]) [3, 4, 5] P UDP + sinapaldehyde-4-O-b-d-glucopyranoside <10> [5]

124

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Coniferyl-alcohol glucosyltransferase

S UDPglucose + sinapic acid <1, 3> (<1>, glucosylated at 21% of the activity with coniferyl alcohol [1]; <3>, glucosylated at 14% of the activity with coniferyl alcohol [4]) (Reversibility: ? <1, 3> [1, 4]) [1, 4] P UDP + ? S UDPglucose + sinapyl alcohol <1-3, 5, 10, 12> (<1>, glucosylated at 74% of the activity with coniferyl alcohol [1]; <2>, glucosylated at 14% of the activity with coniferyl alcohol [3]; <3>, glucosylated at 82% of the activity with coniferyl alcohol [4]; <12>, glucosylated at 96% of the activity with coniferyl alcohol [7]) (Reversibility: ? <1, 2, 3, 10, 12> [1, 3, 4, 5, 7]) [1, 3, 4, 5, 7] P UDP + syringin <10> [5] S UDPglucose + vanillic acid <2> (<2>, glucosylated at 14% of the activity with coniferyl alcohol [3]) (Reversibility: ? <2> [3]) [3] P UDP + vanillic acid-4-O-b-d-glucopyranoside S UDPglucose + vanillin <3> (<3>, glucosylated at 17% of the activity with coniferyl alcohol [4]) (Reversibility: ? <3> [4]) [4] P UDP + vanillin-4-O-b-d-glucopyranoside S Additional information <2> (<2>, TDPglucose can replace UDPglucose with 17% efficiency [3]) [3] P ? Inhibitors EDTA <1> (<1,4-9>, 1 mM, 25% inhibition [1]; <2>, no inhibition [3]) [1] PCMB <1-3> (<1>, 0.1 mM, 70% inhibition [1]; <2>, 0.1 mM, complete inhibition [3]) [1, 3, 4] UDP <2> (<2>, non-competitive, product inhibition [3]) [3] coniferin <2, 12> (<2>, non-competitive, product inhibition [3]; <12>, above 10 mM [7]) [3, 7] coniferyl alcohol <1> (<1>, above 0.08 mM, Paul's scarlet rose [1]) [1] Specific activity (U/mg) 0.036 <1> (<1>, Paul's scarlet rose [1]) [1] 2.436 <2> [3] Km-Value (mM) 0.0033 <1> (coniferyl alcohol) [1] 0.0037 <3> (coniferyl alcohol) [4] 0.0056 <1> (sinapyl alcohol) [1] 0.02 <1> (UDPglucose) [1] 0.025 <3> (UDPglucose) [4] 0.065 <1> (coniferyl aldehyde) [1] 0.1196 <10> (coniferyl alcohol) [5] 0.153 <10> (sinapyl alcohol) [5] 0.22 <2> (UDPglucose) [2, 3] 0.2417 <10> (sinapaldehyde) [5] 0.25 <2> (coniferyl alcohol) [2, 3] 0.3174 <10> (coniferyl aldehyde) [5] 0.58 <2> (sinapyl alcohol) [3] 0.64 <2> (vanillin) [3]

125

Coniferyl-alcohol glucosyltransferase

2.4.1.111

pH-Optimum 7.5 <1-3> (<1,3>, Tris-HCl buffer [1,4]; <2>, Tris-HCl, 0.1-0.2 M, or phosphate buffer [3]) [1, 3, 4] 7.6 <12> [8] 7.8 <12> [7] 8 <1, 3> (<1,3>, phosphate buffer [1,4]) [1, 4] pH-Range 5.5-8.2 <2> (<2>, pH 5.5: about 50% of maximal activity, pH 8.2: about 70% of maximal activity, phosphate buffer [3]) [3] 6.5-8.2 <2> (<2>, about half-maximal activity at pH 6.5 and 83% of maximal activity at pH 8.2, Tris buffer [3]) [3] 6.5-8.4 <1> (<1>, about half-maximal activity at pH 6.5 and 8.4, Tris buffer [1]) [1] 7-9.2 <1> (<1>, about half-maximal activity at pH 7 and 9.2, phosphate buffer [1]) [1] Temperature optimum ( C) 36 <2> [3] 40 <12> [7, 8] Temperature range ( C) 35-45 <12> (<12>, 35 C: 96% of maximal activity, 45 C: 83% of maximal activity [7]) [7]

4 Enzyme Structure Molecular weight 44000 <2> (<2>, gel filtration [3]) [3] 52000 <1> (<1>, gel filtration [1]) [1] Subunits monomer <2> (<2>, 1 * 50000, SDS-PAGE [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue bark <11> [6] callus <1, 4, 5, 7, 8> [1] cambium <2, 10, 12> (<12>, during dormancy activity is not detected in the cambium. Enzyme becomes weakly active in springtime when fusiform cells of the lateral meristem changed from densely protoplasmic to highly vacuolated states, just prior to resumption of cell-division activity. During cambial growth and xylogenesis enzyme activity in cambial derivatives is greater than that found in the cambial zone. Activity disappears when the cambium enters dormancy in August, prior to completion of lignification in the last differentiating latewood tracheids [7]; <12>, initialization in the cambium in early 126

2.4.1.111

Coniferyl-alcohol glucosyltransferase

spring with resumption of cell division activity, not detectable in the early autumn at the time when cell division has ceased and wood formation is still occuring [8]) [3, 5, 7, 8] cell suspension culture <1, 4-7, 9> (<1>, 10-day-old, dark-grown suspension culture [1]) [1] hypocotyl <2> (<2>, predominantly located in epidermal and subepidermal layers, vascular bundles [2]) [2] seedling <2> [2] stem <3> (<3>, segment [4]) [4] Localization cytoplasm <2> (<2>, parietal cytoplasmic layer [2]) [2] Purification <1> [1] <2> [3] <3> (partial [4]) [4] <12> [8]

6 Stability General stability information <2>, addition of high molarity buffer, 200 mM Tris/HCl, or 2-mercaptoethanol, optimal concentration 42 mM, increases stability considerably [3] <2>, in dilute solutions addition of 10-20% glycerol or ethylene glycol increases stability [3] Storage stability <1>, -20 C, addition of 28 mM 2-mercaptoethanol and 10% ethyleneglycol are required for stability, about 20% loss of activity within several weeks [1] <2>, -20 C, 200 mM Tris/HCl, pH 7.5, 15% glycerol, stable for several months [3] <2>, 0-2 C, several weeks [3] <2>, glycerol, 15%, stabilizes during storage [3]

References [1] Ibrahim, R.K.; Grisebach, H.: Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension cultures of Pauls scarlet rose. Arch. Biochem. Biophys., 176, 700-708 (1976) [2] Schmid, G.; Hammer, D.K.; Ritterbusch, A.; Grisebach, H.: Appearance and immunohistochemical localization of UDP-glucose:coniferyl alcohol glucosyltransferase in spruce (Picea albies (L.) Karst.) Seedlings. Planta, 156, 207-212 (1982) [3] Schmid, G.; Grisebach, H.: Enzymic synthesis of lignin precursors. Purification and properties of UDPglucose:coniferyl-alcohol glucosyltransferase

127

Coniferyl-alcohol glucosyltransferase

2.4.1.111

from cambial sap of spruce (Picea abies L.). Eur. J. Biochem., 123, 363-370 (1982) [4] Ibrahim, R.K.: Glucosylation of lignin precursors by uridine diphosphate glucose. Coniferyl alcohol glucosyltransferase in higher plants. Z. Pflanzenphysiol., 85, 253-262 (1977) [5] Steeves, V.; Foerster, H.; Pommer, U.; Savidge, R.: Coniferyl alcohol metabolism in conifers. I. Glucosidic turnover of cinnamyl aldehydes by UDPG:coniferyl alcohol glucosyltransferase from pine cambium. Phytochemistry, 57, 1085-1093 (2001) [6] Yamamoto, E.; Inciong, M.E.J.; Davin, L.B.; Lewis, N.G.: Formation of cisconiferin in cell-free extracts of Fagus grandifolia ehrh bark. Plant Physiol., 94, 209-213 (1990) [7] Savidge, R.A.; Foerster, H.: Seasonal activity of uridine 5'-diphosphoglucose:coniferyl alcohol glucosyltransferase in relation to cambial growth and dormancy in conifers. Can. J. Bot., 76, 486-493 (1998) [8] Foerster, H.; Savidge, R.A.: Characterization and seasonal activity of UDPG: coniferyl alcohol glucosyl transferase linked to cambial growth in jack pine. Proc. Plant Growth Reg. Soc. Am., 22nd, 402-407 (1995)

128

a-1,4-Glucan-protein synthase (UDP-forming)

2.4.1.112

1 Nomenclature EC number 2.4.1.112 Systematic name UDP-glucose:protein 4-a-glucosyltransferase Recommended name a-1,4-glucan-protein synthase (UDP-forming) Synonyms UDP-glucose protein transglucosylase UDP-glucose-protein glucosyltransferase UDP-glucose:protein glucosyltransferase UDPGlc:protein transglucosylase UPTG glucosyltransferase, uridine diphosphoglucose-protein glucosyltransferase, uridine diphosphoglucose-protein 4-aglycogen initiator synthase proglycogen synthase uridine diphosphate glucose-protein transglucosylase I (different from glucose-1-phosphate dependent UDP-glucose-protein transglucosylase, different enzymes or a single enzyme that occurs in 2 conformational states) uridine diphosphoglucose protein transglucosylase I uridine diphosphoglucose-protein 4-a-glucosyltransferase uridine diphosphoglucose-protein glucosyltransferase CAS registry number 152478-54-1 39369-27-2

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

Escherichia coli [1] Rattus norvegicus [2] Solanum tuberosum [3-6, 8-10] Zea mays (hybrid Dekalb 636 [7,8]) [7, 8]

129

a-1,4-Glucan-protein synthase (UDP-forming)

2.4.1.112

3 Reaction and Specificity Catalyzed reaction UDP-glucose + protein = UDP + a-d-glucosyl-protein (The enzyme builds up a-1,4-glucan chains covalently bound to protein, thus acting as an initiator of glycogen synthesis.) Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + protein <1-4> (<4> initiation of glucan synthesis without additional primer [7]; <1,3,4> the enzyme builds up a-1,4-glucan chains covalently bound to protein, thus acting as an initiator of glycogen synthesis [1,5,7-10]; <1> when the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by an ADP-glucose-dependent enzyme [1]; <2> complex product of the reaction acts as a precursor for the synthesis of glycogen by EC 2.4.1.11 [2]) (Reversibility: r <3> [10]; ? <1-4> [1, 2, 5, 7-9]) [1, 2, 5, 7-10] P UDP + a-d-glucosyl-protein <1-4> [1, 2, 5, 7-10] Substrates and products S UDP-glucose + protein <1-4> (<3> recombinant enzyme accepts also UDP-xylose and UDP-galactose as donor substrates, but then the reaction is inhibited by Mn2+ [10]; <3> high specificity for UDPglucose [3,4,9]; <3> purified UPTG undergoes selfglucosylation in an UDP-glucose and Mn2+ -dependent reaction, UPTG is the enzyme and at the same time the priming protein required for the biogenesis of protein bound a-glucan in potato tuber [5]) (Reversibility: r <3> [10]; ? <1-4> [1-9]) [1-10] P UDP + a-d-glucosyl-protein <1-4> (<2> a-1,4-glucan protein [2]; <3> glucose is attached by O-linkage to the amino acid residue, serine or threonine, of the protein substrate [3,6]) [1-10] S Additional information <3, 4> (<3,4> enzyme performs autoglucosylation [5,7,9,10]; <3,4> purified UPTG undergoes selfglucosylation in an UDP-glucose and Mn2+ -dependent reaction, UPTG is the enzyme and at the same time the priming protein required for the biogenesis of protein bound a-glucan [5-10]) [5-10] P ? Inhibitors Brij-58 <1> (<1> 1%, weak [1]) [1] Lubrol <1> [1] endogenous inhibitor protein <3, 4> (<3,4> MW 80000 Da, purified from maize endosperm, not found in potato tubers [8]) [8] p-chloromercuribenzoate <3> (<3> complete inactivation [4]) [4]

130

2.4.1.112

a-1,4-Glucan-protein synthase (UDP-forming)

Metals, ions Mg2+ <3> (<3> 3fold activation at 4 mM [4]) [4] Mn2+ <3, 4> (<3,4> absolutely required [7,9]; <3> 5fold stimulation at 5 mM [5,9]; <3> 3fold activation at 4 mM [4,10]) [4, 5, 7, 9, 10] Na2 EDTA <2> (<2> enzyme requires presence of some salts e.g. Na2 EDTA at high concentrations [2]) [2] Additional information <3> (<3> no effect of K+ , Li+ , Na+ and NH+4 [4]) [4] Specific activity (U/mg) 2.23 <3> (<3> purified enzyme [9]) [9] Km-Value (mM) 0.0007 <3> (UDP-glucose) [9] 0.004 <3> (UDP-glucose) [4] pH-Optimum 5-9 <3> (<3> broad optimum [4]) [4] 7.4 <3> (<3> assay at [5]) [5] 8 <4> (<4> assay at [8]) [8] Temperature optimum ( C) 30 <1, 3, 4> (<1,3,4> assay at [1,5,6,8]) [1, 5, 6, 8] 37 <3> (<3> very rapid reaction [4]) [4] Additional information <4> (<4> heating to 65 C before the enzyme assay increased the activity by about 14.5fold [7]) [7] Temperature range ( C) 25-37 <3> (<3> half-maximal activity at 25 C and 37 C [4]) [4]

4 Enzyme Structure Subunits ? <3, 4> (<3,4> x * 38000, SDS-PAGE [5,7,9]) [5, 7, 9] Posttranslational modification glycoprotein <1, 3, 4> (<3> binds to concanavalin A [6]; <3,4> autoglucosylation [5,7-9]; <1,3> contains a-1,4-glucosidic bonds [1,5]) [1, 5-9]

5 Isolation/Preparation/Mutation/Application Source/tissue endosperm <4> [7, 8] leaf <3> [10] liver <2> [2] petiole <3> [10] root <3> [10] seedling <4> [8] shoot <3> [10] 131

a-1,4-Glucan-protein synthase (UDP-forming)

2.4.1.112

stem <3> [10] stolon <3> [10] tuber <3> [3-6, 8-10] Localization membrane <1-4> [1-7] Purification <3> (partial [3]) [3, 5, 9] <4> (partial [7]) [7] Cloning <3> (from genetic library, functional expression in Escherichia coli, DNA and amino acid sequence determination and analysis [10]) [10]

6 Stability Temperature stability 45 <3> (<3> 2 min, about 20% loss of activity [4]) [4] 65 <4> (<4> 50% remaining activity after 15 min [7]) [7] Storage stability <1>, 4 C, activity is increased after 7 days of storage, then declines to the starting level, lag phase [1] <3>, -10 C, rapidly destroyed [4] <3>, 0-4 C, stable for 2-3 weeks [4] <3>, 0 C, great loss of activity within 24 h [5] <3>, 0 C, loss of 50% activity within 24 h without Mn2+ , purified enzyme is stable for 30 days with Mn2+ [9] <4>, -20 C, 20 days, about 10fold increased activity [7]

References [1] Barengo, R.; Krisman, C.R.: Initiation of glycogen biosynthesis in Escherichia coli. Studies of the properties of the enzymes involved. Biochim. Biophys. Acta, 540, 190-196 (1978) [2] Krisman, C.R.; Barengo, R.: A precursor of glycogen biosynthesis: a-1,4glucan-protein. Eur. J. Biochem., 52, 117-123 (1975) [3] Moreno, S.; Tandecarz, J.S.: Potato tuber glucosyl transferases: partial characterization of the solubilized enzymes. FEBS Lett., 139, 313-316 (1982) [4] Lavintman, N.; Tandecarz, J.S.; Carceller, M.; Mendiara, S.; Cardini, C.E.: Role of uridine diphosphate glucose in the biosynthesis of starch. Mechanism of formation and enlargement of a glucoproteic acceptor. Eur. J. Biochem., 50, 145-155 (1974) [5] Ardila, F.J.; Tandecarz, J.S.: Potato tuber UDP-glucose:protein transglucosylase catalyzes its own glucosylation. Plant Physiol., 99, 1342-1347 (1992)

132

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a-1,4-Glucan-protein synthase (UDP-forming)

[6] Moreno, S.; Cardini, C.E.; Tandecarz, J.S.: a-Glucan synthesis on a protein primer, uridine diphosphoglucose: protein transglucosylase I. Separation from starch synthetase and phosphorylase and a study of its properties. Eur. J. Biochem., 157, 539-545 (1986) [7] Rothschild, A.; Tandecarz, J.S.: UDP-glucose: protein transglucosylase in developing maize endosperm. Plant Sci., 97, 119-127 (1994) [8] Rothschild, A.; Wald, F.A.; Bocca, S.N.; Tandecarz, J.S.: Inhibition of UDPglucose: protein transglucosylase by a maize endosperm protein factor. Cell. Mol. Biol., 42, 645-651 (1996) [9] Bocca, S.N.; Rothschild, A.; Tandecarz, J.S.: Initiation of starch biosynthesis: purification and characterization of UDP-glucose:protein transglucosylase from potato tubers. Plant Physiol. Biochem., 35, 205-212 (1997) [10] Bocca, S.N.; Kissen, R.; Rojas-Beltran, J.A.; Noel, F.; Gebhardt, C.; Moreno, S.; Jardin, P.d.; Tandecarz, J.S.: Molecular cloning and characterization of the enzyme UDP-glucose: protein transglucosylase from potato. Plant Physiol. Biochem., 37, 809-819 (1999)

133

a-1,4-Glucan-protein synthase (ADP-forming)

2.4.1.113

1 Nomenclature EC number 2.4.1.113 Systematic name ADP-glucose:protein 4-a-d-glucosyltransferase Recommended name a-1,4-glucan-protein synthase (ADP-forming) Synonyms ADP-glucose:protein glucosyltransferase adenosine diphosphoglucose-protein glucosyltransferase glucosyltransferase, adenosine diphosphoglucose-protein CAS registry number 67053-99-0

2 Source Organism <1> Escherichia coli [1] <2> Thermococcus hydrothermalis (AL662 [2]) [2]

3 Reaction and Specificity Catalyzed reaction ADP-glucose + protein = ADP + a-d-glucosyl-protein (The enzyme builds up a-1,4-glucan chains covalently bound to protein, thus acting as an initiator of glycogen synthesis.) Reaction type hexosyl group transfer Natural substrates and products S ADP-glucose + protein <1, 2> (<1> the enzyme builds up a-1,4-glucan chains covalently bound to protein thus acting as an initiator of glycogen synthesis, once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme [1]) (Reversibility: ? <1, 2> [1, 2]) [1, 2] P ADP + a-d-glucosyl-protein <1, 2> [1, 2]

134

2.4.1.113

a-1,4-Glucan-protein synthase (ADP-forming)

Substrates and products S ADP-glucose + protein <1, 2> (<2> best donor substrate, enzyme can also use UDP-glucose as donor substrate [2]) (Reversibility: ? <1, 2> [1, 2]) [1, 2] P ADP + a-d-glucosyl-protein <1, 2> [1, 2] Inhibitors Brij-58 <1> [1] Lubrol <1> [1] Km-Value (mM) 0.9 <2> (ADP-glucose) [2] pH-Optimum 5.5 <2> [2] pH-Range 5.2-7.5 <2> (<2> half-maximal activity at pH 5.2 and pH 7.5 [2]) [2] Temperature optimum ( C) 30 <1> (<1> assay at [1]) [1] 80 <2> [2] Temperature range ( C) 70-90 <2> (<2> 55% activity at 70 C and 90 C [2]) [2] Additional information <2> (<2> no activity at 25 C and 110 C [2]) [2]

4 Enzyme Structure Molecular weight 85000 <2> (<2> gel filtration [2]) [2] Subunits dimer <2> (<2> 2 * 42000, SDS-PAGE [2]) [2] Posttranslational modification glycoprotein <1> (<1> contains a-1,4-glucosidic bonds [1]) [1]

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

135

a-1,4-Glucan-protein synthase (ADP-forming)

2.4.1.113

6 Stability Temperature stability 80 <2> (<2> stable for at least 2 h [2]) [2] 90 <2> (<2> half-life: 26 min, and 52 min in presence of 3.2 mM ADP-glucose [2]) [2] 100 <2> (<2> half-life: 3 min, and 9.5 min in presence of 3.2 mM ADP-glucose [2]) [2] 110 <2> (<2> inactivated, but in presence of 3.2 mM ADP-glucose the enzyme shows a half-life of 6 min [2]) [2] General stability information <1>, 1% Brij-58 totally inhibited the enzyme activity of the stored enzyme preparation after 50 min [1] Storage stability <1>, 4 C, loss of about 90% activity after 30 days [1]

References [1] Barengo, R.; Krisman, C.R.: Initiation of glycogen biosynthesis in Escherichia coli. Studies of the properties of the enzymes involved. Biochim. Biophys. Acta, 540, 190-196 (1978) [2] Gruyer, S.; Legin, E.; Bliard, C.; Ball, S.; Duchiron, F.: The endopolysaccharide metabolism of the hyperthermophilic archeon Thermococcus hydrothermalis: polymer structure and biosynthesis. Curr. Microbiol., 44, 206-211 (2002)

136

2-Coumarate O-b-glucosyltransferase

2.4.1.114

1 Nomenclature EC number 2.4.1.114 Systematic name UDP-glucose:trans-2-hydroxycinnamate O-b-d-glucosyltransferase Recommended name 2-coumarate O-b-glucosyltransferase Synonyms UDPG:o-coumaric acid O-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-o-coumarate CAS registry number 73665-97-1

2 Source Organism <1> Melilotus alba (cucubb genotype [1]; var. White Blossom [2]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + trans-2-hydroxycinnamate = UDP + trans-b-d-glucosyl-2-hydroxycinnamate Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + 2-hydroxycinnamate <1> (<1>, involved in the biosynthesis of coumarin [1,2]) (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + trans-b-d-glucosyl-o-hydroxycinnamic acid <1> [1, 2] Substrates and products S UDPglucose + 2-hydroxycinnamate <1> (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + trans-b-d-glucosyl-o-hydroxycinnamic acid <1> [1, 2] Inhibitors Cu2+ <1> (<1>, 1 mM, 63% inhibition [1]) [1] Zn2+ <1> (<1>, 1 mM, 77% inhibition [1]) [1] 137

2-Coumarate O-b-glucosyltransferase

2.4.1.114

sodium citrate <1> (<1>, 4 mM, 37% inhibition [1]) [1] Additional information <1> (<1>, not inhibitory: 4 mM EDTA or 0.8 mM 2coumaric acid glucoside [1]) [1] Activating compounds 2-mercaptoethanol <1> (<1>, stimulation [1]) [1] cysteine <1> (<1>, stimulation [1]) [1] pH-Optimum 7.6-8.3 <1> [1] pH-Range 6.5-8.8 <1> (<1>, 25% of maximal activity at pH 6, 10% of maximal activity at pH 9 [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [1, 2] Localization Additional information <1> (<1>, not associated with chloroplasts [2]) [2] Purification <1> (partial [1]) [1]

6 Stability Temperature stability 4 <1> (<1>, 1-2 d, complete loss of activity [1]) [1] General stability information <1>, dialysis overnight inactivates [1] <1>, freezing results in a complete loss of activity [1] Storage stability <1>, 4 C, complete loss of activity within 1-2 days [1]

References [1] Kleinhofs, A.; Haskins, F.A.; Gorz, H.J.: trans-o-Hydroxycinnamic acid glucosylation in cell-free extracts of Meliotus alba. Phytochemistry, 6, 1313-1318 (1967) [2] Poulton, J.E.; McRee, D.E.; Conn, E.E.: Intracellular localization of two enzymes involved in coumarin biosynthesis in Meliotus alba. Plant Physiol., 65, 171-175 (1980)

138

Anthocyanidin 3-O-glucosyltransferase

2.4.1.115

1 Nomenclature EC number 2.4.1.115 Systematic name UDP-glucose:anthocyanidin 3-O-d-glucosyltransferase Recommended name anthocyanidin 3-O-glucosyltransferase Synonyms UDP-glucose:anthocyanidin/flavonol 3-O-glucosyltransferase UDP-glucose:cyanidin-3-O-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-anthocyanidin 3-OCAS registry number 65607-32-1

2 Source Organism <1> Silene dioica [1] <2> Hippeastrum sp. (cv. Dutch Red Hybrid [2]) [2] <3> Tulipa sp. (tulip, var. Most Miles [2]) [2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + anthocyanidin = UDP + anthocyanidin-3-O-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + anthocyanidin <1> (<1>, involved in anthocyanine biosynthesis [1]) (Reversibility: ? <1> [1]) [1] P UDP + anthocyanidin-3-O-glucoside Substrates and products S UDPglucose + cyanidin <1> (<1>, best substrate [1]) (Reversibility: ? <1> [1]) [1] P UDP + cyanidin 3-O-glucoside <1> [1]

139

Anthocyanidin 3-O-glucosyltransferase

2.4.1.115

S UDPglucose + delphinidin <1> (<1>, more slowly than cyanidin [1]) (Reversibility: ? <1> [1]) [1] P UDP + delphinidin 3-O-glucoside <1> [1] S UDPglucose + malvidin <2, 3> (Reversibility: ? <2, 3> [2]) [2] P UDP + malvidin 3-O-glucoside S UDPglucose + pelargonidin <1> (<1>, more slowly than cyanidin [1]) (Reversibility: ? <1> [1]) [1] P UDP + pelargonidin 3-O-glucoside <1> [1] Inhibitors CaCl2 <1> (<1>, 2 mM, 18% inhibition [1]) [1] HgCl2 <1> (<1>, 0.1 mM, 85% inhibition [1]) [1] NEM <1> (<1>, 1.25 mM, 30% inhibition [1]) [1] PCMB <1> (<1>, 1.25 mM, 93% inhhibition, 2-mercaptoethanol or cysteine restores activity [1]) [1] ZnCl2 <1> (<1>, 2 mM, 22% inhibition [1]) [1] Activating compounds 2-mercaptoethanol <1> (<1>, detergent required for maximal activity [1]) [1] 2-methoxyethanol <1> (<1>, activation [1]) [1] Triton X-100 <1> (<1>, activation [1]) [1] Tween 20 <1> (<1>, detergent required for maximal activity [1]) [1] Tween 80 <1> (<1>, detergent required for maximal activity [1]) [1] cetrimide <1> (<1>, activation [1]) [1] cysteine <1> (<1>, detergent required for maximal activity [1]) [1] Specific activity (U/mg) 0.0252 <1> [1] Km-Value (mM) 0.07 <1> (cyanidin) [1] 0.13 <1> (delphinidin) [1] 0.13 <1> (pelargonidin) [1] 0.41 <1> (UDPglucose) [1] pH-Optimum 7.5 <1> [1] pH-Range 6.6-8 <1> (<1>, 50% of maximal activity at pH 6.6 and pH 8.0 [1]) [1]

4 Enzyme Structure Molecular weight 125000 <1> (<1>, gel filtration [1]) [1]

140

2.4.1.115

Anthocyanidin 3-O-glucosyltransferase

Subunits dimer <1> (<1>, 2 * 60000, can exist as monomer or dimer, SDS-PAGE [1]) [1] monomer <1> (<1>, 1 * 60000, can exist as monomer or dimer, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue calyx <1> [1] leaf <2, 3> [2] leaf <1> (<1>, young rosette leaves [1]) [1, 2] petal <1, 2, 3> (<1>, highest activity in petals of opening flowers of young plants [1]) [1, 2] Localization cytosol <2, 3> [2] soluble <2, 3> [2] Additional information <2, 3> (<2,3>, no activity in the vacuole [2]) [2] Purification <1> (partial [1]) [1]

6 Stability General stability information <1>, repeated freeze-thawing inactivates [1] Storage stability <1>, -17 C, about 40% loss of activity within 3 months [1] <1>, 4 C, about 40% loss of activity within 1 week [1]

References [1] Kamsteeg, J.; van Brederode, J.; van Nigtevecht, G.: Identification and properties of UDP-glucose:cyanidin-3-O-glucosyltransferase isolated from petals of the red campion (Silene dioica). Biochem. Genet., 16, 1045-1058 (1978) [2] Hrazdina, G.; Wagner, G.J.; Siegelman, H.W.: Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts. Phytochemistry, 17, 53-56 (1978)

141

Cyanidin-3-rhamnosylglucoside 5-Oglucosyltransferase

2.4.1.116

1 Nomenclature EC number 2.4.1.116 Systematic name UDP-glucose:cyanidin-3-O-d-rhamnosyl-1,6-d-glucoside 5-O-d-glucosyltransferase Recommended name cyanidin-3-rhamnosylglucoside 5-O-glucosyltransferase Synonyms glucosyltransferase, uridine diphosphoglucose-cyanidin 3-rhamnosylglucoside 5-OCAS registry number 70248-66-7

2 Source Organism <1> Silene dioica [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + cyanidin-3-O-d-rhamnosyl-1,6-d-glucoside = UDP + cyanidin-3-O-[d-rhamnosyl-1,6-d-glucoside]-5-O-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + cyanidin-3-O-d-rhamnosyl-1,6-d-glucoside <1> (<1>, involved in anthocyan biosynthesis. Activity is controlled by a single dominant gene [1]) [1] P UDP + cyanidin-3-O-[d-rhamnosyl-1,6-d-glucoside]-5-O-d-glucoside Substrates and products S UDPglucose + cyanidin 3-O-d-glucoside <1> (Reversibility: ? <1> [2]) [2] P UDP + ? <1> (<1>, depending upon pH, the enzyme catalyzes the formation of either anthocyanidin 3-rhamnosylglucoside or 3,5-diglucoside.

142

2.4.1.116

S P

S P S P

Cyanidin-3-rhamnosylglucoside 5-O-glucosyltransferase

Maximal formation of anthocyanidin 3-rhamnosylglucoside-5-glucoside at pH 7.4, maximal formation of anthocyanidin 3,5-diglucoside at pH 6.5. Formation of cyanidin 3-rhamnosylglucoside-5-glucoside proceeds 5-10fold faster than the cyanidin 3,5-diglucoside formation [2]) [2] UDPglucose + cyanidin 3-O-d-rhamnosylglucoside <1> (Reversibility: ? <1> [2]) [2] UDP + ? <1> (<1>, depending upon pH, the enzyme catalyzes the formation of either anthocyanidin 3-rhamnosylglucoside or 3,5-diglucoside. Maximal formation of anthocyanidin 3-rhamnosylglucoside-5-glucoside at pH 7.4, maximal formation of anthocyanidin 3,5-diglucoside at pH 6.5 [2]) [2] UDPglucose + cyanidin-3-O-d-rhamnosyl-1,6-d-glucoside <1> (<1>, no activity with ADPglucose [1]) (Reversibility: ? <1> [1]) [1] UDP + cyanidin-3-O-[d-rhamnosyl-1,6-d-glucoside]-5-O-d-glucoside <1> [1] UDPglucose + pelargonidin-3-O-rhamnosylglucoside <1> (Reversibility: ? <1> [1]) [1] UDP + ? <1> [1]

Inhibitors EDTA <1> (<1>, 1 mM, 57% inhibition [1]; <1>, 1 mM, pH 7.5, inhibits formation of cyanidin 3-rhamnosylglucoside-5-glucoside but has no effect on the formation of cyanidin 3,5-diglucoside at pH 6.5 [2]) [1, 2] Hg2+ <1> [1, 2] NEM <1> (<1>, 3 mM, 25% inhibition, 2-mercpatoethanol or cysteine does not protect [1]) [1] PCMB <1> (<1>, 3 mM, 25% inhibition, 2-mercpatoethanol or cysteine does not protect [1]) [1] Zn2+ <1> [2] Activating compounds Ca2+ <1> (<1>, 1 mM, highest stimulation of divalent cations [1]; <1>, 1 mM CaCl2 stimulates 5-O-glucosylation of cyanidin 3-rhamnosyl-glucoside 1.6fold, pH 7.4 [2]) [1, 2] Co2+ <1> (<1>, 1 mM activates [1,2]) [1, 2] Mg2+ <1> (<1>, 1 mM activates [1,2]) [1, 2] Mn2+ <1> (<1>, 1 mM activates [1,2]) [1, 2] Additional information <1> (<1>, glucosylation of cyanidin 3-glucoside at pH 6.5 is not stimulated by divalent metal ions [2]) [2] Specific activity (U/mg) 0.00953 <1> [1] Km-Value (mM) 0.5 <1> (UDPglucose, <1>, reaction with cyanidin 3-rhamnosylglucoside at pH 7.4 [1]) [1, 2] 0.59 <1> (UDPglucose, <1>, reaction with + cyanidin 3-rhamnosylglucoside at pH 6.5 [2]) [2]

143

Cyanidin-3-rhamnosylglucoside 5-O-glucosyltransferase

2.4.1.116

0.67 <1> (UDPglucose, <1>, reaction with cyanidin-3-glucoside at pH 6.5 [2]) [2] 3.6 <1> (cyanidin-3-rhamnosylglucoside) [1, 2] 23.4 <1> (cyanidin-3-glucoside) [2] pH-Optimum 6.5 <1> (<1>, activity with cyanidin-3-glucoside [2]) [2] 7.4 <1> (<1>, activity with cyanidin-3-rhamnosylglucoside [2]) [1, 2] pH-Range 6-7.3 <1> (<1>, pH 6.0: about 80% of maximal activity, pH 7.3: about 50% of half-maximal activity, reaction with cyanidin 3-glucoside [2]) [2] 6.9-8.8 <1> (<1>, about 50% of maximal activity at pH 6.9 and at pH 8.8 [1]) [1] 7-8.5 <1> (<1>, about half-maximal activity at pH 7 and 8.5, reaction with cyanidin-3-rhamnosylglucoside [2]) [2]

4 Enzyme Structure Molecular weight 55000 <1> (<1>, gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue petal <1> (<1>, not in green parts of the plant [1]) [1, 2] Purification <1> (partial [1]) [1]

6 Stability pH-Stability 7.6 <1> (<1>, 30 min, stable below [2]) [2] 8 <1> (<1>, 30 min, about 10% loss of activity [2]) [2] 8.5 <1> (<1>, 30 min, about 40% loss of activity [2]) [2] General stability information <1>, repeated freeze-thawing inactivates [1] Storage stability <1>, -20 C, about 60-80% loss of activity within 3 months [1]

144

2.4.1.116

Cyanidin-3-rhamnosylglucoside 5-O-glucosyltransferase

References [1] Kamsteeg, J.; van Brederode, J.; van Nigtevecht, G.: Identification, properties, and genetic control of UDP-glucose: cyanidin-3-rhamnosyl-(1 ! 6)-glucoside-5-O-glucosyltransferase isolated from petals of the red campion (Silene dioica). Biochem. Genet., 16, 1059-1071 (1978) [2] Kamsteeg, J.; van Brederode, J.; van Nigtevecht, G.: The pH-dependent substrate specificity of UDP-glucose:anthocyandin 3-rhamnosylglucoside, 5-Oglucosyltransferase in petals of Silene dioica: the formation of anthocyanidin 3,5-diglucosides. Z. Pflanzenphysiol., 96, 87-93 (1980)

145

Dolichyl-phosphate b-glucosyltransferase

2.4.1.117

1 Nomenclature EC number 2.4.1.117 Systematic name UDP-glucose:dolichyl-phosphate b-d-glucosyltransferase Recommended name dolichyl-phosphate b-glucosyltransferase Synonyms UDP-glucose dolichyl-phosphate glucosyltransferase UDP-glucose:dolichol phosphate glucosyltransferase UDP-glucose:dolicholphosphoryl glucosyltransferase UDP-glucose:dolichyl monophosphate glucosyltransferase UDP-glucose:dolichyl phosphate glucosyltransferase UDP-glucose:dolichylphosphate glucosyltransferase glucosyltransferase, uridine diphosphoglucose-dolichol polyprenyl phosphate:UDP-d-glucose glucosyltransferase CAS registry number 71061-42-2

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

Acanthamoeba castellanii [1] Bos taurus (calf [2]) [2] Rattus norvegicus (rat [3]) [3, 11] Zea mays (L. inbred A 636) [4, 8] Homo sapiens [5, 6, 10] Phaseolus aureus (mung bean [7]) [7] Candida albicans (strain ATCC 26555 [9]) [9, 13, 14] Saccharomyces cerevisiae [12]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + dolichyl phosphate = UDP + dolichyl b-d-glucosyl phosphate (<5> sequential mechanism [6])

146

2.4.1.117

Dolichyl-phosphate b-glucosyltransferase

Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + dolichyl phosphate <1> (Reversibility: r <1> [1]) [1] P UDP + dolichyl b-d-glucosyl phosphate S Additional information <1, 6> (<1> synthesis of dolichyl d-glucosyl phosphate, a glucosyl donor in the formation of lipid-linked core oligosaccharides [1]; <6> enzyme is involved in the biosynthesis of the lipidlinked oligosaccharides that are precursors of N-linked glycoproteins [7]) [1, 7] P ? Substrates and products S UDP-glucose + dolichyl phosphate <1-8> (Reversibility: r <1, 7> [1, 13]; ? <2-8> [2-9, 12]) [1-9, 12, 13] P UDP + dolichyl b-d-glucosyl phosphate <1-4> [1-4] S UDP-glucose + ficaprenyl phosphate <1> (Reversibility: r <1> [1]) [1] P UDP + ficaprenyl b-d-glucosyl phosphate S UDP-glucose + solanesyl phosphate <1> (Reversibility: r <1> [1]) [1] P UDP + solanesyl b-d-glucosyl phosphate S Additional information <1> (<1> no substrate: GDP-d-glucose, UDP-dglucuronic acid, UDP-N-acetyl-d-glucosamine, UDP-d-xylose [1]) [1] P ? Inhibitors Ca2+ <7> (<7> slight inhibition [9]) [9] EDTA <1, 6> (<1> inhibition reversed by MgCl2 [1]) [1, 7] Mn2+ <7> (<7> slight inhibition [9]) [9] MnCl2 <6> (<6> stimulates at 1 mM, inhibits at higher concentration [7]) [7] UDP <1, 5> (<1,5> competitive [1,5]) [1, 5, 6] UDP-glucuronic acid <4> [8] UMP <1, 7> (<1> reversible [1]) [1, 9, 13] amphomycin <7> (<7> IC50 0.04 mM [9]) [9] tunicamycin <4> (<4> 50% inhibition at 0.04 mg per ml [8]) [4, 8] Activating compounds Triton X-100 <4> (<4> required for optimal activity, 0.01-0.015% [4]) [4] detergent <4> (<4> required for maximum activity, optimum for Triton X100: 0.015% [4,8]) [4, 8] phospholipid <5> (<5> required, phosphatidylethanolamine most effective [5]) [5] reducing agent <1> (<1> required [1]) [1] Metals, ions Ca2+ <4, 5> (<5> divalent cation required, maximum stimulation in presence of Ca2+ . Co2+, Mg2+ or Mn2+ can replace Ca2+ [6]; <4> divalent cation required, efficiency of activiation in descending order: Mg2+ , Mn2+ , Ca2+ ,

147

Dolichyl-phosphate b-glucosyltransferase

2.4.1.117

Co2+, Zn2+ [4]; <6> CaCl2 cannot replace MgCl2 or MnCl2 [7]; <7> no stimulation [9,13]) [4, 6, 7] Co2+ <4, 5> (<5> divalent cation required, maximum stimulation in presence of Ca2+ . Co2+, Mg2+ or Mn2+ can replace Ca2+ [6]; <4> divalent cation required, efficiency of activiation in descending order: Mg2+ , Mn2+ , Ca2+ , Co2+, Zn2+ [4]) [4, 6] Mg2+ <1, 4, 5, 7> (<5> divalent cation required, maximum stimulation in presence of Ca2+ . Co2+ , Mg2+ or Mn2+ can replace Ca2+ [6]; <4> divalent cation required, efficiency of activiation in descending order: Mg2+ , Mn2+ , Ca2+ , Co2+, Zn2+ [4]; <4> Km : 0.7 mM [4, 8]; <1> divalent cation required, either Mg2+ or Mn2+ , Mn2+ stimulates the enzyme to a greater extent than Mg2+ at low concentrations, Mg2+ stimulates to a greater extent than Mn2+ at high concentration, but each is maximally effective at a concentration around 8 mM [1]; <7> Mg2+ required, not Mn2+ , Ca2+ [9]; <7> Mg2+ stimulates [13]) [1, 4, 6, 8, 9, 13] Mn2+ <1, 4-6> (<5> divalent cation required, maximum stimulation in presence of Ca2+ . Co2+, Mg2+ or Mn2+ can replace Ca2+ [6]; <4> divalent cation required, efficiency of activiation in descending order: Mg2+ , Mn2+ , Ca2+ , Co2+, Zn2+ [4]; <1> divalent cation required, either Mg2+ or Mn2+ , Mn2+ stimulates the enzyme to a greater extent than Mg2+ at low concentrations, Mg2+ stimulates to a greater extent than Mn2+ at high concentration, but each is maximally effective at a concentration around 8 mM [1]; <6> MnCl2 stimulates at 1 mM, inhibits at higher concentration [7]; <7> no stimulation [9,13]) [1, 4, 6, 7] Zn2+ <4> (<4> divalent cation required, efficiency of activiation in descending order: Mg2+ , Mn2+ , Ca2+ , Co2+, Zn2+ [4]) [4] Specific activity (U/mg) Additional information <5, 6> [5, 7] Km-Value (mM) 0.00015 <5> (UDP-glucose, <5> pH 7.2 [6]) [6] 0.00033 <5> (UDP-glucose, <5> pH 5.3 [6]) [6] 0.0005 <4> (UDP-glucose) [8] 0.0012 <4> (dolichyl phosphate) [8] 0.002 <6> (dolichyl phosphate) [7] 0.0045 <1> (dolichyl phosphate) [1] 0.0091 <1> (UDP-glucose) [1] 0.027 <6> (UDP-glucose) [7] 0.05 <7> (UDP-glucose) [9] 0.104 <7> (UDP-glucose) [13] Additional information <4> [4, 8] Ki-Value (mM) 0.0076 <4> (UDP-glucuronic acid) [8] 0.1 <5> (UDP, <5> at pH 7.2 [6]) [6] 0.1 <7> (UMP) [13] 0.14 <4> (UDP) [4]

148

2.4.1.117

Dolichyl-phosphate b-glucosyltransferase

0.15 <4> (UDP) [8] 0.17 <5> (UDP, <5> at pH 5.3 [6]) [6] 0.3 <7> (UMP) [9] 0.65 <4> (UMP) [8] 0.66 <4> (UMP) [4] pH-Optimum 5.3 <5> (<5> second optimum at pH 7.2 [6]) [6] 6-7 <6> [7] 6.5 <4> [4] 7 <1> [1] 7.2 <5> (<5> second optimum at pH 5.3 [6]) [6] 7.5 <4, 7> (<7> Tris/HCl buffer [9]) [8, 9] pH-Range 5.6-8.8 <1> (<1> pH 5.6: 25% of maximum activity, pH 8.8: about 40% of maximum activity [1]) [1] Temperature optimum ( C) 28 <7> [9] 30 <1> [1] 37 <6> (<6> assay at [7]) [7]

4 Enzyme Structure Subunits ? <5, 6, 8> (<5> x * 36000, SDS-PAGE [5]; <6> x * 39000, SDS-PAGE after photoaffinity labeling, catalytic subunit [7]; <8> x * 38000, deduced from gene sequence [12]) [5, 7, 12]

5 Isolation/Preparation/Mutation/Application Source/tissue cyst <1> [1] endosperm <4> (<4> culture [4]) [4, 8] liver <3, 5> [3, 5, 6, 10] pancreas <2> [2] Localization endoplasmic reticulum <5, 8> [10, 12] microsome <2-6> (<4> membrane [4]; <3> active centres are cytoplasmically oriented, activation by detergent may be conformation-dependent [11]) [2-8, 11] soluble <1> [1]

149

Dolichyl-phosphate b-glucosyltransferase

2.4.1.117

Purification <4> (partial [8]) [8] <5> [5] <6> (partial [7]) [7] <7> (partial [14]) [14] Renaturation <5> (5 mM uridine or UDPglucuronic acid reconstitutes inactivated enzyme [5]) [5] Cloning <8> [12]

6 Stability Temperature stability 30 <1> (<1> 2 h, 50% loss of activity [1]) [1] 52 <1> (<1> 10 min, complete loss of activity [1]) [1] General stability information <5>, uridine or UDPglucuronic acid, 5 mM, protects solubilized enzyme against rapid inactivation [5] Storage stability <1>, -20 C, indefinitely stable below [1] <1>, 4 C, stable for several days [1] <4>, -70 C, up to 4 weeks [8]

References [1] Villemez, C.L.; Carlo, P.L.: Properties of a soluble polyprenyl phosphate: UDP-d-glucose glucosyltransferase. J. Biol. Chem., 254, 4814-4819 (1979) [2] Herscovics, A.; Bugge, B.; Jeanloz, R.W.: Glucosyltransferase activity in calf pancreas microsomes. Formation of dolichyl d[14 C]glucosyl phosphate and 14 C-labeled lipid-linked oligosaccharides from UDP-d-[14 C]glucose. J. Biol. Chem., 252, 2271-2277 (1977) [3] Behrens, N.H.; Leloir, L.F.: Dolichol monophosphate glucose: an intermediate in glucose transfer in liver. Proc. Natl. Acad. Sci. USA, 66, 153-159 (1970) [4] Riedell, W.E.; Miernyk, J.A.: Glycoprotein synthesis in maize endosperm cells. The nucleoside diphosphate-sugar:dolichol-phosphate glycosyltransferase. Plant Physiol., 87, 420-426 (1988) [5] Matern, H.; Bolz, R.; Matern, S.: Isolation and characterization of UDP-glucose dolichyl-phosphate glucosyltransferase from human liver. Eur. J. Biochem., 190, 99-105 (1990) [6] Matern, H.; Matern, S.: Control of dolichyl phosphoglucose formation in human liver microsomes. Kinetic and inhibition studies of nucleosides, nu150

2.4.1.117

[7] [8] [9]

[10] [11] [12] [13]

[14]

Dolichyl-phosphate b-glucosyltransferase

cleotides and analogues of UDPglucose. Biochim. Biophys. Acta, 1004, 6772 (1989) Drake, R.R.; Kaushal, G.P.; Pastuszak, I.; Elbein, A.D.: Partial purificaton, photoaffinity labeling, and properties of mung bean UDP-glucose:dolicholphosphate glucosyltransferase. Plant Physiol., 97, 396-401 (1991) Miernyk, J.A.; Riedell, W.E.: Characterization of maize endosperm UDPglucose:dolicholphosphate glucosyltransferase. Phytochemistry, 30, 28652867 (1991) Arroyo-Flores, B.L.; Rodriguez-Bonilla, J.; Villagomez-Castro, J.C.; CalvoMendez, C.; Flores-Carreon, A.; Lopez-Romero, E.: Biosynthesis of glycoproteins in Candida albicans: Activity of mannosyl and glucosyl transferases. Fungal Genetics and Biology, 30, 127-133 (2000) Gartung, C.; Matern, S.; Matern, H.: The submicrosomal localization of uridine 5'-diphosphate-glucose dolichyl-phosphate glucosyltransferase and bile acid glucosyltransferase in the human liver. J. Hepatol., 20, 32-40 (1994) Bossuyt, X.; Blanckaert, N.: Topology of nucleotide-sugar:dolichyl phosphate glycosyltransferases involved in the dolichol pathway for protein glycosylation in native rat liver microsomes. Biochem. J., 296, 627-632 (1993) Heesen, S.; Lehle, L.; Weissmann, A.; Aebi, M.: Isolation of the ALG5 locus encoding the UDP-glucose:dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae. Eur. J. Biochem., 224, 71-79 (1994) Rodriguez-Bonilla, J.; Vargas-Rodriguez, L.; Calvo-Mendez, C.; Flores-Carreon, A.; Lopez-Romero, E.: Biosynthesis of glycoproteins in Candida albicans: Biochemical characterization of dolichol phosphate glucose synthase. Antonie Leeuwenhoek, 73, 373-380 (1998) Lopez-Romero, E.; Flores-Carreon, A.; Arroyo-Flores, B.L.; Torre-Bouscoulet, M.E.; Bravo-Torres, J.C.; Villagomez-Castro, J.C.; Balcazar-Orozco, R.: Glycosyl transferases and glycosidases of glycoprotein biosynthesis with emphasis on Candida albicans and Entamoeba histolytica. Recent Res. Dev. Microbiol., 4, 667-681 (2000)

151

Cytokinin 7-b-glucosyltransferase

2.4.1.118

1 Nomenclature EC number 2.4.1.118 Systematic name UDP-glucose:zeatin 7-glucosyltransferase Recommended name cytokinin 7-b-glucosyltransferase Synonyms cytokinin 7-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-zeatin 7CAS registry number 72103-03-8

2 Source Organism <1> Raphanus sativus [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + N6 -alkylaminopurine = UDP + N6 -alkylaminopurine-7-b-dglucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + N6 -alkylaminopurine <1> (<1>, involved in regulation of cytokinin activity [2]) [2] P UDP + N6 -alkylaminopurine-7-b-d-glucoside Substrates and products S TDPglucose + zeatin <1> (<1>, as effective as UDPglucose [2]) (Reversibility: ? <1> [2]) [2] P TDP + zeatin 7-b-d-glucoside S UDPglucose + 6-benzylaminopurine <1> (<1>, activity is 15% higher for 6-benzylaminopurine than for zeatin [2]) (Reversibility: ? <1> [2]) [2]

152

2.4.1.118

Cytokinin 7-b-glucosyltransferase

UDP + 6-benzylaminopurine-7-b-d-glucoside UDPglucose + N6 -alkylaminopurine <1> (Reversibility: ? <1> [1,2]) [1, 2] UDP + N6 -alkylaminopurine-7-b-d-glucoside <1> [1] UDPglucose + dihydroxyzeatin <1> (Reversibility: ? <1> [2]) [2] UDP + dihydroxyzeatin 7-b-d-glucoside UDPglucose + zeatin <1> (<1>, i.e. N6 -(4-hydroxy-3-methylbut-trans-2enylamino)purine [2]) (Reversibility: ? <1> [1,2]) [1, 2] P UDP + zeatin 7-b-d-glucoside <1> (<1>, + zeatin 9-glucoside [2]) [1, 2]

P S P S P S

Inhibitors 2-chloro-6-(5-hydroxypentylamino)-7-methylpurine <1> [3] 2-chloro-6-(5-hydroxypentylamino)-9-methylpurine <1> [3] 3-methyl-7-pentylaminopyrazolo[4,3-d]pyrimidine <1> (<1>, competitive [3]) [3] 4-cyclopentylamino-2-methylthiopyrrolo[2,3-d]pyrimidine <1> [3] 4-furfurylaminopyrazolo[3,4-d]pyrimidine <1> [3] 6-(5-hydroxypentylamino)-9-methylpurine <1> [3] 6-benzylamino-2-(2-hydroxyethylamino)-7-methylpurine <1> [3] 6-benzylamino-2-(2-hydroxyethylamino)-9-methylpurine <1> [3] 6-benzylamino-9-(3-hydroxyprolyl)purine <1> [3] N-(3-chlorophenyl)-N'-phenylurea <1> [3] N-benzyl-N'-phenylurea <1> [3] theophylline <1> [3] Specific activity (U/mg) 0.00046 <1> [2] Km-Value (mM) 0.15 <1> (zeatin) [2] 0.19 <1> (UDPglucose) [2] Ki-Value (mM) 0.0033 <1> (6-benzylamino-2-(2-hydroxyethylamino)-9-methylpurine) [3] 0.022 <1> (3-methyl-7-pentylaminopyrazolo[4,3-d]pyrimidine) [3] 0.027 <1> (4-cyclopentylamino-2-methylthiopyrrolo[2,3-d]pyrimidine) [3] 0.048 <1> (2-chloro-6-(5-hydroxypentylamino)-9-methylpurine) [3] 0.061 <1> (6-benzylamino-2-(2-hydroxyethylamino)-7-methylpurine) [3] 0.075 <1> (N-(3-chlorophenyl)-N'-phenylurea) [3] 0.1 <1> (N-benzyl-N'-phenylurea) [3] 0.2 <1> (6-benzylamino-9-(3-hydroxyprolyl)purine) [3] 0.22 <1> (2-chloro-6-(5-hydroxypentylamino)-7-methylpurine) [3] 0.23 <1> (6-(5-hydroxypentylamino)-9-methylpurine) [3] 0.28 <1> (4-furfurylaminopyrazolo[3,4-d]pyrimidine) [3] 0.38 <1> (theophylline) [3] pH-Optimum 7.3-7.4 <1> [2]

153

Cytokinin 7-b-glucosyltransferase

2.4.1.118

4 Enzyme Structure Molecular weight 46500 <1> (<1>, gel filtration [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue cotyledon <1> (<1>, expanded [1]) [1-3] Localization soluble <1> [1] Purification <1> (partial [1,2]) [1, 2]

References [1] Entsch, B.; Letham, D.S.: Enzymic glucosylation of the cytokinin 6-benzylaminopurine. Plant Sci. Lett., 14, 205-212 (1979) [2] Entsch, B.; Parker, C.W.; Letham, D.S.; Summons, R.E.: Preparation and characterization, using high-performance liquid chromatography, of an enzyme forming glucosides of cytokinins. Biochim. Biophys. Acta, 570, 124-139 (1979) [3] Parker, C.W.; Entsch, B.; Letham, D.S.: Inhibitors of two enzymes which metabolize cytokinins. Phytochemistry, 25, 303-310 (1986)

154

Dolichyl-diphosphooligosaccharide-protein glycotransferase

2.4.1.119

1 Nomenclature EC number 2.4.1.119 Systematic name dolichyl-diphosphooligosaccharide:protein-l-asparagine oligopolysaccharidotransferase Recommended name dolichyl-diphosphooligosaccharide-protein glycotransferase Synonyms OST asparagine N-glycosyltransferase dolichyldiphosphooligosaccharide-protein oligosaccharyltransferase glycosyltransferase, dolichyldiphosphooligosaccharide-protein glycosyltransferase, dolichylpyrophosphodiacetylchitobiose-protein oligomannosyltransferase oligosaccharide transferase oligosaccharyltransferase, dolichyldiphosphoryloligosaccharide-protein CAS registry number 75302-32-8

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

Gallus gallus (hen [1,2]) [1, 2, 7, 10, 11, 20] Mus musculus [2] Rattus norvegicus [2, 4, 5] Canis familiaris [2, 8, 16, 20] Oryctolagus cuniculus [2] Sus scrofa [9, 21] Bos taurus (calf [6]) [6] Trypanosoma cruzi [5] Leptomonas samueli [5] Crithidia fasciculata [5] Blastocrithidia culicis [5] Drosophila melanogaster [2] Saccharomyces cerevisiae (yeast, strain X-2180-1B [5]; wild-type strain X2180-1A [12]) [2, 5, 12, 15, 22]

155

Dolichyl-diphosphooligosaccharide-protein glycotransferase

2.4.1.119

<14> yeast [3, 14, 17, 20] <15> Sus scrofa [13, 19, 20] <16> Homo sapiens [18, 20]

3 Reaction and Specificity Catalyzed reaction dolichyl diphosphooligosaccharide + protein l-asparagine = dolichyl diphosphate + a glycoprotein with the oligosaccharide chain attached by N-glycosyl linkage to protein l-asparagine Reaction type hexosyl group transfer Natural substrates and products S lipid-linked oligosaccharide + unfolded nascent polypeptide chain <1, 2, 3, 4, 5, 12, 13, 14> (<1, 4>, central enzyme in synthesis of N-linked glycoproteins [7,8]; <1, 2, 3, 4, 5, 12, 13>, central enzyme in the pathway of glycoprotein assembly [2]; <4>, the DAD1 subunit is a defender against apoptotic cell death [16]; <14>, the enzyme catalyzes the glycosylation of selected asparagine residues of nascent polypeptide chains as they are translocated into the lumen of the endoplasmic reticulum [17]; <6, 14>, N-glycosylation of eukaryotic secretory and membrane-bound proteins is an essential and highly conserved protein modification. The key step in this pathway is the en bloc transfer of the high mannose core oligosaccharide Gly3 Man9 GlcNAc2 from the lipid carries dolichyl phosphate to selected Asn-X-Ser/Thr sequences of nascent polypeptide chains during their translocation across the endoplasmic reticulum membrane [20, 21]; <14>, dolichyl-diphosphochitobiose-Man9 Glc3 is the preferred glycosyl donor both in vivo and in vitro [20]) (Reversibility: ? <1, 2, 3, 4, 5, 12, 13, 14> [2, 7, 8, 17]) [2, 7, 8, 16, 17, 20] P ? Substrates and products S dolichyl diphosphate-di-N-acetylchitobiose + Tyr-Asn-Leu-Thr-Ser-Val <3> (Reversibility: ? <3> [4]) [4] P ? S dolichyl diphosphochitobiose-(mannosyl)9-(glucosyl)3 + synthetic hexapeptide <3, 13> (<13>, e.g. Tyr-Asn-Leu-Thr-Ser-Val [12]; <13>, glycosyl-donors are also dolichyl diphosphochitobiose or dolichyl diphosphochitobiose-mannose, no substrates are dolichyl diphosphochitobiose(mannosyl)9 or dolichyl diphospho-N-acetyl-d-glucosamine [12]) (Reversibility: ? <3, 13> [5, 12]) [5, 12] P ? S dolichyl diphosphooligosaccharide + protein l-asparagine <1-16> (<1>, (Glc)n -(Man)x -GlcNAc2 -diphosphoryldolichol as oligosaccharyl donor and tryptic peptide consisting of residues 29-59 from bovine a-lactalbu-

156

2.4.1.119

P S

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

Dolichyl-diphosphooligosaccharide-protein glycotransferase

min as acceptor [1]; <13>, glycosyl donor specificity [12]; <1-6, 12, 13>, peptide acceptor specificity [2, 4, 9, 10, 11]; <1, 2, 3, 4, 5, 12, 13>, catalyzes reaction between b-amido nitrogen of Asn-residue and oligosaccharyl-diphosphodolichol [2]; <1, 3>, Asn-Xaa-Thr/Ser is a necessary and sufficient prerequisite for N-glycosylation [4, 10]; <1>, reaction with oligosaccharide-lipid present in hen oviduct microsomes and Na -Ac-AsnLeu-Thr-NHCH3 . Kinetic study with intact microsomal membrane [10]; <14>, dolichyl-diphosphochitobiose-Man9 Glc3 is the preferred glycosyl donor both in vivo and in vitro. The minimal glycosyl donor is dolichyldiphosphochitobiose [20]; <6>, highly stereospecific for the conformation of the 3-carbon atom in the hydroxy amino acid. Binding of the threonine b-methyl group by the enzyme is also specific, with serine, l-threo-b-hydroxynorvaline and l-b-hydroxynorleucine containing tripeptides all bound less efficiently than the threonine CH3 -CH(OH) group [21]) (Reversibility: ? <1-16> [1-21]) [1-21] dolichyl diphosphate + glycoprotein with the oligosaccharide chain attached by N-glycosyl-linkage to protein l-asparagine <1-16> [1-21] dolichyl diphosphooligosaccharide + synthetic tripeptides <1, 4, 6> (<4>, Asn-Tyr-Thr [8]; <6>, Ac-Asn-Leu-Thr-NH2 , Ac-Asn-Ala-Thr-NH2 and benzoyl-Asn-Leu-N-methylamide [9]; <6>, peptides that serve as acceptors show a secondary structural motif that involves the interaction between the asparagine side-chain carboxamide and the backbone amide of the threonine [9]; <1>, Asn-Leu-Thr and its N-terminal acetyl-, benzoyl-, octanoyl- or t-butoxycarbonyl-derivatives [10]; <1>, peptide hydrophobicity increases its acceptor activity [10]; <1>, no substrates are Asn-LeuThr-derivatives containing asparagine modifications or substitution [10]) (Reversibility: ? <1, 4, 6> [3, 8, 9, 10]) [3, 8, 9, 10] ? dolichyl-diphosphochitobiose + Arg-Asn-Gly-Thr-Ala-Val-methylester <7> (Reversibility: ? <7> [6]) [6] ? dolichyl-diphosphochitobiose + N-benzoyl-Asn-Gly-Thr-NHCH3 <15> (Reversibility: ? <15> [13]) [13] ? dolichyl-diphosphochitobiose + Tyr-Asn-Leu-Thr-Ser-Val <13> (Reversibility: ? <13> [12]) [12] ? dolichyl-diphosphochitobiose-Man1 + Tyr-Asn-Leu-Thr-Ser-Val <13> (Reversibility: ? <13> [12]) [12] ? dolichyl-diphosphochitobiose-Man7 Glc3 + protein-l-asparagine <8, 9, 10, 11> (Reversibility: ? <8, 9, 10, 11> [5]) [5] ? dolichyl-diphosphochitobiose-Man9 + Na -Ac-Asn-Tyr-Thr-NH2 <14> (Reversibility: ? <14> [14]) [14] ?

157

Dolichyl-diphosphooligosaccharide-protein glycotransferase

2.4.1.119

S dolichyl-diphosphochitobiose-Man9 Glc3 + Na -Ac-As-Lys(Ne -p-azidobenzoyl)-Thr-NH2 <1> (Reversibility: ? <1> [7]) [7] P ? S dolichyl-diphosphochitobiose-Man9 Glc3 + Tyr-Asn-Leu-Thr-Ser-Val <13> (Reversibility: ? <13> [12]) [12] P ? S dolichyl-diphosphochitobiose-Man9 Glc3 + a-Ac-Asn-Tyr-Thr-NH2 <14> (Reversibility: ? <14> [14]) [14] P ? S dolichyl-diphosphochitobiose-Man9 Glc3 + protein-l-asparagine <3, 8, 9, 10, 11, 13> (Reversibility: ? <3, 8, 9, 10, 11, 13> [5]) [5] P ? S Additional information <14> (<14>, evidence against either enol lactone or ketone formation as an intermediate [3]; <14>, allosteric communication between regulatory and catalytic dolichol-linked oligosaccharide binding sites [14]) [3, 14] P ? Inhibitors Arg-Asn-Gly-(Gly-allyl)-Ala-Val-methyl ester <7> [6] Arg-Asn-Gly-(Gly-allylepoxide)-Ala-Val-methyl ester <7> [6] Arg-Asn-Gly-(Gly-epoxyethylglycine)-Ala-Val-methylester <7> [6] Arg-Asn-Gly-(Gly-vinyl)-Ala-Val-methyl ester <7> [6] Ca2+ <1> [10] Cu2+ <1> [10] EDTA <1> [1, 10] l-threoninamide, (2S)-2-(benzoylamino)-4-[(phenylmethyl)amino]butanoylglycyl-l-threonyl-l-valyl <13> [22] l-threoninamide, (2S)-2-(benzoylamino)-4-[[(4-methoxyphenyl)methyl]amino]butanoylglycyl-l-threonyl-l-valyl <13> [22] l-threoninamide, (2S)-2-(benzoylamino)-4-[[(4-nitrophenyl)methyl]amino]butanoylglycyl-l-threonyl-l-valyl <13> [22] l-threoninamide, (2S)-2-[(6-mercapto-1-oxohexyl)amino]-4-[(2-naphthalenylmethyl)amino]butanoyl-l-cysteinyl-l-threonyl-l-valyl-, cyclic (1-2)-thioether <13> [22] l-threoninamide, (2S)-2-[(6-mercapto-1-oxohexyl)amino]-4-[(2-naphthalenylmethyl)amino]butanoyl-l-cysteinyl-l-threonyl-l-valyl-N-(2-phenylethyl)-, cyclic (1-2)-thioether <13> [22] l-threoninamide, (2S)-2-[(6-mercapto-1-oxohexyl)amino]-4-[(2-naphthalenylmethyl)amino]butanoyl-l-cysteinyl-l-threonyl-l-valyl-N-[2-(4-nitrophenyl)ethyl]-, cyclic (1-2)-thioether <13> [22] l-threoninamide, (2S)-4-(decylamino)-2-[(6-mercapto-1-oxohexyl)amino]butanoyl-l-cysteinyl-l-threonyl-l-valyl-, cyclic (1-2)-thioether <13> [22] l-threoninamide, (2S)-4-amino-2-[(6-mercapto-1-oxohexyl)amino]butanoyll-cysteinyl-l-threonyl-l-valyl-, cyclic (1-2)-thioether <13> [22]

158

2.4.1.119

Dolichyl-diphosphooligosaccharide-protein glycotransferase

l-threoninamide, (2S)-4-amino-2-[(6-mercapto-1-oxohexyl)amino]butanoyll-cysteinyl-l-threonyl-l-valyl-N-(2-phenylethyl)-, cyclic (1-2)-thioether <13> [22] l-threoninamide, (2S)-4-amino-2-[(6-mercapto-1-oxohexyl)amino]butanoyll-cysteinyl-l-threonyl-l-valyl-N-(4-phenylbutyl)-, cyclic (1-2)-thioether <13> [22] l-threoninamide, (2S)-4-amino-2-[(6-mercapto-1-oxohexyl)amino]butanoyll-cysteinyl-l-threonyl-l-valyl-N-[2-(4-nitrophenyl)ethyl]-, cyclic (1-2)-thioether <13> [22] Mg2+ <1> (<1>, above 3 mM [1]; <1>, no inhibition [10]) [1] N-benzoyl-Asn-Leu-Thr <1> [10] N-dinitrobenzoal-Arg-Asn-Ala-epoxyethylglycine-Ala-Val <15> (<15>, Asn residue of the inhibitor is glycosylated during inactivation [13]) [13] N-octanoyl-Asn-Leu-Thr <1> [10] N-t-butoxycarbonyl-Asn-Leu-Thr <1> [10] Zn2+ <1> [10] digitonin <16> (<16>, above 0.1% [18]) [18] octylglucoside <13> (<13>, concentration 1% [12]) [12] Activating compounds digitonin <16> (<16>, purified enzyme requires 0.1% digitonin for maximal activity [18]) [18] phosphatidylcholine <16> (<16>, optimal concentration: 2 mM [18]) [18] Metals, ions Mg2+ <1, 13, 14> (<1>, absolute requirement for a divalent cation, Mg2+ is 30% as effective as Mn2+ [1]; <13>, divalent cation required. Mg2+ , at concentration of 10 mM yields only one third the activity observed with Mn2+ [12]; <1>, 30% as effective as Mn2+ [1]; <1>, inhibition above 3 mM [1]; <14>, absolute requirement for divalent cations that can adopt an octahedral coordination geometry with preference for Mn2+ over Mg2+ [20]) [1, 12, 20] Mn2+ <1, 13, 14> (<1>, requirement [1,7,10]; <1>, absolute requirement for a divalent cation, 3 mM Mn2+ [1]; <13>, divalent cation required, 1 mM Mn2+ is optimal [12]; <14>, absolute requirement for divalent cations that can adopt an octahedral coordination geometry with preference for Mn2+ over Mg2+ [20]) [1, 7, 10, 12, 20] Specific activity (U/mg) 0.000985 <4> [8] Additional information <13, 16> [12, 18] Km-Value (mM) 0.00015 <14> (Na -Ac-Asn-Tyr-Thr-NH2 ) [14] 0.0005 <13> (dolichyl diphosphochitobiose-(Man)9 -(Glc)3 ) [12] 0.0012 <13> (dolichyl diphosphochitobiose) [12] 0.021 <14> (dolichyl-diphosphochitobiose-(Man)9 -(Glc)3 ) [14] 0.0315 <14> (dolichyl-diphosphochitobiose-(Man)9 ) [14] 0.05 <13> (Tyr-Asn-Leu-Thr-Ser-Val, <13>, reaction with dolichyl diphosphochitobiose-Man9 Glc3 [12]) [12] 159

Dolichyl-diphosphooligosaccharide-protein glycotransferase

2.4.1.119

0.072 <1> (diphenyl-Ala-Leu-Glu-Asn-Ala-Thr-Arg-NH2 , <1>, membranebound enzyme [11]) [11] 0.08 <1> (Tyr-Gln-Ser-Asn-Ser-Thr-Met, <1>, membrane-bound enzyme [11]) [11] 0.08 <1> (acetyl-Asn-Ala-Thr, <1>, membrane-bound enzyme [11]) [11] 0.127 <1> (Tyr-Gln-Ser-Asn-Ser-Thr-Met, <1>, solubilized enzyme [11]) [11] 0.143 <1> (acetyl-Asn-Ala-Thr, <1>, solubilized enzyme [11]) [11] 0.16 <6> (N-benzoyl-Asn-Gly-Thr-NHCH3 ) [21] 0.23 <1> (diphenyl-Ala-Leu-Glu-Asn-Ala-Thr-Arg-NH2 , <1>, solubilized enzyme [11]) [11] 0.23 <1> (diphenyl-AlaLeu-Glu-Asn-Ala-Thr-Arg-NH2 ) [11] 0.25 <6> (benzoyl-Asn-Leu-Thr-N-methyl-threonine) [9] 0.278 <1> (acetyl-Asn-Lys-Thr) [11] 0.29 <3> (Tyr-Asn-Leu-Thr-Ser-Val) [4] 0.3 <1> (Ala-Leu-Gln-Asn-Ala-Thr-Arg, <1>, solublilized enzyme [11]) [11] 0.3 <1> (Ala-Leu-Glu-Asn-Ala-Thr-Arg-NH2 ) [11] 0.358 <1> (Ala-Leu-Gln-Asn-Ala-Thr-Arg, <1>, membrane bound enzyme [11]) [11] 0.56 <1> (Asn-Ala-Thr, <1>, solubilized enzyme [11]) [11] 0.6 <6> (N-benzoyl-Asn-Gly-Ser-NHCH3 ) [21] 0.6 <13> (Tyr-Asn-Leu-Thr-Ser-Val, <13>, reaction with dolichyl diphosphochitobiose [12]) [12] 1 <6> (Ac-Asn-Leu-Thr-NH2 ) [9] 2 <6> (Ac-Asn-Ala-Thr-NH2 ) [9] 2.09 <1> (Asn-Ala-Thr, <1>, membrane-bound enzyme [11]) [11] 3.3 <6> (Asn-Asp-Thr) [9] 4.3 <6> (N-benzoyl-Asn-Gly-l-threo-b-hydroxynorvaline-NHCH3 ) [21] 12.5 <6> (N-benzoyl-Asn-Gly-d-allo-Thr-NHCH3 ) [21] Additional information <1> (<1>, kinetic study with intact microsomal membrane. Km -value for Na -Ac-Asn-Leu-Thr-NHCH3 is 0.01 mM [10]) [10] pH-Optimum 6.5-7.5 <14> [20] 6.5-7.7 <13> [12] 7-7.5 <1> [1] pH-Range 6-8 <1> (<1>, pH 6.0: about 30% of maximal activity, pH 8.0: about 20% of maximal activity [1]) [1]

4 Enzyme Structure Subunits ? <1, 4, 13, 14, 15> (<4>, x * 66000 + x * 63000 + x * 48000, SDS-PAGE [8]; <1>, x * 60000, SDS-PAGE [2,7]; <13>, x * 78000, Stt3p, + x * 64000, Ost1p, + x * 45000, Wbp1p, + x * 34000, Ost3p, + x * 30000, Swp1p, + x * 16000, Ost2p, + x * 9500, Ost5p, + x * 3600, Ost4p, the enzyme is composed of 160

2.4.1.119

Dolichyl-diphosphooligosaccharide-protein glycotransferase

equimolar amounts of eight subunits, the enzyme is composed of the following three subcomplexes: 1. Stt3p-Ost4p-Ast3p, 2. Swp1p-Wbp1p-Ost2p0, 3. Ost1p-Aso5p, SDS-PAGE [15]; <13>, x * 80000, SDS-PAGE [2]; <15>, x * 48900, OST48, + x * 68700, ribophorin I, + x * 69300, ribophorin II + ?, calculation from nucleotide sequence [19]; <14>, x * 62000-64000, Ost1p, + x * 6000, Stt3p, + x * 47000, Wbp1p, + x * 34000, Ost3p, + x * 32000, Ost6p, + x * 30000, Swp1p, + x * 16000, Ost2p, + x * 9500, Ost5p, + x * 3600, Ost4p [20]) [2, 7, 15, 19, 20] Additional information <4, 14, 16> (<4>, oligosaccharyltransferase activity is mediated by a protein complex composed of ribophorin I, 66000 Da, and ribophorin II, 63000 Da, and a 48000 Da protein [8]; <4>, the 12500 Da DAD1 protein is a tightly associated subunit of the OST both in intact membrane and in purified enzyme [16]; <14>, heteromeric complex of five subunits [17]; <16>, after SDS-PAGE the most active fractions contain four predominant protein bands with apparent molecular weight in the 50000 Da to 65000 Da range. N-terminal sequence analysis identifies the protein as ribophorin I, ribophorin II, and a 50000 Da homologue of Wbp1, a yeast protein essential for N-glycosylation [18]) [8, 16, 17, 18] Posttranslational modification glycoprotein <1> (<1>, N-linked [7]) [7]

5 Isolation/Preparation/Mutation/Application Source/tissue FAZA cell <3> (<3>, hepatoma cell line [2]) [2] brain <1> [2] epimastigote <8> [5] intestine <1> [2] kidney <1> [2] liver <1-3, 6, 7, 15> [2, 4, 5, 6, 9, 13, 19, 20, 21] lymphocyte <16> [18] oviduct <1> [1, 2, 7, 10, 20] pancreas <4> [2, 8, 16, 20] thyroid gland <1> [11] uterus <5> [2] Localization endoplasmic reticulum <15> (<15>, OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed to towards the lumen of the endoplasmic reticulum [19]) [19] membrane <7-11, 13, 14, 15> (<7, 8, 9, 10, 11, 13, 14>, bound to [3, 5, 6, 12, 20]; <15>, OST48, ribophorin I and ribophorin II possess a type I membrane topolyogy with the bulk of their polypeptide chains directed towards the lumen of the endoplasmic reticulum [19]) [3, 5, 6, 12, 17, 19, 20] microsome <1, 3, 4, 7, 13, 14, 16> (<1>, luminal face of membrane [10]; <4>, rough [16]) [3, 4, 6, 7, 10, 11, 16, 18, 22] 161

Dolichyl-diphosphooligosaccharide-protein glycotransferase

2.4.1.119

rough endoplasmic reticulum <1, 2, 3, 4, 5, 12, 13> (<1, 2, 3, 4, 5, 12, 13>, integral membrane protein, tightly associated to luminal side of membrane [2]; <4>, ribophorin I and II are integral membrane glycoproteins [8]; <1>, the enzyme is oriented at the luminal face of the endoplasmic membrane [10]) [1, 2, 7, 8, 10, 11, 16] Purification <1> [1, 2, 11] <4> (partial [8]) [8] <13> (partial [12]) [12] <14> (purification of an affinity-tagged version of the enzyme complex from a membrane protein fraction [17]) [17] <16> [18] Cloning <14> (creation of a yeast strain in which the essential 64000 Da glycoprotein Nlt1p subunit of the oligosaccharyl transferase is modified by the addition of a 22-residue carboxy-terminal affinity tag, the tag includes both an 8-residue FLAG epitope and a 6-residue histidine motif [17]) [17] <15> (cloning of OST48, ribophorin I, ribophorin II and expression in COS-1 cells [19]) [19]

6 Stability Temperature stability 30 <16> (<16>, 3 h, stable [18]) [18] 50 <1> (<1>, enzyme in endoplasmic reticulum preparations is stable below [7]) [7] 60 <1> (<1>, inactivation by heating of endoplasmic reticulum preparations [7]) [7] General stability information <1>, solubilization with detergents inactivates [7] <4>, phosphatidylcholine stabilizes detergent-solubilized enzyme [8] <13>, glycerol, 25%, stabilizes [12] <14>, strongly stabilized by addition of phospholipids upon detergent solubilization, phosphatidylcholine is twice as effective as phosphatidylethanolamine [20] Storage stability <1>, 4 C, less than 20% loss of activity within 1 week [1] <6>, -85 C, membrane preparation, 30% glycerol, several months [9] <13>, -20 C, Nonidet-solubilized crude enzyme, 25% glycerol, 0.01% 2-mercaptoethanol, 18% loss of activity after 1 month and 87% after 5 months [12] <13>, -70 C, Nonidet-solubilized crude enzyme, 25% glycerol, 0.01% 2-mercaptoethanol, retains 90% of original activity after 1 month and 55% after 5 months [12]

162

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Dolichyl-diphosphooligosaccharide-protein glycotransferase

References [1] Das, R.C.; Heath, E.C.: Dolichyldiphosphoryloligosaccharide-protein oligosaccharyltransferase; solubilization, purification, and properties. Proc. Natl. Acad. Sci. USA, 77, 3811-3815 (1980) [2] Kaplan, H.A.; Welply, J.K.; Lennarz, W.J.: Oligosaccharyl transferase: the central enzyme in the pathway of glycoprotein assembly. Biochim. Biophys. Acta, 90, 161-173 (1987) [3] Lee, J.; Coward, J.K.: Oligosaccharyltransferase: synthesis and use of deuterium-labeled peptide substrates as mechanistic probes. Biochemistry, 32, 6794-6801 (1993) [4] Bause, E.: Studies on the acceptor specificity of asparagine-N-glycosyltransferase from rat liver. FEBS Lett., 103, 296-299 (1979) [5] Bosch, M.; Trombetta, S.; Engstrom, U.; Parodi, A.J.: Characterization of dolichol diphosphate oligosaccharide: protein oligosaccharyltransferase and glycoprotein-processing glucosidases occurring in trypanosomatid protozoa. J. Biol. Chem., 263, 17360-17365 (1988) [6] Bause, E.: Active-site-directed inhibition of asparagine N-glycosyltransferases with epoxy-peptide derivatives. Biochem. J., 209, 323-330 (1983) [7] Welply, J.K.; Shenbagamurthi, P.; Naider, F.; Park, H.R.; Lennarz, W.J.: Active site-directed photoaffinity labeling and partial characterization of oligosaccharyltransferase. J. Biol. Chem., 260, 6459-6465 (1985) [8] Kelleher, D.J.; Kreibich, G.; Gilmore, R.: Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins I and II and a 48 kd protein. Cell, 69, 55-65 (1992) [9] Imperiali, B.; Shannon, K.L.: Differences between Asn-Xaa-Thr-containing peptides: a comparison of solution conformation and substrate behavior with oligosaccharyltransferase. Biochemistry, 30, 4374-4380 (1991) [10] Welply, J.K.; Shenbagamurthi, P.; Lennarz, W.J.; Naider, F.: Substrate recognition by oligosaccharyltransferase. Studies on glycosylation of modified Asn-X-Thr/Ser tripeptides. J. Biol. Chem., 258, 11856-11863 (1983) [11] Ronin, C.; Granier, C.; Caseti, C.; Bouchilloux, S.; van Rietschoten, J.: Synthetic substrates for thyroid oligosaccharide transferase. Effects of peptide chain length and modifications in the Asn-Xaa-Thr-region. Eur. J. Biochem., 118, 159-164 (1981) [12] Sharma, C.B.; Lehle, L.; Tanner, W.: N-Glycosylation of yeast proteins. Characterization of the solubilized oligosaccharyl transferase. Eur. J. Biochem., 116, 101-108 (1981) [13] Bause, E.; Wesemann, M.; Bartoschek, A.; Breuer, W.: Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site. Biochem. J., 322, 95-102 (1997) [14] Karaoglu, D.; Kelleher, D.J.; Gilmore, R.: Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeast oligosaccharyltransferase. Biochemistry, 40, 12193-12206 (2001)

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Dolichyl-diphosphooligosaccharide-protein glycotransferase

2.4.1.119

[15] Karaoglu, D.; Kelleher, D.J.; Gilmore, R.: The highly conserved Stt3 protein is a subunit of the yeast oligosaccharyltransferase and forms a subcomplex with Ost3p and Ost4p. J. Biol. Chem., 272, 32513-32520 (1997) [16] Kelleher, D.J.; Gilmore, R.: DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase. Proc. Natl. Acad. Sci. USA, 94, 4994-4999 (1997) [17] Pathak, R.; Imperiali, B.: A dual affinity tag on the 64-kDa Nlt1p subunit allows the rapid characterization of mutant yeast oligosaccharyl transferase complexes. Arch. Biochem. Biophys., 338, 1-6 (1997) [18] Kumar, V.; Heinemann, F.S.; Ozols, J.: Interleukin-2 induces N-glycosylation in T-cells: characterization of human lymphocyte oligosaccharyltransferase. Biochem. Biophys. Res. Commun., 247, 524-529 (1998) [19] Hardt, B.; Aparicio, R.; Bause, E.: The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation. Glycoconjugate J., 17, 767-779 (2001) [20] Knauer, R.; Lehle, L.: The oligosaccharyltransferase complex from yeast. Biochim. Biophys. Acta, 1426, 259-273 (1999) [21] Breuer, W.; Klein, R.A.; Hardt, B.; Bartoschek, A.; Bause, E.: Oligosaccharyltransferase is highly specific for the hydroxy amino acid in Asn-XaaThr/Ser. FEBS Lett., 501, 106-110 (2001) [22] Ufret, M.d.L.; Imperiali, B.: Probing the extended binding determinants of oligosaccharyl transferase with synthetic inhibitors of asparagine-linked glycosylation. Bioorg. Med. Chem. Lett., 10, 281-284 (2000)

164

Sinapate 1-glucosyltransferase

2.4.1.120

1 Nomenclature EC number 2.4.1.120 Systematic name UDP-glucose:sinapate d-glucosyltransferase Recommended name sinapate 1-glucosyltransferase Synonyms HCA-GT SGT UDP-glucose:sinapic acid glucosyltransferase UDPglucose:sinapic acid glucosyltransferase glucosyltransferase, uridine diphosphoglucose-sinapate uridine 5'-diphosphoglucose-hydroxycinnamic acid acylglucosyltransferase uridine 5'-diphosphoglucose:hydroxycinnamic acid acylglucosyltransferase Additional information (cf. EC 2.4.1.126) CAS registry number 74082-53-4

2 Source Organism <1> Raphanus sativus [1, 2, 3] <2> Brassica napus [4]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + sinapate = UDP + 1-sinapoyl-d-glucose (<1>, sequential mechanism [3]; <2>, mechanism fits a random bi bi model [4]) Reaction type hexosyl group transfer

165

Sinapate 1-glucosyltransferase

2.4.1.120

Natural substrates and products S UDPglucose + sinapic acid <1> (<1>, detoxification of sinapic acid and activation of sinapic acid for the subsequent sinapoyltransferase reaction leading to sinapoylmalate [1]) (Reversibility: r <1> [1, 2]) [1, 2] P UDP + 1-sinapoylglucose Substrates and products S TDPglucose + sinapic acid <1, 2> (<2>, 96% of the activity with UDPglucose [4]) (Reversibility: r <2> [4]; ? <1> [2]) [2, 4] P TDP + sinapoyl-d-glucose S UDPglucose + 3,5-dihydroxybenzoic acid <1> (<1>, 5% of activity compared to sinapic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + 3,5-dihydroxybenzoyl-d-glucose S UDPglucose + 4-hydroxybenzoic acid <1> (<1>, 31% of activity compared to sinapic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + 4-hydroxybenzoyl-d-glucose S UDPglucose + 5-hydroxyferulic acid <2> (<2>, 39% of the activity with sinapic acid [4]) (Reversibility: ? <2> [4]) [4] P UDP + 5-hydroxyferuloyl-d-glucose S UDPglucose + anthranilic acid <1> (<1>, 31% of activity compared to sinapic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + anthraniloyl-d-glucose S UDPglucose + benzoic acid <1> (<1>, 39% of activity compared to sinapic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + benzoyl-d-glucose S UDPglucose + caffeic acid <1, 2> (<1>, 38% of activity compared to sinapic acid [1]; <2>, 14% of the activity with sinapic acid [4]) (Reversibility: ? <1, 2> [1, 4]) [1, 4] P UDP + caffeoyl-d-glucose S UDPglucose + cinnamic acid <1, 2> (<1>, 16% of activity compared to sinapic acid [1]; <2>, 24% of the activity with sinapic acid [4]) (Reversibility: ? <1, 2> [1, 4]) [1, 4] P UDP + cinnamoyl-d-glucose S UDPglucose + ferulic acid <1, 2> (<1>, 38% of activity compared to sinapic acid [1]; <2>, 77% of the activity with sinapid acid [4]) (Reversibility: ? <1, 2> [1, 4]) [1, 4] P UDP + feruloyl-d-glucose S UDPglucose + p-coumaric acid <1> (<1>, 40% of activity compared to sinapic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + p-coumaroyl-d-glucose S UDPglucose + p-coumaric acid <2> (<2>, 21% of the activity with sinapic acid [4]) (Reversibility: ? <2> [4]) [4] P UDP + p-coumaroyl-d-glucose S UDPglucose + sinapic acid <1, 2> (Reversibility: r <1, 2> [1, 2, 3, 4]) [1, 2, 3, 4] P UDP + 1-sinapoyl-d-glucose <1, 2> [1, 2, 3, 4]

166

2.4.1.120

Sinapate 1-glucosyltransferase

S UDPglucose + syringic acid <1, 2> (<1>, 35% of activity compared to sinapic acid [1]; <2>, 10% of the activity with sinapic acid [4]) (Reversibility: ? <1, 2> [1, 4]) [1, 4] P UDP + syringoyl-d-glucose S UDPglucose + vanillic acid <1> (<1>, 36% of activity compared to sinapic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + vanilloyl-d-glucose Inhibitors Cu2+ <2> [4] Hg2+ <2> [4] PCMB <1> (<1>, 100% inhibition at 1 mM, activity can be restored up to 50% by addition of 10 mM dithiothreitol, 24% inhibition at 0.1 mM, activity can be completely restored by 10 mM dithiothreitol [1]) [1] TDP <2> [4] UDP <1, 2> (<1>, strong [1]) [1, 4] UDPmannose <2> (<2>, weak inhibition [4]) [4] UDPxylose <2> (<2>, weak inhibition [4]) [4] Zn2+ <2> [4] p-hydroxymercuribenzoate <2> [4] Activating compounds 2-mercaptoethanol <1> (<1>, stimulation [1]) [1] dithiothreitol <1> (<1>, 10 mM, 3-fold stimulation [1]) [1] Additional information <1> (<1>, SH-group required [1,2]) [1, 2] Metals, ions Mg2+ <2> (<2>, modest stimulation [4]) [4] Mn2+ <2> (<2>, modest stimulation [4]) [4] Specific activity (U/mg) 0.0094 <1> (<1>, reaction with sinapic acid [2]) [2] Km-Value (mM) 0.03 <1> (sinapic acid) [2] 0.055 <1> (UDPglucose) [2] 0.16 <2> (sinapic acid) [4] 2.4 <2> (UDPglucose) [4] pH-Optimum 5.8 <1> (<1>, potassium phosphate buffer [2]) [2] 6 <1, 2> (<1,2>, MES buffer [2,4]) [2, 4] 7 <1> [1] pH-Range 4.5-8.5 <2> (<2>, no activity detected below pH 4.5 and above pH 8.5 [4]) [4] 5-7 <1> (<1>, pH 5.0: 85% of maximal activity, pH 7.0: 63% of maximal activity [2]) [2]

167

Sinapate 1-glucosyltransferase

2.4.1.120

Temperature optimum ( C) 32 <2> [4] 40 <1> [1] Temperature range ( C) 25-45 <1> (<1>, 25 C: 50% of maximal activity, 45 C: 80% of maximal activity [1]) [1] 32-50 <2> (<2>, 32 C: maximal activity, 50 C: no activity above [4]) [4]

4 Enzyme Structure Molecular weight 42000 <2> (<2>, gel filtration [4]) [4]

5 Isolation/Preparation/Mutation/Application Source/tissue seedling <1, 2> [1, 2, 3, 4] Localization cytosol <2> [4] Purification <1> (partial [2,3]) [2, 3] <2> [4]

6 Stability Temperature stability 4 <2> (<2>, 2 d, less than 10% loss of activity [4]) [4] 25 <2> (<2>, 24 h more than 90% loss of activity [4]) [4] Storage stability <1>, 4 C, 8 days, 23% loss of activity [2] <1>, enzyme is less stable at -20 C than at 4 C [2] <2>, -20 C, litte loss of activity after 1 month [4] <2>, 4 C, less than 10% loss of activity after 2 d, more than 90% loss of activity after 1 month [4]

References [1] Strack, D.: Enzymatic synthesis of 1-sinapoylglucose from free sinapic acid and UDP-glucose by a cell-free system from Raphanus saticus seedlings. Z. Naturforsch. C, 35, 204-208 (1979)

168

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Sinapate 1-glucosyltransferase

[2] Nurmann, G.; Strack, D.: Formation of 1-sinapoylglucose by UDP-glucose: sinapic acid glucosyltransferase from cotyledons of Raphanus sativus. Z. Pflanzenphysiol., 102, 11-17 (1981) [3] Mock, H.P.; Strack, D.: Energetics of the uridine 5'-diphosphoglucose:hydroxycinnamic acid acyl-glucosyltransferase reaction. Phytochemistry, 32, 575-579 (1993) [4] Wang, S.X.; Ellis, B.E.: Enzymology of UDP-glucose:sinapic acid glucosyltransferase from Brassica napus. Phytochemistry, 49, 307-318 (1998)

169

Indole-3-acetate b-glucosyltransferase

2.4.1.121

1 Nomenclature EC number 2.4.1.121 Systematic name UDP-glucose:indole-3-acetate b-d-glucosyltransferase Recommended name indole-3-acetate b-glucosyltransferase Synonyms IAA-glucose synthase IAGlc synthase IAGlu synthase UDP-glucose:indol-3-ylacetate glucosyl-transferase UDP-glucose:indol-3-ylacetate glucosyltransferase UDPG-indol-3-ylacetyl glucosyl transferase UDPglucose:indole-3-acetate b-d-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-indoleacetate indol-3-ylacetylglucose synthase CAS registry number 74082-56-7

2 Source Organism <1> Zea mays [1-6] <2> Arabidopsis sp. [7]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + indole-3-acetate = UDP + indole-3-acetyl-b-1-d-glucose Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + indole-3-acetate <1> (<1>, first reaction in a series leading to the ester conjugates of indole-3-acetic acid [2,5]; <1>, first enzyme-catalyzed reaction leading from indole-3-acetic acid to the myo-inositol ester 170

2.4.1.121

Indole-3-acetate b-glucosyltransferase

of indole-3-acetate [3]; <1>, first enzyme in pathway synthesizing ester conjugates of indoleacetate, constitutive in maize vegetative tissue, can be increased by incubation of tissue with 1-naphthalene acetic acid [6]) (Reversibility: ? <1> [2, 3, 5, 6]) [2, 3, 5, 6] P ? Substrates and products S UDPglucose + 2,5-dimethoxycinnamic acid <1> (<1>, 4.3% of the activity with indole-3-acetate [5]) (Reversibility: ? <1> [5]) [5] P UDP + 2,5-dimethoxycinnamoyl-b-1-d-glucose S UDPglucose + 3,4-dimethoxycinnamic acid <1> (<1>, 6.1% of the activity with indole-3-acetate [5]) (Reversibility: ? <1> [5]) [5] P UDP + 3,4-dimethoxycinnamoyl-b-1-d-glucose S UDPglucose + 3-hydroxy-4-methoxy cinnamic acid <1> (<1>, 9.4% of the activity with indole-3-acetate [5]) (Reversibility: ? <1> [5]) [5] P UDP + 3-hydroxy-4-methoxycinnamoyl-b-1-d-glucose S UDPglucose + ferulic acid <1> (<1>, 7.8% of the activity with indole-3acetate [5]) (Reversibility: ? <1> [5]) [5] P UDP + feruloyl-b-1-d-glucose S UDPglucose + indole butyric acid <2> (Reversibility: ? <2> [7]) [7] P UDP + indole butanoyl-b-1-d-glucose S UDPglucose + indole propionic acid <2> (Reversibility: ? <2> [7]) [7] P UDP + indole propanoyl-b-1-d-glucose S UDPglucose + indole-3-acetate <1> (<1>, equilibrium away from ester formation and towards formation of indole-3-acetate [4]; <1>, specific for UDPglucose [1,4]) (Reversibility: r <1> [4]; ? <1> [1-3, 5]) [1-5] P UDP + indole-3-acetyl-b-1-d-glucose <1> (<1>, i.e. 1-O-indol-3-ylacetylb-d-glucose [1]) [1-5] S UDPglucose + m-hydroxycinnamic acid <1> (<1>, 4.3% of the activity with indole-3-acetate [5]) (Reversibility: ? <1> [5]) [5] P UDP + m-hydroxycinnamoyl-b-1-d-glucose S UDPglucose + naphthalene-1-acetic acid <1> (<1>, 70% of the activity with indole-3-acetate [1]) (Reversibility: ? <1> [1, 6]) [1, 6] P UDP + naphthyl-1-acetyl-b-1-d-glucose S UDPglucose + o-hydroxycinnamic acid <1> (<1>, 7.6% of the activity with indole-3-acetate [5]) (Reversibility: ? <1> [5]) [5] P UDP + o-hydroxycinnamoyl-b-1-d-glucose S UDPglucose + p-coumaric acid <1> (<1>, 10.2% of the activity with indole-3-acetate [5]) (Reversibility: ? <1> [5]) [5] P UDP + p-coumaroyl-b-1-d-glucose S UDPglucose + phenylacetic acid <1> (Reversibility: ? <1> [6]) [6] P UDP + phenylacetyl-b-1-d-glucose Inhibitors 2,4-dichlorophenoxy acetic acid <1> [4] UDP <2> [7] abscisic acid <1> (<1>, weak [4]) [4] diphosphate <1> [4] 171

Indole-3-acetate b-glucosyltransferase

2.4.1.121

gibberellic acid <1> (<1>, weak [4]) [4] indole-3-acetate glucan <1> [4] indole-3-acetate-myo-inositol <1> [4] kinetin <1> (<1>, weak [4]) [4] naphthylphthalamic acid <1> [4] phosphate <1> (<1>, 100 mM phosphate buffer, 50% inhibition [3]) [3] phosphatidyl ethanolamine <1> [4] zeatin <1> [4] Activating compounds calmodulin <1> (<1>, stimulates [4]) [4] thiol compounds <1> (<1>, stimulate [4]) [4] Metals, ions Ca2+ <1, 2> (<1,2>, stimulates [4,7]) [4, 7] Mg2+ <2> (<2>, stimulates [7]) [7] Mn2+ <2> (<2>, stimulates [7]) [7] Specific activity (U/mg) 0.798 <1> [3] 4.029 <1> [5] Additional information <1> [1] Km-Value (mM) 0.14 <2> (indole propionic acid) [7] 0.15 <2> (indole butyric acid) [7] 0.18 <2> (cinnamic acid) [7] 0.24 <2> (indole-3-acetate) [7] 0.25 <2> (UDPglucose) [7] 1.08 <1> (indole-3-acetate) [1] 2.5 <1> (UDPglucose) [1] pH-Optimum 7.1 <1> [1] 7.3-7.6 <1> [3] pH-Range 6.7-7.6 <1> (<1>, pH 7.3-7.6: maximal activity, no activity below pH 6.7 [3]) [3]

4 Enzyme Structure Molecular weight 46500 <1> (<1>, gel filtration [3]) [3] 52000 <1> (<1>, gel filtration [4]) [4] Subunits monomer <1> (<1>, 1 * 51000, SDS-PAGE [5]) [5]

172

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Indole-3-acetate b-glucosyltransferase

5 Isolation/Preparation/Mutation/Application Source/tissue embryo <1> [4] endosperm <1> (<1>, cultured [4]; <1>, immature [6]) [3, 4, 6] kernel <1> (<1>, immature [1]) [1, 2, 3, 5, 6] seedling <1> [6] Purification <1> (partial [3,5]) [3, 5] <2> (recombinant enzyme [7]) [7]

6 Stability General stability information <1>, loses activity during column chromatographic procedures [3] <1>, retention of 79% of the initial activity after 16 h dialysis [3] <1>, stable to lyophilization [3] Storage stability <1>, -20 C, stable for 15 d, 15% loss of activity after 35 d [3]

References [1] Michalczuk, L.; Bandurski, R.S.: Enzymic synthesis of 1-O-indol-3-ylacetylb-d-glucose and indol-3-ylacetyl-myo-inositol. Biochem. J., 207, 273-281 (1982) [2] Kowalczyk, S.; Bandurski, R.S.: Isomerization of 1-O-indol-3-ylacetyl-b-dglucose. Enzymatic hydrolysis of 1-O-, 4-O-, and 6-O-indol-3-ylacetyl-b-dglucose and the enzymatic synthesis of indole-3-acetyl glycerol by a hormone metabolizing complex. Plant Physiol., 94, 4-12 (1990) [3] Leznicki, A.J.; Bandurski, R.S.: Enzymic synthesis of indole-3-acetyl-1-O-bd-glucose. Plant Physiol., 88, 1474-1480 (1988) [4] Leznicki, A.J.; Bandurski, R.S.: Enzymic synthesis of indole-3-acetyl-1-O-bd-glucose. Plant Physiol., 88, 1481-1485 (1988) [5] Kowalczyk, S.; Bandurski, R.S.: Enzymic synthesis of 1-O-(indol-3-ylacetyl)b-d-glucose. Purification of the enzyme from Zea mays, and preparation of antibodies to the enzyme. Biochem. J., 279, 509-514 (1991) [6] Kowalczyk, S.; Jakubowska, A.; Bandurski, R.S.: 1-Naphtalene acetic acid induces indole-3-ylacetylglucose synthase in Zea mays seedling tissue. Plant Growth Regul., 38, 127-134 (2002) [7] Jackson, R.G.; Lim, E.K.; Li, Y.; Kowalczyk, M.; Sandberg, G.; Hoggett, J.; Ashford, D.A.; Bowles, D.J.: Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase. J. Biol. Chem., 276, 4350-4356 (2001)

173

Glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase

2.4.1.122

1 Nomenclature EC number 2.4.1.122 Systematic name UDP-galactose:glycoprotein-N-acetyl-d-galactosamine 3-b-d-galactosyltransferase Recommended name glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase Synonyms 3bGal-T4 <5, 6> [7] O-glycan core 1 UDPgalactose:N-acetyl-a-galactosaminyl-R b3-galactosyltransferase <3, 4> [5] UDP-Gal:GalNAc-a-Ser/Thr b3-galactosyltransferase <4> [8] UDP-Gal:N-acetylgalactosaminide mucin:b1,3-galactosyltransferase <3, 4> [1, 3] core 1 b3-Gal-T <4> [6, 8] core 1 b3-Gal-transferase <3, 4> [5] galactosyltransferase, uridine diphosphogalactose-mucin b-(1-3)uridine diphosphogalactose-mucin b-(1-3)-galactosyltransferase CAS registry number 97089-61-7

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

174

Gallus gallus [4] Homo sapiens [2, 10] Sus scrofa [1, 5] Rattus norvegicus [3, 5, 6, 8] Homo sapiens [7] Homo sapiens (EST clone, contains full length sequence [7]) [7] Homo sapiens (form 1 [10]) [9, 10] Mus musculus [9] Caenorhabditis elegans [9] Rattus norvegicus [9] Homo sapiens (form 2 [10]) [10]

2.4.1.122

Glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase

3 Reaction and Specificity Catalyzed reaction UDP-galactose + glycoprotein N-acetyl-d-galactosamine = UDP + glycoprotein d-galactosyl-1,3-N-acetyl-d-galactosamine (the non-reducing O-serinelinked N-acetylgalactosamine residues in mucin glycoproteins can act as acceptors; <4> charge-charge interactions between enzyme and substrate [6]) Reaction type hexosyl group transfer Natural substrates and products S UDP-galactose + N-acetylgalactosaminide mucin <3> (<3> participates in synthesis of the core portion of O-serine- and O-threonine-linked oligosaccharides in respiratory acidic mucins [1]) [1] P UDP + b-d-galactosyl-1,3-N-acetylgalactosaminide mucin S UDP-galactose + glycophorin <2> [2] P ? S UDP-galactose + glycoprotein N-acetyl-d-galactosamine <3, 4, 11> (<11> enzyme is important in O-glycan biosynthesis in digestive organs [10]; <4> key enzyme in biosynthesis of core 1 O-glycans [8]; <4> major control factor in the biosynthesis of O-glycans [3]; <3,4> involved in O-glycan biosynthesis [5]) [5, 8, 10] P UDP + glycoprotein d-galactosyl-1,3-N-acetyl-d-galactosamine Substrates and products S UDP-galactose + A-P-N-acetyl-d-galactosaminyl-T-S-S-A <4> (<4> synthetic peptide [5]) (Reversibility: ? <4> [5]) [5] P UDP + b-d-galactosyl-1,3-N-acetylgalactosyl-(A-P-N-acetyl-d-galactosaminyl-T-S-S-A) S UDP-galactose + A-P-N-acetyl-d-galactosaminyl-T-S-S-S <3> (<3> synthetic peptide [5]) (Reversibility: ? <3> [5]) [5] P UDP + b-d-galactosyl-1,3-N-acetylgalactosyl-(A-P-N-acetyl-d-galactosaminyl-T-S-S-S) S UDP-galactose + N-acetyl-d-galactosamine <1, 3> (Reversibility: ? <1,3> [1,4]) [1, 4] P UDP + b-d-galactosyl-1,3-N-acetyl-d-galactosamine <1, 3> [1, 4] S UDP-galactose + N-acetyl-d-glucosamine <1> (Reversibility: ? <1> [4]) [4] P UDP + b-d-galactosyl-1,3-N-acetyl-d-glucosamine S UDP-galactose + N-acetyl-a-d-galactosaminyl-benzyl <3, 4> (<4> removal or substitution of the 3-OH group or removal of the 4-OH group abolishes enzyme activity, removal or substitution of the 6-OH reduces activity slightly leading to competititve substrates [3]) (Reversibility: ? <3,4> [3,5]) [3, 5] P UDP + b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-benzyl <3, 4> [3, 5]

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2.4.1.122

S UDP-galactose + N-acetyl-a-d-galactosaminyl-phenyl <4, 7> (Reversibility: ? <4,7> [8,9]) [8, 9] P UDP + b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-phenyl <4, 7> [8, 9] S UDP-galactose + N-acetylgalactosaminide mucin <1-4> (Reversibility: ? <1-4> [1-4]) [1-4] P UDP + b-d-galactosyl-1,3-N-acetylgalactosaminide mucin <3> [1] S UDP-galactose + N-acetylgalactosaminitol <3> (Reversibility: ? <3> [1]) [1] P UDP + b-d-galactosyl-1,3-N-acetylgalactosaminitol <3> [1] S UDP-galactose + asialo bovine submaxillary mucin <4> (Reversibility: ? <4> [8]) [8] P UDP + b-d-galactosyl-1,3-N-acetylgalactosyl-asialo bovine submaxillary mucin <4> [8] S UDP-galactose + asialo ovine submaxillary mucin <1, 2> (<1> best substrate [4]) (Reversibility: ? <1,2> [2,4]) [2, 4] P UDP + b-d-galactosyl-1,3-N-acetylgalactosyl-asialo ovine submaxillary mucin <1, 2> [2, 4] S UDP-galactose + asialo-agalacto-a1 -acid glycoprotein <1> (Reversibility: ? <1> [4]) [4] P UDP + galactosyl-b-1,3-asialo-agalacto-a1 -acid glycoprotein S UDP-galactose + asialo-agalacto-glycophorin <2> (Reversibility: ? <2> [2]) [2] P UDP + b-d-galactosyl-1,3-N-acetylgalactosyl-asialo-agalacto-glycophorin <2> [2] S UDP-galactose + asialo-mucin of Cowper's gland <3> (Reversibility: ? <3> [1]) [1] P UDP + b-d-galactosyl-1,3-N-acetylgalactosyl-asialo-mucin of Cowper's gland <3> [1] S UDP-galactose + ganglioside GM2 <1, 5> (<5> i.e. GalNAcb1,4-(NeuAca2,3-)Galb1,4-Glcb1-ceramide [7]) (Reversibility: ? <1,5> [4,7]) [4, 7] P UDP + galactosyl-b-1,3-ganglioside GM2 <5> (<5> i.e. ganglioside GM1 [7]) [7] S UDP-galactose + ganglioside Gg3 <5> (<5> i.e. GalNAcb1,4-Galb1,4Glcb1-ceramide [7]) (Reversibility: ? <5> [7]) [4, 7] P UDP + galactosyl-b-1,3-ganglioside Gg3 <5> [7] S UDP-galactose + glycoprotein N-acetyl-d-galactosamine <1-5, 7, 11> (<4> linked to serine or threonine of the peptide/protein [8]; <4> Galb1,3-GalNAca residues adjacent to Thr(GalNAc) reduces the activity [6]; <4,7> threonine a-d-galactosamine is preferred to serine a-d-galactosamine [6,9]; <4> substrates with negatively charged amino acids on the N-terminal side are highly efficient substrates [6]; <3> most active acceptor: macromolecular mucin glycopeptides with free N-acetylgalactosyl residues linked to polypeptide chain [1]; <3,7> high specificity for Nacetylgalactosaminyl residues linked to serine or threonine [1,9]; <3,4> glycosyl acceptor specificity study [1,3,5,6]; <3> glycopeptides with less

176

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P S P S

P

Glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase

than 5 amino acids containing terminal N-acetylgalactosaminyl residues [1]) (Reversibility: ? <1-5,7,11> [1-10]) [1-10] UDP + glycoprotein d-galactosyl-1,3-N-acetyl-d-galactosamine <1-5, 7, 11> (<4,11> i.e. core 1 structure [8,10]) [1-10] UDP-galactose + p-nitrophenyl-b-N-acetylgalactosamide <3, 11> (Reversibility: ? <3,11> [1,10]) [1, 10] UDP + b-d-galactosyl-1,3-N-acetylgalactosyl-p-nitrophenyl Additional information <1, 3, 5> (<5> no acceptor substrates: ovalbumin, b-N-acetyl-d-glucosaminyl-benzyl [7]; <3> no acceptor substrates: type A and type 0 blood group glycoprotein, ovomucoid, ovalbumin, and fetuin devoid of sialic acid and galactose [1]; <1,3> poor substrates are low-molecular weight acceptors, e.g. N-acetylgalactosamine [1,4]; <3> terminal a-2,6-linked sialic acid residues inhibit the enzyme [1]) [1, 4, 7] ?

Inhibitors 6-O-(4,4-azo)phenyl-GalNAc-a-benzyl <4> [3] UDP <4> (<4> slight inhibition [8]) [8] a-lactalbumin <1> [4] Additional information <4> (<4> proline on the C-terminal side of the peptide substrate adjacent to Thr(GalNAc) is inhibitory [6]) [6] Activating compounds Triton X-100 <3> (<3> activation and stabilization, unstable without [1]) [1] Metals, ions Mn2+ <1-4> (<4> required [8]; <1> activation, 5-10 mM [4]) [1, 2, 4, 8] Specific activity (U/mg) 0.0000028 <5> (<5> recombinant in Sf9 cells [7]) [7] 0.000042 <4> (<4> liver [8]) [8] 0.00088 <3> (<3> microsomal membranes, substrate: N-acetyl-d-galactosaminyl-a-benzyl [5]) [5] 0.00134 <3> (<3> microsomal membranes, substrate: A-P-N-acetyl-d-galactosaminyl-T-S-S-S [5]) [5] 0.00428 <1> (<1> purified enzyme [4]) [4] 0.0071 <4> (<4> partially purified enzyme, substrate: N-acetyl-d-galactosaminyl-a-benzyl [5]) [5] 0.0194 <3> (<3> Golgi membranes [1]) [1] 0.031 <4> (<4> partially purified enzyme, substrate: A-P-N-acetyl-d-galactosaminyl-T-S-S-A [5]) [5] 0.81 <3> (<3> purified enzyme [1]) [1] 3.43 <4> (<4> purified enzyme [8]) [8] Additional information <1> [4] Km-Value (mM) 0.00035 <3> (asialo Cowper's gland mucin) [1] 0.02 <3> (UDP-galactose) [1] 0.025 <1> (UDP-galactose) [4]

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2.4.1.122

0.09 <3> (asialo Cowper's gland mucin, <3> calculated on the basis of terminal N-galactosaminyl residues [1]) [1] 0.55 <4> (A-P-N-acetyl-d-galactosaminyl-T-S-S-A) [5] 0.63 <4> (UDP-galactose, <4> with N-acetyl-d-galactosaminyl-a-phenyl [8]) [8] 0.76 <4> (N-acetyl-a-d-galactosaminyl-phenyl) [8] 1.7 <4> (N-acetyl-a-d-galactosaminyl-benzyl) [5] 5 <1> (asialo ovine submaxillary mucin, <1> in terms of N-acetylgalactosamine equivalents [4]) [4] pH-Optimum 5-7 <1> (<1> broad [4]) [4] 6.5 <4> (<4> assay at [8]) [8] 6.9 <3> [1] Additional information <1> (<1> pI: 6.40 [4]) [4] pH-Range 5.5-7.9 <3> [1] 6.5-7.4 <7, 11> (<7,11> assays at [10]) [10] Temperature optimum ( C) 37 <1-4, 7, 11> (<1-4,7,11> assay at [1,2,4,5,8,10]) [1, 2, 4, 5, 8, 10]

4 Enzyme Structure Molecular weight 84000-86000 <4> (<4> gel filtration [8]) [8] 90000 <3> (<3> gel filtration in the presence of Triton X-100 [1]) [1] Subunits ? <1> (<1> x * 68000, SDS-PAGE [4]) [4] monomer <3, 4> (<4> 1 * 42000-43000, SDS-PAGE and amino acid sequence determination [8]; <3> 1 * 82000, SDS-PAGE [1]) [1, 8]

5 Isolation/Preparation/Mutation/Application Source/tissue brain <5-7> (<5,6> low content [7]) [7, 9] colon <7> (<7> low content [9]) [9] colonic mucosa <3, 4> [5] embryo <1> [4] erythrocyte <2> [2] heart <5, 7> (<7> high content [9]) [7, 9] kidney <5, 7, 11> (<7> high content [9]; <5> low content [7]) [7, 9, 10] leukocyte <7> [9] liver <1, 4, 5, 7> (<7> high content [9]; <4> high content [8]) [3-9] lung <5, 7> [7, 9] 178

2.4.1.122

Glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase

pancreas <5> [7] placenta <5, 7> (<7> high content [9]) [7, 9] salivary gland <11> [10] skeletal muscle <5, 7> [7, 9] small intestine <7, 11> (<7,11> low content [9,10]) [9, 10] spleen <7, 11> (<7> low content [9]) [9, 10] stomach <3, 11> [5, 10] testis <11> [10] thymus <7, 11> (<7> low content [9]) [9, 10] tracheal mucosa <3> [1] Additional information <2> (<2> no endogenous activity in Jurkat and LSC, a colorectal cell line, cells [10]) [10] Localization Golgi apparatus <3> (<3> major part [1]) [1] endoplasmic reticulum <3> [1] membrane <1-4> (<4> associated [8]; <3> inner surface [1]) [1, 2, 4, 5, 8] microsome <1, 3, 11> [1, 4, 10] Additional information <3, 7, 11> (<7,11> type II transmembrane protein [9,10]; <3> subcellular distribution [1]) [1, 9, 10] Purification <1> (solubilized with 1% Triton X-100 in 0.1 M NaCl [4]) [4] <2> (partial, solubilized with 3% Triton X-100 [2]) [2] <3> (affinity chromatography on Sepharose 4B containing covalently bound mucin substrate [1]) [1] <4> (partial [3,5]; affinity chromatography on immobilized asialo-bovine submaxillary gland mucin [8]) [3, 5, 8] <7> (partial, recombinant soluble epitope-tagged enzyme from 293T cell medium [9]) [9] Cloning <5> (functional expression in Spodoptera frugiperda Sf9 cells via baculovirus infection [7]) [7] <7> (functional expression in human LSC cells [10]; DNA and amino acid sequence determination and analysis, transient functional expression in human 293T cells as wild-type and soluble, i.e. secreted into the medium, epitope-tagged protein, stable functional expression of the soluble epitopetagged enzyme in CHO-K1 and Lec1-CHO cells [9]) [9, 10] <8> (DNA and amino acid sequence determination [9]) [9] <9> (DNA and amino acid sequence determination [9]) [9] <10> (DNA and amino acid sequence determination [9]) [9] <11> (DNA sequence determination, functional expression in human LSC cells [10]) [10]

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Glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase

2.4.1.122

6 Stability General stability information <3>, stabilization by Triton X-100 or Nonidet P-40 required, unstable without [1] Storage stability <3>, 3 C, 0.1% Triton X-100 and 0.1 mg/ml albumin, at least 3 months [1]

References [1] Mendicino, J.; Sivakami, S.; Davila, M.; Chandrasekaran, E.V.: Purification and properties of UDP-Gal:N-acetylgalactosaminide mucin:b1,3-galactosyltransferase from swine trachea mucosa. J. Biol. Chem., 257, 3987-3994 (1982) [2] Hesford, F.J.; Berger, E.G.; Van den Eijnden, D.H.: Identification of the product formed by human erythrocyte galactosyltransferase. Biochim. Biophys. Acta, 659, 302-311 (1981) [3] Brockhausen, I.; Möller, G.; Pollex-Kruger, A.; Rutz, V.; Paulsen, H.; Matta, K.L.: Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-a-R b3-galactosyltransferase from rat liver. Biochem. Cell Biol., 70, 99-108 (1992) [4] Furukawa, K.; Roth, S.: Co-purification of galactosyltransferases from chick-embryo liver. Biochem. J., 227, 573-582 (1985) [5] Brockhausen, I.; Möller, G.; Merz, G.; Adermann, K.; Paulsen, H.: Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide substrates influences the activity of O-glycan core 1 UDPgalactose:N-acetyl-a-galactosaminyl-R b3-galactosyltransferase. Biochemistry, 29, 10206-10212 (1990) [6] Granovsky, M.; Bielfeldt, T.; Peters, S.; Paulsen, H.; Meldal, M.; Brockhausen, J.; Brockhausen, I.: UDPgalactose:glycoprotein-N-acetyl-d-galactosamine 3-b-d-galactosyltransferase activity synthesizing O-glycan core 1 is controlled by the amino acid sequence and glycosylation of glycopeptide substrates. Eur. J. Biochem., 221, 1039-1046 (1994) [7] Amado, M.; Almeida, R.; Carneiro, F.; Levery, S.B.; Holmes, E.H.; Nomoto, M.; Hollingsworth, M.A.; Hassan, H.; Schwientek, T.; Nielsen, P.A.; Bennett, E.P.; Clausen, H.: A family of human b3-galactosyltransferases. Characterization of four members of a UDP-galactose:b-N-acetyl-glucosamine/b-Nacetyl-galactosamine b-1,3-galactosyltransferase family. J. Biol. Chem., 273, 12770-12778 (1998) [8] Ju, T.; Cummings, R.D.; Canfield, W.M.: Purification, characterization, and subunit structure of rat core 1 b1,3-galactosyltransferase. J. Biol. Chem., 277, 169-177 (2002) [9] Ju, T.; Brewer, K.; D'Souza, A.; Cummings, R.D.; Canfield, W.M.: Cloning and expression of human core 1 b1,3-galactosyltransferase. J. Biol. Chem., 277, 178-186 (2002)

180

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Glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase

[10] Kudo, T.; Iwai, T.; Kubota, T.; Iwasaki, H.; Takayma, Y.; Hiruma, T.; Inaba, N.; Zhang, Y.; Gotoh, M.; Togayachi, A.; Narimatsu, H.: Molecular cloning and characterization of a novel UDP-Gal:GalNAca peptide b1,3-galactosyltransferase (C1Gal-T2), an enzyme synthesizing a core 1 structure of Oglycan. J. Biol. Chem., 277, 47724-47731 (2002)

181

Inositol 1-a-galactosyltransferase

2.4.1.123

1 Nomenclature EC number 2.4.1.123 Systematic name UDP-galactose:myo-inositol 1-a-d-galactosyltransferase Recommended name inositol 1-a-galactosyltransferase Synonyms GOLS GS GolS UDP-d-galactose:inositol galactosyltransferase galactinol synthase galactosyltransferase, uridine diphosphogalactose-inositol uridine diphosphogalactose-inositol galactosyltransferase Additional information <1> (<1> cf. EC 2.4.1.67 and EC 2.4.1.82 [1]) [1] CAS registry number 79955-89-8

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

182

Cucumis sativus (cucumber, cv. Chipper [1]) [1] Phaseolus vulgaris (var. Big Red [2]) [2] Cucurbita pepo (var. Black Beauty [2]) [2] Lycopersicon esculentum (tomato, cv. Moneymaker, full length cDNA of LeGOLS-1 [3]) [3] Arabidopsis thaliana (ecotype Columbia, isoenzyme AtGolS1 [4]) [4] Ajuga reptans (GolS-1, 2 isoforms: GolS-1 and GolS-2 [5]) [5] Ajuga reptans (GolS-2, 2 isoforms: GolS-1 and GolS-2 [5]) [5] Arabidopsis thaliana (ecotype Columbia, isoenzyme AtGolS2 [4]) [4] Arabidopsis thaliana (ecotype Columbia, isoenzyme AtGolS3 [4]) [4] Lycopersicon esculentum (tomato, cv. Moneymaker, genomic sequence of LeGOLS-1 [3]) [3]

2.4.1.123

Inositol 1-a-galactosyltransferase

3 Reaction and Specificity Catalyzed reaction UDP-galactose + myo-inositol = UDP + 1-O-a-d-galactosyl-d-myo-inositol Reaction type hexosyl group transfer Natural substrates and products S UDP-galactose + myo-inositol <1-10> (<1> responsible for galactinol synthesis, involved in raffinose and stachyose biosynthesis, cf. EC 2.4.1.67 and EC 2.4.1.82 [1]; <2> key enzyme in controlling galactose oligosaccharide biosynthesis, which plays a role in seed desiccation tolerance [2]; <4,6,10> first committed enzyme in biosynthesis of raffinose family oligosaccharides, which play a role in plant stress tolerance [3,5]; <5,8,9> catalyzes the first step in the biosynthesis of raffinose family oligosaccharides, raffinose and galactinol are involved in tolerance to drought, high salinity and cold stress and may function as osmoprotectants in droughtstress tolerance, stress inducible enzyme plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions [4]; <6,7> galactinol synthesis, which is the galactosyl donor for the synthesis of raffinose and stachyose, at least 2 isoforms: GolS-1 is mainly involved in the synthesis of storage raffinose family oligosaccharides and GolS-2 in the synthesis of transport raffinose family oligosaccharides, GolS-2 expression is much lower than that of GolS-1, galactinol may be catabolized by the reverse reaction [5]) [1-5] P UDP + 1-O-a-d-galactosyl-d-myo-inositol Substrates and products S UDP-galactose + myo-inositol <1-10> (<2> high specific affinity for UDP-Gal and myo-inositol [2]; <6,7> reaction is probably reversible [5]) (Reversibility: r <6,7> [5]; ? <1-5,8-10> [1-4]) [1-5] P UDP + 1-O-a-d-galactosyl-d-myo-inositol <1-10> (<1-10> identical with galactinol [1-5]) [1-5] Activating compounds dithiothreitol <2> (<2> absolute requirement [2]) [2] Additional information <4-10> (<4,10> osmotic or dehydration stress induces LeGOLS-1 expression, cold induces gene expression only in vegetative tissues [3]; <5,8> AtGolS1 and 2 expression are induced by drought and highsalinity stresses and weakly induced by abscisic acid, but not by cold stress [4]; <9> AtGolS3 is induced by cold stress, but not by drought or salt stress or by abscisic acid [4]; <6,7> cold-inducible GolS-1 and 2 genes [5]) [3-5] Metals, ions Mn2+ <1, 2> (<1> concentration influences pH-optimum [1]; <2> absolute requirement [2]) [1, 2]

183

Inositol 1-a-galactosyltransferase

2.4.1.123

Specific activity (U/mg) 8.75 <2> [2] 32.1 <3> [2] Additional information <1> [1] Km-Value (mM) 0.4 <2> (UDP-galactose) [2] 4.5 <2> (myo-inositol) [2] pH-Optimum 5.5 <1> (<1> at 7 mM Mn2+ [1]) [1] 5.6 <1> (<1> assay at [1]) [1] 6.2 <1> (<1> at 2 mM Mn2+ [1]) [1] 7 <1, 2> (<1> at 0.2 mM Mn2+ , broad activity optimum centered around pH 7 [1]) [1, 2] Additional information <1> (<1> Mn2+ -concentration influences pH-optimum [1]) [1] pH-Range 5-7 <1> (<1> about half-maximal activity at pH 5 and 7, at 7 mM Mn2+ [1]) [1] 5.1-7.8 <1> (<1> about half-maximal activity at pH 5.1 and 7.8, at 2.0 mM Mn2+ [1]) [1] 5.2-8.6 <1> (<1> about half-maximal activity at pH 5.2 and 8.6, at 0.2 mM Mn2+ [1]) [1] Temperature optimum ( C) 30 <1-10> (<1-10> assay at [1-5]) [1-5]

4 Enzyme Structure Subunits ? <2, 3> (<2> x * 38000, SDS-PAGE [2]; <3> x * 36000, SDS-PAGE [2]) [2] Posttranslational modification no modification <8, 9> (<8,9> no putative serine phosphorylation site [4]) [4] phosphoprotein <5, 6> (<5> AtGolS1 protein has a putative serine phosphorylation site at position 270 [4]; <6> putative serine phosphorylation site at GolS-1 codon position 263 [5]) [4, 5]

5 Isolation/Preparation/Mutation/Application Source/tissue cotyledon <2> (<2> highest activity among all of the components of seed, much poorer source than Cucurbita pepo mature leaves [2]) [2]

184

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Inositol 1-a-galactosyltransferase

endosperm <4, 10> (<4,10> LeGOLS-1 mRNA is present in endosperm caps [3]) [3] leaf <1, 3, 6, 7> (<1> 7th node from the growing tip, from fruiting plants [1]; <3> mature leaves, much richer source than Phaseolus vulgaris cotyledons [2]; <6,7> source and sink leaves, GolS-1 is source leaf-specific, equally weak expression of GolS-2 in all leaf types [5]) [1, 2, 5] mesophyll <6> (<6> GolS-1 is primarily expressed in the mesophyll, the site of raffinose family oligosaccharides storage [5]) [5] phloem <7> (<7> GolS-2 is primarily expressed in the phloem-associated intermediary cells known for their role in raffinose family oligosaccharides phloem loading [5]) [5] radicle <4, 10> (<4,10> LeGOLS-1 mRNA is most abundant in radicle tips [3]) [3] seed <4, 5, 8-10> (<5,8,9> high level of AtGolS1 and 2 expression in mature seeds, but very low of AtGolS3 [4]) [3, 4] Localization Additional information <1> (<1> not in lysates of chloroplasts from leaves [1]) [1] Purification <1> (partial, 41fold [1]) [1] <2> (1591fold, copurified with a 41 and 43 kDa peptide [2]) [2] <3> (2472fold [2]) [2] <5, 8, 9> (purification of glutathione S-transferase fusion proteins GTS-AtGolS1, 2 and 3, overexpressed in Escherichia coli [4]) [4] Cloning <4, 10> (LeGOLS-1 gene encoding a 318-amino acids peptide is cloned, gene and cDNA structure, LeGOLS-1 expression pattern in seeds and seedlings during seed maturation and germination under various conditions, hormonal control of transcription of LeGOLS-1 in the absence of gibberellin and abscisic acid, up-regulation of gene expression before maturation desiccation and again after imbibition whenever radicle protrusion is prevented [3]) [3] <5, 8, 9> (7 AtGolS genes, cloning of AtGolS1, 2 and 3, AtGolS1 and 2 are induced by drought and high-salinity stresses, but not by cold stress, AtGolS3 is induced by cold stress, but not by drought or salt stress, overexpression of glutathione S-transferase fusion proteins GTS-AtGolS1, 2 and 3 in Escherichia coli, overexpression of AtGolS2 in transgenic Arabidopsis improves drought tolerance, AtGolS3 is controlled by the transcription factor DREB1A [4]) [4] <6, 7> (cold-inducible GolS-1 and -2 genes encode 2 distinct galactinol synthases, cloning and sequencing of the GolS-1 and -2 genes, deduced amino acid sequences, expression of GolS-1 cDNA in Escherichia coli as functional enzyme [5]) [5]

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Inositol 1-a-galactosyltransferase

2.4.1.123

6 Stability General stability information <1>, 70-80% loss of activity during purification [1] <2>, ammonium sulfate fractionation causes 32% loss of enzyme activity [2] <2>, bovine serum albumin is essential to preserve enzyme activity during the assay [2] Storage stability <2>, -20 C, enzyme in ammonium sulfate precipitate form, 6-8 weeks, stable [2]

References [1] Pharr, D.M.; Sox, H.N.; Locy, R.D.; Huber, S.C.: Partial characterization of the galactinol forming enzyme from leaves of Cucumis sativus L.. Plant Sci. Lett., 23, 25-33 (1981) [2] Liu, J.J.; Odegard, W.; de Lumen, B.O.: Galactinol synthase from kidney bean cotyledon and zucchini leaf. Purification and N-terminal sequences. Plant Physiol., 109, 505-511 (1995) [3] Downie, B.; Gurusinghe, S.; Dahal, P.; Thacker, R.R.; Snyder, J.C.; Nonogaki, H.; Yim, K.; Fukanaga, K.; Alvarado, V.; Bradford, K.J.: Expression of a galactinol synthase gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented. Plant Physiol., 131, 1347-1359 (2003) [4] Taji, T.; Ohsumi, C.; Luchi, S.; Seki, M.; Kasuga, M.; Kobayashi, M.; Yamaguchi-Shinozaki, K.; Shinozaki, K.: Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J., 29, 417-426 (2002) [5] Sprenger, N.; Keller, F.: Allocation of raffinose family oligosaccharides to transport and storage pools in Ajuga reptans: the roles of two distinct galactinol synthases. Plant J., 21, 249-258 (2000)

186

2.4.1.124

1 Nomenclature EC number 2.4.1.124 (deleted, now included with EC 2.4.1.87)

187

Sucrose-1,6-a-glucan 3(6)-a-glucosyltransferase

2.4.1.125

1 Nomenclature EC number 2.4.1.125 Systematic name sucrose:1,6-a-d-glucan 3(6)-a-d-glucosyltransferase Recommended name sucrose-1,6-a-glucan 3(6)-a-glucosyltransferase Synonyms GTF-S glucosyltransferase, sucrose-1,6-a-glucan 3(6)-asucrose:1,6-, 1,3-a-d-glucan 3-a- and 6-a-d-glucosyltransferase sucrose:1,6-a-d-glucan 3-a- and 6-a-glucosyltransferase sucrose:1,6-a-glucan 3(6)-a-glucosyltransferase water-soluble-glucan synthase CAS registry number 81725-87-3

2 Source Organism <1> Streptococcus mutans (6715, serotype g [1,4,6]; Ingbritt, serotype c [2]; HS6, serotype a [3,5]) [1-6] <2> Streptococcus gordonii [7, 8]

3 Reaction and Specificity Catalyzed reaction sucrose + (1,6-a-d-glucosyl)n = d-fructose + (1,6-a-d-glucosyl)n+1 Reaction type hexosyl group transfer Substrates and products S sucrose + (1,3-a-d-glucosyl)n <2> (Reversibility: ? <2> [8]) [8] P d-fructose + (1,3-a-d-glucosyl)n+1 S sucrose + (1,6-a-d-glucosyl)n <1, 2> (<1> also transfers glucosyl residues to the 3-position on glucose residues in glucans, producing a

188

2.4.1.125

Sucrose-1,6-a-glucan 3(6)-a-glucosyltransferase

highly-branched 1,6-a-d-glucan [1-3, 5]) (Reversibility: ? <1, 2> [1-8]) [1-8] P d-fructose + (1,6-a-d-glucosyl)n+1 <1> (<1> 1,6-a-d-glucan with highly (35%) branched structure of 1,3,6-linked glucose residues [1]; <1> 1,6-ad-glucan with 17.7% of 1,3,6-branching structure [2]; <1> glucan consists of 49.1 mol% 1,6-a-linked glucose and 33.9 mol% 1,3-a-linked glucose with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-a-branched glucose [3]; <1> 1,6-a-d-glucan with 20 and 24.5 mol% 1,3,6-branch points [5]; <1> enzyme exhibits 87% 1,6-a-bond-, 6% 1,3-a-bond- and 7% 1,3,6branch-forming activities [6]) [1-6] Inhibitors 6-deoxysucrose <1> (<1> competitive [4]) [4] 6-thiosucrose <1> [4] Activating compounds dextran T10 <1> (<1> activation [2]; <1> no stimulation in the range 0.012.0 mg/ml [3]) [2] Specific activity (U/mg) 5.86-6.8 <1> [5] 9.8 <1> [2] 34.9 <1> [1] 89.7 <1> [3] Km-Value (mM) 0.0071 <1> (dextran, <1> pH 6.5, 37 C [2]) [2] 1.3 <1> (sucrose, <1> pH 6.5, 37 C [5]) [5] 2.4 <1> (sucrose, <1> pH 6.5, 37 C [1]) [1] 4.3 <1> (sucrose, <1> pH 6.5, 37 C [2]) [2] 4.9 <1> (sucrose, <1> pH 6.5, 37 C [3]) [3] pH-Optimum 5.5 <1> [1, 5] 6 <1> [3] 6.5 <1> [2] Temperature optimum ( C) 37 <1> (<1> assay at [1,3]) [1, 3]

4 Enzyme Structure Molecular weight 149000 <1> (<1> sedimentation equilibrium studies [1]) [1] 174000 <2> (<2> calculated from necleotide sequence [7]) [7]

189

Sucrose-1,6-a-glucan 3(6)-a-glucosyltransferase

2.4.1.125

Subunits monomer <1, 2> (<1> 1 * 151000, SDS-PAGE [2]; <1> 1 * 159000, SDS-PAGE [3]; <1> 1 * 161000, enzyme I, SDS-PAGE [5]; <1> 1 * 174000, enzyme II, SDS-PAGE [5]; <2> 1 * 174000, SDS-PAGE [7]) [2, 3, 5, 7] Posttranslational modification Additional information <1> (<1> carbohydrate content: less than 1% w/w [1]; <1> 1.5% [3]) [1, 3]

5 Isolation/Preparation/Mutation/Application Source/tissue culture supernatant <1> [1, 3, 5] Localization extracellular <1, 2> [1, 3, 5-7] Purification <1> (6715 [1]; Ingbritt, serotype c, 3 isoenzymes [2]; HS6, serotype a [3,5]; 2 isoenzymes: I and II [5]) [1-3, 5] Cloning <2> (expression in strain CH1 and CH107 [8]) [8] <2> (expression of mutants with different numbers of repeats in gtfG in strain CH1 [7]) [7]

References [1] Shimamura, A.; Tsumori, H.; Mukasa, H.: Purification and properties of Streptococcus mutans extracellular glucosyltransferase. Biochim. Biophys. Acta, 702, 72-80 (1982) [2] Mukasa, H.; Shimamura, A.; Tsumori, H.: Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c. Biochim. Biophys. Acta, 719, 81-89 (1982) [3] Tsumori, H.; Shimamura, A.; Mukasa, H.: Purification and properties of extracellular glucosyltransferase synthesizing 1,6-, 1,3-a-d-glucan from Streptococcus mutans serotype a. J. Gen. Microbiol., 131, 3347-3353 (1985) [4] Binder, T.P.; Robyt, J.F.: Inhibition of Streptococcus mutans 6715 glucosyltransferases by sucrose analogs modified at positions 6 and 6' Carbohydr. Res., 140, 9-20 (1985) [5] Tsumori, H.; Shimamura, A.; Mukasa, H.: Purification and properties of extracellular glucosyltransferases from Streptococcus mutans serotype a. J. Gen. Microbiol., 129, 3251-3259 (1983) [6] Shimamura, A.; Tsumori, H.; Mukasa, H.: Three kinds of extracellular glucosyltransferases from Streptococcus mutans 6715 (serotype g). FEBS Lett., 157, 79-84 (1983)

190

2.4.1.125

Sucrose-1,6-a-glucan 3(6)-a-glucosyltransferase

[7] Vickerman, M.M.; Sulavik, M.C.; Minick, P.E.; Clewell, D.B.: Changes in the carboxyl-terminal repeat region affect extracellular activity and glucan products of Streptococcus gordonii glucosyltransferase. Infect. Immun., 64, 5117-5128 (1996) [8] Vickerman, M.M.; Clewell, D.B.: Deletions in the carboxyl-terminal region of Streptococcus gordonii glucosyltransferase affect cell-associated enzyme activity and sucrose-associated accumulation of growing cells. Appl. Environ. Microbiol., 63, 1667-1673 (1997)

191

Hydroxycinnamate 4-b-glucosyltransferase

2.4.1.126

1 Nomenclature EC number 2.4.1.126 Systematic name UDP-glucose:trans-4-hydroxycinnamate 4-O-b-d-glucosyltransferase Recommended name hydroxycinnamate 4-b-glucosyltransferase Synonyms UDP-glucose-hydroxycinnamate glucosyltransferase UDPglucose:trans-4-hydroxycinnamate 4-O-b-d-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-hydroxycinnamate hydroxycinnamoyl glucosyltransferase uridine diphosphoglucose-hydroxycinnamate glucosyltransferase CAS registry number 77848-85-2

2 Source Organism <1> Lycopersicon esculentum (var. cerasiforme [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + trans-4-hydroxycinnamate = UDP + 4-O-b-d-glucosyl-4-hydroxycinnamate Reaction type hexosyl group transfer Substrates and products S UDPglucose + aesculin <1> (Reversibility: ? <1> [1]) [1] P UDP + 4-O-b-d-glucosyl-aesculin S UDPglucose + caffeic acid <1> [1] P UDP + 4-O-glucosyl-caffeic acid <1> (<1>, no glucose ester is formed [1]) [1] S UDPglucose + daphnetin <1> (Reversibility: ? <1> [1]) [1]

192

2.4.1.126

Hydroxycinnamate 4-b-glucosyltransferase

P UDP + 4-O-b-d-glucosyl-daphnetin S UDPglucose + ferulic acid <1> (Reversibility: ? <1> [1]) [1] P UDP + feruloyl-b-d-glucose + 4-O-glucosyl-ferulic acid <1> (<1>, a mixture of the glucoside and the glucose ester is formed [1]) [1] S UDPglucose + kaempferol <1> (Reversibility: ? <1> [1]) [1] P UDP + 4-O-b-d-glucosyl-kaempferol S UDPglucose + p-coumaric acid <1> (Reversibility: ? <1> [1]) [1] P UDP + p-coumaroyl-b-d-glucose + 4-O-glucosyl-p-coumaric acid <1> (<1>, a mixture of the glucoside and the glucose ester is formed [1]) [1] S UDPglucose + quercetin <1> (Reversibility: ? <1> [1]) [1] P UDP + 4-O-b-d-glucosyl-quercetin S UDPglucose + scopoletin <1> (Reversibility: ? <1> [1]) [1] P UDP + 4-O-b-d-glucosyl-scopoletin S UDPglucose + sinapic acid <1> (Reversibility: ? <1> [1]) [1] P UDP + 4-O-b-d-glucosyl-sinapic acid + sinapoyl-b-d-glucose <1> (<1>, a mixture of the glucoside and the glucose ester is formed [1]) [1] Inhibitors Ca2+ <1> (<1>, 0.1-10 mM: 20-50% inhibition [1]) [1] EDTA <1> (<1>, 0.001 mM: 30% inhibition, 0.01 mM: complete inactivation [1]) [1] Mg2+ <1> (<1>, 0.1-10 mM: 20-50% inhibition [1]) [1] Mn2+ <1> (<1>, 0.1-10 mM: 20-80% inhibition [1]) [1] PCMB <1> (<1>, 0.001-0.01 mM: 50-70% inhibition [1]) [1] UDP <1> (<1>, 1 mM, 70% inhibition after 60 min [1]) [1] iodoacetate <1> (<1>, 0.001-0.01 mM: 50-70% inhibition [1]) [1] Metals, ions MnCl2 <1> (<1>, 0.006-0.01 mM: stimulation of about 10% [1]) [1] Specific activity (U/mg) 0.00112 <1> (<1>, reaction with ferulic acid [1]) [1] Km-Value (mM) 0.0008 <1> (p-coumaric acid) [1] 0.0014 <1> (ferulic acid) [1] 0.0015 <1> (caffeic acid) [1] 0.0025 <1> (sinapic acid) [1] 0.01 <1> (UDPglucose, <1>, reaction with ferulic acid [1]) [1] pH-Optimum 7 <1> (<1>, formation of glucose ester of p-coumaric acid [1]) [1] 8 <1> (<1>, formation of glucoside of p-coumaric acid [1]) [1]

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

193

Hydroxycinnamate 4-b-glucosyltransferase

2.4.1.126

Purification <1> (partial [1]) [1]

6 Stability General stability information <1>, b-mercaptoethanol, 10 mM, stabilizes, 80% restorage of activity by its addition after treatment with p-chloromercuribenzoate [1] Storage stability <1>, -20 C, purified enzyme, complete loss of activity after 12-24 h [1] <1>, 0-4 C, purified enzyme, 40% loss of activity after 24 h, 70% loss of activity after 48 h [1]

References [1] Fleuriet, A.; Macheix, J.J.: Partial purification and some properties of a hydroxycinnamoyl glucosyltransferase from tomato fruits. Z. Naturforsch. C, 35, 967-972 (1980)

194

Monoterpenol b-glucosyltransferase

2.4.1.127

1 Nomenclature EC number 2.4.1.127 Systematic name UDP-glucose:(-)-menthol O-b-d-glucosyltransferase Recommended name monoterpenol b-glucosyltransferase Synonyms UDPglucose:monoterpenol glucosyltransferase glucosyltransferase, uridine diphosphoglucose-monoterpenol uridine diphosphoglucose-monoterpenol glucosyltransferase CAS registry number 78990-64-4

2 Source Organism <1> Mentha x piperita (cv. Black Mitcham [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + (-)-menthol = UDP + (-)-menthyl O-b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + l-menthol <1> (<1>, enzyme is involved in metabolism of l-menthone [1]) (Reversibility: ? <1> [1]) [1] P UDP + l-menthyl-b-d-glucoside <1> [1] Substrates and products S UDPglucose + d-neomenthol <1> (Reversibility: ? <1> [1]) [1] P UDP + d-neomenthyl-b-d-glucoside <1> [1] S UDPglucose + l-menthol <1> (Reversibility: ? <1> [1]) [1] P UDP + l-menthyl-b-d-glucoside <1> [1]

195

Monoterpenol b-glucosyltransferase

2.4.1.127

Inhibitors Ca2+ <1> (<1>, 5 mM, strong [1]) [1] CdCl2 <1> (<1>, strong [1]) [1] Co2+ <1> (<1>, 5 mM, strong [1]) [1] HgCl2 <1> (<1>, strong [1]) [1] Ni2+ <1> (<1>, 5 mM, strong [1]) [1] Zn2+ <1> (<1>, 5 mM, strong [1]) [1] p-hydroxymercuribenzoate <1> (<1>, strong [1]) [1] Activating compounds 2-mercaptoethanol <1> (<1>, without addition 73% decrease of activity, suggesting a thiol function necessary for activity [1]) [1] Metals, ions Mg2+ <1> (<1>, 20 mM, about 30% stimulation [1]) [1] MnCl2 <1> (<1>, slight stimulation [1]) [1] Km-Value (mM) 0.048 <1> (d-neomenthol) [1] 0.059 <1> (l-menthol) [1] 0.2 <1> (UDPglucose, <1>, reaction with d-neomenthol [1]) [1] 0.25 <1> (UDPglucose, <1>, reaction with l-menthol [1]) [1] pH-Optimum 7.3 <1> (<1>, 70 mM Tris-sodium maleate buffer [1]) [1] 7.7 <1> (<1>, 70 mM Na-phosphate buffer [1]) [1] pH-Range 6-8.5 <1> (<1>, about 50% of maximal activity at pH 6.0 and pH 8.5, 70 mM Tris-sodium maleate buffer [1]) [1] 7.3-8.3 <1> (<1>, about 50% of maximal activity at pH 7.3 and pH 8.3, 70 mM Na-phosphate buffer [1]) [1]

4 Enzyme Structure Molecular weight 46000 <1> (<1>, gel filtration [1]) [1]

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

196

2.4.1.127

Monoterpenol b-glucosyltransferase

References [1] Martinkus, C.; Croteau, R.: Metabolism of monoterpenes. Evidence for compartmentation of l-menthone metabolism in peppermint (Mentha piperita) leaves. Plant Physiol., 68, 99-106 (1981)

197

Scopoletin glucosyltransferase

2.4.1.128

1 Nomenclature EC number 2.4.1.128 Systematic name UDP-glucose:scopoletin O-b-d-glucosyltransferase Recommended name scopoletin glucosyltransferase Synonyms SGTase UDP-glucose:scopoletin glucosyltransferase glucosyltransferase, uridine diphosphoglucose-scopoletin CAS registry number 81210-69-7

2 Source Organism <1> Nicotiana tabacum (Bright Yellow [1]) [1, 3] <2> Duboisia myoporoides [2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + scopoletin = UDP + scopolin Reaction type hexosyl group transfer Substrates and products S UDPglucose + scopoletin <1, 2> (Reversibility: ? <1, 2> [1, 2]) [1, 2] P UDP + scopolin <1, 2> [1, 2] Activating compounds 2,4-dichlorophenoxyacetic acid <1> (<1> 0.005 mM, 10fold activation [1]) [1]

198

2.4.1.128

Scopoletin glucosyltransferase

Specific activity (U/mg) 0.00084 <2> (<2> activity in crude extracts [2]) [2] 0.0116 <1> [1] 0.114 <1> (<1> cells cultured in the presence of 0.005 mM 2,4-dichlorophenoxyacetic acid [1]) [1] pH-Optimum 7.5 <1> (<1> assay at [1]) [1] Temperature optimum ( C) 30 <1> (<1> assay at [1]) [1]

4 Enzyme Structure Molecular weight 45000 <1> (<1> native PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue callus <1> (<1> cell culture [1]) [1] suspension culture <2> [2] Localization cytosol <1> (<1> absent from isolated vacuoles, most probably located in the cytosol [3]) [3] Purification <1> (ammonium sulfate, Sephadex G-100, DEAE-cellulose, hydroxyapatite [1]) [1]

References [1] Hino, F.; Okazaki, M.; Miura, Y.: Effect of 2,4-dichlorophenoxyacetic acid on glucosylation of scopoletin to scopolin in tobacco tissue culture. Plant Physiol., 69, 810-813 (1982) [2] Betry, P.; Fliniaux, M.A.; Mackova, M.; Gillet, F.; Macek, T.; Jacquin-Dubreuil, A.: Scopoletin-glucosyltransferase activity from Duboisia myoporoides; improvement of cultivation conditions and crude extract preparation procedure. J. Plant Physiol., 146, 503-507 (1995) [3] Taguchi, G.; Fujikawa, S.; Yazawa, T.; Kodaira, R.; Hayashida, N.; Shimosaka, M.; Okazaki, M.: Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells. Plant Sci., 151, 153-161 (2000)

199

Peptidoglycan glycosyltransferase

2.4.1.129

1 Nomenclature EC number 2.4.1.129 Systematic name undecaprenyldiphospho-(N-acetyl-d-glucosaminyl-(1!4)-(N-acetyl-d-muramoylpentapeptide):undecaprenyldiphospho-(N-acetyl-d-glucosaminyl(1!4)-N-acetyl-d-muramoylpentapeptide) disaccharidetransferase Recommended name peptidoglycan glycosyltransferase Synonyms PBP1b PG-II bactoprenyldiphospho-N-acetylmuramoyl-(N-acetyl-d-glucosaminyl)-pentapeptide:peptidoglycan N-acetylmuramoyl-N-acetyl-d-glucosaminyltransferase glycosyltransferase, peptidoglycan penicillin-binding protein 1B penicillin-binding protein 3 peptidoglycan transglycosylase CAS registry number 79079-04-2

2 Source Organism <1> Bacillus megaterium (strain 899 [1]) [1] <2> Escherichia coli (K12, strains JST975srev61/pLC26-6 (F- mrcB mreA recA) [2]; strain JA200/pLC19-19 [4]) [2, 4, 5, 6] <3> Micrococcus luteus (strain SM1, synonym M. lysodeikticus [3]) [3] <4> Staphylococcus aureus (strain SAK 101 [3]) [3] <5> Streptococcus pneumoniae (strain R6cwl [5]) [5, 6]

200

2.4.1.129

Peptidoglycan glycosyltransferase

3 Reaction and Specificity Catalyzed reaction [GlcNAc-(1!4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-d-Ala)]n -diphosphoundecaprenol + GlcNAc-(1!4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Alad-Ala)-diphosphoundecaprenol = [GlcNAc-(1!4)-Mur2Ac(oyl-l-Ala-g-dGlu-l-Lys-d-Ala-d-Ala)]n+1 -diphosphoundecaprenol + undecaprenyl diphosphate Reaction type hexosyl group transfer Natural substrates and products S [GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-d-Ala)]n -diphosphoundecaprenol + GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-dAla-d-Ala)-diphosphoundecaprenol <2, 3, 4> (<2>, peptidoglycan-synthetic enzyme activities of penicillin-binding protein 3 may by involved in the process of cell division [2]; <3,4>, gram-positive cocci have cell wall peptidoglycan which seems to be synthesized by penicillin-binding protein transpeptidases and a separate transglycosylase [3]; <2>, enzyme is involved in synthesis of peptidoglycan [6]) (Reversibility: ? <2, 3, 4> [2, 3, 6]) [2, 3, 6] P [GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-d-Ala)]n+1 -diphosphoundecaprenol + undecaprenyl diphosphate Substrates and products S [GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-d-Ala)]n -diphosphoundecaprenol + GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-dAla-d-Ala)-diphosphoundecaprenol <1-5> (<1>, enzyme synthesizes lysozame-sensitive peptidoglycan from undecaprenyldiphosphoryl-disaccharide-pentapeptide [1]) (Reversibility: ? <1-5> [1-5]) [1-5] P [GlcNAc-(1-4)-Mur2Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-d-Ala)]n+1 -diphosphoundecaprenol + undecaprenyl diphosphate S Additional information <2, 5> (<2>, the enzyme shows both transglycosylase and transpeptidase activities [5]; <5>, the enzyme possesses peptidoglycan transglycosylase activity that lacks penicillin-binding activity [5]) [5] P ? Inhibitors Ca2+ <2> [4] Co2+ <2> [4] EDTA <2> (<2>, in the absence of detergents, stimulates in the presence of high concentrations of methanol and detergents [4]) [4] Fe2+ <2> [4] Mn2+ <2> [4] Ni2+ <2> [4] Triton X-100 <2> (<5>, up to 0.6% [5]; <2>, inhibits at 0.1% [4]) [4]

201

Peptidoglycan glycosyltransferase

2.4.1.129

Zn2+ <2> [4] dimethylsulfoxide <2> (in the presence of 0.05% sarkosyl [4]) [4] enramycin <2> [4] macarbomycin <2-4> (<4>, strong [3]; <3>, weak [3]) [2, 3, 4] moenomycin <2, 4, 5> (<3>, no inhibition [3]) [2, 3, 4, 5, 6] sodium 1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid <2> (<2>, in the absence of detergents, stimulates in the presence of high concentrations of methanol and detergents [4]) [4] sodium deoxycholate <2> (<2>, in the presence of methanol, inhibits at 0.5% [4]) [4] vancomycin <1, 2, 5> [1, 4, 5] Activating compounds EDTA <2> (<2>, stimulation in the presence of high concentrations of methanol and detergents [4]) [4] Triton X-100 <2, 5> (<2>, activation, 0.05%, [4]; <5>, up to 0.6% [5]; <2>, inhibits at 0.1% [4]) [4, 5] benzylpenicillin <2> (<2>, stimulation, in the presence of 15% methanol, not at higher methanol concentrations or in the presence of deoxycholate [4]) [4] deoxycholate <1> [1] dimethylsulfoxide <2> (<2>, activation, in the absence of methanol, inhibits in the presence of 0.05% sarkosyl [4]) [4] imipenem <2> (<2>, stimulation, in the presence of 15% methanol, not at higher methanol concentrations or in the presence of deoxycholate [4]) [4] sarkosyl <2> (<2>, activation [4]) [4] sodium 1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid <2> (<2>, stimulation in the presence of high concentrations of methanol and detergents [4]) [4] sodium deoxycholate <2> (<2>, 0.05-0.1%, activation [4]) [4] Additional information <2> (<2>, no activation by cephalexin, nocardicin A or mecillinam [4]) [4] Metals, ions Mg2+ <2> (<2>, slight stimulation [4]; <1,3,4>, no stimulation [1,3]) [4] Additional information <2> (<2>, no divalent cation requirement [4]) [4] pH-Optimum 7-8.2 <3> [3] 7-9 <2> (<2>, broad, in the presence of 0.1% deoxycholate [4]) [4] 7.2-7.5 <4> [3] 8.5 <2> (<2>, Tris-HCl buffer without deoxycholate [4]) [4] Temperature optimum ( C) 37 <2> [4] Temperature range ( C) 20-45 <2> (<2>, about 60% of maximal activity at 20 C and about half-maximal activity at 45 C [4]) [4]

202

2.4.1.129

Peptidoglycan glycosyltransferase

4 Enzyme Structure Molecular weight 61000 <1> (<1>, gel filtration [1]) [1] Subunits ? <2> (<2>, x * 60000, SDS-PAGE [2]; <2>, x * 90000, E. coli penicillin binding protein 1B a, b or g [4]) [2, 4] Additional information <2> (<2>, enzyme is present as a monomer and as a dimer [6]) [6]

5 Isolation/Preparation/Mutation/Application Localization cell envelope <2> [6] membrane <1-5> [1-5] Purification <1> [1] <2> (3 isozymes [4]; penicillin-binding protein 3 [2]) [2, 4] <3> (partial [3]) [3] <4> (partial [3]) [3] <5> (partial [5]) [5] Cloning <2> (Escherichia coli structural gene mrcB, recloned from plasmid pLC19-19 to high copy number plasmid pBr322, yielding plasmid pTM13) [4]

6 Stability Temperature stability 60 <2> (<2>, up to 87% loss of activity within 10 min [4]) [4] Storage stability <2>, -80 C, in concentrated PEG 6000 solution, stable for several months [4]

References [1] Taku, A.; Stuckey, M.; Fan, D.P.: Purification of the peptidoglycan transglycosylase of Bacillus megaterium. J. Biol. Chem., 257, 5018-5022 (1982) [2] Ishino, F.; Matsuhashi, M.: Peptidoglycan synthetic enzyme activities of highly purified penicillin-binding protein 3 in Escherichia coli: a septumforming reaction sequence. Biochem. Biophys. Res. Commun., 101, 905-911 (1981)

203

Peptidoglycan glycosyltransferase

2.4.1.129

[3] Park, W.; Matsuhashi, M.: Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins. J. Bacteriol., 157, 538-544 (1984) [4] Nakagawa, J.; Tamaki, S.; Tomioka, S.; Matsuhashi, M.: Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides. Penicillin-binding protein 1Bs of Escherichia coli with activities of transglycosylase and transpeptidase. J. Biol. Chem., 259, 13937-13946 (1984) [5] Park, W.; Seto, H.; Hakenbeck, R.; Matsuhashi, M.: Major peptidoglycan transglycosylase activity in Streptococcus pneumoniae that is not a penicillin-binding protein. FEMS Microbiol. Lett., 27, 45-48 (1985) [6] Van Heijenoort, J.: Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology, 11, 25R-36R (2001)

204

Dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase

2.4.1.130

1 Nomenclature EC number 2.4.1.130 Systematic name dolichyl-phosphate-d-mannose:glycolipid a-d-mannosyltransferase Recommended name dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase Synonyms Dol-P-Man:Man7 GlcNAc2 -PP-Dol mannosyltransferase <6, 9> [5] dolichol phosphomannose-oligosaccharide-lipid mannosyltransferase dolichyl-P-Man:Man7 GlcNAc2 -PP-dolichyl a6-mannosyltransferase <2> [2] mannosyltransferase, dolichol phosphomannose-oligosaccharide-lipid oligomannosylsynthase CAS registry number 77967-76-1

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

Mus musculus (Thy-1-mutant [1]) [1] Homo sapiens (gene hALG12 [2]) [2] Homo sapiens (EST clone [2]) [2] Saccharomyces cerevisiae (gene ALG12 [4]; gene ALG3 [3]) [3, 4] Saccharomyces cerevisiae (gene PMT4 [6]) [6] Homo sapiens (gene ALG12 [4,5]) [4, 5] Homo sapiens (gene ALG3 [5]) [5] Saccharomyces cerevisiae (gene PMT3 [6]) [6] Homo sapiens (mutant Dol-P-Man:Man7 GlcNAc2 -PP-Dol mannosyltransferase [5]) [5]

3 Reaction and Specificity Catalyzed reaction transfers an a-d-mannosyl residue from dolichyl-phosphate d-mannose into membrane lipid-linked oligosaccharide

205

Dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase

2.4.1.130

Reaction type hexosyl group transfer Natural substrates and products S dolichol-phosphomannose + lipid-linked oligosaccharide <1, 2, 4, 6> (<2,4,6> lipid carrier is dolichyl diphosphate [2-5]; <1,2,6> pathway leading to biosynthesis of asparagine-linked oligosaccharides of mammalian glycolipids [1-5]) [1-5] P dolichol phosphate + lipid-linked mannosyl-oligosaccharide S Additional information <2, 6, 7> (<2,6,7> enzyme deficiency leads to congenital disorders of glycosylation type Ig, i.e. CDG1 [2,4,5]) [2, 4, 5] P ? Substrates and products S dolichol-phosphomannose + (d-mannose)5 -(N-acetyl-d-glucosaminyl)2 dolichyl-diphosphate <2, 4, 6, 7> (<7> ALG3 enzyme catalyses this step, but not the further elongation of the oligosaccharide chain [5]) (Reversibility: ? <2,4,6,7> [2-5]) [2-5] P dolichol phosphate + (d-mannose)6 -(N-acetyl-d-glucosaminyl)2 -dolichyldiphosphate <2, 4, 6, 7> [2-5] S dolichol-phosphomannose + (d-mannose)6 -(N-acetyl-d-glucosaminyl)2 dolichyl-diphosphate <2, 4, 6> (Reversibility: ? <2,4,6> [2-5]) [2-5] P dolichol phosphate + (d-mannose)7 -(N-acetyl-d-glucosaminyl)2 -dolichyldiphosphate <2, 4, 6> [2-5] S dolichol-phosphomannose + (d-mannose)7 -(N-acetyl-d-glucosaminyl)2 dolichyl-diphosphate <2, 4, 6> (<6> no activity in ALG12 mutant T67M/ R146Q [4]; <2> no activity in ALG12 mutant F142V [2]) (Reversibility: ? <2,4,6> [2-5]) [2-5] P dolichol phosphate + (d-mannose)8 -(N-acetyl-d-glucosaminyl)2 -dolichyldiphosphate <2, 4, 6> [2-5] S dolichol-phosphomannose + (d-mannose)8 -(N-acetyl-d-glucosaminyl)2 dolichyl-diphosphate <2, 4, 6> (<6> no activity in ALG12 mutant T67M/ R146Q [4]; <2> no activity in ALG12 mutant F142V [2]) (Reversibility: ? <2,4,6> [2-5]) [2-5] P dolichol phosphate + (d-mannose)9 -(N-acetyl-d-glucosaminyl)2 -dolichyldiphosphate <2, 4, 6> [2-5] S dolichol-phosphomannose + lipid-linked oligosaccharide <1, 2, 4, 6, 8> (<2,4-6,8> lipid carrier is dolichyl diphosphate [2-6]; <4> no activity with GDP-mannose as donor substrate [3]; <1> strict donor specificity, less specific for oligosaccharide acceptor [1]; <1,2,4> transfers 4 mannosyl-residues to lipid-linked oligosaccharide, the latter containing already 5 mannosyl-residues [1-5]) (Reversibility: ? <1,2,4,6,8> [1-6]) [1-6] P dolichol phosphate + lipid-linked mannosyl-oligosaccharide <1, 2, 4, 6, 8> [1-6] S Additional information <4, 7> (<4,7> transfer of a mannosyl-residue to (d-mannose)5 -(N-acetyl-d-glucosaminyl)2 -dolichyl-diphosphate is cata-

206

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Dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase

lysed by ALG3 gene product, which probably is a different enzyme than that catalysing the transfer to (d-mannose)6 -8-(N-acetyl-dglucosaminyl)2 -dolichyl-diphosphate [3,5]) [3, 5] P ? Inhibitors Cd2+ <4> (<4> inhibits the addition of the 7th to 9th mannosyl residue to the dolichyl-linked oligosaccharide, but the addition of the 6th mannosyl residue [3]) [3] EDTA <4> (<4> inhibits the addition of the 7th to 9th mannosyl residue to the dolichyl-linked oligosaccharide, but the addition of the 6th mannosyl residue [3]) [3] Zn2+ <4> (<4> inhibits the addition of the 7th to 9th mannosyl residue to the dolichyl-linked oligosaccharide, but the addition of the 6th mannosyl residue [3]) [3] Metals, ions Ca2+ <4> (<4> can partly substitute for Mn2+ [3]) [3] Mg2+ <4> (<4> can partly substitute for Mn2+ [3]) [3] Mn2+ <4> (<4> required for the addition of the 7th to 9th mannosyl residue to the dolichyl-linked oligosaccharide, but for addition of the 6th mannosyl residue [3]; <4> can be partly substituted by Mg2+ or Ca2+ [3]) [3] pH-Optimum 6.5 <4, 6> (<4,6> assay at [3,5]) [3, 5] 7.4 <1> (<1> assay at [1]) [1] Temperature optimum ( C) 24 <6> (<6> assay at [5]) [5] 25 <4> (<4> assay at [3]) [3] 37 <1> (<1> assay at [1]) [1]

4 Enzyme Structure Posttranslational modification glycoprotein <5, 8> (<8> contains 4 potential N-glycosylation sites [6]; <5> contains 2 potential N-glycosylation sites [6]) [6]

5 Isolation/Preparation/Mutation/Application Source/tissue BW-5147 cell <1> [1] lymphoma cell line <1> (<1> BW-5147 cell line and mutant BW-5147.3.(Thy1- E)10 cell line [1]) [1] skin fibroblast <2, 6, 7> (<2> from biopsy od CDG1 patients or as immortalized cell line [2]) [2, 5]

207

Dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase

2.4.1.130

Localization endoplasmic reticulum <4, 6, 8, 9> (<4,8> lumenal side [3,6]) [3, 4-6] membrane <1, 4> [1, 3] rough microsome <4> [3] Cloning <2> (DNA and amino acid sequence determination and analysis, expression in immortalized ME normal fibroblasts and immortalized mutant fibroblasts of the CDG1 patient [2]) [2] <3> (DNA sequence determination [2]) [2] <4> (functional expression of HA-epitope-tagged fusion protein in inactive alg3-mutant, complementation [3]) [3] <5> (DNA sequence determination and analysis, chromosome mapping [6]) [6] <6> (functional expression in enzyme deficient yeast, complementation [4]; expression of T67M/R146Q mutant gene in enzyme deficient yeast, very weak complementation [4]) [4] <6> (retroviral expression of the wild-type ALG12 in fibroblasts of the natural L158P ALG12-mutant, complementation [5]) [5] <8> (DNA sequence determination and analysis, chromosome mapping [6]) [6] Engineering F142V <2> (<2> natively occurring point mutation leading to the congenital disorder of glycosylation type Ig, i.e. CDG1, in the patient, i.e. deficiency in the capacity to add the 8th mannose residue onto the lipid-linked oligosaccharide precursor, accumulation of (d-mannose)7 -(N-acetyl-d-glucosaminyl)2 -dolichyl-diphosphate [2]) [2] L158P <6> (<6> natural ALG12 mutant, patient with CDG1 syndrome, additional point mutation leading to a prematur stop resulting in loss of 74 amino acid residues at the C-terminus [5]) [5] R161Q <4> (<4> introduction of mutation corresponding to the native mutation R146Q in the human gene, no severe effects [4]) [4] T61M <4> (<4> introduction of mutation corresponding to the native mutation T67M in the human gene, altered phenotype, growth defect [4]) [4] T61M/R161Q <4> (<4> introduction of mutation corresponding to the native mutation T67M/R146Q in the human gene, altered phenotype [4]) [4] T67M/R146Q <6> (<6> natively occurring point mutations leading to the congenital disorder of glycosylation type Ig, i.e. CDG1, in the patient, i.e. deficiency in the capacity to add the 8th mannose residue onto the lipidlinked oligosaccharide precursor, accumulation of (d-mannose)7 -(N-acetyld-glucosaminyl)2 -dolichyl-diphosphate [4]) [4] Additional information <4, 5, 8> (<4> inactive alg3-mutant [3]; <5,8> homozygous diploid double disruption null mutants of PMT3 and PMT4, lethal phenotype when combined with a disruption mutation in dolichyl-phosphate-mannose-protein mannosyltransferase, EC 2.4.1.109, genes [6]) [3, 6]

208

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Dolichyl-phosphate-mannose-glycolipid a-mannosyltransferase

References [1] Rearick, J.I.; Fujimoto, K.; Kornfeld, S.: Identification of the mannosyl donors involved in the synthesis of lipid-linked oligosaccharides. J. Biol. Chem., 256, 3762-3769 (1981) [2] Levrat, C.; Louisot, P.; Morelis, R.: Topological investigations. Study of the trypsin sensitivity of the N-acetylglucosaminyl and mannosyl-transferase activities located in the outer mitochondrial membrane. J. Biochem., 106, 133138 (1989) [3] Chantret, I.; Dupre, T.; Delenda, C.; Bucher, S.; Dancourt, J.; Barnier, A.; Charollais, A.; Heron, D.; Bader-Meunier, B.; Danos, O.; Seta, N.; Durand, G.; Oriol, R.; Codogno, P.; Moore, S.E.H.: Congenital disorders of glycosylation type Ig is defined by a deficiency in dolichyl-P-mannose:Man7 GlcNAc2 PP-dolichyl mannosyltransferase. J. Biol. Chem., 277, 25815-25822 (2002) [4] Grubenmann, C.E.; Frank, C.G.; Kjaergaard, S.; Berger, E.G.; Aebi, M.; Hennet, T.: ALG12 mannosyltransferase defect in congenital disorder of glycosylation type Ig. Hum. Mol. Genet., 11, 2331-2339 (2002) [5] Thiel, C.; Schwarz, M.; Hasilik, M.; Grieben, U.; Hanefeld, F.; Lehle, L.; von Figura, K.; Koerner, C.: Deficiency of dolichyl-P-Man:Man7 GlcNAc2 -PP-dolichyl mannosyltransferase causes congenital disorder of glycosylation type Ig. Biochem. J., 367, 195-201 (2002) [6] Immervoll, T.; Gentzsch, M.; Tanner, W.: PMT3 and PMT4, two new members of the protein-O-mannosyltransferase gene family of Saccharomyces cerevisiae. Yeast, 11, 1345-1351 (1995)

209

Glycolipid 2-a-mannosyltransferase

2.4.1.131

1 Nomenclature EC number 2.4.1.131 Systematic name GDP-mannose:glycolipid 1,2-a-d-mannosyltransferase Recommended name glycolipid 2-a-mannosyltransferase Synonyms GDP-mannose-oligosaccharide-lipid mannosyltransferase guanosine diphosphomannose-oligosaccharide-lipid mannosyltransferase mannosyltransferase, guanosine diphosphomannose-oligosaccharide-lipid oligosaccharide-lipid mannosyltransferase CAS registry number 74506-43-7

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

Oryctolagus cuniculus (New Zealand White rabbits [1]) [1, 3] Sus scrofa [2] Bos taurus (cow [4]) [4] Saccharomyces cerevisiae [5]

3 Reaction and Specificity Catalyzed reaction transfers an a-d-mannosyl residue from GDP-mannose into lipid-linked oligosaccharide, forming an a-1,2-d-mannosyl-d-mannose linkage Reaction type hexosyl group transfer Natural substrates and products S GDPmannose + oligosaccharide-lipid acceptor <1, 3> (<1> biosynthesis of oligosaccharide-lipids [1,3]; <3> involved in biosynthesis of lipidlinked oligosaccharides up to (Man)5 -(GlcNAc)2 , involved in the production of two 1,2-linked mannosyl residues in mammalian lipid-linked tet-

210

2.4.1.131

Glycolipid 2-a-mannosyltransferase

radecasaccharide of the structure (Glc)3 (Man)9 (GlcNAc)2 , the major precursor for eukaryotic en-bloc-glycosylation of asparagin-linked glycoproteins [4]) [1, 3, 4] P GDP + a-mannosyl-1,2-oligosaccharide-lipid S Additional information <4> (<4> involved in asparagine-linked glycosylation, reaction proceeds in the endoplasmatic reticulum, role of the ALG9 step in downstream ER and Golgi N-glycan processing events [5]) [5] P ? Substrates and products S GDP-mannose + lipid-linked Mana(1-3)Manb-GlcNAcb-GlcNAc <3> (<3> reaction via penta- and hexasaccharide intermediates to a lipidlinked heptasaccharide, presumably multiple mannosyltransferases involved [4]) (Reversibility: ? <3> [4]) [4] P GDP + lipid-linked Mana(1-2)Mana(1-2)Mana(1-3)(Mana(1-6))ManbGlcNAcb-GlcNAc <3> [4] S GDP-mannose + oligosaccharide-lipid acceptor <1-3> (<1,2> acceptor isolated from pig liver [1-3]; <3> acceptor isolated from baby hamster kidney cells, i.e. BHK cells [4]; <2> oligosaccharide portion: a mixture of trisaccharide and pentasaccharide, acceptor lipids are probably ManbGlcNAc-GlcNAc-pyrophosphoryldolichol and Man2 a-Manb-GlcNAcGlcNAc-diphosphoryldolichol [2]; <1> oligosaccharide-P-P-lipid: major oligosaccharide component is Man2 GlcNAc2 [3]; <3> acceptors can be mannose or Man-(GlcNAc)2 to (Man)5 -(GlcNAc)2 , lipid-linked tetrasaccharide Mana(1-3)Manb-GlcNAcb-GlcNAc, presumably multiple mannosyltransferases involved [4]) (Reversibility: ? <1-3> [1-4]) [1-4] P GDP + a-mannosyl-1,2-oligosaccharide-lipid <1-3> (<1> presumably a heptasaccharide with a 1,6-linked branched glycosyl unit with 65% of the newly added mannosyl units in a terminal nonreducing position, structure may be Mana(1-2)Man-Man(Man(1-6))Man-GlcNAc-GlcNAc [1]; <2> heptasaccharide-lipid as product, probably Man5 GlcNAc2 as heptasaacharide [2]; <1> penta-, hexa-, and heptasaccharide as oligosaccharide portion, formed by the stepwise addition of mannose to the growing oligosaccharide chain [3]) [1-4] S Additional information <1-4> (<1,2> no substrate: dolichol-phosphat [1,2]; <2> no sugar donor: mannosylphosphoryldolichol [2]; <3> no sugar donor: mannosylphosphorylretinol [4]; <4> (Man)6 -(GlcNAc)2 precursor of enzyme [5]) [1, 2, 4, 5] P Additional information <4> (<4> Mana(1-2)Mana(1-2)Mana(13)(Mana(1-2)Mana(1-3)Mana(1-6))Manb(1-4)GlcNAcb(1-4)GlcNAca/b is the product of the middle-arm terminal a1,2-mannosyltransferase Alg9p [5]) [5] Inhibitors ADP <1> (<1> 2 mM, less than 15% inhibition [1]) [1] ATP <1> (<1> 2 mM, less than 15% inhibition [1]) [1] Ca2+ <3> (<3> weak [4]) [4] Cu2+ <3> (<3> strong [4]) [4] 211

Glycolipid 2-a-mannosyltransferase

2.4.1.131

GDP <1> (<1> 0.02 mM, 84% inhibition [1]) [1] GMP <1> (<1> 0.02 mM, 30% inhibition [1]) [1] GTP <1> (<1> 0.02 mM, 40% inhibition [1]) [1] Mn2+ <3> [4] UDP <1> (<1> 2 mM, less than 15% inhibition [1]) [1] UDP-N-acetylglucosamine <1> (<1> 2 mM, less than 15% inhibition [1]) [1] UDP-galactose <1> (<1> 2 mM, less than 15% inhibition [1]) [1] UDP-glucose <1> (<1> 2 mM, less than 15% inhibition [1]) [1] deoxycholate <1> [1] taurocholate <1> [1] Additional information <1-3> (<2,3> not inhibited by amphomycin [2,4]; <1-3> not inhibited by EDTA [1,2,4]) [1, 2, 4] Activating compounds EDTA <3> (<3> slight stimulation [4]) [4] endogen lipids <3> (<3> soluble in CHCl3 /CH3 OH, 2:1, activation [4]) [4] Metals, ions Additional information <1-3> (<1-3> no requirement of divalent cations [1,2,4]) [1, 2, 4] Specific activity (U/mg) Additional information <1> [1, 3] Km-Value (mM) 0.00064 <1> (oligosaccharide-lipid acceptor, <1> based on the assumption of incorporation of only a single mannosyl unit into the product [1]) [1] 0.0047 <1> (GDPmannose) [1] pH-Optimum 6.5-7.5 <1> [1] 6.8-7.6 <3> (<3> broad [4]) [4] Temperature optimum ( C) 37 <1, 2> (<1,2> assay at [1-3]) [1-3]

5 Isolation/Preparation/Mutation/Application Source/tissue aorta <2> (<2> intimal layer [2]) [2] liver <1> [1, 3] mammary gland <3> (<3> lactating mammary tissue [4]) [4] Localization membrane <1-3> (<1-3> membrane-bound [2-4]) [2-4] microsome <1, 3> [1, 3, 4] Purification <1> (solubilized with 0.15% v/v Nonidet P-40, partial [1]) [1]

212

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Glycolipid 2-a-mannosyltransferase

Cloning <4> (ALG9 gene encodes a middle-arm terminal a1,2-mannosyltransferase Alg9p [5]) [5]

6 Stability Storage stability <1>, 0-4 C, solubilized enzyme, at least 2 weeks, stable [1] <1>, 0 C, partially purified enzyme, 5 days, stable [1] <3>, 4 C, solubilized enzyme, 2 weeks: about 40% loss of activity, 7 days: about 20% loss of activity [4]

References [1] Schutzbach, J.S.; Springfield, J.D.; Jensen, J.W.: The biosynthesis of oligosaccharide-lipids. Formation of an a-1,2-mannosyl-mannose linkage. J. Biol. Chem., 255, 4170-4175 (1980) [2] Spencer, J.P.; Elbein, A.D.: Transfer of mannose from GDP-mannose to lipidlinked oligosaccharide by soluble mannosyl transferase. Proc. Natl. Acad. Sci. USA, 77, 2524-2527 (1980) [3] Jensen, J.W.; Springfield, J.D.; Schutzbach, J.S.: The biosynthesis of oligosaccharide-lipids. Isolation of an oligosaccharide-P-P-lipid acceptor. J. Biol. Chem., 255, 11268-11272 (1980) [4] Prakash, C.; Katial, A.; Kang, M.S.; Vijay, I.K.: Solubilization of mannosyltransferase activities for the biosynthesis of mammary glycoproteins. Elongation of tetrasaccharide-lipid to heptasaccharide-lipid by a solubilized enzyme preparation. Eur. J. Biochem., 139, 87-93 (1984) [5] Cipollo, J.F.; Trimble, R.B.: The Saccharomyces cerevisiae alg12d mutant reveals a role for the middle-arm a1,2Man- and upper-arm a1,2Mana1,6Manresidues of Glc(3)Man(9)GlcNAc(2)-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus. Glycobiology, 12, 749-762 (2002)

213

Glycolipid 3-a-mannosyltransferase

2.4.1.132

1 Nomenclature EC number 2.4.1.132 Systematic name GDP-mannose:glycolipid 1,3-a-d-mannosyltransferase Recommended name glycolipid 3-a-mannosyltransferase Synonyms GDP-Man:Dol-PP-GlcNAc2 Man2 a-1,3-mannosyltransferase <2> [15] GDP-mannose-oligosaccharide-lipid mannosyltransferase II a-1,3-mannosyltransferase mannosyltransferase II mannosyltransferase, guanosine diphosphomannose-oligosaccharide-lipid II CAS registry number 81181-76-2

2 Source Organism <1> Oryctolagus cuniculus [1-3] <2> Saccharomyces cerevisiae (haploid strain yg414, Alg3p and MNN1 [7]; Mnn1p [4,6,11,13,14]; Mnt2p and Mnt3p [6]; ALG2 protein [8,15]; A) [48, 11, 13-16] <3> Homo sapiens (hALG2 [8]) [8] <4> Acetobacter xylinum (recombinant AceA [9]) [9] <5> Cryptococcus neoformans (pathogenic fungus [10,12]) [10, 12] <6> Bos taurus (calf [17]) [17]

3 Reaction and Specificity Catalyzed reaction transfers an a-d-mannosyl residue from GDP-mannose into lipid-linked oligosaccharide, forming an a-1,3-d-mannosyl-d-mannose linkage Reaction type hexosyl group transfer

214

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Glycolipid 3-a-mannosyltransferase

Natural substrates and products S GDPmannose + tetrasaccharide-diphosphoryl-lipid <1, 2> (<1> involved in the biosynthesis of asparagine-linked saccharide chains of mammalian glycoproteins [1,2]; <1> the 1,3-linked mannosyl residue in mammalian lipid-linked oligosaccharide of the structure Glc3 Man9 GlcNAc2 is produced by this enzyme [1,2]) [1, 2, 4-6] P GDP + mannosyl-a-1,3-tetrasaccharide-diphosphoryl-lipid S GDPmannose + undecaprenyl-diphosphate-linked cellobiose <4> (<4> natural substrate [9]) [9] P ? S Additional information <2, 3, 5> (<2> regulatory role for the Alg3p-dependent a-1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation, it provides structural information that potentiates the Alg6p, Alg8p and Alg10p ER glucosyltransferases and Gls1p and Gls2p trimming glucosidases [7]; <3> hALG2: early steps of dolichollinked oligosaccharide biosynthesis [8]; <5> role in the synthesis of the glucuronoxylomannan of the capsule [12]; <2> Mnn1p is required for the complex glycosylation of secreted proteins [11]; <2> involved in oligosaccharide-lipid synthesis [16]) [7, 8, 11, 12, 16] P ? Substrates and products S GDPmannose + ManGlcNAc2 -PP-dolichol <3> (<3> hALG2 [8]) (Reversibility: ? <3> [8]) [8] P GDP + Mana(1-3)ManGlcNAc2 -PP-dolichol <3> [8] S GDPmannose + Mana(1-2)Mana(1-2)Mana(1-3)(Mana(1-6))Manb(14)GlcNAcb(1-4)GlcNAc-PP-dolichol <2> (<2> cytosolic alg3 Man5 GlcNAc2 precursor [7]; <2> alg3 Man5 GlcNAc2 precursor [16]) (Reversibility: ? <2> [7,16]) [7, 16] P GDP + Mana(1-2)Mana(1-2)Mana(1-3)(Mana(1-3)Mana(1-6))Manb(14)GlcNAcb(1-4)GlcNAc-PP-dolichol <2> (<2> product of the Alg3p step [7]) [7] S GDPmannose + Manb(1-4)GlcNAcb(1-4)GlcNAc-PP-dolichol <6> (Reversibility: ? <6> [17]) [17] P GDP + Mana(1-3)Manb(1-4)GlcNAcb(1-4)GlcNAc-PP-dolichol <6> [17] S GDPmannose + acceptor containing mannose a-1,3-linked at the non-reducing terminus <5> (Reversibility: ? <5> [10]) [10] P ? S GDPmannose + a-1,3-dimannoside acceptor <5> (Reversibility: ? <5> [10]) [10] P GDP + a-1,3-trimannoside product <5> (<5> enzyme forms a second a1,3-linkage [12]) [12] S GDPmannose + phytanyl-pyrophosphate-linked cellobiose <4> (<4> synthetic substrate for recombinant a-1,3-mannosyltransferase AceA, product is a trisaccharide [9]) (Reversibility: ? <4> [9]) [9] P ?

215

Glycolipid 3-a-mannosyltransferase

2.4.1.132

S GDPmannose + tetrasaccharide-diphosphoryl-lipid <1, 2> (<1> no acceptor is dolichol-phosphate [1]) (Reversibility: ? <1,2> [1-6]) [1-6] P GDP + mannosyl-a-1,3-tetrasaccharide-diphosphoryl-lipid <1> [1-3] S Additional information <2> (<2> MNN1 encoded enzyme adds at least the penultimate a-1,3-linked Man of the terminal Mana(1-3)Mana(1-3)-disaccharide on O-linked glycans [7]; <2> Mnn1p family of a-1,3-mannosyltransferases is responsible for adding the terminal mannose residues of Olinked oligosaccharides, MNT2 and MNT3 genes in combination with MNN1 have overlapping roles in the addition of the fourth and fifth a-1,3linked mannose residues to form Man4 and Man5 oligosaccharides [6]; <2> Mnn1p has 2 conserved aspartate residues necessary for activity [14]; <2> primary and secondary structure of the ALG2 protein [15]) [6, 7, 14, 15] P ? Inhibitors ADP <1> (<1> weak [1]) [1] ATP <1> (<1> weak [1]) [1] EDTA <1> [1] GDP <1, 5> (<1> 50% inhibition at 0.0006 mM [1]) [1, 12] GDPglucose <1> [1] GMP <1> (<1> 50% inhibition at 0.094 mM [1]) [1] GTP <1> (<1> 50% inhibition at 0.006 mM [1]) [1] NiCl2 <1> [1] UDP <1> (<1> weak [1]) [1] UDP-N-acetylglucosamine <1> (<1> weak [1]) [1] UDPglucose <1> (<1> weak [1]) [1] UTP <1> (<1> weak [1]) [1] ZnCl2 <1> [1] glycerol <1> (<1> 1-2% v/v [1]) [1] mannose-1-phosphate <5> [12] phospholipids <1> (<1> stable bilayer forming, e.g. phosphatidylcholine, restorable by addition of cholesterol, dolichol and dolichol-derivatives [2]) [2] Additional information <1, 5, 6> (<1> no inhibition by amphomycin [1]; <5> method of screening for a-1,3-mannosyltransferase inhibitors [10]; <5> not inhibited by amphomycin or tunicamycin [12]; <6> active in presence of 10 mM EDTA [17]) [1, 10, 12, 17] Activating compounds Nonidet P-40 <1> (<1> activation, up to 0.0225% v/v [1]) [1] cardiolipin <1> (<1> activation, 35% as effective as phosphatidylethanolamine [3]) [3] dolichol <1> (<1> stimulates enzyme in the presence of inhibitory concentrations of phosphatidylcholine [1]) [1] dolichol-phosphate <1> (<1> stimulates enzyme in the presence of inhibitory concentrations of phosphatidylcholine [1]) [1] phosphatidylethanolamine <1> (<1> activation [2,3]; <1> phosphoethanolamine containing unsaturated acylchains [3]; <1> from various eukaryotic and prokaryotic sources [3]; <1> and mixtures with other phospholipids 216

2.4.1.132

Glycolipid 3-a-mannosyltransferase

forming non-bilayer phospholipid phases e.g. hexagonal phases [3]; <1> in the absence of detergent [3]; <1> enzyme-phospholipid-complex formation [3]; <1> no activation by phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, lysophosphocholine or lysophosphoethanolamine [3]) [2, 3] phosphatidylserine <1> (<1> activation, 16-43% as effective as phosphatidylethanolamine [3]) [3] Metals, ions Ca2+ <1> (<1> activation, about 60% as effective as Mg2+ [1]) [1] Co2+ <1> (<1> activation, about 4% as effective as Mg2+ [1]) [1] Mg2+ <1> (<1> requirement [1,3]; <1> 10 mM [1]) [1, 3] Mn2+ <1> (<1> activation, about 40% as effective as Mg2+ [1]) [1] Additional information <6> (<6> no require of divalent cations [17]) [17] Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 0.00021 <1> (tetrasaccharide-diphosphoryl-lipid, <1> detergent-stimulated [1]) [1] 0.00065 <1> (GDPmannose, <1> detergent-stimulated [1]) [1] 0.00096 <1> (tetrasaccharide-diphosphoryl-lipid) [3] 0.00142 <1> (GDPmannose) [3] pH-Optimum 6.8-7.3 <1> [1] Temperature optimum ( C) 27 <2> (<2> assay at [7]) [7] 37 <1> (<1> assay at [1-3]) [1-3]

4 Enzyme Structure Molecular weight 85000 <2> (<2> SDS-PAGE [4]) [4] 98500 <2> (<2> SDS-PAGE, MW slowly increases due to glycosylation [5]) [5] Posttranslational modification glycoprotein <2> (<2> N- and O-linked oligosaccharides [5]) [4, 5]

5 Isolation/Preparation/Mutation/Application Source/tissue liver <1> [1-3] pancreas <6> [17] skin fibroblast <3> [8] 217

Glycolipid 3-a-mannosyltransferase

2.4.1.132

Localization Golgi apparatus <2> [11, 13] Golgi membrane <2> (<2> colocalizes with guanosine diphosphatase [5]) [4, 5] membrane <2, 5> (<2> type II transmembrane proteins [6,11]) [6, 11, 12] microsome <1, 6> [1-3, 17] Purification <1> (partial [1]) [1] Cloning <2> (Mnn1p [4]; wild type and mutant that lacks NH2 -terminal cytoplasmic tail [5]; MNN1, MNT2 and MNT3 genes encode a-1,3-mannosyltransferases, wild type and mutants carrying single and multiple combinations of the disrupted gene [6]; alg3 encodes enzyme responsible for the a-1,3-Man middlearm addition, MNN1 encoded enzyme adds at least the penultimate a-1,3linked Man of the terminal Mana(1-3)Mana(1-3)-disaccharide on O-linked glycans [7]; ALG2 gene encodes GDP-Man:Dol-PP-GlcNAc2 Man2 a-1,3-mannosyltransferase, amino acid sequence [15]; MNN1 gene encoding Mnn1p is cloned and sequenced [11]) [4-7, 11, 15] <3> (hALG2 encodes an a-1,3-mannosyltransferase resulting in Mana(13)ManGlcNAc2 -PP-dolichol [8]) [8] Engineering Additional information <2, 3> (<2,3> ALG2 mutant with severely reduced enzyme activity [8]; <2> lumenal domain retention mutants of Mnn1p [13]; <2> mutagenesis of Mnn1p by altering either of the 2 conserved aspartates eliminates all enzymic activity, but does not affect the overall folding and assembly of Mnn1p [14]; <2> Mnn1 mutant strain [11]; <2> alg3 mutant [16]) [8, 11, 13, 14, 16] Application medicine <3> (<3> hALG2 is the cause of a new type of congenital disorders of glycosylation designated CDG-Ii [8]) [8] pharmacology <5> (<5> sreening for a-1,3-mannosyltransferase inhibitors and anti-fungal therapeutics [10]; <5> target for antifungal drug discovery [12]) [10, 12]

6 Stability Temperature stability 37 <1> (<1> 5 min, in 0.0225% Nonidet P-40 without substrate, about 80% loss of activity. In phosphatidylethanolamine without substrate: at least 5 min stable, after 25 min about 15% loss of activity [3]) [3] Additional information <1> (<1> phosphatidylethanolamine increases heatstability [3]) [3]

218

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Glycolipid 3-a-mannosyltransferase

General stability information <1>, glycerol, 0.5% v/v, stabilizes during storage and purification [1] Storage stability <1>, -20 C, in 0.1% v/v Nonidet P-40 and 10% v/v glycerol, at least 3 months [3] <1>, 0-4 C, in 0.1% v/v Nonidet P-40 and 10% v/v glycerol, 24 h [3] <1>, glycerol, 10% v/v, stabilizes during storage [2, 3]

References [1] Jensen, J.W.; Schutzbach, J.S.: The biosynthesis of oligosaccharide-lipids. Partial purification and characterization of mannosyltransferase II. J. Biol. Chem., 256, 12899-12904 (1981) [2] Jensen, J.W.; Schutzbach, J.S.: Activation of mannosyltransferase II by nonbilayer phospholipids. Biochemistry, 23, 1115-1119 (1984) [3] Jensen, J.W.; Schutzbach, J.S.: The biosynthesis of oligosaccharide-lipids. Activation of mannosyltransferase II by specific phospholipids. J. Biol. Chem., 257, 9025-9029 (1982) [4] Graham, T.R.; Krasnov, V.A.: Sorting of yeast a1,3 mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein. Mol. Biol. Cell, 6, 809-824 (1995) [5] Graham, T.R.; Seeger, M.; Payne, G.S.; MacKay, V.L.; Emr, S.D.: Clathrindependent localization of a 1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae. J. Cell. Biol., 127, 667-678 (1994) [6] Romero, P.A.; Lussier, M.; Veronneau, S.; Sdicu, A.M.; Herscovics, A.; Bussey, H.: Mnt2p and Mnt3p of Saccharomyces cerevisiae are members of the Mnn1p family of a-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides. Glycobiology, 9, 1045-1051 (1999) [7] Cipollo, J.F.; Trimble, R.B.: The accumulation of Man6 GlcNAc2 -PP-dolichol in the Saccharomyces cerevisiae Dalg9 mutant reveals a regulatory role for the Alg3p a1,3-Man middle-arm addition in downstream oligosaccharidelipid and glycoprotein glycan processing. J. Biol. Chem., 275, 4267-4277 (2000) [8] Thiel, C.; Schwarz, M.; Peng, J.; Grzmil, M.; Hasilik, M.; Braulke, T.; Kohlschuetter, A.; von Figura, K.; Lehle, L.; Koerner, C.: A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis. J. Biol. Chem., 278, 22498-22505 (2003) [9] Lellouch, A.C.; Watt, G.M.; Geremia, R.A.; Flitsch, S.L.: Phytanyl-pyrophosphate-linked substrate for a bacterial a-mannosyltransferase. Biochem. Biophys. Res. Commun., 272, 290-292 (2000) [10] Doering, T.: a-1,3-Mannosyltransferase from Cryptococcus neoformans and other pathogenic fungi and method of screening for a-1,3-mannosyl-

219

Glycolipid 3-a-mannosyltransferase

[11]

[12] [13] [14] [15]

[16] [17]

220

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transferase inhibitors and anti-fungal therapeutics. PCT Int. Appl., 2000, 38pp (2000) Yip, C.L.; Welch, S.K.; Klebl, F.; Gilbert, T.; Seidel, P.; Grant, F.; O'Hara, P.J.; MacKay, V.L.: Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins. Proc. Natl. Acad. Sci. USA, 91, 2723-2727 (1994) Doering, T.: A unique a-1,3-mannosyltransferase of the pathogenic fungus Cryptococcus neoformans. J. Bacteriol., 181, 5482-5488 (1999) Reynolds, T.B.; Hopkins, B.D.; Lyons, M.R.; Graham, T.R.: The high osmolarity glycerol response (HOG) MAP kinase pathway controls localization of a yeast Golgi glycosyltransferase. J. Cell Biol., 143, 935-946 (1998) Wiggins, C.A.R.; Munro, S.: Activity of the yeast MNN1 a-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc. Natl. Acad. Sci. USA, 95, 7945-7940 (1998) Shpakov, A.O.; Derkach, K.V.: Yeast dolichol-coupled mannosyltransferases. Theoretical analysis of primary structure and identification of sites homologous to other enzymes of the dolichol cycle. Zh. Evol. Biokhim. Fiziol., 32, 3-18 (1996) Verostek, M.F.; Atkinson, P.H.; Trimble, R.B.: Structure of Saccharomyces cerevisiae alg3,sec18 mutant oligosaccharides. J. Biol. Chem., 266, 55475551 (1991) Herscovics, A.; Warren, C.D.; Jeanloz, R.W.: Solubilization of an a-(1-3)-dmannosyltransferase from pancreas which utilizes synthetic dolichyl pyrophosphate trisaccharide b-Man-(1-4)-b-GlcNAc-(1-4)-GlcNAc as substrate. FEBS Lett., 156, 298-302 (1983)

Xylosylprotein 4-b-galactosyltransferase

2.4.1.133

1 Nomenclature EC number 2.4.1.133 Systematic name UDP-galactose:O-b-d-xylosylprotein 4-b-d-galactosyltransferase Recommended name xylosylprotein 4-b-galactosyltransferase Synonyms UDP-d-galactose:d-xylose galactosyltransferase UDP-d-galactose:xylose galactosyltransferase UDPgalactose:O-b-d-xylosylprotein 4-b-d-galactosyltransferase galactosyltranferase I galactosyltransferase, uridine diphosphogalactose-xylose CAS registry number 52227-72-2

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

Gallus gallus [1-3, 5] Bos taurus [4, 5] Drosophila melanogaster (S2 cells [6]) [6] Homo sapiens [7, 9, 10, 13] Mus musculus [8, 11, 12]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + O-b-d-xylosylprotein = UDP + 4-b-d-galactosyl-O-b-d-xylosylprotein Reaction type hexosyl group transfer Natural substrates and products S UDPgalactose + N-acetyl-b-d-glucosamine <5> [11] P UDP + b-d-galactosyl-1,4-N-acetyl-b-d-glucosamine

221

Xylosylprotein 4-b-galactosyltransferase

2.4.1.133

S UDPgalactose + O-b-d-xylosylprotein <1> (<1> involved in chondroitin sulfate biosynthesis [1-3]) [1-3] P UDP + 4-b-d-galactosyl-O-b-d-xylosylprotein Substrates and products S UDPgalactose + 4-methylumbelliferyl-b-d-xyloside <4> (<4> assay method for measuring the galactosyltransferase I activity with d-galactal as inhibitor for endogenous b-galactosidase [7]) (Reversibility: ? <4> [7,9]) [7, 9] P UDP + b-d-galactosyl-1,4-b-d-xylosyl-1-O-(4-methylumbelliferone) S UDPgalactose + N-acetyl-b-d-glucosamine <5> (Reversibility: ? <5> [11]) [11] P UDP + b-d-galactosyl-1,4-N-acetyl-b-d-glucosamine <5> (<5> or polyb4-N-acetyllactosamine structures in glycoconjugates [11]) [11] S UDPgalactose + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6-b-dmannosyl-octyl <5> (Reversibility: ? <5> [8]) [8] P UDP + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl1,6-b-d-mannosyl-octyl S UDPgalactose + N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2)-a-d-mannosyl-1,6-b-d-mannosyl-octyl <5> (Reversibility: ? <5> [8]) [8] P UDP + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-dglucosaminyl-1,2)-a-d-mannosyl-1,6-b-d-mannosyl-octyl S UDPgalactose + N-acetyl-b-d-glucosaminyl-1,6-(b-galactosyl-1,4-N-acet<5> yl-b-d-glucosaminyl-1,2)-a-d-mannosyl-1,6-b-d-mannosyl-octyl (Reversibility: ? <5> [8]) [8] P UDP + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,6-(b-galactosyl1,4-N-acetyl-b-d-glucosaminyl-1,2)-a-d-mannosyl-1,6-b-d-mannosyl-octyl S UDPgalactose + N-acetyl-b-d-glucosaminyl-1,6-a-d-mannosyl-1,6-b-dmannosyl-octyl <5> (Reversibility: ? <5> [8]) [8] P UDP + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,6-a-d-mannosyl1,6-b-d-mannosyl-octyl S UDPgalactose + b-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,6-(N-acet<5> yl-b-d-glucosaminyl-1,2)-a-d-mannosyl-1,6-b-d-mannosyl-octyl (Reversibility: ? <5> [8]) [8] P UDP + b-d-galactosyl-1,4-b-galactosyl-1,4-N-acetyl-b-d-glucosaminyl1,6-(N-acetyl-b-d-glucosaminyl-1,2)-a-d-mannosyl-1,6-b-d-mannosyloctyl S UDPgalactose + p-nitrophenyl N-acetyl-b-d-glucosaminide <3> (Reversibility: ? <3> [6]) [6] P UDP + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminide-1-O-nitrophenol S UDPgalactose + p-nitrophenyl b-d-xylopyranoside <3, 4> (Reversibility: ? <3,4> [6,10]) [6, 10] P UDP + b-d-galactopyranosyl-1,4-xylopyranosyl-1-O-nitrophenol S UDPgalactose + p-nitrophenyl b-d-xylose <3> (Reversibility: ? <3> [6]) [6]

222

2.4.1.133

P S P S P

Xylosylprotein 4-b-galactosyltransferase

UDP + b-d-galactosyl-1,4-b-d-xylosyl-1-O-nitrophenol UDPgalactose + xylose <1> (Reversibility: ? <1> [1-3]) [1-3] UDP + 4-O-b-d-galactosyl-b-d-xylose <1> [1-3] UDPgalactose + xylosylserine <1> (Reversibility: ? <1> [2]) [2] UDP + b-d-galactosyl-1,4-b-d-xylosylserine

Inhibitors cycloheximide <2> (<2> first order kinetics [5]) [5] Activating compounds ATP <4> (<4> results suggest that a protein kinase in the 100000 x g supernatant activates galactosyltransferase I [9]) [9] Metals, ions Co2+ <3> (<3> slightly activation [6]) [6] Mg2+ <3> (<3> very slightly activation [6]) [6] Mn2+ <1, 3> (<1> requirement, 15 mM [1,2]; <3> optimal concentration: 20 mM [6]) [1, 2, 6] Ni2+ <3> (<3> very slightly activation [6]) [6] Specific activity (U/mg) 1.9 <4> (<4> the protA-tagged b4GalT [13]) [13] 240 <5> (<5> transgenic line: b4GalT[-1270/-619]LacZ-930 [11]) [11] 400 <5> (<5> transgenic line: b4GalT[-1085/-628]LacZ-641 and b4GalT[-756/-743]LacZ-377 [11]) [11] 600 <5> (<5> nontransgenic [11]) [11] 650 <5> (<5> transgenic line: b4GalT[-1270/-619]LacZ-915 and b4GalT[-1085/-474]LacZ-310 [11]) [11] 700 <5> (<5> transgenic line: b4GalT[-1085/-628]LacZ-642 [11]) [11] 800 <5> (<5> transgenic line: b4GalT[-1085/-628]LacZ-648 [11]) [11] 1400 <5> (<5> transgenic line: b4GalT[-756/-743]LacZ-2226 [11]) [11] 2400 <5> (<5> transgenic line: b4GalT[-756/-743]LacZ-378 [11]) [11] 2600 <5> (<5> transgenic line: b4GalT[-756/-743]LacZ-2211 [11]) [11] 3300 <5> (<5> transgenic line: b4GalT[-756/-743]LacZ-380 [11]) [11] 4000 <5> (<5> transgenic line: b4GalT[-756/-743]LacZ-374 [11]) [11] 4200 <5> (<5> transgenic line: b4GalT[-756/-743]LacZ-369 [11]) [11] 5500 <5> (<5> transgenic line: b4GalT[-793/-707]LacZ-330 [11]) [11] 6700 <5> (<5> transgenic line: b4GalT[-793/-707]LacZ-349 [11]) [11] 43000 <5> (<5> transgenic line: b4GalT[-1085/-628]LacZ-42 [11]) [11] 45000 <5> (<5> transgenic line: b4GalT[-1085/-474]LacZ-328 [11]) [11] 47000 <5> (<5> transgenic line: b4GalT[-1270/-619]LacZ-923 [11]) [11] 49000 <5> (<5> transgenic line: b4GalT[-1270/-474]LacZ-140 [11]) [11] 50000 <5> (<5> transgenic line: b4GalT[-1085/-474]LacZ-330 and b4GalT[-1085/-474]LacZ-346 [11]) [11] 53000 <5> (<5> transgenic line: b4GalT[-1085/-628]LacZ-58 [11]) [11] 56000 <5> (<5> transgenic line: b4GalT[-793/-707]LacZ-320 [11]) [11] 68000 <5> (<5> transgenic line: b4GalT[-793/-707]LacZ-337 [11]) [11] 161000 <5> (<5> transgenic line: b4GalT[-793/-707]LacZ-339 [11]) [11]

223

Xylosylprotein 4-b-galactosyltransferase

2.4.1.133

Km-Value (mM) 0.05 <1, 3> (UDPgalactose, <1> pH 6.5, 30 C [1]; <3> 37 C [6]) [1, 6] 0.33 <4> (4-methylumbelliferyl-b-d-xyloside, <4> pH 5.5, 37 C after preincubation with ATP [9]) [9] 0.33 <5> (N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2)a-d-mannosyl-1,6-b-d-mannosyl-octyl, <5> pH 7, 37 C [8]) [8] 0.34 <4> (UDPgalactose, <4> pH 5.5, 37 C after preincubation with ATP [9]) [9] 0.41 <4> (UDPgalactose, <4> pH 5.5, 37 C before preincubation with ATP [9]) [9] 0.5 <4> (4-methylumbelliferyl-b-d-xyloside, <4> pH 5.5, 37 C before preincubation with ATP [9]) [9] 0.88 <5> (N-acetyl-b-d-glucosaminyl-1,6-a-d-mannosyl-1,6-b-d-mannosyloctyl, <5> pH 7, 37 C [8]) [8] 0.91 <5> (N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6-b-d-mannosyloctyl, <5> pH 7, 37 C [8]) [8] 1.2 <3> (p-nitrophenyl b-d-xylose, <3> 37 C [6]) [6] 1.41 <5> (b-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2)-a-d-mannosyl-1,6-b-d-mannosyl-octyl, <5> pH 7, 37 C [8]) [8] 2.29 <5> (N-acetyl-b-d-glucosaminyl-1,6-(b-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,2)-a-d-mannosyl-1,6-b-d-mannosyl-octyl, <5> pH 7, 37 C [8]) [8] pH-Optimum 6.5 <1, 3> (<1> assay at [3]) [3, 6] Temperature optimum ( C) 37 <1> (<1> assay at [1-3]) [1-3]

4 Enzyme Structure Molecular weight 55000 <2> (<2> gel filtration [5]) [5] Posttranslational modification glycoprotein <5> [11]

5 Isolation/Preparation/Mutation/Application Source/tissue brain <5> [11] cartilage <1, 2> (<1> epiphyses from embryonic femurs and tibiae [1,2,5]; <2> articular cartilage [5]) [1-3, 5] heart <5> [11] intestine <5> [11] kidney <5> [11] liver <5> [11] lung <5> [11]

224

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Xylosylprotein 4-b-galactosyltransferase

muscle <5> (<5> sceletal [11]) [11] skin fibroblast <4> [7, 9] spermatid <5> (<5> round spermatid [11]) [11] spermatocyte <5> (<5> pachytene spermatocyte [11]) [11] spleen <5> [11] testis <5> [11] Localization Golgi apparatus <3> [6] Golgi trans-face <5> (<5> type II membrane-bound protein [11]) [11] membrane <1> [1-3] Purification <1> (solubilized and partially purified, affinity chromatography [1-3]) [1-3] <2> [5] <4> [10, 13] <5> [12] Cloning <2> (expression in Escherichia coli [4]) [4] <3> (expression in Spodoptera frugiperda SF9 cells [6]) [6] <4> (expression in Spodoptera frugiperda SF9 cells as prot-A-b4-GalT fusion protein [13]) [13] <4> (transient expression in mouse fibroblast L cells and chinese hamster ovary K-1 cells [10]) [10] <5> (expression in COS-1 cells [8]) [8] <5> (expression in Xenopus laevis oocytes [12]) [12] <5> (expression in transgenic mice [11]) [11]

6 Stability General stability information <1>, Nonidet P-40 stabilizes solubilized enzyme [2] <1>, dialysis against detergent-free buffer, +/- 0.25 M KCl inactivates, dialysis against 1% Nonidet P-40 and 0.25 M KCl restores activity, even after a period of 18 h in the absence of detergent [2] <2>, repeated freezing and thawing, collagenase treatment and sonication result in a significant loss of activity [5] Storage stability <1>, -17 C, concentrated enzyme preparation of 1 mg/ml, at least 6 months [1]

References [1] Schwartz, N.B.; RodØn, L.: Biosynthesis of chondroitin sulfate. Solubilization of chondroitin sulfate glycosyltransferases and partial purification of

225

Xylosylprotein 4-b-galactosyltransferase

[2] [3] [4]

[5] [6] [7]

[8]

[9]

[10]

[11]

[12] [13]

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uridine diphosphate-d-galactose:d-xylose galactosyltrans. J. Biol. Chem., 250, 5200-5207 (1975) Schawrtz, N.: Biosynthesis of chondroitin sulfate. Role of phospholipids in the activity of UDP-d-galactose:d-xylose galactosyltransferase. J. Biol. Chem., 251, 285-291 (1976) Schwartz, N.B.; RodØn, L.; Dorfman, A.: Biosynthesis of chondroitin sulfate: interaction between xylosyltransferase and galactosyltransferase. Biochem. Biophys. Res. Commun., 56, 717-724 (1974) Gunasekaran, K.; Ma, B.; Ramakrishnan, B.; Qasba, P.K.; Nussinov, R.: Interdependence of backbone flexibility, residue conservation, and enzyme function: a case study on b1,4-galactosyltransferase-I. Biochemistry, 42, 3674-3687 (2003) Parker, G.J.; Handley, C.J.; Robinson, H.C.: An assay for galactosyltransferase-I activity in articular cartilage. Anal. Biochem., 226, 154-160 (1995) Vadaie, N.; Hulinsky, R.S.; Jarvis, D.L.: Identification and characterization of a Drosophila melanogaster ortholog of human b1,4-galactosyltransferase VII. Glycobiology, 12, 589-597 (2002) Higuchi, T.; Tamura, S.; Takagaki, K.; Nakamura, T.; Morikawa, A.; Tanaka, K.; Tanaka, A.; Saito, Y.; Endo, M.: A method for determination of galactosyltransferase I activity synthesizing the proteoglycan linkage region. J. Biochem. Biophys. Methods, 29, 135-142 (1994) Ujita, M.; McAuliffe, J.; Hindsgaul, O.; Sasaki, K.; Fukuda, M.N.; Fukuda, M.: Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of i-extension enzyme and b1,4-galactosyltransferase I. J. Biol. Chem., 274, 16717-16726 (1999) Higuchi, T.; Tamura, S.; Tanaka, K.; Takagaki, K.; Saito, Y.; Endo, M.: Effects of ATP on regulation of galactosyltransferase-I activity responsible for synthesis of the linkage region between the core protein and glycosaminoglycan chains of proteoglycans. Biochem. Cell Biol., 79, 159-164 (2001) Okajima, T.; Yoshida, K.; Kondo, T.; Furukawa, K.: Human homolog of Caenorhabditis elegans sqv-3 gene is galactosyltransferase I involved in the biosynthesis of the glucosaminoglycan-protein linkage region of proteoglycans. J. Biol. Chem., 274, 22915-22918 (1999) Charron, M.; Shaper, N.L.; Rajput, B.; Shaper, J.H.: A novel 14-base-pair regulatory element is essential for in vivo expression of murine b4-galactosyltransferase-I in late pachytene spermatocytes and round spermatids. Mol. Cell. Biol., 19, 5823-5832 (1999) Shi, X.; Amindari, S.; Paruchuru, K.; Skalla, D.; Burkin, H.; Shur, B.D.; Miller, D.J.: Cell surface b1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling. Development, 128, 645-654 (2001) Zhou, D.; Malissard, M.; Berger, E.G.; Hennet, T.: Secretion and purification of recombinant b1-4 galactosyltransferase from insect cells using pFmelprotA, a novel transposition-based baculovirus transfer vector. Arch. Biochem. Biophys., 374, 3-7 (2000)

Galactosylxylosylprotein 3-b-galactosyltransferase

2.4.1.134

1 Nomenclature EC number 2.4.1.134 Systematic name UDP-galactose:4-b-d-galactosyl-O-b-d-xylosylprotein 3-b-d-galactosyltransferase Recommended name galactosylxylosylprotein 3-b-galactosyltransferase Synonyms UDPgalactose:4-b-d-galactosyl-O-b-d-xylosylprotein 3-b-d-galactosyltransferase galactosyltransferase II galactosyltransferase, uridine diphosphogalactose-galactosylxylose Additional information (cf. EC 2.4.1.133 and EC 2.4.1.135) CAS registry number 56626-19-8 56626-21-2

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

Gallus gallus [1, 2] Caenorhabditis elegans [3] Haemophilus ducreyi [4] Mus musculus [5, 6] Homo sapiens [6]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + 4-b-d-galactosyl-O-b-d-xylosylprotein = UDP + 3-b-d-galactosyl-4-b-d-galactosyl-O-b-d-xylosylprotein Reaction type hexosyl group transfer

227

Galactosylxylosylprotein 3-b-galactosyltransferase

2.4.1.134

Natural substrates and products S UDP-galactose + 4-b-d-galactosyl-O-b-d-xylosylprotein <1> (<1> involved in biosynthesis of chondroitin sulfate [1]) [1] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-1,4-b-d-xylosylprotein Substrates and products S UDP-galactose + 4-b-d-galactosyl-O-b-d-xylose <1> (Reversibility: ? <1> [1,2]) [1, 2] P UDP + 3-b-d-galactosyl-4-b-d-galactosyl-O-b-d-xylose <1> [1] S UDP-galactose + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-O-naphthalenemethanol <2> (Reversibility: ? <2> [3]) [3] P UDP + b-d-galactosyl-1,3-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosylO-naphthalenemethanol S UDP-galactose + b-d-galactosyl-1,3-N-acetyl-a-d-glucosaminyl-O-naphthalenemethanol <4, 5> (Reversibility: ? <4,5> [6]) [6] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-1,3-N-acetyl-a-d-glucosaminylO-naphthalenemethanol S UDP-galactose + b-d-galactosyl-1,3-N-acetyl-b-d-glucosaminyl-O-naphthalenemethanol <2> (Reversibility: ? <2> [3]) [3] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-1,3-N-acetyl-b-d-glucosaminylO-naphthalenemethanol S UDP-galactose + b-d-galactosyl-1,3-b-galactosyl-O-naphthalenemethanol <2, 4, 5> (Reversibility: ? <2,4,5> [3,6]) [3, 6] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-1,3-b-galactosyl-O-naphthalenemethanol S UDP-galactose + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-O-naphthalenemethanol <2, 4, 5> (Reversibility: ? <2,4,5> [3,6]) [3, 6] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminylO-naphthalenemethanol S UDP-galactose + b-d-galactosyl-1,4-b-d-xylosyl-O-benzyl <2, 4, 5> (Reversibility: ? <2,4,5> [3,6]) [3, 6] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-1,4-b-d-xylosyl-O-benzyl S UDP-galactose + b-d-galactosyl-O-naphthalenemethanol <4, 5> (Reversibility: ? <4,5> [6]) [6] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-O-naphthalenemethanol S UDP-galactose + b-galactosides <1> (<1> e.g. phenyl or pyridine 3-O-bgalactosides, poor substrate: pyridine 2-S-b-thiogalactoside, no substrate: d-galactose [2]) [2] P ? S UDP-galactose + galactosyl-xylosylserine <1> (Reversibility: ? <1> [2]) [2] P UDP + 3-b-d-galactosyl-4-b-d-galactosyl-O-b-d-xylosylserine <1> [2] S UDP-galactose + pyridine 3-O-b-d-galactoside <1> (Reversibility: ? <1> [2]) [2] P UDP + ? Metals, ions Mn2+ <1> (<1> requirement [1]) [1] 228

2.4.1.134

Galactosylxylosylprotein 3-b-galactosyltransferase

Specific activity (U/mg) 1 <2> (<2> pmol/h/ml medium, b-d-galactosyl-1,3-N-acetyl-b-d-glucosaminyl-O-naphthalenemethanol as substrate [3]) [3] 1 <4, 5> (<4,5> pmol/h/ml medium, b-d-galactosyl-1,3-b-d-galactosyl-Onaphthalenemethanol as substrate [6]) [6] 3 <2> (<2> pmol/h/ml medium, b-d-galactosyl-1,3-b-d-galactosyl-Onaphthalenemethanol as substrate [3]) [3] 4 <4, 5> (<4,5> pmol/h/ml medium, b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-O-naphthalenemethanol as substrate [6]) [6] 6 <2> (<2> pmol/h/ml medium, N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-O-naphthalenemethanol as substrate [3]) [3] 6 <4, 5> (<4,5> pmol/h/ml medium, b-d-galactosyl-1,3-N-acetyl-a-d-glucosaminyl-O-naphthalenemethanol as substrate [6]) [6] 18 <4, 5> (<4,5> pmol/h/ml medium, b-d-galactosyl-O-naphthalenemethanol as substrate [6]) [6] 84 <4, 5> (<4,5> pmol/h/ml medium, b-d-galactosyl-1,4-b-xylosyl-O-benzyl as substrate [6]) [6] 2660 <2> (<2> pmol/h/ml medium, b-d-galactosyl-1,4-b-xylosyl-O-benzyl as substrate [3]) [3] Km-Value (mM) 1.05 <1> (UDPgalactose, <1> pH 6, 37 C, + pyridine 3-O-b-galactoside [2]) [2] 100 <1> (pyridine 3-O-b-galactoside, <1> pH 6, 37 C, value above [2]) [2] pH-Optimum 6 <1> (<1> broad, b-galactosides [2]) [2] Temperature optimum ( C) 37 <1> (<1> assay at [1,2]) [1, 2]

4 Enzyme Structure Molecular weight 33400 <3> (<3> calculated from nucleotide sequence [4]) [4]

5 Isolation/Preparation/Mutation/Application Source/tissue brain <4> (<4> m-RNA expression [5]) [5] cartilage <1> (<1> epiphyses from embryonic femurs and tibiae [1,2]) [1, 2] testis <4> (<4> m-RNA expression [5]) [5] Localization Golgi apparatus <4, 5> (<4,5> medial [6]) [6] membrane <1> [1]

229

Galactosylxylosylprotein 3-b-galactosyltransferase

2.4.1.134

Purification <1> (partial [1]) [1] Cloning <2> (expression in Caenorhabditis elegans [3]) [3] <2> (transient expression as a fusion protein with protein A in chinese hamster ovary cells COS-1 [3]) [3] <3> (expression in Haemophilus ducreyi strain A77 [4]) [4] <4> (expression in Spodoptera frugiperda SF9 cells [5]) [5] <4> (transient expression as a fusion protein with protein A in chinese hamster ovary cells COS-1 [6]) [6] <5> (transient expression as a fusion protein with protein A in chinese hamster ovary cells COS-1 [6]) [6]

6 Stability Temperature stability 50 <1> (<1> 60 min, about 40% loss of activity, b-galactosides [2]) [2] 60 <1> (<1> t1=2 : 7 min, b-galactosides [2]) [2] Storage stability <1>, -20 C, microsomal enzyme preparation, at least 12 months [2]

References [1] Schwartz, N.B.; RodØn, L.: Biosynthesis of chondroitin sulfate. Solubilization of chondroitin sulfate glycosyltransferases and partial purification of uridine diphosphate-d-galactose:d-xylose galactosyltrans. J. Biol. Chem., 250, 52005207 (1975) [2] Robinson, J.A.; Robinson, H.C.: Initiation of chondroitin sulphate synthesis by b-d-galactosides. Substrates for galactosyltransferase II. Biochem. J., 227, 805-814 (1985) [3] Hwang, H.-Y.; Olson, S.K.; Brown, J.R.; Esko, J.D.; Horvitz, H.R.: The Caenorhabditis elegans genes sqv-2 and sqv-6, which are required for vulval morphogenesis, encode glycosaminoglycan galactosyltransferase II and xylosyltransferase. J. Biol. Chem., 278, 11735-11738 (2003) [4] Sun, S.; Schilling, B.; Tarantino, L.; Tullius, M.V.; Gibson, B.W.; Munson, R.S., Jr.: Cloning and characterization of the lipooligosaccharide galactosyltransferase II gene of Haemophilus ducreyi. J. Bacteriol., 182, 2292-2298 (2000) [5] Nakamura, N.; Yamakawa, N.; Sato, T.; Tojo, H.; Tachi, C.; Furukawa, K.: Differential gene expression of b-1,4-galactosyltransferases I, II and V during mouse brain development. J. Neurochem., 76, 29-38 (2001) [6] Bai, X.; Zhou, D.; Brown, J.R.; Crawford, B.E.; Hennet, T.; Esko, J.D.: Biosynthesis of the linkage region of glycosaminoglycans. Cloning and activity of galactosyltransferase II, the sixth member of the b1,3-galactosyltransferase family (b3GalT6). J. Biol. Chem., 276, 48189-48195 (2001) 230

Galactosylgalactosylxylosylprotein 3-b-glucuronosyltransferase

2.4.1.135

1 Nomenclature EC number 2.4.1.135 Systematic name UDP-glucuronate:3-b-d-galactosyl-4-b-d-galactosyl-O-b-d-xylosylprotein dglucuronosyltransferase Recommended name galactosylgalactosylxylosylprotein 3-b-glucuronosyltransferase Synonyms Galb1,3-glucuronosyltransferase GlcAT-I UDP-GlcUA:Gal b-1,3-Gal-R glucuronyltransferase UDP-GlcUA:glycoprotein b-1,3-glucuronyltransferase UDP-glucuronic acid:Galb1,3Gal-R glucuronsyltransferase b1,3-glucuronyltransferase I glucuronosyltransferase I CAS registry number 9030-08-4

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

Gallus gallus [1] Mus musculus [2] Homo sapiens [3, 4, 5, 6, 7] Cricetulus griseus (mutant lacking glucuronosyltransferase I [8]) [8]

3 Reaction and Specificity Catalyzed reaction UDP-glucuronate + 3-b-d-galactosyl-4-b-d-galactosyl-O-b-d-xylosylprotein = UDP + 3-b-d-glucuronosyl-3-b-d-galactosyl-4-b-d-galactosyl-O-b-d-xylosylprotein Reaction type hexosyl group transfer

231

Galactosylgalactosylxylosylprotein 3-b-glucuronosyltransferase

2.4.1.135

Natural substrates and products S UDPglucuronate + 3-b-d-galactosyl-4-b-d-galactosyl-O-b-d-xylosylprotein <1, 2> (<2>, involved in the biosynthesis of the heparin-polypeptide linkage region [2]; <1>, involved in the biosynthesis of the polysaccharide-protein-linkage region of proteochondroitin sulfate [1]) (Reversibility: ? <1, 2> [1, 2]) [1, 2] P ? S UDPglucuronate + Galb(1-3)-Galb(1-4)Xyl <3> (<3>, high expression of the GlcAT-I gene renders the cells capable of synthesizing the HNK-1 epitope [4]) (Reversibility: ? <3> [4]) [4] P UDP + Glc-Galb(1-3)-Galb(1-4)Xyl S Additional information <3, 4> (<3>, enzyme is involved in heparan sulfate and chondroitin sulfate biosynthesis [3]; <3>, the enzyme is involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans [4]; <3>, central enzyme in the initial steps of proteoglycan synthesis [5]; <3>, expressed in Pichia pastoris [6]; <3>, the enzyme forms the glycosaminoglycan-protein linkage region, GlcAb(1-3)Galb(13)Galb(1-4)Xylb1-O-Ser, of the proteoglycans [7]; <4>, mutant lacking glucuronosyltransferase I fails to make both heparan sulfate and chondroitin sulfate [8]) [3, 4, 5, 6, 7, 8] P ? Substrates and products S UDPglucuronate + 3-b-d-galactosyl-d-galactose <1, 2> (<1>, transfer to non-reducing terminal [1]) (Reversibility: ? <1, 2> [1, 2]) [1, 2] P UDP + 3-b-d-glucuronosyl-3-b-d-galactosyl-d-galactose <1, 2> [1, 2] S UDPglucuronate + 3-b-d-galactosyl-d-galactosyl-4-xylose <1> (Reversibility: ? <1> [1]) [1] P UDP + 3-b-d-glucuronosyl-3-b-d-galactosyl-d-galactosyl-4-xylose <1> [1] S UDPglucuronate + 3-b-d-galactosyl-d-galactosyl-4-xylosylserine <1> (Reversibility: ? <1> [1]) [1] P UDP + 3-b-d-glucuronosyl-3-b-d-galactosyl-d-galactosyl-4-xylosylserine <1> [1] S UDPglucuronate + Galb(1-3)-Galb(1-4)Xyl <3> (Reversibility: ? <3> [4]) [4] P UDP + Glc-Galb(1-3)-Galb(1-4)Xyl S Additional information <1, 3> (<1>, galactosyl-b-1,4-galactose is a less effective acceptor substrate [1]; <1,2>, galactosyl-b-1,6-galactose is a less effective acceptor substrate [1,2]; <2>, galactosyl-b-1,4-xylose, galactosylb-1,4-glucose is a less effective acceptor substrate [2]; <3>, negligible activity with Galb(1-3)Galb1-O-benzyl, Galb(1-4)GlcNAc and Galb(1-4)Glc [4]) [1, 2, 4] P ?

232

2.4.1.135

Galactosylgalactosylxylosylprotein 3-b-glucuronosyltransferase

Inhibitors 3-O-b-d-galactosyl-galactose <1> (<1>, with 3-galactosyl-4-galactosyl-xylose as substrate [1]) [1] N-phenylmaleimide <3> [6] Additional information <1> (<1>, no inhibition by chondroitin 6-sulfate trisaccharides or pentasaccharides containing N-acetylglucosamine 6-sulfate at their non-reducing termini [1]) [1] Metals, ions Cu2+ <3> (<3>, divalent cations essential, 2.7% as effective as Mn2+ [4]) [4] Mg2+ <3> (<3>, divalent cations essential, 5.5% as effective as Mn2+ [4]) [4] Mn2+ <1, 2, 3> (<1,2> required [1,2]; <1>, 15 mM [1]; <2>, 20 mM [2]; <3>, divalent cations essential, Mn2+ exhibits highest activity, optimal concentration: 2 mM [4]) [1, 2, 4] Specific activity (U/mg) Additional information <2> [2] Km-Value (mM) 0.00025 <2> (UDPglucuronate) [2] 0.0091 <2> (3-b-galactosyl-galactose) [2] 0.0293 <3> (UDPglucuronate) [4] 0.0804 <3> (Galb(1-3)Galb(1-4)Xyl) [4] 0.09489 <3> (UDPglucuronate, <3>, wild-type enzyme [6]) [6] 0.287 <3> (UDPglucuronate, <3>, mutant enzyme C33A [6]) [6] Additional information <3> [6] pH-Optimum 5-7.5 <1> [1] 6.5 <3> [4] 7.5 <2> [2] pH-Range 6.5-8.5 <2> (<2>, about 50% of maximal activity at pH 6.5 and pH 8.5 [2]) [2]

4 Enzyme Structure Molecular weight 85000 <3> (<3>, SDS-PAGE under nonreducing conditions [6]) [6] Subunits dimer <3> (<3>, 2 * 43000, SDS-PAGE after disulfide reduction [6]) [6] Posttranslational modification glycoprotein <3> [6]

233

Galactosylgalactosylxylosylprotein 3-b-glucuronosyltransferase

2.4.1.135

5 Isolation/Preparation/Mutation/Application Source/tissue cartilage <1> [1] mastocytoma cell <2> [2] placenta <3> [7] Localization membrane <1, 2> (<1,2>, bound [1,2]) [1, 2] microsome <2> [2] Purification <2> (partial [2]) [2] Crystallization <3> (crystal structure in presence of the donor substrate UDP-glucuronic acid [3]; crystal structure of the enzyme at 2.3 A in the presence of the UDP, Mn2+ and Galb(1-3)Galb(1-4)Xyl [5]) [3, 5] Cloning <3> (expression of full-length GlcAT-I in COS-1 cells [4]; expression in Pichia pastoris [6]; expression in COS-1 cells [7]) [4, 6, 7] Engineering C301A <3> (<3>, mutant is not N-glycosylated, molecular weight is about 4000 Da less than that of the wild-type protein, enzyme is completely inactive [6]) [6] C33A <3> (<3>, mutation abolishes the ability of the protein to form dimers [6]) [6]

References [1] Helting, T.; Roden, L.: Biosynthesis of chondroitin sulfate. II. Glucuronosyl transfer in the formation of the carbohydrate-protein linkage region. J. Biol. Chem., 244, 2799-2805 (1969) [2] Helting, T.: Biosynthesis of heparin. Solubilization and partial purification of uridine diphosphate glucuronic acid: acceptor glucuronosyltransferase from mouse mastocytoma. J. Biol. Chem., 247, 4327-4332 (1972) [3] Pedersen, L.C.; Darden, T.A.; Negishi, M.: Crystal structure of b1,3-glucuronyltransferase I in complex with active donor substrate UDP-GlcUA. J. Biol. Chem., 277, 21869-21873 (2002) [4] Tone, Y.; Kitagawa, H.; Imiya, K.; Oka, S.; Kawasaki, T.; Sugahara, K.: Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. FEBS Lett., 459, 415-420 (1999) [5] Pedersen, L.C.; Tsuchida, K.; Kitagawa, H.; Sugahara, K.; Darden, T.A.; Negishi, M.: Heparan/chondroitin sulfate biosynthesis. Structure and mechan-

234

2.4.1.135

Galactosylgalactosylxylosylprotein 3-b-glucuronosyltransferase

ism of human glucuronyltransferase I. J. Biol. Chem., 275, 34580-34585 (2000) [6] Ouzzine, M.; Gulberti, S.; Netter, P.; Magdalou, J.; Fournel-Gigleux, S.: Structure/function of the human Ga1b1,3-glucuronosyltransferase. Dimerization and functional activity are mediated by two crucial cysteine residues. J. Biol. Chem., 275, 28254-28260 (2000) [7] Kitagawa, H.; Tone, Y.; Tamura, J.; Neumann, K.W.; Ogawa, T.; Oka, S.; Kawasaki, T.; Sugahara, K.: Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. J. Biol. Chem., 273, 6615-6618 (1998) [8] Bai, X.; Wei, G.; Sinha, A.; Esko, J.D.: Chinese hamster ovary cell mutants defective in glycosaminoglycan assembly and glucuronosyltransferase I. J. Biol. Chem., 274, 13017-13024 (1999)

235

Gallate 1-b-glucosyltransferase

2.4.1.136

1 Nomenclature EC number 2.4.1.136 Systematic name UDP-glucose:gallate b-d-glucosyltransferase Recommended name gallate 1-b-glucosyltransferase Synonyms UDP-glucose-vanillate 1-glucosyltransferase UDP-glucose:gallate glucosyltransferase UDP-glucose:vanillate 1-O-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-vanillate 1CAS registry number 89700-30-1

2 Source Organism <1> Quercus rubra [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + gallate = UDP + 1-galloyl-b-d-glucose Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + gallate <1> (<1>, physiological role probably is the formation of b-glucogallin, the putative first intermediate in the biosynthesis of gallotannins [1,2]) (Reversibility: r <1> [1, 2]) [1, 2] P UDP + 1-O-galloyl-b-d-glucose <1> (i.e. b-glucogallin) [1, 2] Substrates and products S UDPglucose + 3,4,5-trimethoxybenzoate <1> (<1>, 7% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-3,4,5-trimethoxybenzoyl-b-d-glucose

236

2.4.1.136

Gallate 1-b-glucosyltransferase

S UDPglucose + anisate <1> (<1>, 38% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-anisoyl-b-d-glucose S UDPglucose + benzoate <1> (<1>, 16% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + benzoyl-b-d-glucose S UDPglucose + cinnamate <1> (<1>, 9% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-p-cinnamoyl-b-d-glucose S UDPglucose + ferulate <1> (<1>, 35% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-feruloyl-b-d-glucose S UDPglucose + gallate <1> (<1>, 52% of activity compared to vanillate [1]) (Reversibility: r <1> [1, 2]) [1, 2] P UDP + 1-O-galloyl-b-d-glucose <1> (i.e. b-glucogallin) [1, 2] S UDPglucose + m-coumarate <1> (<1>, 14% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-m-coumaroyl-b-d-glucose S UDPglucose + p-coumarate <1> (<1>, 32% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-p-coumaroyl-b-d-glucose S UDPglucose + p-hydroxybenzoate <1> (<1>, 48% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-hydroxybenzoyl-b-d-glucose S UDPglucose + protocatechuate <1> (<1>, 22% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + protocatechoyl-b-d-glucose S UDPglucose + sinapate <1> (<1>, 13% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-sinapoyl-b-d-glucose S UDPglucose + vanillate <1> (<1>, best substrate [1]) (Reversibility: ? <1> [1]) [1] P UDP + vanilloyl-b-d-glucose S UDPglucose + veratrate <1> (<1>, 71% of activity compared to vanillate [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1-O-veratroyl-b-d-glucose Inhibitors UDP <1> (<1>, strong [1]) [1] dithiothreitol <1> (<1>, 4 mM, slight [1]) [1] Specific activity (U/mg) 0.0117 <1> (<1>, reaction with gallate [1]) [1] Km-Value (mM) 0.57 <1> (vanillate) [1] 0.72 <1> (veratrate) [1] 1.1 <1> (gallate) [2]

237

Gallate 1-b-glucosyltransferase

2.4.1.136

1.11 <1> (gallate) [1] 2.3 <1> (UDPglucose, <1>, reaction with gallate [2]) [2] 3.45 <1> (p-hydroxybenzoate) [1] pH-Optimum 6.5-7 <1> [1] 7 <1> (<1>, Tris-HCl or KHPO4 - buffer [2]) [2] pH-Range 4.7-9 <1> (<1>, 50% of maximal activity at pH 4.7 and 9.0 [1]) [1] 5.5-8.5 <1> (<1>, 50% of maximal activity at pH 5.5 and 8.5 [2]) [2] Temperature optimum ( C) 42 <1> [1]

4 Enzyme Structure Molecular weight 68000 <1> (<1>, gel filtration [1]) [1]

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

6 Stability Oxidation stability <1>, O2 -sensitive, mercaptoethanol stabilizes [1] General stability information <1>, 2-mercaptoethanol in extraction buffers stabilizes [1] <1>, diluted enzyme is unstable [1] Storage stability <1>, 0-4 C, 1-2 weeks [1]

References [1] Gross, G.: Partial purification and properties of UDP-glucose: vanillate 1-Oglucosyl transferase from oak leaves. Phytochemistry, 22, 2179-2182 (1983) [2] Gross, G.: Synthesis of b-glucogallin from UDP-glucose and gallic acid by an enzyme preparation from oak leaves. FEBS Lett., 148, 67-70 (1982) 238

sn-Glycerol-3-phosphate 2-a-galactosyltransferase

2.4.1.137

1 Nomenclature EC number 2.4.1.137 Systematic name UDP-galactose:sn-glycerol-3-phosphate 2-a-d-galactosyltransferase Recommended name sn-glycerol-3-phosphate 2-a-galactosyltransferase Synonyms FPS UDP-galactose, sn-3-glycerol phosphate:1-2' galactosyltransferase UDP-galactose:sn-glycerol-3-phosphate-2-d-galactosyl transferase floridoside phosphate synthase floridoside-phosphate synthase synthetase, floridoside phosphate Additional information (cf. EC 2.4.1.96) CAS registry number 80747-34-8

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

Porphyra perforata [2] Dumontia incrassata (O.F. Müller, Lamour. [1]) [1] Chondrus crispus (Stackh. [1]) [1] Cystoclonium purpureum (Lightf., Batt. [1]) [1] Corallina officinalis [1] Porphyra umbilicalis [1] Lomentaria umbellata (H. u. H., Gendo [1]) [1] Catenella nipea (Zan. [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + sn-glycerol 3-phosphate = UDP + 2-(a-d-galactosyl)-snglycerol 3-phosphate

239

sn-Glycerol-3-phosphate 2-a-galactosyltransferase

2.4.1.137

Reaction type hexosyl group transfer Natural substrates and products S UDPgalactose + sn-glycerol 3-phosphate <1-8> (<2-8> involved in biosynthesis of floridoside, a typical low-molecular weight red algal carbohydrate [1]; <1> key enzyme in floridoside biosynthesis [2]) [1, 2] P UDP + 2-(a-d-galactosyl)-sn-glycerol 3-phosphate Substrates and products S UDPgalactose + sn-glycerol 3-phosphate <1-8> (<1> high substrate specificity [2]) (Reversibility: ? <1-8> [1,2]) [1, 2] P UDP + 2-(a-d-galactosyl)-sn-glycerol 3-phosphate <2-8> (<2-8> the product is hydrolyzed by a phosphatase to floridoside [1]) [1] Inhibitors UDP <1> (<1> 5 mM, competitive [2]) [2] Cofactors/prosthetic groups Additional information <1> (<1> no cofactor or activator found [2]) [2] Activating compounds Additional information <1> (<1> no cofactor or activator found [2]) [2] Specific activity (U/mg) 0.177 <1> [2] Km-Value (mM) 2.7 <1> (UDPgalactose) [2] 8 <1> (sn-glycerol 3-phosphate) [2] pH-Optimum 7.5 <2-8> (<2-8> assay at [1]) [1] Temperature optimum ( C) 20 <2-8> (<2-8> assay at [1]) [1]

4 Enzyme Structure Molecular weight 120000 <1> (<1> HPLC gel filtration [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue thallus <2-8> [1] Purification <1> (25fold [2]) [2]

240

2.4.1.137

sn-Glycerol-3-phosphate 2-a-galactosyltransferase

References [1] Kremer, B.P.; Kirst, G.O.: Biosynthesis of 2-O-d-glycerol-a-d-galactopyranoside (floridoside) in marine Rhodophyceae. Plant Sci. Lett., 23, 349-357 (1981) [2] Meng, J.; Srivastava, L.M.: Partial purification and characterization of floridoside phosphate synthase from Porphyra perforata. Phytochemistry, 30, 1763-1766 (1991)

241

2.4.1.138

Mannotetraose 2-a-N-acetylglucosaminyltransferase

1 Nomenclature EC number 2.4.1.138 Systematic name UDP-N-acetyl-d-glucosamine:mannotetraose transferase

a-N-acetyl-d-glucosaminyl-

Recommended name mannotetraose 2-a-N-acetylglucosaminyltransferase Synonyms acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine mannoside a1 -2a-N-acetylglucosaminyltransferase uridine diphosphoacetylglucosamine mannoside a1-2-acetylglucosaminyltransferase CAS registry number 81032-47-5

2 Source Organism <1> Kluyveromyces lactis (strain KL8 [2]; yeast, wild-type strains Y-58a his 4c or his 3 and mutant strains mnn1, mnn2-1 or mnn2-2 [1]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + 1,3-a-d-mannosyl-1,2-a-d-mannosyl-1,2-ad-mannosyl-d-mannose = UDP + 1,3-a-d-mannosyl-1,2-(N-acetyl-a-d-glucosaminyl-a-d-mannosyl)-1,2-a-d-mannosyl-d-mannose Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + 1,3-a-d-mannosyl-1,2-a-d-mannosyl1,2-a-d-mannosyl-d-mannose <1> (<1> involved in biosynthesis of mannoprotein side-chain units [1,2]) (Reversibility: ? <1> [1, 2]) [1, 2]

242

2.4.1.138

Mannotetraose 2-a-N-acetylglucosaminyltransferase

P UDP + 1,3-a-d-mannosyl-1,2-(N-acetyl-a-d-glucosaminyl-a-d-mannosyl)-1,2-a-d-mannosyl-d-mannose <1> [1, 2] Substrates and products S UDP-N-acetyl-d-glucosamine + 1,3-a-d-mannosyl-1,2-a-d-mannosyl1,2-a-d-mannosyl-d-mannose <1> (<1> i.e. mannotetraose acceptor: acceptor substrates are mannoprotein, saccharides with more than 25 mannose-units, with 15 to 25 units, with 8 to 15 units, with 5 to 8 units and below 5 units, the latter being the best substrates [1]) (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + 1,3-a-d-mannosyl-1,2-(N-acetyl-a-d-glucosaminyl-a-d-mannosyl)-1,2-a-d-mannosyl-d-mannose <1> [1, 2] S Additional information <1> (<1> enzyme depends on the UDPase, which generates UDP as antiporter to transport UDP-N-acetyl-d-glucosamine into the Golgi lumen [2]) [2] P ? Inhibitors Co2+ <1> (<1> strong, 1 mM, even in the presence of Mn2+ , wild-type [1]) [1] Cu2+ <1> (<1> wild-type [1]) [1] Mn2+ <1> (<1> above 10 mM, wild-type [1]) [1] NaCl <1> (<1> weak [1]) [1] NaN3 <1> [1] Tris <1> (<1> wild-type [1]) [1] Tween 80 <1> [1] UDP <1> (<1> wild-type [1]) [1, 2] UDP-hexylamine <1> (<1> wild-type [1]) [1] imidazole <1> (<1> wild-type [1]) [1] phosphate <1> (<1> wild-type, complete inhibition at 40 mM [1]) [1] Metals, ions Mn2+ <1> (<1> requirement, 10 mM, inhibits at higher concentrations, Mg2+ or Ca2+ cannot replace Mn2+ [1]) [1] Specific activity (U/mg) 0.0056 <1> [1] Additional information <1> (<1> activity of wild-type and mutant strains with different acceptors [1]) [1] Km-Value (mM) 0.0364 <1> (UDP-N-acetylglucosamine, <1> mutant 2-2 [1]) [1] 0.0447 <1> (UDP-N-acetylglucosamine, <1> wild-type [1]) [1] 11 <1> (mannotetraose acceptor, <1> solubilized enzyme [1]) [1] 13 <1> (mannotetraose acceptor, <1> membrane-bound enzyme [1]) [1] pH-Optimum 6.5 <1> [1] Additional information <1> (<1> pI: 4.9, in the presence of Triton X-100 and 2 M urea [1]) [1] 243

Mannotetraose 2-a-N-acetylglucosaminyltransferase

2.4.1.138

4 Enzyme Structure Molecular weight 300000 <1> (<1> gel filtration in the presence of Triton X-100, native MW may be lower [1]) [1] Posttranslational modification glycoprotein <1> (<1> possesses no N-acetylglucosamine residues, enzyme does not bind to wheat germ agglutinin [1]) [1]

5 Isolation/Preparation/Mutation/Application Localization Golgi membrane <1> (<1> catalytic reaction takes place in the lumen of Golgi vesicles [2]) [2] endoplasmic reticulum <1> (<1> integral membrane protein [1]) [1] protoplast <1> [1] Purification <1> (partial [1]) [1] Engineering Additional information <1> (<1> enzyme activity in KL8 null mutant, which has no remaining UDPase activity, is reduced due to lack of UDP-N-acetyl-dglucosaminose [2]; <1> analysis of mutations in strain mnn2-2 [1]) [1, 2]

6 Stability Temperature stability 35 <1> (<1> 2 h, inactivation [1]) [1] 45 <1> (<1> t1=2 : 4 min [1]) [1] Organic solvent stability Additional information <1> (<1> organic solvent extraction, unstable to [1]) [1] General stability information <1>, Triton X-100 stabilizes solubilized enzyme [1] Storage stability <1>, 4 C, in imidazole-glycerol buffer, 2% Triton X-100, t1=2 : 80 days [1]

244

2.4.1.138

Mannotetraose 2-a-N-acetylglucosaminyltransferase

References [1] Douglas, R.H.; Ballou, C.E.: Purification of an a-N-acetylglucosaminyltransferase from the yeast Kluyveromyces lactis and a study of mutants defective in this enzyme activity. Biochemistry, 21, 1561-1570 (1982) [2] Lopez-Avalos, M.D.; Uccelletti, D.; Abeijon, C.; Hirschberg, C.B.: The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles. Glycobiology, 11, 413-422 (2001)

245

Maltose synthase

2.4.1.139

1 Nomenclature EC number 2.4.1.139 Systematic name a-d-glucose-1-phosphate:a-d-glucose-1-phosphate 4-a-d-glucosyltransferase (dephosphorylating) Recommended name maltose synthase Synonyms synthase, maltose CAS registry number 81669-74-1

2 Source Organism <1> Spinacia oleracea (spinach) [1]

3 Reaction and Specificity Catalyzed reaction 2 a-d-glucose 1-phosphate = maltose + 2 phosphate Reaction type hexosyl group transfer Natural substrates and products S a-d-glucose 1-phosphate + a-d-glucose 1-phosphate <1> (Reversibility: ? <1> [1]) [1] P maltose + 2 phosphate <1> [1] Substrates and products S Additional information <1> (<1>, exchange of phosphate group of a-dglucose 1-phosphate with phosphate is possible, neither maltose 1-phosphate nor free phosphate can be detected as an intermediate [1]) [1] P ?

246

2.4.1.139

Maltose synthase

Inhibitors ADPglucose <1> (<1>, strong [1]) [1] ATP <1> [1] d-glucono-1,5-lactone <1> (<1>, competitive [1]) [1] GDPglucose <1> (<1>, strong [1]) [1] UDPglucose <1> (<1>, strong [1]) [1] b-d-glucose 1-phosphate <1> [1] phosphate <1> [1] Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 1.5 <1> (a-d-glucose 1-phosphate) [1] pH-Optimum 6.8 <1> [1] pH-Range 6.2-7.5 <1> (<1>, 30% of maximal activity at pH 6.2, 45% of maximal activity at pH 7.5 [1]) [1]

4 Enzyme Structure Molecular weight 95000 <1> (<1>, gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue cotyledon <1> [1] Purification <1> (partial [1]) [1]

6 Stability General stability information <1>, during purification addition of 15 mM a-d-glucose 1-phosphate to the isolation buffer is necessary for stabilization [1] <1>, purification has to be carried out at 4 C [1]

References [1] Schilling, N.: Characterization of maltose biosynthesis from a-d-gluccose-1phosphate in Spinacia oleracea L.. Planta, 154, 87-93 (1982)

247

Alternansucrase

2.4.1.140

1 Nomenclature EC number 2.4.1.140 Systematic name sucrose:1,6(1,3)-a-d-glucan 6(3)-a-d-glucosyltransferase Recommended name alternansucrase Synonyms glucosyltransferase, sucrose-1,6(3)-a-glucan 6(3)-asucrose:1,6-, 1,3-a-d-glucan 3-a- and 6-a-d-glucosyltransferase CAS registry number 100630-46-4

2 Source Organism <1> Leuconostoc mesenteroides (NRRL B-1355 [1,2,5,7]; strains: R15, R1579, R1622 and R1535 [7]; NRRL B-23192 [10]) [1, 2, 5, 6, 7, 8, 9, 10] <2> Streptococcus mutans (Ingbritt, serotype c [3]; HS6 serotype a [4]) [3, 4]

3 Reaction and Specificity Catalyzed reaction transfers alternately an a-d-glucosyl residue from sucrose to the 6-position and the 3-position of the non-reducing terminal residue of an a-d-glucan, thus producing a glucan having alternating a-1,6- and a-1,3-linkages Reaction type hexosyl group transfer Substrates and products S sucose + butyl-a-D-glucopyranoside <1> (Reversibility: ? <1> [9]) [9] P ? S sucose + octyl-a-d-glucopyranoside <1> (Reversibility: ? <1> [9]) [9] P ? S sucrose + a-d-glucan <1, 2> (Reversibility: ? <1, 2> [1-10]) [1-10]

248

2.4.1.140

Alternansucrase

P alternating-1,6-1,3-a-d-glucan <1, 2> (<2>, the product is an insoluble dglucan that consists of 76 mol% 1,3-a-linked glucose and 24 mol% 1,6-alinked glucose [3]; <2>, glucan consists of 49.1 mol% 1,6-a-linked glucose and 33.9 mol% 1,3-a-linked glucose with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-a-branched glucose [4]) [1-4] S sucrose + cellobiose <1> (Reversibility: ? <1> [10]) [10] P oligosaccharides <1> (<1>, a-d-glucopyranosyl-(1,2)-(b-d-glucopyranosyl-(1,4))-d-glucopyranose + a-d-glucopyranosyl-(1,6)-b-d-glucopyranosyl-(1,4)-d-glucopyranose, the last compound in turn can be glycosylated leading to the synthesis of a tetrasaccharide with an additional a-(1,6)linkage at the non-reducing end [10]) [10] S sucrose + isomaltose <1> (Reversibility: ? <1> [2]) [2] P oligoalternan <1> [2] S sucrose + maltose <1> (Reversibility: ? <1> [2, 5]) [2, 5] P oligoalternan <1> (<1>, panose is the first acceptor product [2]) [2] S sucrose + methyl-a-d-glucoside <1> (Reversibility: ? <1> [2]) [2] P oligoalternan <1> [2] Inhibitors 2-aminoethanol <1> [1] 3-deoxy-3-fluoro-a-d-glucopyranosyl fluoride <1> [1] SDS <1> [1] Tris(hydroxymethyl)aminomethane <1> [1] octyl b-d-glucopyranoside <1> [1] Additional information <2> (<2>, below a sodium phosphate buffer concentration of 50 mM the activity is reduced by 75% [3]) [3] Activating compounds dextran T10 <2> (<2>, 1.7fold stimulation [3]) [3] Specific activity (U/mg) 7.31 <2> [3] 89.7 <2> [4] Km-Value (mM) 4.9 <2> (sucrose) [4] 16.3 <2> (sucrose) [3] pH-Optimum 5.5 <1> [1] 5.6 <1> [2] 6 <2> [4] 6.5 <2> [3] pH-Range 4-7.2 <1> (<1>, pH 4.0: about 75% of maximal activity, pH 7.2: about 60% of maximal activity [1]) [1] 4.6-6.4 <1> (<1>, about 50% of activity maximum at pH 4.6 and 6.4 [2]) [2]

249

Alternansucrase

2.4.1.140

Temperature optimum ( C) 40 <1> [2] Temperature range ( C) 30-50 <1> (<1>, 30 C: about 65% of maximal activity, 50 C: about 55% of maximal activity [2]) [2]

4 Enzyme Structure Subunits ? <2> (<2>, x * 158000, SDS-PAGE with and without 2-mercaptoethanol [3]; <2>, x * 159000, SDS-PAGE [4]) [3, 4] Additional information <1> (<1>, cultures of strains B-1355 and R15 have a principal alternansucrase band of 204000 Da. These strains also produce minor alternansucrase bands. Cultures of strains R1579, R1622 and R1535 produce principal alternansucrase activity bands of 191000 Da, 161000 Da and 150000 Da, respectively and numerous minor bands [7]) [7] Posttranslational modification glycoprotein <2> (<2>, 1.5% carbohydrate [4]) [4]

5 Isolation/Preparation/Mutation/Application Source/tissue culture medium <1, 2> [1, 2, 4] Localization cell associated <2> [3] extracellular <1, 2> [1, 4] particle-bound <1> (<1>, the concentration of the enzyme in the particulate fraction suggests that it is associated primarily with the cell wall or membrane [6]) [6] Purification <1> (NRLL B-1355 [2]) [2] <2> [3, 4] Cloning <1> (expression in Escherichia coli [5]) [5] Application synthesis <1> (<1>, modification of bacterial cellulose using sucrose, dextransucrase from Leuconostoc mesenteroides B-742CMB and alternansucrase from Leuconostoc B-1355C. This modification method produces a new bacterial cellulose that has a unique structure and probably new property [8]) [8]

250

2.4.1.140

Alternansucrase

6 Stability Temperature stability 40 <1> (<1>, pH 5.4, half-life: 2 days [2]) [2]

References [1] Cote, G.L.; Robyt, J.F.: Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1-6),(1-3)-a-d-glucan. Carbohydr. Res., 101, 57-74 (1982) [2] Lopez-Munguia, A.; Pelenc, V.; Remaud, M.; Biton, J.; Michel, J.M.; Lang, C.; Paul, F.; Monsan, P.: Production and purification of alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355, for the synthesis of oligoalternan. Enzyme Microb. Technol., 15, 77-85 (1993) [3] Mukasa, H.; Shimamura, A.; Tsumori, H.: Purification and characterization of cell-associated glucosyltransferase synthesizing insoluble glucan from Streptococcus mutans serotype c. J. Gen. Microbiol., 135, 2055-2063 (1989) [4] Tsumori, H.; Shimamura, A.; Mukasa, H.: Purification and properties of extracellular glucosyltransferase synthesizing 1,6-, 1,3-a-d-glucan from Streptococcus mutans serotype a. J. Gen. Microbiol., 131, 3347-3353 (1985) [5] Arguello-Morales, M.A.; Remaud-Simeon, M.; Pizzut, S.; Sarcabal, P.; Willemot, R.M.; Monsan, P.: Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. FEMS Microbiol. Lett., 182, 81-85 (2000) [6] Zahnley, J.C.; Smith, M.R.: Cellular association of glucosyltransferases in Leuconostoc mesenteroides and effects of detergent on cell association. Appl. Biochem. Biotechnol., 87, 57-70 (2000) [7] Smith, M.R.; Zahnley, J.C.: Leuconostoc mesenteroides B-1355 mutants producing alternansucrases exhibiting decreases in apparent molecular mass. Appl. Environ. Microbiol., 63, 581-586 (1997) [8] Kim, D.; Kim, Y.M.; Park, M.R.; Park, D.H.: Modification of Acetobacter xylinum bacterial cellulose using dextransucrase and alternansucrase. J. Microbiol. Biotechnol., 9, 704-708 (1999) [9] Richard, G.; Morel, S.; Willemot, R.M.; Monsan, P.; Remaud-Simeon, M.: Glucosylation of a-butyl- and a-octyl-d-glucopyranosides by dextransucrase and alternansucrase from Leuconostoc mesenteroides. Carbohydr. Res., 338, 855-864 (2003) [10] Arguello Morales, M.A.; Remaud-Simeon, M.; Willemot, R.M.; Vignon, M.R.; Monsan, P.: Novel oligosaccharides synthesized from sucrose donor and cellobiose acceptor by alternansucrase. Carbohydr. Res., 331, 403-411 (2001)

251

N-Acetylglucosaminyldiphosphodolichol N-acetylglucosaminyltransferase

2.4.1.141

1 Nomenclature EC number 2.4.1.141 Systematic name UDP-N-acetyl-d-glucosamine:N-acetyl-d-glucosaminyl-diphosphodolichol N-acetyl-d-glucosaminyltransferase Recommended name N-acetylglucosaminyldiphosphodolichol N-acetylglucosaminyltransferase Synonyms N,N'-diacetylchitobiosylpyrophosphoryldolichol synthase UDP-GlcNAc:dolichyl-pyrophosphoryl-GlcNAc GlcNAc transferase uridine diphosphoacetylglucosamine-dolichylacetylglucosamine pyrophosphate acetylglucosaminyltransferase CAS registry number 75536-54-8

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

Homo sapiens [1] Rattus norvegicus [1] Vigna radiata (var. radiata [2]) [2] Cricetulus griseus [3] Gallus gallus (15-16 days old [4,5]) [4, 5] Sus scrofa [6]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-diphosphodolichol = UDP + N,N'-diacetylchitobiosyl-diphosphodolichol Reaction type hexosyl group transfer

252

2.4.1.141

N-Acetylglucosaminyldiphosphodolichol N-acetylglucosaminyltransferase

Natural substrates and products S N-acetyl-d-glucosaminyl-diphosphodolichol + UDPglucosyl-glucuronic acid <2, 3> (Reversibility: ? <2, 3> [1, 2]) [1, 2] P glucosyl-glucuronic acid-glucosyl-N-acetyl-diphosphodolichol + UDP <2, 3> [1, 2] S UDP-N-acetyl-d-glucosamine + dolichol-phosphate <2> (Reversibility: ? <2> [1]) [1] P N-acetyl-d-glucosaminyl-diphosphodolichol + UMP <2> [1] Substrates and products S N-acetyl-d-glucosaminyl-diphosphodolichol + UDP-N-acetyl-d-glucosamine <3, 5> (Reversibility: ? <3,5> [2,4]) [2, 4] P diphosphodolichyl-N-acetyl-d-glucosaminyl-N-acetyl-d-glucosamine + UDP <3> [2] S N-acetyl-d-glucosaminyl-diphosphodolichol + UDPglucosyl-glucuronic acid <2> (Reversibility: ? <2> [1]) [1] P glucosyl-glucuronic acid-glucosyl-N-acetyl-diphosphodolichol + UDP <2> [1] S UDP-N-acetyl-d-glucosamine + dolichol-phosphate <2, 5> (Reversibility: ? <2,5> [1,4]) [1, 4] P N-acetyl-d-glucosaminyl-diphosphodolichol + UMP <2, 5> [1, 4] Inhibitors EDTA <3, 5> [2, 4] Mg2+ <5> (<5> concentrations above 6.7 mM [4]) [4] N-acetyl-d-glucosaminyl-diphosphodolichol <5> (<5> exogenously added, in presence of mannosyl-phosphodolichol [5]) [5] P1-dolichyl P2-(2-O-ethyl-a-d-glucopyranosyl) diphosphate <6> (<6> inhibitors when studied in competition with N-acetyl-d-glucosaminyl-diphosphodolichol [6]) [6] P1-dolichyl P2-(2-deoxy-2-fluoro-a-d-glucopyranosyl) diphosphate <6> (<6> inhibitors when studied in competition with N-acetyl-d-glucosaminyldiphosphodolichol [6]) [6] P1-dolichyl P2-(2-deoxy-2-trifluoroacetamido-a-d-glucopyranosyl) diphosphate <6> (<6> inhibitors when studied in competition with N-acetyl-d-glucosaminyl-diphosphodolichol [6]) [6] UDP <3, 5> [2, 4] UDPglucose <3, 5> [2, 4] UMP <3, 5> [2, 4] UTP <3, 5> [2, 4] amphomycin <5> (<5> partial inhibition [4]) [4] bacitracin <3> [2] diphosphodolichyl-N-acetyl-d-glucosaminyl-N-acetyl-d-glucosamine <5> (<5> exogenously added, extensive inhibition in absence and presence of +mannosyl-phosphodolichol and showdomycin [5]) [5] diumycin <3, 5> [2, 4] showdomycin <5> (<5> complete inhibition [4,5]) [4, 5]

253

N-Acetylglucosaminyldiphosphodolichol N-acetylglucosaminyltransferase

2.4.1.141

Activating compounds TX-100 <5> (<5> 0.15% [4]) [4] Triton X-100 <3, 4> (<3,4> strong requirement [2,3]) [2, 3] mannosyl-phosphodolichol <5> [5] Metals, ions Mg2+ <3, 5> (<3> stimulates [2]; <5> slight enhancement in activity at 6.7 mM [4]) [2, 4] Mn2+ <3, 5> (<3,5> stimulates, not as effective as Mg2+ [2,4]) [2, 4] Km-Value (mM) 0.0002 <4> (N-acetyl-d-glucosaminyl-diphosphodolichol) [3] 0.00025 <3> (UDP-N-acetyl-d-glucosamine) [2] 0.0022 <3> (N-acetyl-d-glucosaminyl-diphosphodolichol) [2] 0.00264 <5> (N-acetyl-d-glucosaminyl-diphosphodolichol) [4] 0.0571 <5> (UDP-N-acetyl-d-glucosamine) [4] Ki-Value (mM) 0.0028 <5> (diphosphodolichyl-N-acetyl-d-glucosaminyl-N-acetyl-d-glucosamine) [5] 0.0044 <5> (N-acetyl-d-glucosaminyl-diphosphodolichol) [5] pH-Optimum 7 <6> [6] 7.4 <4> [3] 7.5 <5> [4] pH-Range 7.4-7.6 <3> [2] Temperature optimum ( C) 37 <3, 4, 5> (<3,4,5> assay at [2-5]) [2-5]

5 Isolation/Preparation/Mutation/Application Source/tissue fibroblast <1> [1] liver <6> [6] lung <1, 2> [1] ovary <4> [3] retina <5> [4, 5] seedling <3> [2] Localization membrane <4> [3] microsome <2, 3, 5, 6> [1, 2, 4, 5, 6] Purification <3> (solubilized enzyme purified on DE-52 column [2]) [2]

254

2.4.1.141

N-Acetylglucosaminyldiphosphodolichol N-acetylglucosaminyltransferase

6 Stability Storage stability <3>, -20 C, 0.5 mM DTT, pH 7.2, 20% glycerol, 10% loss of activity after 1 month [2] <4>, 4 C, Tris-buffered saline, pH 7.4, 48 h [3] <6>, -80 C, 30% glycerol, several months [6]

References [1] Turco, S.J.; Heath, E.C.: Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations. J. Biol. Chem., 252, 2918-2928 (1977) [2] Kaushal, G.P.; Elbein, A.D.: Purification and properties of UDP-GlcNAc:dolichyl-pyrophosphoryl-GlcNAc transferase from mung bean seedling. Plant Physiol., 81, 1086-1091 (1986) [3] McLachlan, K.R.; Krag, S.S.: Three enzymes involved in oligosaccharide-lipid assembly in chinese hamster ovary cells differ in lipid substrate preference. J. Lipid Res., 35, 1861-1868 (1994) [4] Kean, E.L.; Niu, N.: Kinetics of formation of GlcNAc-GlcNAc-P-P-dolichol by microsomes from the retina of the embryonic chick. Glycoconjugate J., 15, 11-17 (1998) [5] Kean, E.L.; Wei, Z.; Anderson, V.E.; Zhang, N.; Sayre, L.M.: Regulation of the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol, feedback and product inhibition. J. Biol. Chem., 274, 34072-34082 (1999) [6] Tai, V.W.F.; O'Reilly, M.K.; Imperiali, B.: Substrate specificity of N-acetylglucosaminyl(diphosphodolichol) N-acetylglucosaminyl transferase, a key enzyme in the dolichol pathway. Bioorg. Med. Chem., 9, 1133-1140 (2001)

255

Chitobiosyldiphosphodolichol b-mannosyltransferase

2.4.1.142

1 Nomenclature EC number 2.4.1.142 Systematic name GDP-mannose:chitobiosyldiphosphodolichol b-d-mannosyltransferase Recommended name chitobiosyldiphosphodolichol b-mannosyltransferase Synonyms GDP-mannose-dolichol diphosphochitobiose mannosyltransferase guanosine diphosphomannose-dolichol diphosphochitobiose mannosyltransferase CAS registry number 83380-85-2

2 Source Organism <1> Sus scrofa [1] <2> Glycine max [2]

3 Reaction and Specificity Catalyzed reaction GDP-mannose + chitobiosyldiphosphodolichol = GDP + b-1,4-d-mannosylchitobiosyldiphosphodolichol Reaction type hexosyl group transfer Natural substrates and products S GDP-mannose + diphosphodolichyl-N-acetyl-d-glucosaminyl-N-acetyl-dglucosamine <1> [1] P GDP + mannose-b-diphosphodolichyl-N-acetyl-d-glucosaminyl-N-acetyld-glucosamine

256

2.4.1.142

Chitobiosyldiphosphodolichol b-mannosyltransferase

Substrates and products S GDP-mannose + diphosphodolichyl-N-acetyl-d-glucosaminyl-N-acetyl-dglucosamine <1, 2> (Reversibility: ? <1,2> [1,2]) [1, 2] P GDP + mannose-b-diphosphodolichyl-N-acetyl-d-glucosaminyl-N-acetyld-glucosamine <1, 2> [1, 2] Inhibitors GDP <1, 2> [1, 2] GDP-glucose <1, 2> [1, 2] GMP <1, 2> [1, 2] GTP <1, 2> [1, 2] p-chloromercuribenzenesulfonic acid <1> [1] Activating compounds NP-40 <1, 2> (<1> optimum activity at 0.1% [1]) [1, 2] Triton X-100 <2> (<2> optimum activity at 0.08-0.1% [2]) [2] phosphatidic acid <2> [2] phosphatidylcholine <2> [2] phosphatidylserine <2> [2] phospholipid <2> [2] Metals, ions Ca2+ <1> (<1> less stimulatory than Mg2+ [1]) [1] Mg2+ <1, 2> (<1> absolute requirement for activity, optimum activity at 5mM [1]; <2> requirement for activity [2]) [1, 2] Mn2+ <1> (<1> less stimulatory than Mg2+ [1]) [1] Specific activity (U/mg) Additional information <1, 2> [1, 2] Km-Value (mM) 0.0005 <1> (GDP-mannose) [1] 0.0017 <2> (GDP-mannose) [2] pH-Optimum 6.9-7 <2> [2] 7 <1> [1] Temperature optimum ( C) 37 <1> (<1> assay at [1]) [1]

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

257

Chitobiosyldiphosphodolichol b-mannosyltransferase

2.4.1.142

Purification <1> (DEAE-cellulose, epoxy-activated-Sepharose [1]) [1] <2> (DEAE-cellulose, GDP-affinity column [2]) [2]

6 Stability Storage stability <1>, -18 C, 0.5 mM DTT, 20% glycerol, several weeks [1] <2>, frozen, Tris-buffer, pH 7.0, 0.5 mM DTT, 20% glycerol, 0.1% detergent, 1 month [2]

References [1] Kaushal, G.P.; Elbein, A.D.: Purification and properties of b-mannosyltransferase that synthesizes Man-b-GlcNAc-GlcNAc-pyrophosphoryl-dolichol. Arch. Biochem. Biophys., 250, 38-47 (1986) [2] Kaushal, G.P.; Elbein, A.D.: Partial purification and characterization of bmannosyltransferase from suspension-cultured soybean cells. Biochemistry, 26, 7953-7960 (1987)

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1 Nomenclature EC number 2.4.1.143 Systematic name UDP-N-acetyl-d-glucosamine:6-(a-d-mannosyl)-b-d-mannosyl-glycoprotein 2-b-N-acetyl-d-glucosaminyltransferase Recommended name a-1,6-mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase Synonyms N-acetylglucosaminyltransferase II N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase II UDP-GlcNAc:mannoside a1-6 acetylglucosaminyltransferase acetylglucosaminyltransferase II a-1,6-mannosyl-glycoprotein b-1,2-N-acetylglucosaminyltransferase a-1,6-mannosylglycoprotein b-1-2-N-acetylglucosaminyltransferase uridine diphosphoacetylglucosamine-a-1,6-mannosylglycoprotein b-1-2-Nacetylglucosaminyltransferase uridine diphosphoacetylglucosamine-a-d-mannoside b1-2-acetylglucosaminyltransferase uridine diphosphoacetylglucosamine-mannoside a1!6-acetylglucosaminyltransferase Additional information (cf. EC 2.4.1.101, EC 2.4.1.144, EC 2.4.1.145, EC 2.4.1.155, EC 2.4.1.201) CAS registry number 105913-04-0 91755-74-7 91847-11-9

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

Bos taurus [2, 6] Mesocricetus auratus [4] Gallus gallus (hen) [4] Rattus norvegicus [1, 7, 11, 13, 20, 21] Sus scrofa [5, 19] Acer pseudoplatanus (sycamore) [8]

259

a-1,6-Mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase

<7> <8> <9> <10> <11> <12> <13> <14> <15>

2.4.1.143

Phaseolus aureus (mung bean) [3] Arabidopsis thaliana [9] Bombyx mori (Bm-N [10]) [10] Mamestra brassicae (IZD-Mb-0503 [10]) [10] Spodoptera frugiperda (Sf9 and Sf12 [10]) [10] Homo sapiens [12-15] Mus musculus [16] Oncorhynchus mykiss (CHO-cells [17]) [17] Oryctolagus cuniculus [18]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + 6-(a-d-mannosyl)-b-d-mannosyl-R = UDP + 6(2[N-acetyl-b-d-glucosaminyl]-a-d-mannosyl)-b-d-mannosyl-R Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + a-d-mannosyl-1,6-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-1,4-N-acetylglucosaminyl-R <4> (<4> essential for biosynthesis of complex N-linked oligosaccharides [1]) (Reversibility: ? <4> [1]) [1] P UDP + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-1,4-N-acetylglucosaminyl-R <4> [1] Substrates and products S UDP-N-acetyl-d-glucosamine + 3-O-methyl-a-d-mannosyl-1,6-(N-acetylb-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl <15> (Reversibility: ? <15> [18]) [18] P UDP + N-acetyl-b-d-glucosamyl-1,2-(3-O-methyl-a-d-mannosyl)-1,6-(Nacetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl S UDP-N-acetyl-d-glucosamine + 3-hydroxy-a-d-mannosyl-1,6-(N-acetylb-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl <15> (Reversibility: ? <15> [18]) [18] P UDP + N-acetyl-b-d-glucosamyl-1,2-(3-hydroxy-a-d-mannosyl)-1,6-(Nacetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl S UDP-N-acetyl-d-glucosamine + 4-O-methyl-a-d-mannosyl-1,6-(N-acetylb-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl <4, 15> (Reversibility: ? <4,15> [18,20]) [18, 20] P UDP + N-acetyl-b-d-glucosamyl-1,2-(4-O-methyl-a-d-mannosyl)-1,6-(Nacetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl S UDP-N-acetyl-d-glucosamine + 5-deoxy-a-d-mannosyl-1,6-(N-acetyl-bd-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl <4> (Reversibility: ? <4> [20]) [20]

260

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a-1,6-Mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransferase

P UDP + N-acetyl-b-d-glucosamyl-1,2S UDP-N-acetyl-d-glucosamine + 6-O-methyl-a-d-mannosyl-1,6-(N-acetylb-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl <15> (Reversibility: ? <15> [18]) [18] P UDP + N-acetyl-b-d-glucosamyl-1,2-(6-O-methyl-a-d-mannosyl)-1,6-(Nacetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl S UDP-N-acetyl-d-glucosamine + a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-1,4-N-acetylglucosaminylR <1-15> (<4> R represents the remainder of the N-oligosaccharide core (n fucose), R: H or 1,4-(+/-)-fucose-1,6-N-acetylglucosaminyl-1-N-asparagine [1]; <6> or 1,4-(+/-)-fucose-1,6-N-acetylglucosamine-pyridinylamine (with or without xylose in 1,2-b-linkage to the b-mannosyl residue) [8]; <7> best acceptor, high specificity [3]; <4> acceptors are free and protein-matrix-bound glycans [7]; <7> UDP-N-acetyl-d-glucosamine cannot be replaced by UDP-N-acetylgalactosamine, UDPgalactose, UDPxylose, UDPglucose or GDPfucose [3]; <7> no acceptor substrates: overview [3]) (Reversibility: ? <1-15> [1-21]) [1-21] P UDP + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-1,4-N-acetylglucosaminyl-R <1-5, 7> [1, 3-6] S UDP-N-acetyl-d-glucosamine + a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl <4> (Reversibility: ? <4> [20]) [20] P UDP + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-1,4-N-acetylglucosaminyl-R S UDP-N-acetyl-d-glucosamine + dideoxytetrasaccharide <4> (Reversibility: ? <4> [11]) [11] P UDP + dideoxypentasaccharide Inhibitors 2-deoxy-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl1,3)-b-d-mannosyl-O-octyl <15> (<15> reversible inhibition [18]) [18] 5-Hg-UDP <4> (<4> reversible [1]) [1] 5-Hg-UDP-N-acetylglucosamine <4> (<4> reversible [1]) [1] 6-deoxy-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl1,3)-b-d-mannosyl-O-octyl <4> [20] EDTA <5> [5] N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3-b-d-mannosyl-octyl <4> [20] O-(4,4-azo)pentyl-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-dmannosyl-1,3)-b-d-mannosyl-O-octyl <4, 15> (<15> irreversible inhibition [18]) [18, 20] O-(5-amino)pentyl-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-dmannosyl-1,3)-b-d-mannosyl-O-octyl <15> (<15> reversible inhibition [18]) [18]

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O-(5-iodoacetamido)pentyl-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl <15> (<15> irreversible inhibition [18]) [18] O-pentyl-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl1,3)-b-d-mannosyl-O-octyl <15> (<15> reversible inhibition [18]) [18] Activating compounds CHAPS <14> [17] Triton X-100 <7, 14> (<7> activation, 0.1% [3]) [3, 17] Zwittergent 3-12 <14> [17] Metals, ions Cd2+ <7> (<7> activation, can replace Mn2+ to some extent, 0.5-1 mM [3]) [3] Mn2+ <2, 7, 14> (<2,7> requirement [3,4]; <7> 2-3 mM [3]; <2> 10-15 mM [4]) [3, 4, 17] Additional information <7, 14> (<7> Ca2+ , Co2+ , Cu2+ , Hg2+ , Mg2+ or Zn2+ cannot replace Mn2+ , at 1 or 2.5 mM each [3]; <14> Ca2+ , Co2+ , Cu2+ and Mg2+ cannot replace Mn2+ [17]) [3, 17] Specific activity (U/mg) 7.8 <4> (<4> a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl as substrate [20]) [20] 9.3 <7> [3] 11.2 <4> (<4> 4-O-methyl-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl1,2-a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl as substrate [20]) [20] 20 <12> [12] 21.4 <4> (<4> 5-deoxy-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2a-d-mannosyl-1,3)-b-d-mannosyl-O-octyl as substrate [20]) [20] 27.5 <4> [1, 4] Km-Value (mM) 0.0166 <7> (a-d-mannosyl-1,6-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl1,3)-b-d-mannosyl-1,4-N-acetylglucosaminyl-R, pH 6.5 [3]) [3] 0.018 <7> (UDP-N-acetylglucosamine, pH 6.5 [3]) [3] 0.1 <1> (a-d-mannosyl-1,6-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl1,3)-b-d-mannosyl-1,4-N-acetylglucosaminyl-R, <1> 37 C [6]) [6] 0.13 <4> (a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl1,3)-b-d-mannosyl-O-octyl, <4> 37 C [20]) [20] 0.16 <4> (4-O-methyl-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-ad-mannosyl-1,3)-b-d-mannosyl-O-octyl, <4> 37 C [20]) [20] 0.19 <2> (a-d-mannosyl-1,6-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl1,3)-b-d-mannosyl-1,4-N-acetylglucosaminyl-R, <2> pH 6.5, 37 C [4]) [4] 0.23 <14> (UDP-N-acetylglucosamine, <14> pH 6.5, 37 C [17]) [17] 0.55 <4> (5-deoxy-a-d-mannosyl-1,6-(N-acetyl-b-d-glucosaminyl-1,2-a-dmannosyl-1,3)-b-d-mannosyl-O-octyl, <4> 37 C [20]) [20] 0.96 <2> (UDP-N-acetylglucosamine, <2> pH 6.5, 37 C [4]) [4]

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pH-Optimum 5-8 <14> [17] 6-6.5 <2, 5> [4, 5] 6.5-7 <7> [3] Temperature optimum ( C) 30 <4> (<4> assay at [11]) [11] 37 <1-5> (<1-5> assay at [1,4-7]) [1, 4-7]

4 Enzyme Structure Molecular weight 51100 <5> (<5> calculated from nucleotide sequence [19]) [19] 56000 <12> (<12> gel filtration, fusion protein [12]) [12] Additional information <4> (<4> rat enzyme in crude extract exists in 2 forms, high-molecular weight form: above MW 200000, low-molecular weight form: MW 43000-48000, gel filtration [1]) [1] Subunits ? <4> (<4> x * 43000, x * 48000, rat, SDS-PAGE, one or both of these bands represent the enzyme [1]; <4> x * 42000, SDS-PAGE [20,21]) [1, 20, 21]

5 Isolation/Preparation/Mutation/Application Source/tissue brain <5> [19] cell suspension culture <6> [8] colostrum <1> [2, 6] kidney <2> (<2> baby hamster [4]) [4] liver <4, 5> [1, 5, 7, 11, 19-21] ovary <2, 14> [4, 14, 17] oviduct <3> [4] seedling <7> [3] tracheal mucosa <5> [5] Localization Golgi apparatus <4, 6, 8, 12> (<8> type II transmembrane topology [9]) [7-9, 12, 14, 15, 21] membrane <2-4, 7> [1, 3, 4, 7] microsome <5, 7> [3, 5] Purification <4> (solubilized with Triton X-100/NaCl, enzyme in crude extract exists in 2 molecular weight forms: purification of low-molecular weight form, affinity chromatography on Hg-UDP-N-acetylglucosamine-thiopropyl-Sepharose [1]) [1, 21] <7> (solubilized with Triton X-100 [3]) [3] <12> [12] 263

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2.4.1.143

Cloning <4> (transient expression in COS-7 cells, linked in frame to a cDNA segment encoding the cleavable signal sequence of the human IL-2 receptor [21]) [21] <5> (expression in Escherichia coli [19]) [19] <8> (expression in Spodoptera frugiperda Sf-9 cells [9]) [9] <12> (expression as fusion protein vHis-GlcNAc-TII in Spodoptera frugiperda Sf-9 cells [12]) [12]

6 Stability General stability information <2>, bovine serum albumin stabilizes during purification [1, 4] <4>, Triton X-100 stabilizes [1] <5>, acetone precipitation, unstable to [5] Storage stability <4>, 4 C, at least 6 months in 20% glycerol, 10 mM EDTA and 0.1% Triton X100 [1] <7>, -20 C, several weeks in 20% glycerol, 0.0005 mM DTT and 0.1% Triton X-100 [3] <7>, 0 C, at least 1 week [3]

References [1] Bendiak, B.; Schachter, H.: Control of glycoprotein synthesis. Purification of UDP-N-acetylglucosamine:a-d-mannoside b1-2 N-acetylglucosaminyltransferase II from rat liver. J. Biol. Chem., 262, 5775-5783 (1987) [2] Rogers, G.N.; Paulsen, J.C.; Daniels, R.S.; Skehel, J.J.; Wilson, I.A.; Wiley, D.C.: Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature, 304, 76-78 (1983) [3] Szumilo, T.; Kaushal, G.P.; Elbein, A.D.: Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants. Biochemistry, 26, 5498-5505 (1987) [4] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and O-glycan synthesis. Methods Enzymol., 179, 351-397 (1989) [5] Oppenheimer, C.L.; Eckhardt, A.E.; Hill, R.L.: The nonidentity of porcine N-acetylglucosaminyltransferases I and II. J. Biol. Chem., 256, 11477-11482 (1981) [6] Harpaz, N.; Schachter, H.: Control of glycoprotein synthesis. Bovine colostrum UDP-N-acetylglucosamine:a-d-mannoside b2-N-acetylglucosaminyltransferase I. Separation from UDP-N-acetylglucosamine:a-d-mannoside b 2-N-acetylglucosaminyltransferase II, partial purification, and substrate specificity. J. Biol. Chem., 255, 4885-4893 (1980)

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[7] Shao, M.-C.; Wold, F.: The effect of the protein matrix on glycan processing in glycoproteins. Kinetic analysis of three rat liver Golgi enzymes. J. Biol. Chem., 263, 5771-5774 (1988) [8] Tezuka, K.; Hayashi, M.; Ishihara, H.; Akazawa, T.; Takahashi, N.: Studies on synthetic pathway of xylose-containing N-linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.). Eur. J. Biochem., 203, 401-413 (1992) [9] Strasser, R.; Steinkellner, H.; Boren, M.; Altmann, F.; Mach, L.; Glössl, J.; Mucha, J.: Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana. Glycoconjugate J., 16, 787-791 (2000) [10] Altmann, F.; Kornfeld, G.; Dalik, T.; Staudacher, E.; Glössl, J.: Processing of asparagine-linked oligosaccharides in insect cells. N-acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells. Glycobiology, 3, 619-625 (1993) [11] Alton, G.; Srivastava, G.; Kaur, K.J.; Hindsgaul, O.: Use of N-acetylglucosaminyltransferases I and II in the synthesis of a dideoxypentasaccharide. Bioorg. Med. Chem., 2, 675-680 (1994) [12] Tan, J.; D'Agostaro, G.A.F.; Bendiak, B.; Reck, F.; Sarkar, M.; Squire, J.A.; Leong, P.; Schachter, H.: The human UDP-N-acetylglucosamine:a-6-dmannoside-b-1,2-N-acetylglucosaminyltransferase II gene (MGAT2). Cloning of genomic DNA, localization to chromosome 14q21, expression in insect cells and purification of the recombinant protein. Eur. J. Biochem., 231, 317-328 (1995) [13] Schachter, H.; Chen, S.H.; Zhou, S.; Tan, J.; Yip, B.; Sarkar, M.; Spence, A.: Structure and function of the genes encoding N-acetylglucosaminyltransferases which initiate N-glycan antennae. Biochem. Soc. Trans., 25, 875880 (1997) [14] Chen, S.-H.; Zhou, S.; Tan, J.; Schachter, H.: Transcriptional regulation of the human UDP-GlcNAc:a-6lpha-d-mannoside b-1-2-N-acetylglucosaminyltransferase II gene (MGAT2) which controls complex N-glycan synthesis. Glycoconjugate J., 15, 301-308 (1998) [15] Zhang, W.; Revers, L.; Pierce, M.; Schachter, H.: Regulation of expression of the human b-1,2-N-acetylglucosaminyltransferase II gene (MGAT2) by Ets transcription factors. Biochem. J., 347, 511-518 (2000) [16] Wang, Y.; Schachter, H.; Marth, J.D.: Mice with a homozygous deletion of the Mgat2 gene encoding UDP-N-acetylglucosamine:a-6-d-mannoside b1,2-N-acetylglucosaminyltransferase II: a model for congenital disorder of glycosylation type IIa. Biochim. Biophys. Acta, 1573, 301-311 (2002) [17] Fritz, T.A.; Gabb, M.M.; Wei, G.; Esko, J.D.: Two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate. J. Biol. Chem., 269, 28809-28814 (1994) [18] Reck, F.; Springer, M.; Paulsen, H.; Brockhausen, I.; Sarkar, M.; Schachter, H.: Synthesis of tetrasaccharide analogs of the N-glycan substrate of b(1!2)-N-acetylglucosaminyltransferase II using trisaccharide precursors and recombinant b-(1!2)-N-acetylglucosaminyltransferase I. Carbohydr. Res., 259, 93-101 (1994)

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[19] Leeb, T.; Kriegesmann, B.; Baumgartner, B.G.; Klett, C.; Yerle, M.; Hameister, H.; Brenig, B.: Molecular cloning of the porcine b-1,2-N-acetylglucosaminyltransferase II gene and assignment to chromosome 1q23-q27. Biochim. Biophys. Acta, 1336, 361-366 (1997) [20] Reck, F.; Meinjohanns, E.; Springer, M.; Wilkens, R.; Van Dorst, J.A.; Paulsen, H.; Moller, G.; Brockhausen, I.; Schachter, H.: Synthetic substrate analogues for UDP-GlcNAc: Man a 1-6R b(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme. Glycoconjugate J., 11, 210-216 (1994) [21] D'Agostaro, G.A.F.; Zingoni, A.; Moritz, R.L.; Simpson, R.J.; Schachter, H.; Bendiak, B.: Molecular cloning and expression of cDNA encoding the rat UDP-N-acetylglucosamine:a-6-d-mannoside b-1,2-N-acetylglucosaminyltransferase II. J. Biol. Chem., 270, 15211-15221 (1995)

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b-1,4-Mannosyl-glycoprotein 4-b-Nacetylglucosaminyltransferase

2.4.1.144

1 Nomenclature EC number 2.4.1.144 Systematic name UDP-N-acetyl-d-glucosamine:b-d-mannosyl-glycoprotein 4-b-N-acetyl-dglucosaminyltransferase Recommended name b-1,4-mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase Synonyms GlcNAc-transferase-III <4> [15] GnT-III <1, 2, 4, 5, 7> [2, 5-8, 10-14, 16, 17] GnTIII <6> [9] N-acetyl-glucosaminyltransferase III <4> [14] N-acetylglucosaminyltransferase III N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase III acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-glycopeptide b4-, III b-1,4-mannosyl-glycoprotein b-1,4-N-acetylglucosaminyltransferase b-d-mannoside b-1,4-N-acetylglucosaminyltransferase <4> [14] b1,4-N-acetylglucosaminyltransferase III <1, 2, 4, 5> [13, 16, 17] uridine diphosphate (UDP)-N-acetylglucosamin/b-d-mannoside b-1,4-Nacetylglucosaminyltransferase III <4> [15] uridine diphosphoacetylglucosamine-glycopeptide b4-acetylglucosaminyltransferase III CAS registry number 83744-93-8

2 Source Organism <-1> no activity in Mus musculus (lung melanoma cell line B16-hm [8]) [8] <1> Gallus gallus (hen, White Leghorn [1,3]) [1, 3, 11, 16] <2> Rattus norvegicus [3, 4, 7, 11, 13, 16, 17] <3> Cricetulus griseus [2, 3] <4> Homo sapiens (single copy gene, no isoforms [6,16]) [2, 3, 5, 6, 8, 10-12, 14-17] <5> Rattus norvegicus (male Donryu [2]) [2, 11, 16]

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b-1,4-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

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<6> Mesocricetus auratus (forming normal prion protein PrPC and pathogenic prion protein PrPSc [9]) [9] <7> Mus musculus [11]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + b-d-mannosyl-R = UDP + 4-(N-acetyl-b-dglucosaminyl)-b-d-mannosyl-R (<2> sequential, random or partially random mechanism [16]; R represents the remainder of the N-linked oligosaccharide in the glycoprotein acceptor. The action of this enzyme probably prevents further attachment of N-acetylglucosamine residues to the growing carbohydrate chain.) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + (N-acetyl-b-d-glucosaminyl-1,2-a-dmannosyl-1,3)-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6)-b-dmannosyl-1,4-N-acetyl-b-d-glucosaminyl-R <1-5, 7> (<2, 4> important role in carcinogenesis and cancer metastasis [16]; <2,4,5> key regulatory role in N-glycan biosynthesis, responsible for cancer-associated alterations [16]; <1-5, 7> R represents the remainder of the N-oligosaccharide core (+/-fucose), essential for biosynthesis of complex N-linked oligosaccharides [1, 3, 11, 16]; <2, 4> numerous target proteins, multifacial glycosyltransferase [11, 16]; <2, 4, 5, 7> key enzyme in the biosynthesis of Nglycans since it inhibits the other involved glycosyltransferases by adding the GlcNAc-residue in an b-1,4- linkage [11, 16]; <7> enzyme plays an important role in the progression of preneoplastic foci to primary hepatoma [11]) [1, 3, 5, 11, 16] P UDP + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,6)-(N-acetyl-b-d-glucosaminyl-1,4)-bd-mannosyl-1,4-N-acetyl-b-d-glucosaminyl-R S UDP-N-acetyl-d-glucosamine + E-cadherin <4> (<4> recombinant enzyme expressed in murine lung melanoma cells [11,16]) [11, 16] P UDP + E-cadherin with bisecting N-acetyl-d-glucosamine S Additional information <2, 4, 6> (<2,4> biosynthetic pathway of the core structures of Asn-linked sugar chains, overview [16]; <4> the biantennary structure of a core mannose is twisted in presence of bisecting GlcNAc, probably responsible for the substrate inaccessibility to N-acetylglucosamine transferase V to form the b-1,6 structure [11]; <4> key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue, modification of N-glycans by the enzyme affects a number of intracellular signalling pathways [10,11]; <6> in brain cells with down-regulated enzyme activity, normal prion proteins PrPC get

268

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conversed to pathogenic prion protein PrPSc due to a lower amount of glycans with bisecting N-acetylglucosamine residues [9]) [9-11, 16] P ? Substrates and products S UDP-d-glucose + (N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-(Nacetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6)-b-d-mannosyl-1,4-Nacetyl-b-d-glucosaminyl-R <2> (<2> 0.2% activity compared to donor substrate UDP-N-acetyl-d-glucosamine [13,16]; <2> pyridylaminated biantennary sugar chain [13]) (Reversibility: ? <2> [13,16]) [13, 16] P UDP + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,6)-(b-d-glucosyl-1,4)-b-d-mannosyl1,4-N-acetyl-b-d-glucosaminyl-R S UDP-N-acetyl-d-galactosamine + (N-acetyl-b-d-glucosaminyl-1,2-a-dmannosyl-1,3)-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6)-b-dmannosyl-1,4-N-acetyl-b-d-glucosaminyl-R <2> (<2> 0.1% activity compared to donor substrate UDP-N-acetyl-d-glucosamine [13,16]; <2> pyridylaminated biantennary sugar chain [13]) (Reversibility: ? <2> [13,16]) [13, 16] P UDP + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,6)-(N-acetyl-b-d-galactosaminyl-1,4)b-d-mannosyl-1,4-N-acetyl-b-d-glucosaminyl-R S UDP-N-acetyl-d-glucosamine + (N-acetyl-b-d-glucosaminyl-1,2-a-dmannosyl-1,3)-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6)-b-dmannosyl-1,4-N-acetyl-b-d-glucosaminyl-N-acetyl-d-glucoaminyl-2-aminopyridine <4> (Reversibility: ir <4> [6]) [6] P UDP + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,6)-(N-acetyl-b-d-glucosaminyl-1,4)-bd-mannosyl-1,4-N-acetyl-b-d-glucosaminyl-N-acetyl-d-glucoaminyl-2aminopyridine <4> [6] S UDP-N-acetyl-d-glucosamine + (N-acetyl-b-d-glucosaminyl-1,2-a-dmannosyl-1,3)-(N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,6)-b-dmannosyl-1,4-N-acetyl-b-d-glucosaminyl-R <1-7> (<2> absolute requirement for the conserved residues Asp321 and Asp323 as part of a D-X-D motif [17]; <2-4, 7> R represents the remainder of the N-oligosaccharide core, (+/-)-fucose, favourite substrate [3, 11, 16]; <2-4> b1,4-galactosylation of N-acetylglucosaminyl-1,2-a-mannosyl-1,3-b-mannosyl moiety prevents transferase III action [3, 16]; <1, 2, 4> b1,2-GlcNAc linked to a1,3-Man is required for activity, b1,2-GlcNAc linked to a1,6-Man is not required [16]; <1, 2, 4> any agalacto form of the bi-, tri- and tetraantennary sugar chains is capable of serving as ana cceptor substrate [16]; <2, 4> structural requirements, stereochemistry [16]; <1, 2, 4> substrate specificity [3, 16]; <2, 4> pyridylaminated biantennary sugar chain [4, 13, 15]) (Reversibility: ir <1, 2, 4, 5> [1, 2, 5, 6, 15, 16]; ? <1-5, 7> [3, 4, 7, 8, 11, 13, 14, 17]) [1-9, 11, 13-17] P UDP + N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,6)-(N-acetyl-b-d-glucosaminyl-1,4)-bd-mannosyl-1,4-N-acetyl-b-d-glucosaminyl-R <1-7> (<1-7> incorporates

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S P

S P S P S P S

P

a N-acetylglucosamine residue in b-1,4-linkage to b-linked mannosyl, i.e. bisecting N-acetylglucosamine [1-3, 5, 8, 9, 11, 14, 16, 17]) [1-6, 8, 9, 11, 14, 16, 17] UDP-N-acetyl-d-glucosamine + GlcNAc-b-1,2-Man-a-1,6-(GlcNAc-b-1,2Man-a-1,3)-Man-b-1,4-GlcNAc-b-1,4-(Fuc-a-1,6)-GlcNAc-Asn <1> (Reversibility: ir <1> [1]) [1] UDP + GlcNAc-b-1,2-Man-a-1,6-(GlcNAc-b-1,2-Man-a-1,3)(GlcNAc-b1,4)-Man-b-1,4-GlcNAc-b-1,4-(Fuc-a-1,6)-GlcNAc-Asn <1> (<1> incorporates a N-acetylglucosamine residue in b-1,4-linkage to b-linked mannosyl, i.e. bisecting N-acetylglucosamine, both terminal b-1,2-linked Nacetylglucosamine residues required for maximum activity, removal of one or both of them reduces activity by 85 or 93%, respectively [1]) [1] UDP-N-acetyl-d-glucosamine + GlcNAc-b-1,2-Man-a-1,6-(GlcNAc-b-1,2Man-a-1,3)-Man-b-1,4-GlcNAc-b-1,4-GlcNAc-2-aminopyridine <2> (Reversibility: ? <2> [7]) [7] UDP + GlcNAc-b-1,2-Man-a-1,6-(GlcNAc-b-1,2-Man-a-1,3)(GlcNAc-b1,4)-Man-b-1,4-GlcNAc-b-1,4-GlcNAc-2-aminopyridine UDP-N-acetyl-d-glucosamine + g-glutamyltranspeptidase <2> (Reversibility: ? <2> [11]) [11] UDP + g-glutamyltranspeptidase with bisecting N-acetyl-d-glucosamine <2> [11] UDP-N-acetyl-d-glucosamine + transferrin <2> (Reversibility: ? <2> [16]) [16] UDP + transferrin with bisecting N-acetyl-d-glucosamine <2> [16] Additional information <2> (<2> specificity towards the sugar moiety of the donor substrate appears to be critically determined during the catalytic process but not during binding to the enzyme in ground state [16]; <2> UDP-galactose is no substrate [13,16]; <2> ADP-, CDP-, GDP-, and TDP-glucose are no donor substrates [13]) [13, 16] ?

Inhibitors ADP <2> [13] ADP-glucose <2> (<2> competitive [13]) [13] CDP <2> [13] CDP-glucose <2> (<2> competitive [13]) [13] EDTA <4> [8] GDP <2> [13] GDP-glucose <2> (<2> competitive [13]) [13] N-acetyl-d-glucosamine <2> [13] TDP <2> [13] TDP-glucose <2> (<2> competitive [13]) [13] UDP <2> [13]

270

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against UDP-N-acetyl-d-glucosamine against UDP-N-acetyl-d-glucosamine

against UDP-N-acetyl-d-glucosamine

against UDP-N-acetyl-d-glucosamine

2.4.1.144

b-1,4-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

a-N-acetyl-d-glucosamine-1-phosphate <2> [13] castanospermine <2> (<2> activity is not affected, but the enzyme is not localized in the Golgi apparatus [7]) [7] hepatitis B virus <4> (<4> selective suppression of enzyme activity on transcription level in transfected cells, e.g. cell line Hb611 [5]) [5] inactive rat mutant D323A enzyme <4> (<4> acts as an inhibitor of endogenous enzyme activity when expressed in human Huh6 cells [17]) [17] tunicamycin <2> (<2> completely abolishes the enzyme activity due to deglycosylation of the enzyme, not localized in the Golgi apparatus [7]) [7] Additional information <4> (<4> transgenic expression of the enzyme in hepatitis B virus infected cells suppress the virus expression, cell line HB611 [8,11]) [8, 11] Activating compounds Triton X-100 <1, 2, 4, 5> (<1,5> activation, about 8fold at 0.13-1.3% v/v [13]) [1-3, 6, 14, 15, 17] all-trans retinoic acid <4> (<4> activation [14]) [14] deoxymannojirimycin <4> (<4> increase in activity, HepG2 and Hep3B cells [15]) [15] forskolin <4> (<4> induction of the enzyme in hepatoma cells [11]) [11] tunicamycin <4> (<4> increase in activity, HepG2 and Hep3B cells [15]) [15] Additional information <4> (<4> not affected by swainsonine [15]; <4> not affected by cAMP [14]; <4> interferon-a-2a leads to an increase in enzyme activity in hepatitis B virus infected cells due to reduction of virus expression [5]) [5, 14] Metals, ions Mn2+ <1, 2, 4> (<2> Asp321 and Asp323 are absolutely required for the coordination of Mn2+ during enzyme reaction [17]; <4> HepG2 cells: maximal at 12 mM, Hep3B cells: maximal at 15 mM [15]; <1,2> broad optimum at 12 mM [1,3,4]) [1, 3, 4, 6, 14-17] Specific activity (U/mg) 0.000000022 <4> (<4> hepatoma cells [12]) [12] 0.0000001 <4> (<4> HB611 cells [5,8]) [5, 8] 0.00000013 <4> (<4> normal liver [15]) [15] 0.0000004 <4> (<4> HB611 cells treated with interferon-a-2a [5]) [5] 0.0000009 <5> (<5> recombinant enzyme in HeLa cells [2]) [2] 0.0000021 <4> (<4> Huh6 cells treated with interferon-a-2a [5]) [5] 0.0000023 <4> (<4> Huh6 cells [5,8]) [5, 8] 0.0000025 <4> (<4> hepatoma cell line 7721 [14]) [14] 0.000004 <4> (<4> about, hepatoma cell line 7721, all-trans retinoic acid treated [14]) [14] 0.0000096 <4> (<4> fetal primary hepatocytes [15]) [15] 0.000012 <4> (<4> purified enzyme [12]) [12] 0.0000124 <4> (<4> hepatic carcinoma cells [15]) [15] 0.000017 <2> (<2> wild-type and mutant D329A [17]) [17] 0.000018 <4> (<4> HepG2 cells [15]) [15]

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0.000024 <4> (<4> Hep3B cells [15]) [15] 0.00006 <5> (<5> recombinant enzyme in COS-1 cells [2]) [2] 0.000083 <1> [1] 0.02 <2> (<2> purified recombinant enzyme [13]) [13] 5.52 <5> (<5> purified enzyme [2]) [2] Additional information <4> (<4> activity and expression level in various leukemic cells [6]; <4> transcript amount is not correlated to enzyme activity [6]) [6] Km-Value (mM) 0.021 <2> (pyridylaminated acceptor substrate, <2> with UDP-GlcNAc as donor substrate [13]; <2> recombinant enzyme [13]) [13] 0.023 <2> (pyridylaminated acceptor substrate, <2> with UDP-Glc as donor substrate [13]; <2> recombinant enzyme [13]) [13] 0.024 <2> (pyridylaminated acceptor substrate, <2> with UDP-GalNAc as donor substrate [13]; <2> recombinant enzyme [13]) [13] 0.19 <2> (pyridylaminated acceptor substrate) [4] 0.23 <1> ((N-acetyl-b-d-glucosaminyl-1,2-a-d-mannosyl-1,3)-(N-acetyl-b-dglucosaminyl-1,2-a-d-mannosyl-1,6)-b-d-mannosyl-1,4-N-acetyl-b-d-glucosaminyl-R) [1, 3] 0.42 <2> (UDP-N-acetyl-d-glucosamine, <2> recombinant enzyme [13,16]) [13, 16] 1 <2> (UDP-d-glucose, <2> recombinant enzyme [13,16]) [13, 16] 1.1 <1> (UDP-N-acetyl-d-glucosamine) [1, 3] 1.1 <4> (pyridylaminated acceptor substrate, <4> Hep3B cells [15]) [15] 3.1 <2> (UDP-N-acetyl-d-glucosamine, <2> + pyridylaminated acceptor substrate [4]) [4] 3.4 <4> (pyridylaminated acceptor substrate, <4> HepG2 cells [15]) [15] 3.6 <2> (UDP-N-acetyl-d-galactosamine, <2> recombinant enzyme [13,16]) [13, 16] 4.7 <4> (UDP-N-acetyl-d-glucosamine, <4> Hep3B cells [15]) [15] 6.8 <4> (UDP-N-acetyl-d-glucosamine, <4> HepG2 cells [15]) [15] Ki-Value (mM) 0.75 <2> (ADP-glucose, <2> recombinant enzyme [13]) [13] 1 <2> (GDP-glucose, <2> recombinant enzyme [13]) [13] 3.9 <2> (CDP-glucose, <2> recombinant enzyme [13]) [13] pH-Optimum 6 <4> (<4> HepG2 cells [15]) [15] 6-7 <1> [3] 6.3 <2, 4> (<2,4> assay at [6,14,17]) [4, 6, 14, 17] 6.5 <4> (<4> Hep3B cells [15]) [15] 7 <1> [1] Temperature optimum ( C) 37 <2, 4> (<2,4> assay at [6,7,14]) [6, 7, 14] 40 <4> (<4> HepG2 cells [15]) [15] 43 <4> (<4> Hep3B cells [15]) [15] 272

2.4.1.144

b-1,4-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

4 Enzyme Structure Molecular weight 110000 <4> (<4> about, PAGE [12]) [12] Additional information <5> (<5> type II transmembrane protein [2,16]; <5> amino acid and DNA sequence [2,16]) [2, 16] Subunits ? <2, 5> (<2> x * 60000 + x * 50000, SDS-PAGE [13]; <2> x * 68100, wildtype enzyme, SDS-PAGE [7]; <5> x * 62000 + x * 52000, SDS-PAGE [2]) [2, 7, 13] dimer <4> (<4> 1 * 63000 + 1 * 53000, SDS-PAGE [12]) [12] Posttranslational modification glycoprotein <2> (<2> 3 N-glycosylation sites: Asn243, Asn261, Asn399, essential for activity, tunicamycin-treated enzyme, which is not glycosylated, shows no activity [7,16,17]; <2> requires core glycosylation but not glucose trimming [7]; <5> enzyme binds to concanavalin A [2]; <5> enzyme contains 3 putative N-glycosylation sites [2]) [2, 7, 16, 17]

5 Isolation/Preparation/Mutation/Application Source/tissue CHO cell <3> (<3> LEC10 ovary cell line [3]; <3> ricin resistant mutant [3]) [2, 3] HB611 cell <4> (<4> hepatoblastoma cell line, derived from Huh6 by transfection of 3 copies of hepatitis B virus genome, decreased transcription level [5,8,11]) [5, 8, 11] HeLa cell <4> [2] Hep-3B cell <4> (<4> hepatoma cell line [15]) [15] Hep-G2 cell <4> (<4> hepatoma cell line [15]) [15] Huh-6 cell <4> (<4> hepatoblastoma cell line [5,8,17]) [5, 8, 17] M13 cell <2> (<2> induction of the enzyme impairs the sorting of membranous proteins towards the cell surface [11]) [11] bone marrow <4> [6] brain <2, 4, 6, 7> (<6> cells forming normal prion protein PrPC with normal enzyme activity, and pathogenic prion protein PrPSc with reduced enzyme activity [9]) [4, 9, 11, 16] chronic myeloid leukemia cell <4> (<4> chronic myelogenous leukemia granulocyte cell lines, e.g. clone KU812 and subclone KU812F [6]) [6] hepatocyte <2, 4> (<4> different expression level in fetal and adult hepatocytes [15]; <2,4> very low amount [11,15]; <4> primary [15]) [11, 15] hepatoma ascites cell <2> (<2> about 100fold increased activity compared to normal liver tissue [11]) [11]

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b-1,4-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

2.4.1.144

hepatoma cell <2, 4, 7> (<7> low activity [11]; <2> primary, about 100fold increased activity compared to normal liver tissue [11]; <4> 7721 cells, activity during cell-cycle [14]) [3, 4, 11, 12, 14, 15] hepatoma cell line <4> (<4> 7721 cell [14]) [14] kidney <2, 4, 5, 7> [2, 4, 11, 16] leukemia cell <4> (<4> from patients with chronic lymphocytic leukemia CLL or acute lymphoblastic leukemia ALL [6]) [6, 11] liver <1, 2, 4> (<4> high levels in fetal liver, but not in adult liver [11]; <2,4> very low amount [11,15,16]; <1> preneoplastic hepatic nodules [3]) [3, 4, 11, 15, 16] lymphoid cell line <4> [3] myeloid cell line <4> [3] myeloma cell <4> (<4> multiple myeloma [6]) [6] oviduct <1> [1, 3, 11, 16] peripheral blood <4> [6] serum <2> [4] Additional information <2, 4> (<4> multiple promotors allow tissue-specific expression [16]; <2> tissue distribution [4]) [4, 16] Localization Golgi apparatus <2, 4> (<2> wild-type [7]; <2> deglycosylated mutants are not localized in the Golgi apparatus [7,16]) [7, 11, 14, 16] cytosol <4> (<4> minor fraction [15]) [15] endoplasmic reticulum <2> (<2> tunicamycin-treated and castanosperminetreated enzyme, not native enzyme [7]) [7] membrane <1-5> [1-3, 11, 15, 16] microsome <1> [1, 3] Purification <2> (recombinant His-tagged protein from medium of Spodoptera frugiperda Sf 21 cell culture [13]) [13, 16] <4> (recombinant mutant enzyme from E. coli [12]; wild-type, diethyl(2-hydroxypropyl)aminoethyl-Sepharose and immunoaffinity chromatography [12]) [12] <5> (primary structure [2,16]) [2, 11, 16] Cloning <2> (expression of a catalytically inactive D232A mutant in human hepatoblastoma cell line Huh6, leading to suppression of the endogenous enzyme activity, but not to a significant decrease in the expression level of the endogenous enzyme [17]; expression of soluble His-tagged protein in Spodoptera frugiperda Sf 21 insect cells via baculovirus infection, secretion into the medium [13,16]; construction of transgenic mice, disruption of certain functions of apolipoprotein B leading to generation of a aftty liver [11,16]; overexpression in rat pheochromocytoma cell line PC-12, glycoproteins contain elevated levels of bisecting GlcNAc, abolished cell differentiation [11]; expression of wild-type and mutants in Cos-1 cells [7,16,17]) [7, 11, 13, 16, 17]

274

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b-1,4-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

<4> (cloning of cDNA by RT-PCR [15]; expression of mutant aglycosyl-enzyme, lacking the first 23 amino acid residues, in Escherichia coli [12]; expression in erythroleukemia cell line K562, leading to increased resistance to lysis by natural killer cells and enhanced spleen colonization ability [11,16]; overexpression in HeLa cells, leading to suppression of H2 O2 -induced activation of PKCd-JNK1 signalling pathway resulting in inhibition of apoptosis [10]; overexpression in murine lung melanoma cell line B16-hm, leading to suppression of the formation of b-1,6-tri- and tetraantennary N-linked oligosaccharides by inhibiting the responsible enzymes, e.g. N-acetylglucosamine transferase V, EC 2.4.1.155, decrease of the metastatic potential of B16-hm melanoma in vivo, changed phenotype [8,11,16]) [8, 10-12, 15, 16] <5> (cloning from genetic library, DNA sequence determination, functional overexpression in COS-1 or HeLa-cells, transient transfection [2]) [2, 16] Engineering D321A <2> (<2> site-directed mutagenesis, expression level in COS-1 cells similar to wild-type, but no remaining catalytic activity [17]) [17] D323A <2> (<2> site-directed mutagenesis, expression level in COS-1 cells similar to wild-type, but no remaining catalytic activity [17]; <2> expression of a catalytically inactive D232A mutant in human hepatoblastoma cell line Huh6, leading to suppression of the endogenous enzyme activity, but not to a significant decrease in the expression level of the endogenous enzyme [17]) [17] D329A <2> (<2> site-directed mutagenesis, unaltered compared to wild-type [17]) [17] N243Q <2> (<2> site-directed mutagenesis of 1 N-glycosylation site, 40-50% activity compared to the wild-type, slightly increased Km -value for the donor substrate [7]) [7, 16] N243Q/N261Q <2> (<2> site-directed mutagenesis of 2 N-glycosylation sites, 10% activity compared to the wild-type [7]) [7, 16] N243Q/N261Q/N399Q <2> (<2> site-directed mutagenesis of all N-glycosylation sites, no remaining activity [7]) [7, 16] N243Q/N399Q <2> (<2> site-directed mutagenesis of 2 N-glycosylation sites, highly reduced activity [7]) [7, 16] N261Q <2> (<2> site-directed mutagenesis of 1 N-glycosylation site, 40-50% activity compared to the wild-type, slightly increased Km -value for the donor substrate [7]) [7, 16] N261Q/N399Q <2> (<2> site-directed mutagenesis of 2 N-glycosylation sites, highly reduced activity [7]) [7, 16] N399Q <2> (<2> site-directed mutagenesis of 1 N-glycosylation site, 70-80% activity compared to the wild-type [7]) [7, 16]

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b-1,4-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

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6 Stability Storage stability <1>, -20 C, chicken oviduct microsomal preparation, several months in detergent-free buffer [3]

References [1] Narasimhan, S.: Control of glycoprotein synthesis. UDP-GlcNAc:glycopeptide b4-N-acetylglucosaminyltransferase III, an enzyme in hen oviduct which adds GlcNAc in b1-4 linkage to the b-linked mannose of the trimannosyl core of N-glycosyl oligosaccharides. J. Biol. Chem., 257, 10235-10242 (1982) [2] Nishikawa, A.; Ihara, Y.; Hatakeyama, M.; Kangawa, K.; Taniguchi, N.: Purification, cDNA cloning, and expression of UDP-N-acetylglucosamine: b-dmannoside b-1,4N-acetylglucosaminyltransferase III from rat kidney. J. Biol. Chem., 267, 18199-18204 (1992) [3] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and O-glycan synthesis. Methods Enzymol., 179, 351-397 (1989) [4] Taniguchi, N.; Nishikawa, A.; Fujii, S.; Gu, J.: Glycosyltransferase assays using pyridylaminated acceptors: N-acetylglucosaminyltransferase III, IV, and V. Methods Enzymol., 179, 397-408 (1989) [5] Miyoshi, E.; Nishikawa, A.; Ihara, Y.; Hayashi, N.; Fusamoto, H.; Kamada, T.; Taniguchi, N.: Selective suppression of N-acetylglucosaminyltransferase III activity in a human hepatoblastoma cell line transfected with hepatitis B virus. Cancer Res., 54, 1854-1858 (1994) [6] Yoshimura, M.; Nishikawa, A.; Ihara, Y.; Nishiura, T.; Nakao, H.; Kanayama, Y.; Matuzawa, Y.; Taniguchi, N.: High expression of UDP-N-acetylglucosamine:b-D-mannoside b-1,4-N-acetylglucosaminyltransferase III (GnT-III) in chronic myelogenous leukemia in blast crisis. Int. J. Cancer, 60, 443-449 (1995) [7] Nagai, K.; Ihara, Y.; Wada, Y.; Taniguchi, N.: N-Glycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III. Glycobiology, 7, 769-776 (1997) [8] Taniguchi, N.; Yoshimura, M.; Miyoshi, E.; Ihara, Y.; Nishikawa, A.; Kang, R.; Ikeda, Y.: Gene expression and regulation of N-acetylglucosaminyltransferases III and V in cancer tissues. Adv. Enzyme Regul., 38, 223-232 (1998) [9] Rudd, P.M.; Endo, T.; Colominas, C.; Groth, D.; Wheeler, S.F.; Harvey, D.J.; Wormald, M.R.; Serban, H.; Prusiner, S.B.; Kobata, A.; Dwek, R.A.: Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc. Natl. Acad. Sci. USA, 96, 13044-13049 (1999) [10] Shibukawa, Y.; Takahashi, M.; Laffont, I.; Honke, K.; Taniguchi, N.: Downregulation of hydrogen peroxide-induced PKCd activation in N-acetylgluco-

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[11] [12]

[13]

[14]

[15]

[16] [17]

b-1,4-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

saminyltransferase III-transfected HeLaS3 cells. J. Biol. Chem., 278, 31973203 (2003) Taniguchi, N.; Miyoshi, E.; Ko, J.H.; Ikeda, Y.; Ihara, Y.: Implication of Nacetylglucosaminyltransferases III and V in cancer: gene regulation and signaling mechanism. Biochim. Biophys. Acta, 1455, 287-300 (1999) Song, E.-Y.; Kim, K.-A.; Kim, Y.-D.; Kwon, D.-H.; Lee, H.-S.; Kim, C.-H.; Chung, T.-W.; Byun, S.M.: A simplified procedure for the purification of human N-acetylglucosaminyltransferase-III from human hepatocellular carcinoma tissues. Biotechnol. Lett., 22, 201-204 (2000) Ikeda, Y.; Koyota, S.; Ihara, H.; Yamaguchi, Y.; Korekane, H.; Tsuda, T.; Sasai, K.; Taniguchi, N.: Kinetic basis for the donor nucleotide-sugar specificity of b1,4-N-acetylglucosaminyltransferase III. J. Biochem., 128, 609-619 (2000) Guo, H.B.; Jiang, A.L.; Ju, T.Z.; Chen, H.L.: Opposing changes in N-acetylglucosaminyltransferase-V and -III during the cell cycle and all-trans retinoic acid treatment of hepatocarcinoma cell line. Biochim. Biophys. Acta, 1495, 297-307 (2000) Song, E.-Y.; Kang, S.-K.; Lee, Y.-C.; Park, Y.-G.; Chung, T.-H.; Kwon, D.-H.; Byun, S.-M.; Kim, C.-H.: Expression of bisecting N-acetylglucosaminyltransferase-III in human hepatocarcinoma tissues, fetal liver tissues, and hepatoma cell lines of Hep3B and HepG2. Cancer Invest., 19, 799-807 (2001) Ikeda, Y.; Taniguchi, N.: Enzymatic properties and biological functions of b1,4-N-acetylglucosaminyltransferase III. Trends Glycosci. Glycotechnol., 13, 167-176 (2001) Ihara, H.; Ikeda, Y.; Koyota, S.; Endo, T.; Honke, K.; Taniguchi, N.: A catalytically inactive b1,4-N-acetylglucosaminyltransferase III (GnT-III) behaves as a dominant negative GnT-III inhibitor. Eur. J. Biochem., 269, 193201 (2002)

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a-1,3-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

2.4.1.145

1 Nomenclature EC number 2.4.1.145 Systematic name UDP-N-acetyl-d-glucosamine:3-[2-(N-acetyl-b-d-glucosaminyl)-a-d-mannosyl]-glycoprotein 4-b-N-acetyl-d-glucosaminyltransferase Recommended name a-1,3-mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase Synonyms N-acetylglucosaminyltransferase IV N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase IV a-1,3-mannosylglycoprotein b-1,4-N-acetylglucosaminyltransferase b-acetylglucosaminyltransferase IV uridine diphosphoacetylglucosamine-glycopeptide b4-acetylglucosaminyltransferase IV CAS registry number 86498-16-0

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

Gallus gallus [1, 2] Rattus norvegicus [3] Bos taurus [4, 6] Homo sapiens [5, 8, 10] Homo sapiens (isozyme b [7]) [7] Homo sapiens [9]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + 3-(2-[N-acetyl-b-d-glucosaminyl]-a-d-mannosyl)-b-d-mannosyl-R = UDP + 3-(2,4-bis[N-acetyl-b-d-glucosaminyl]-ad-mannosyl)-b-d-mannosyl-R Reaction type hexosyl group transfer

278

2.4.1.145

a-1,3-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

Natural substrates and products S UDP-N-acetyl-d-glucosamine + 3-(2-[N-acetyl-b-d-glucosaminyl]-a-dmannosyl)-b-d-mannosyl-R <1> [1] P UDP + 3-(2,4-bis[N-acetyl-b-d-glucosaminyl]-a-d-mannosyl)-b-d-mannosyl-R Substrates and products S UDP-N-acetyl-d-glucosamine + 3-(2-[N-acetyl-b-d-glucosaminyl]-a-dmannosyl)-b-d-mannosyl-R <1-6> (<1> R represents the remainder of the N-oligosaccharide in the glycoprotein acceptor, the enzyme adds Nacetylglucosamine in b-1,4-linkage to the a-1,3-linked mannosyl residues of the trimannosyl core of N-glycosyloligosaccharides [1]; <1,3> maximal activity requires the presence of both terminal b-1,2-linked N-acetylglucosamine residues in the substrate [1,4]; <2> pyridylaminated sugar chain [3]) (Reversibility: ? <1-6> [1-10]) [1-10] P UDP + 3-(2,4-bis[N-acetyl-b-d-glucosaminyl]-a-d-mannosyl)-b-d-mannosyl-R <1-6> [1-10] Inhibitors Cu2+ <3> [4] EDTA <3> [4] Mn2+ <3> (<3> at high concentration [4]) [4] UDP <3> [4] Additional information <3> (<3> sugar nucleotides having the UDP moiety inhibit activity to different extent [4]) [4] Activating compounds Triton X-100 <1> (<1> maximum activation at 0.125% [1]) [1] Metals, ions Co2+ <3> [4] Mg2+ <3> [4] Mn2+ <1-3> [2-4] Specific activity (U/mg) 1.65 <3> [4] Additional information <1, 4, 5> (<1> activities for different glycopeptides as acceptors [1]; <4> comparison between normal and malignant kidney cortex cells [5]; <5> differences between isozymes expressed in COS7 cells [7]; <4> comparison between normal and malignant pancreatic cells [8]; <4> comparison between normal and malignant chorionic cells [10]) [1, 5, 7, 8, 10] Km-Value (mM) 3.4 <2> (pyridylaminated acceptor substrate) [3] 8.3 <2> (UDP-N-acetylglucosamine, <2> pyridylaminated acceptor substrate [3]) [3] pH-Optimum 7 <1> (<1> in presence of 0.625% Triton X-100 [1]) [1] 7.3 <2, 3> [3, 4]

279

a-1,3-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

2.4.1.145

pH-Range 6.5-7.5 <1> (<1> depends on Triton X-100 concentration [1]) [1] 7-8 <3> [4] Temperature optimum ( C) 37 <1, 2> (<1,2> assay at [1-3]) [1-3]

4 Enzyme Structure Molecular weight 58000 <3> (<3> deduced from SDS-PAGE and gel filtration results [4]) [4] 61610 <3> (<3> deduced from cDNA sequence [6]) [6] 63200 <5> (<5> isozyme b, deduced from cDNA sequence [7]) [7] Subunits monomer <3> (<3> 1 * 58000, SDS-PAGE [4]) [4] Posttranslational modification glycoprotein <3> (<3> Asn-linked sugar chains [4,6]) [4, 6]

5 Isolation/Preparation/Mutation/Application Source/tissue chorion <4> [10] kidney <4> [5] oviduct <1> [1, 2] pancreas <4> [8] small intestine <2, 3> [3, 4] spleen <2> [3] Localization membrane <1> [1, 2] Purification <1> (partial [1,2]) [1, 2] <3> (Q-Sepharose and Cu2+ Sepharose chromatography, followed by UDPhexanolamine-agarose affinity chomatography [4]) [4] Cloning <3> (expression in COS-7 cells [6]) [6] <5> (expression of the isozyme b in COS7 cells [7]) [7] <6> (expression in COS7 cells [9]) [9]

280

2.4.1.145

a-1,3-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

6 Stability Storage stability <1>, -20 C, crude microsomal preparation, several months in detergent-free buffer [2]

References [1] Gleeson, P.A.; Schachter, H.: Control of glycoprotein synthesis. J. Biol. Chem., 258, 6162-6173 (1983) [2] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and O-glycan synthesis. Methods Enzymol., 179, 351-397 (1989) [3] Taniguchi, N.; Nishikawa, A.; Fujii, S.; Gu, J.: Glycosyltransferase assays using pyridylaminated acceptors: N-acetylglucosaminyltransferase III, IV, and V. Methods Enzymol., 179, 397-408 (1989) [4] Oguri, S.; Minowa, M.T.; Ihara, Y.; Taniguchi, N.; Ikenaga, H.; Takeuchi, M.: Purification and characterization of UDP-N-acetylglucosamine: a1,3-dmannoside b1,4-N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase-IV) from bovine small intestine. J. Biol. Chem., 272, 2272122727 (1997) [5] Zhu, T.Y.; Chen, H.L.; Gu, J.X.; Zhang, Y.F.; Zhang, Y.K.; Zhang, R.A.: Changes in N-acetylglucosaminyltransferase III, IV, and V in renal cell carcinoma. J. Cancer Res. Clin. Oncol., 123, 296-299 (1997) [6] Minowa, M.T.; Oguri, S.; Yoshida, A.; Hara, T.; Iwamatsu, A.; Ikenaga, H.; Takeuchi, M.: cDNA cloning and expression of bovine UDP-N-acetylglucosamine: a1,3-d-mannoside b1,4-N-acetylglucosaminyltransferase IV. J. Biol. Chem., 273, 11556-11562 (1998) [7] Yoshida, A.; Minowa, M.T.; Takamatsu, S.; Hara, T.; Ikenaga, H.; Takeuchi, M.: A novel second isoenzyme of the human UDP-N-acetylglucosamine:a1,3-d-mannoside b1,4-N-acetylglucosaminyltransferase family: cDNA cloning, expression, and chromosomal assignment. Glycoconjugate J., 15, 1115-1123 (1998) [8] Nan, B.C.; Shao, D.M.; Chen, H.L.; Huang, Y.; Gu, J.X.; Zhang, Y.B.; Wu, Z.G.: Alteration of N-acetylglucosaminyltransferases in pancreatic carcinoma. Glycoconjugate J., 15, 1033-1037 (1998) [9] Yoshida, A.; Minowa, M.T.; Takamatsu, S.; Hara, T.; Oguri, S.; Ikenaga, H.; Takeuchi, M.: Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine:a1,3-d-mannoside b1,4-N-acetylglucosaminyltransferase. Glycobiology, 9, 303-310 (1999) [10] Takamatsu, S.; Oguri, S.; Minowa, M.T.; Yoshida, A.; Nakamura, K.; Takeuchi, M.; Kobata, A.: Unusually high expression of N-acetylglucosaminyltransferase-IVa in human choriocarcinoma cell lines: a possible enzymatic basis of the formation of abnormal biantennary sugar chain. Cancer Res., 59, 3949-3953 (1999)

281

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

2.4.1.146

1 Nomenclature EC number 2.4.1.146 Systematic name UDP-N-acetyl-d-glucosamine:O-glycosyl-glycoprotein (N-acetyl-d-glucosamine to d-galactose of b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)N-acetyl-d-galactosaminyl-R) b-1,3-N-acetyl-d-glucosaminyltransferase Recommended name b-1,3-galactosyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase Synonyms GlcNAcT-II <4> [7] O-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase II acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-mucin b(1-3)- (elongating) b3GlcNAc-T (elongation) <3> [3] core 1 extension b1,3-N-acetylglucosaminyltransferase <5> [8] core1-b3GlcNAcT <5> [8] elongation 3b-GalNAc transferase <3> [4] elongation 3b-GalNAc-transferase elongation b3 Gn-T <3> [4] uridine diphosphoacetylglucosamine-mucin b(1-3)- (elongating) acetylglucosaminyltransferase Additional information (cf. EC 2.4.1.102, EC 2.4.1.147 and EC 2.4.1.148) CAS registry number 87927-99-9

2 Source Organism <-1> no activity in Canis familiaris (submaxillary gland [1]) [1] <1> Sus scrofa [1, 2] <2> Rattus norvegicus [1] <3> Homo sapiens [3-6] <4> Oryctolagus cuniculus [7] <5> Homo sapiens [8] <6> Mus musculus [8]

282

2.4.1.146

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-R = UDP + N-acetyl-b-d-glucosaminyl1,3-b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-R Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-R <1, 2, 5, 6> (<5,6> enzyme plays essential role in the expression of the MECA-79-positive, high endothelial venule-specific l-selectin ligands required for lymphocyte homing [8]; <1,2,5> involved in elongation of O-glycan cores during mucin biosynthesis [1,8]) [1, 8] P UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,3-(N-acetyl-dglucosaminyl-1,6)-N-acetyl-d-galactosaminyl-R Substrates and products S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-glucosaminyl-1,3-(8-methoxycarbonyloctyl 3,6-di-O-(6-deoxy-a-d-mannopyranosyl)-b-d-mannopyranoside) <4> (<4> synthetic hydrophobic tetrasaccharide substrate [7]) (Reversibility: ? <4> [7]) [7] P UDP + N-acetyl-b-d-glucosaminyl-1,3-(8-methoxycarbonyloctyl 3,6-di-O((N-acetyl-b-d-glucosaminyl-1,6)-6-deoxy-a-d-mannopyranosyl)-b-dmannopyranoside) <4> [7] S UDP-N-acetyl-d-glucosamine + antifreeze glycoprotein <1, 2> (Reversibility: ? <1,2> [1]) [1] P UDP + N-acetyl-b-d-glucosaminyl-1,3-antifreeze glycoprotein <1, 2> [1] S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-R <1-3, 5, 6> (<1-3,5> i.e. core class 2 [1,2,6,8]; <1,2> substrate specificity [1]; <1,2> R. not H [1]; <1,2> polypeptide from mucin or antifreeze glycoprotein [1]) (Reversibility: ? <1-3,5,6> [1-6,8]) [1-6, 8] P UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,3-(N-acetyl-dglucosaminyl-1,6)-N-acetyl-d-galactosaminyl-R <1-3, 5, 6> [1-3, 5, 6, 8] S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-benzyl <1-3> (Reversibility: ? <13> [1,2,4-6]) [1, 2, 4-6] P UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,3-(N-acetyl-dglucosaminyl-1,6)-N-acetyl-d-galactosaminyl-benzyl <1-3> [1, 2, 5, 6] S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-o-nitrophenyl <1, 2> (Reversibility: ? <1,2> [1,2]) [1, 2]

283

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

2.4.1.146

P UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,3-(N-acetyl-dglucosaminyl-1,6)-N-acetyl-d-galactosaminyl-o-nitrophenyl <1, 2> [1, 2] S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-p-nitrophenyl <1-3, 5> (Reversibility: ? <1-3,5> [1-3,8]) [1-3, 8] P UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,3-(N-acetyl-dglucosaminyl-1,6)-N-acetyl-d-galactosaminyl-p-nitrophenyl <1-3, 5> [13, 8] S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-R <3, 5, 6> (<3> elongation [4, 6]; <3, 5> i.e. core class 1 [4, 6, 8]; <3> enzyme competes with core 2 b6-GlcNAc transferase, EC 2.4.1.102, for same substrates [4]) (Reversibility: ? <3, 5, 6> [4, 6, 8]) [4, 6, 8] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-R <3, 5, 6> [6, 8] S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,3-N-acetyl-d-galactosaminyl-p-nitrophenyl <5> (Reversibility: ? <5> [8]) [8] P UDP + b-d-galactosyl-1,3-(N-acetyl-b-d-glucosaminyl-1,6)-N-acetyl-dgalactosaminyl-p-nitrophenyl <5> [8] Inhibitors EDTA <1> (<1> 83% inhibition at 10 mM [1]) [1] Activating compounds Triton X-100 <1> (<1> stimulation, 0.1% v/v [1]) [1] Metals, ions Co2+ <1> (<1> activation [1]) [1] Mn2+ <1> (<3> not required [4]; <1> activation, 10-40 mM [1]) [1] Additional information <1> (<1> no activation by Ca2+ , Mg2+ , Zn2+ [1]) [1] Specific activity (U/mg) 0.0000007 <3> (<3> CaCo-2, colonic adenocarcinoma, cells differentiated to enterocytes [5]) [5] 0.0000015 <3> (<3> undifferentiated CaCo-2, colonic adenocarcinoma, cells [5]) [5] 0.0000033 <3> (<3> below, EBV immortilized B-cell line, from normal patients and such with the Wiskott-Aldrich syndrome [6]) [6] 0.000006 <3> (<3> normal granulocytes [4]) [4] 0.000032 <3> [6] Additional information <5> [8] Km-Value (mM) 0.13 <4> (N-acetyl-b-d-glucosaminyl-1,3-(8-methoxycarbonyloctyl 3,6-di-O(6-deoxy-a-d-mannopyranosyl)-b-d-mannopyranoside)) [7] 0.9 <1> (b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-o-nitrophenyl) [1] 1.2 <1> (b-d-galactosyl-1,3-(N-acetyl-d-glucosaminyl-1,6)-N-acetyl-d-galactosaminyl-benzyl) [1] 1.6 <1> (UDP-N-acetyl-d-glucosamine) [1]

284

2.4.1.146

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

pH-Optimum 6.7 <3> (<3> assay at [3]) [3] 7 <1, 3> (<1,3> assay at [2,6]; <1> sharp optimum [1]) [1, 2, 6] Temperature optimum ( C) 37 <1, 3, 4> (<1,3,4> assay at [2,3,6,7]) [2, 3, 6, 7]

5 Isolation/Preparation/Mutation/Application Source/tissue B-cell <3> (<3> EBV immortilized B-cell line, from normal patients and such with the Wiskott-Aldrich syndrome [3]) [3] CACO-2 cell <3> [5] HT-29 cell <5> (<5> colonic carcinoma cell line [8]) [8] T-lymphocyte <3, 6> [6, 8] blood platelet <3> [6] colon <1, 2, 5> [1, 8] colonic adenocarcinoma cell <3> [5] colorectal adenoma cell <3> (<3> during progression to cancer [6]) [6] enterocyte <3> [5] gastric mucosa <1> [1, 2] granulocyte <3> (<3> low level, in normal, but not in leukemic cells [4]) [4] liver <4> [7] placenta <5> [8] small intestine <5> (<5> low content [8]) [8] vascular tissue <5> (<5> high endothelial venule of secondary lymphoid organs [8]) [8] Additional information <1-3> (<3> not detectable in acute myeloid leukemia blast cell line and chronic myelogenous leukemia granulocyte cell line [4]; <1,2> low activity or absent in rat liver, stomach and submaxillary glands and pig submaxillary glands [1]) [1, 4] Localization membrane <1> [1] microsome <1, 3> [1, 3] Purification <4> (partial [7]) [7] Cloning <5> (isolation and cloning of full length DNA via EST and expression in CHO cells [8]; cloning of a sequence fragment encoding the soluble catalytic domain, expression as His-tagged protein in CHO cells [8]) [8] Application synthesis <5> (<5> use of the recombinant enzyme for construction of the 6sulfo sialyl Lewis x on extended core1 O-glycans [8]) [8]

285

b-1,3-Galactosyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

2.4.1.146

6 Stability Storage stability <1>, -70 C, microsomal pig stomach enzyme preparation, detergent-free 0.25 M sucrose suspension, several years [1]

References [1] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and Oglycan synthesis. Methods Enzymol., 179, 351-397 (1989) [2] Brockhausen, I.; Rachaman, E.S.; Matta, K.L.; Schachter, H.: The separation by liquid chromatography (under elevated pressure) of phenyl, benzyl, and O-nitrophenyl glycosides of oligosaccharides. Analysis of substrates and products for four N-acetyl-d-glucosaminyl-transferases involved in mucin synthesis. Carbohydr. Res., 120, 3-16 (1983) [3] Higgins, E.A.; Siminovitch, K.A.; Zhuang, D.; Brockhausen, I.; Dennis, J.W.: Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome. J. Biol. Chem., 266, 62806290 (1991) [4] Brockhausen, I.; Kuhns, W.; Schachter, H.; Matta K.L.; Sutherland, D.R.; Baker, M.A.: Biosynthesis of O-glycans in leukocytes from normal donors and from patients with leukemia: increase in O-glycan core 2 UDP-GlcNAc:Galb3GalNAca-R (GlcNAc to GalNAc) b(1,6)-N-acetylglucosaminyltransferase in leikemic cells. Cancer Res., 51, 1257-1263 (1991) [5] Brockhausen, I.; Romero, P.A.; Herscovics: Glycosyltransferase changes upon differentation of CaCo-2 human colonic adenocarcinoma cells. Cancer Res., 51, 3136-3142 (1991) [6] Vavasseur, F.; Dole, K.; Yang, J.; Matta, K.L.; Myerscough, N.; Corfield, A.; Paraskeva, C.; Brockhausen, I.: O-glycan biosynthesis in human colorectal adenoma cells during progression to cancer. Eur. J. Biochem., 222, 415-424 (1994) [7] Alton, G.; Srivastava, G.; Kaur, K.J.; Hindsgaul, O.: Use of N-acetylglucosaminyltransferases I and II in the synthesis of a dideoxypentasaccharide. Bioorg. Med. Chem., 2, 675-680 (1994) [8] Yeh, J.-C.; Hiraoka, N.; Petryniak, B.; Nakayama, J.; Ellies, L.G.; Rabuka, D.; Hindsgaul, O.; Marth, J.D.; Lowe, J.B.; Fukuda, M.: Novel sulfated lymphocyte homing receptors and their control by a Core1 extension b1,3-N-acetylglucosaminyltransferase. Cell, 105, 957-969 (2001)

286

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

2.4.1.147

1 Nomenclature EC number 2.4.1.147 Systematic name UDP-N-acetyl-d-glucosamine:O-glycosyl-glycoprotein (N-acetyl-d-glucosamine to N-acetyl-d-galactosaminyl-R) b-1,3-N-acetyl-d-glucosaminyltransferase Recommended name acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase Synonyms O-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase III acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-mucin b(1-3)b3Gn-T6 <6> [5] core 3 b3-GlcNAc-T <2, 3> [4] core 3 synthase <6> [5] core 3b-GlcNAc-transferase mucin core 3 b3-GlcNAc-transferase uridine diphosphoacetylglucosamine-mucin b(1-3)-acetylglucosaminyltransferase Additional information (cf. EC 2.4.1.102, EC 2.4.1.146 and EC 2.4.1.148) CAS registry number 87927-96-6

2 Source Organism <-1> no activity in Canis familiaris (submaxillary glands [1,3]) [1, 3] <1> Sus scrofa [1, 3] <2> Rattus norvegicus [1-4] <3> Homo sapiens [1, 3, 4] <4> monkey [1, 3] <5> Ovis aries [1, 3] <6> Homo sapiens (enzyme form b3-Gn-T6 [5]) [5]

287

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

2.4.1.147

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-galactosaminyl-R = UDP + Nacetyl-b-d-glucosaminyl-1,3-N-acetyl-b-d-galactosaminyl-R Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-galactosaminyl-a-R <2, 6> (<2,6> R: polypeptide derived from mucin, involved in mucin oligosaccharide biosynthesis [1,2,5]; <6> important role in synthesis of mucintype O-glycans in digestive organs [5]) [1, 2, 5] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-b-d-galactosaminyl-R Substrates and products S UDP-N-acetyl-d-glucosamine + 2-azido-2-deoxy-N-acetyl-a-d-galactosaminyl-benzyl <3> (Reversibility: ? <3> [4]) [4] P UDP + N-acetyl-b-d-glucosaminyl-2-azido-2-deoxy-1,3-N-acetyl-a-d-galactosaminyl-benzyl S UDP-N-acetyl-d-glucosamine + N-acetyl-d-galactosyl-ovine submaxillary gland mucin <1> (Reversibility: ? <1> [3]) [3] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-d-galactosyl-mucin of ovine submaxillary gland S UDP-N-acetyl-d-glucosamine + N-acetyl-a-d-galactosaminyl(CH2 )2 NHCO(CH2 )3 CO2 CH3 <3> (Reversibility: ? <3> [4]) [4] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-a-d-galactosaminyl(CH2 )2 NHCO(CH2 )3 CO2 CH3 S UDP-N-acetyl-d-glucosamine + N-acetyl-a-d-galactosaminyl-4-nitrophenyl <3, 6> (<6> recombinant protein, best substrate [5]) (Reversibility: ? <3,6> [4,5]) [4, 5] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-a-d-galactosaminyl-4-nitrophenyl S UDP-N-acetyl-d-glucosamine + N-acetyl-a-d-galactosaminyl-benzyl <2, 3> (<3> best substrate [4]) (Reversibility: ? <2,3> [1-4]) [1-4] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-a-d-galactosaminyl-benzyl <2, 3> [1-4] S UDP-N-acetyl-d-glucosamine + N-acetyl-a-d-galactosaminyl-phenyl <2> (Reversibility: ? <2> [1-3]) [1-3] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-a-d-galactosaminyl-phenyl <2> [1-3] S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-galactosaminyl-4-nitrophenyl <6> (<6> recombinant protein, 15% activity compared to N-acetylb-d-galactosaminyl-a-4-nitrophenyl [5]) (Reversibility: ? <6> [5]) [4, 5] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-b-d-galactosaminyl-4-nitrophenyl

288

2.4.1.147

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-galactosaminyl-MUC1 mucin <6> (Reversibility: ? <6> [5]) [5] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-b-d-galactosaminyl-MUC1 mucin <6> (<6> N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-b-d-galactosamine is bound to the glycoprotein via serine or threonine residues, resulting in a core 3 structure [5]) [1-5] S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-galactosaminyl-R <1-3, 6> (<2,3> substrate specificity is significantly influenced by the structure of the aglycon group [4]; <1-3,6> R: polypeptide derived from mucin [1,5]; <6> activity is restricted to mucins from specific tissues, e.g. stomach, small intestine, and colon [5]; <6> no activity towards a-GalNAc residues of blood group A and the core 6 substrates, and b-GalNAc residues of gangliosides, asialo-, lyso- and GalNAc-b-1,4-(NeuAc-a-2,3-)Gal-b-1,4Glc-b-1-1ceramide, i.e. GM2 [5]; <2> no acceptor substrate: native ovine submaxillary mucin, containing sialyl-2,6-N-acetyl-d-galactosaminylgroups [1,3]) (Reversibility: ? <1-3,6> [1-5]) [1-5] P UDP + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-b-d-galactosaminyl-R <1-3, 6> (<6> N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-b-d-galactosamine is bound to the glycoprotein via serine or threonine residues [5]) [1-5] S UDP-N-acetyl-d-glucosamine + a-d-galactosaminyl-4-nitrophenyl <3> (Reversibility: ? <3> [4]) [4] P UDP + N-acetyl-b-d-glucosaminyl-1,3-a-d-galactosaminyl-4-nitrophenyl S UDP-N-acetyl-d-glucosamine + asialofetuin <6> (<6> recombinant protein [5]; <6> no activity with fetuin [5]) (Reversibility: ? <6> [5]) [5] P UDP + N-acetyl-b-d-glucosaminyl-1,3-asialofetuin S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-a-d-galactosaminyl-4-nitrophenyl <6> (<6> recombinant protein, 2.4% activity compared to N-acetyl-b-d-galactosaminyl-a-4-nitrophenyl [5]) (Reversibility: ? <6> [5]) [4, 5] P UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-N-acetyl-a-dgalactosaminyl-4-nitrophenyl S UDP-N-acetyl-d-glucosamine + bovine submaxillary gland mucin <6> (<6> recombinant protein [5]; <6> no activity with fetuin [5]) (Reversibility: ? <6> [5]) [5] P UDP + N-acetyl-b-d-glucosaminyl-1,3-mucin of bovine submaxillary gland S UDP-N-acetyl-d-glucosamine + ovine submaxillary gland mucin <2, 3> (<2,3> acceptor substrate is desialylated, degalactosylated, defucosylated [4]) (Reversibility: ? <2,3> [4]) [4] P UDP + N-acetyl-b-d-glucosaminyl-1,3-mucin of ovine submaxillary gland Inhibitors Co2+ <1> (<1> in the presence of Mn2+ [3]) [3] EDTA <1> [1] N-iodoacetamido-galactosamine-a-benzyl <3> (<3> colon homogenates, 50% inhibition at 2 mM [4]) [4]

289

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

2.4.1.147

NaCl <2> (<2> slight inhibition at 0.1 M [4]) [4] PMSF <2> (<2> slight inhibition at 1 mM [4]) [4] Triton X-100 <1, 2> (<2> 0.5% reduces activity by 60% [4]; <1> above 0.5% v/v [3]) [3, 4] Triton X-114 <2> (<2> slight stimulation [4]) [4] UDP <2> (<2> 60% reduced activity at 10 mM [4]) [4] deoxycholate <2> (<2> total inactivation [4]) [4] Additional information <1> (<1> no inhibition by 2-mercaptoethanol or Nacetyl-d-glucosamine [3]) [3] Activating compounds NP 40 <2> (<2> activation [4]) [4] Triton X-100 <1, 2> (<1,2> stimulation, 0.1% v/v, inhibits at concentrations above 0.5% v/v [1,3,4]) [1, 3, 4] Metals, ions Mn2+ <1> (<1> absolute requirement, 10-20 mM, cannot be replaced by Co2+, Ca2+ , Mg2+ or Zn2+ [3]) [1, 3] Specific activity (U/mg) 0.00009 <3> (<3> colon mucosa [3]) [3] 0.00033 <2> (<2> colon mucosa [3]) [3] 0.00034 <1> (<1> colon mucosa [3]) [3] Km-Value (mM) 2 <1> (N-acetyl-d-galactosaminyl-a-benzyl) [1, 3] 3.2 <5> (N-acetyl-d-galactosaminyl-a-ovine submaxillary mucin) [1, 3] 5 <1> (N-acetyl-d-galactosaminyl-a-phenyl) [1, 3] pH-Optimum 6.5 <1> (<2> assay at [2]) [1-3] 6.5-7 <2, 3> (<2,3> assay at [4]) [4] 7.2 <6> (<6> assay at [5]) [5] Temperature optimum ( C) 37 <1-3, 6> (<1-3,6> assay at [1-5]) [1-5]

4 Enzyme Structure Subunits ? <6> (<6> x * 43000, amino acid sequence determination [5]; <6> x * 46600, recombinant FLAG-tagged fusion protein, SDS-PAGE [5]) [5] Posttranslational modification glycoprotein <6> (<6> recombinant FLAG-tagged fusion protein is glycosylated in insect cells [5]) [5]

290

2.4.1.147

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

5 Isolation/Preparation/Mutation/Application Source/tissue colon <2, 3, 6> (<6> normal colon, not colon cancer cells [5]) [4, 5] colonic mucosa <1-4> [1-3] skeletal muscle <6> (<6> low amount [5]) [5] small intestine <2, 6> (<2> low activity [1]) [1, 5] stomach <1, 2, 4-6> (<1,2,4,5> low activity [3]) [1, 3, 5] testis <6> (<6> low amount [5]) [5] Additional information <1-3> (<3> no activity in diverse cancer cell lines [4]; <2,3> tissue-specific expression [4]; <1,2> absent from submaxillary glands [1,3]) [1, 3, 4] Localization membrane <2, 3, 6> [4, 5] microsome <1-3> [3, 4] Additional information <6> (<6> type II membrane protein [5]) [5] Purification <6> (recombinant from Sf21 insect cells as FLAG-tagged fusion protein [5]) [5] Cloning <6> (expression in Spodoptera frugiperda Sf21 cells via baculovirus infection as soluble FLAG-tagged fusion protein [5]) [5]

6 Stability General stability information <2>, repeated freezing and thawing results in total loss of activity [4] <2, 3>, enzyme is extremly unstable in solubilized form [4]

References [1] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and Oglycan synthesis. Methods Enzymol., 179, 351-397 (1989) [2] Brockhausen, I.; Rachaman, E.S.; Matta, K.L.; Schachter, H.: The separation by liquid chromatography (under elevated pressure) of phenyl, benzyl, and O-nitrophenyl glycosides of oligosaccharides. Analysis of substrates and products for four N-acetyl-d-glucosaminyl-transferases involved in mucin synthesis. Carbohydr. Res., 120, 3-16 (1983) [3] Brockhausen, I.; Matta, K.L.; Orr, J.; Schachter, H.: Mucin synthesis. UDPGlcNAc:GalNAc-R b3-N-acetylglucosaminyltransferase and UDP-GlcNAc:GlcNAc b1-3GalNAc-R (GlcNAc to GalNAc) b6-N-acetylglucosaminyltransferase from pig and rat colon mucosa. Biochemistry, 24, 1866-1874 (1985)

291

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,3-N-acetylglucosaminyltransferase

2.4.1.147

[4] Vavasseur, F.; Yang, J.-M.; Dole, K.; Paulsen, H.; Brockhausen, I.: Synthesis of O-glycan core 3: characterization of UDP-GlcNAc:GalNAc-R b3-N-acetylglucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines. Glycobiology, 5, 351-357 (1995) [5] Iwai, T.; Inaba, N.; Naundorf, A.; Zhang, Y.; Gotoh, M.; Iwasaki, H.; Kudo, T.; Togayachi, A.; Ishizuka, Y.; Nakanishi, H.; Narimatsu, H.: Molecular cloning and characterization of a novel UDP-GlcNAc:GalNAc-peptide b1,3-N-acetylglucosaminyltransferase (b3Gn-T6), an enzyme synthesizing the core 3 structure of O-glycans. J. Biol. Chem., 277, 12802-12809 (2002)

292

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

2.4.1.148

1 Nomenclature EC number 2.4.1.148 Systematic name UDP-N-acetyl-d-glucosamine:O-oligosaccharide-glycoprotein (N-acetyl-dglucosamine to N-acetyl-d-galactosamine of N-acetyl-b-d-glucosaminyl-1,3N-acetyl-d-galactosaminyl-R) b-1,6-N-acetyl-d-glucosaminyltransferase Recommended name acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase Synonyms O-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase IV acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-mucin b(1!6)-, B core 4 b6-GalNAc-transferase core 6b-GalNAc-transferase B uridine diphosphoacetylglucosamine-mucin b(1!6)-acetylglucosaminyltransferase B Additional information <1, 2, 4-7> (<7,8> core 2 b6-GalNAc-transferase, EC 2.4.1.102, exists in 2 types, type L and type M, the latter shows an additional core 4 b6-GalNAc transferase activity [4,5]; <1,2,4-6> may be identical with EC 2.4.1.102 [3]; cf. EC 2.4.1.102, EC 2.4.1.146 and EC 2.4.1.147) [3-5] CAS registry number 87927-98-8

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

Rattus norvegicus [1-3] Sus scrofa [1, 3] Canis familiaris [1, 3] monkey [1, 3] Homo sapiens [1, 3] Ovis aries [1, 3] Mus musculus [4] bovine Herpes virus type 4 (i.e. BHV-4 [5]) [5]

293

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

2.4.1.148

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-dgalactosaminyl-R = UDP + N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-dglucosaminyl-1,3)-N-acetyl-d-galactosaminyl-R Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-glucosaminyl-1,3-N-acetyla-d-galactosaminyl-R <1-6> (<1-6> involved in mucin oligosaccharide biosynthesis [1-3]) [1-3] P UDP + N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,3)N-acetyl-a-d-galactosaminyl-R Substrates and products S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-glucosaminyl-1,3-N-acetyla-d-galactosaminyl-R <1-7> (<1-8> i.e. core 3 [1,4,5]; <8> enzyme shows both core 2 b6-GalNAc-transferase activity, EC 2.4.1.102, and core 4 b6GalNAc-transferase activity, EC 2.4.1.148, and is termed a type M core 2 b6-GalNAc-transferase [5]; <7> very low activity, recombinant L-type core 2 b6-N-acetylglucosaminyltransferase, EC 2.4.1.102, also shows minor core 4 b6-GalNAc-transferase activity [4]; <1-6> R: polypeptide derived from mucin [1]; <1-6> R is not H [1]) (Reversibility: ? <1-7> [1-4]) [1-5] P UDP + N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,3)N-acetyl-d-a-galactosaminyl-R <1-7> (<1-7> i.e. core 4 [1,4]) [1-4] S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-glucosaminyl-1,3-N-acetyla-d-galactosaminyl-benzyl <1-6> (<7> no activity [4]) (Reversibility: ? <1-6> [1-3]) [1-3] P UDP + N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,3)N-acetyl-a-d-galactosaminyl-benzyl <1-6> [1-3] S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-glucosaminyl-1,3-N-acetyla-d-galactosaminyl-p-nitrophenol <8> (<8> recombinant chimeric soluble enzyme [5]) (Reversibility: ? <8> [5]) [5] P UDP + N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,3)N-acetyl-a-d-galactosaminyl-p-nitrophenol S UDP-N-acetyl-d-glucosamine + N-acetyl-b-d-glucosaminyl-1,3-N-acetyla-d-galactosaminyl-phenyl <1-6> (<7> no activity [4]) (Reversibility: ? <1-6> [1-3]) [1-3] P UDP + N-acetyl-b-d-glucosaminyl-1,6-(N-acetyl-b-d-glucosaminyl-1,3)N-acetyl-a-d-galactosaminyl-phenyl <1-6> [1-3] Inhibitors 2-mercaptoethanol <1> (<1> weak [3]) [3] Ca2+ <1> (<1> weak [3]) [3] Co2+ <1> [3]

294

2.4.1.148

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

EDTA <1> (<1> weak [1,3]) [1, 3] Mg2+ <1> (<1> weak [3]) [3] Zn2+ <1> (<1> inactivation [3]) [3] Cofactors/prosthetic groups AMP <1> (<1> slight stimulation [3]) [3] ATP <1> (<1> slight stimulation [3]) [3] Activating compounds N-acetyl-d-glucosamine <1> (<1> slight stimulation [3]) [3] Triton X-100 <1> (<1> slight stimulation, 0.1% v/v [1,3]) [1, 3] Metals, ions Additional information <1> (<1> no Mn2+ -requirement [1,3]) [1, 3] Specific activity (U/mg) 0.00016 <4> (<4> stomach [3]) [3] 0.00017 <5> (<5> colon [3]) [3] 0.00028 <6> (<6> stomach [3]) [3] 0.0003 <3> (<3> submaxillary gland [3]) [3] 0.00042 <1> (<1> stomach [3]) [3] 0.0018 <1> (<1> colon [3]) [3] 0.00278 <2> (<2> stomach [3]) [3] Km-Value (mM) 0.6 <1> (N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-d-galactosaminyl-a-benzyl, <1> colon [1,3]) [1, 3] 1.8 <2> (N-acetyl-b-d-glucosaminyl-1,3-N-acetyl-d-galactosaminyl-a-benzyl, <2> stomach [1]) [1] pH-Optimum 6.5 <1> (<1> assay at [2]; <1> colon [1,3]) [1-3] Temperature optimum ( C) 37 <1> (<1> assay at [2,3]) [2, 3]

5 Isolation/Preparation/Mutation/Application Source/tissue colonic mucosa <1, 2, 4, 5> (<1> high activity [3]) [1-3] small intestine <1> [1] stomach <1, 2, 4, 6> [1, 3] submaxillary gland <1, 3> [1, 3] Additional information <1, 2> (<1> not in liver [1]; <2> not in submaxillary glands [1]) [1] Localization microsome <1, 2, 4-6> [3]

295

Acetylgalactosaminyl-O-glycosyl-glycoprotein b-1,6-N-acetylglucosaminyltransferase

2.4.1.148

Cloning <8> (functional expression in CHO cells, soluble chimeric enzyme form shows core 4 6b-GalNAc transferase activity, DNA and amino acid sequence determination [5]) [5]

References [1] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and Oglycan synthesis. Methods Enzymol., 179, 351-397 (1989) [2] Brockhausen, I.; Rachaman, E.S.; Matta, K.L.; Schachter, H.: The separation by liquid chromatography (under elevated pressure) of phenyl, benzyl, and O-nitrophenyl glycosides of oligosaccharides. Analysis of substrates and products for four N-acetyl-d-glucosaminyl-transferases involved in mucin synthesis. Carbohydr. Res., 120, 3-16 (1983) [3] Brockhausen, I.; Matta, K.L.; Orr, J.; Schachter, H.: Mucin synthesis. UDPGlcNAc:GalNAc-R b3-N-acetylglucosaminyltransferase and UDP-GlcNAc:GlcNAc b 1-3GalNAc-R (GlcNAc to GalNAc) b 6-N-acetylglucosaminyltransferase from pig and rat colon mucosa. Biochemistry, 24, 1866-1874 (1985) [4] Zeng, S.; Dinter, A.; Eisenkratzer, D.; Biselli, M.; Wandrey, C.; Berger, E.G.: Pilot scale expression and purification of soluble protein A tagged b1,6Nacetylglucosaminyltransferase in CHO cells. Biochem. Biophys. Res. Commun., 237, 653-658 (1997) [5] Vanderplasschen, A.; Markine-Goriaynoff, N.; Lomonte, P.; Suzuki, M.; Hiraoka, N.; Yeh, J.-C.; Bureau, F.; Willems, L.; Thiry, E.; Fukuda, M.; Pastoret, P.-P.: A multipotential b-1,6-N-acetylglucosaminyltransferase is encoded by bovine herpesvirus type 4. Proc. Natl. Acad. Sci. USA, 97, 5756-5761 (2000)

296

N-Acetyllactosaminide b-1,3-Nacetylglucosaminyltransferase

2.4.1.149

1 Nomenclature EC number 2.4.1.149 Systematic name UDP-N-acetyl-d-glucosamine:b-d-galactosyl-1,4-N-acetyl-d-glucosamine b1,3-acetyl-d-glucosaminyltransferase Recommended name N-acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase Synonyms Galb1!4GlcNAc-R b1!3 N-acetylglucosaminyltransferase GnT-i N-acetyllactosamine b(1-3)N-acetylglucosaminyltransferase UDP-GlcNAc:GalR, b-d-3-N-acetylglucosaminyltransferase UDP-GlcNAc:Galb1!4GlcNAcb-Rb1!3-N-acetylglucosaminyltransferase UDP-GlcNAc:Galb1-4Glc(NAc) b-1,3-N-acetylglucosaminyltransferase acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-acetyllactosaminide b1!3b3GnT7 <4> [9] i-GlcNAc transferase i-GlcNAcT iGnT poly-N-acetyllactosamine extension enzyme uridine diphosphoacetylglucosamine-acetyllactosaminide b1!3-acetylglucosaminyltransferase CAS registry number 85638-39-7

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

Rattus norvegicus (Sprague Dawley rats [1,5]) [1, 5, 8] Homo sapiens [2-4, 7, 10, 11] Bos taurus (newborn calf [6]) [6] Mus musculus (b3GnT7 [9]) [9] Cricetulus griseus [12] Homo sapiens [9]

297

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

2.4.1.149

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-d-glucosaminylR = UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-N-acetyl-dglucosaminyl-R Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + N-glycan <2, 3> (<3> physiological acceptors: N-glycans, O-glycans, glycolipids and keratan sulfates [6]; <2> polylactosamine extension occurs on both b1,2- and b1,6-branches of complex N-type glycans [7]) [6, 7] P ? S UDP-N-acetyl-d-glucosamine + O-glycan <3> (<3> physiological acceptors: N-glycans, O-glycans, glycolipids and keratan sulfates [6]) [6] P ? S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-R <1, 2> (<1> might function in the biosynthesis of cell surface polylactosaminoglycans on Novikoff cells and blood group i antigenic structures, enzyme controls the synthesis of linear chain types by adding a Nacetylglucosaminyl residue to a Galb(1-4)GlcNAc-R primer structure present on glycoprotein or glycolipid yielding a GlcNAcb(1-3)Galb(14)GlcNAc-R sequence [1]; <2> involved in the biosynthesis of blood group precursors, enzyme might control the biosynthesis of the linear carbohydrate chain by adding a N-acetylglucosaminyl residue to a Galb(1-4)GlcNAc-R structure present on oligosaccharides, glycosylceramides and glycoproteins [3]; <1> enzyme functions in both the initiation and elongation of linear i-active polylactosaminoglycan chains of Nglycoproteins and possibly other glycoconjugates [5]; <2> enzyme is essential for the formation of poly-N-acetyllactosamines and the i-antigen, responsible for the formation of GlcNAcb(1-3)Galb(1-4)GlcNAc-R structure and poly-N-acetyllactosamine extension [10]) [1, 3, 5, 10] P UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-R S UDP-N-acetyl-d-glucosamine + glycolipid <3> (<3> physiological acceptors: N-glycans, O-glycans, glycolipids and keratan sulfates [6]) [6] P ? S UDP-N-acetyl-d-glucosamine + keratan sulfate <3> (<3> physiological acceptors: N-glycans, O-glycans, glycolipids and keratan sulfates [6]) [6] P ? S UDP-N-acetyl-d-glucosamine + poly-N-acetyllactosamine <1-3, 5> (<3> involved in the initiation and extension of poly-N-acetyllactosamine biosynthesis, may act as a key enzyme [6]; <2> poly-N-acetyllactosamine biosynthesis, polylactosamine extension occurs on both b1,2- and b1,6-

298

2.4.1.149

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

branches of complex N-type glycans [7]; <1> biosynthesis of poly-N-acetyllactosamine chains on N-linked oligosaccharides, catalyzes a rate-limiting reaction in the expression of poly-N-acetyllactosamine chains, especially in pheochromocytoma cells [8]; <2> enzyme is capable of elongation of polylactosamine i-chains [11]; <5> synthesis of N-acetyllactosamine repeats in Asn-linked oligosaccharides is enhanced by an increase of enzyme [12]) [5-8, 10, 11, 12] P ? S Additional information <2, 4> (<2> the enzyme together with N-acetyllactosamine synthase, EC 2.4.1.90, catalyzes formation of linear glycans containing alternating b-3-O-substituted residues of d-galactose and b-4O-substituted residues of N-acetyl-d-glucosamine, structures are present among others in glycosphingolipids or H-II type, polyglycosylceramides and polyglycosylpeptides, e.g. erythroglycan [2]; <2> biosynthesis of lacto-series core chains, enzyme is activated in association with oncogenesis in colonic epithelia [4]; <4> b3GnT7 may play a role in preventing cells from migrating out of the original tissues and invading surrounding tissues [9]) [2, 4, 9] P ? Substrates and products S UDP-N-acetyl-d-glucosamine + GalNAcb(1-4)GlcNAcb(1-3)Galb(1-4)Glc <2> (Reversibility: ? <2> [11]) [11] P UDP + GlcNAcb(1-3)GalNAcb(1-4)GlcNAcb(1-3)Galb(1-4)Glc <2> (<2> mixture of the pentasaccharide product, 27 mol%, and the unreacted tetrasaccharide, 73 mol% [11]) [11] S UDP-N-acetyl-d-glucosamine + GalNAcb(1-4)GlcNAcb(1-3)Galb1-OMe <2> (Reversibility: ? <2> [11]) [11] P UDP + GlcNAcb(1-3)GalNAcb(1-4)GlcNAcb(1-3)Galb1-OMe <2> [11] S UDP-N-acetyl-d-glucosamine + GalNAcb(1-4)GlcNAcb1-OMe <2> (Reversibility: ? <2> [11]) [11] P UDP + GlcNAcb(1-3)GalNAcb(1-4)GlcNAcb1-OMe <2> [11] S UDP-N-acetyl-d-glucosamine + GalNAcb(1-4)GlcNAcb1-OR <2> (<2> i.e. N,N'-diacetyllactosediamine-OR [11]) (Reversibility: ? <2> [11]) [11] P UDP + GlcNAcb(1-3)GalNAcb(1-4)GlcNAcb1-OR <2> (<2> only 12.5% of the substrate is converted into the product, which is unusually resistant towards jackbean b-N-acetylhexosaminidase [11]) [11] S UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb(1-3)Galb(1-4)Glc <2, 3> (<3> i.e. lacto-N-neotetraose, 117% of activity with N-acetyllactosamine [6]) (Reversibility: ? <2,3> [6,10]) [6, 10] P UDP + GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)Galb(1-4)Glc <2, 3> [6, 10] S UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb(1-4)GlcNAc-2-aminopyridine <1> (Reversibility: ? <1> [8]) [8] P UDP + GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-4)GlcNAc-2-aminopyridine <1> [8] S UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb(1-6)(Galb(1-4)GlcNAcb(1-2))Mana(1-6)Manb-octyl <2> (<2> regioselectivity of en-

299

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

P S P S

P S

P S

P S P

300

2.4.1.149

zyme, favored site of GlcNAc addition is the lower b1,2-branch over the b1,6-branch by a 3:1 ratio resulting in a mixture of heptasaccharides [7]) (Reversibility: ? <2> [7]) [7] ? UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb-p-nitrophenol <2> (Reversibility: ? <2> [7]) [7] UDP + GlcNAcb(1-3)Galb(1-4)GlcNAcb-p-nitrophenol UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-R <1, 2> (<1> acts on b-galactosyl-1,4-N-acetylglucosaminyl termini on asialo-a1 -acid glycoproteins and other glycoproteins and oligosaccharides, GlcNAc residues are introduced to position C-3 of the terminal galactose of the glycoprotein [1]; <1> highly specific for acceptor oligosaccharides and glycoproteins carrying a terminal Galb(1-4)GlcNAcb1 -R unit, catalyzes the formation of GlcNAcb(1-3)Galb(1-4)GlcNAcb-R sequence, Galb(1-4)GlcNAcb(1-2)(Galb(1-4)GlcNAcb(1-6))Man pentasaccharide in the acceptor structure is a requirement for optimal activity, branch specificity, branches of this pentasaccharide structure, when contained in tri- and tetraantennary oligosaccharides, are highly preferred over other branches for attachment of the 1st and 2nd mol of GlcNAc into the acceptor molecule, enzyme also shows activity towards oligosaccharides related to blood group I- and i-active polylactosaminoglycans [5]; <2> iGnT has a unique characteristic in binding to acceptor substrates, preferentially adding poly-N-acetyllactosamine to membrane glycoproteins [10]) (Reversibility: ? <1,2> [1-3,5,10,11]) [1-3, 5, 10, 11] UDP + N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-R <1, 2> [1-3, 5, 10, 11] UDP-N-acetylglucosamine + N-acetyllactosamine <1-3, 5> (<2> more physiological acceptor than lactose [2]; <1,2> more effective acceptor than lactose [3,5]; <3> transfer of one GlcNAc to position C-3 of the terminal Gal residue of lactose or N-acetyllactosamine in the b linkage [6]) (Reversibility: ? <1-3,5> [2,3,5,6,12]) [2, 3, 5, 6, 12] UDP + GlcNAcb(1-3)Galb(1-4)GlcNAc <1, 2> [2, 3, 5] UDP-N-acetylglucosamine + asialo-a1 -acid glycoprotein <1-3> (<1> GlcNAc residues are introduced to position C-3 of the terminal galactose of the glycoprotein, relative high activity towards asialo-a1 -acid glycoprotein in Novikoff ascites tumor cells [1]; <1> much more effective than asialo-transferrin and asialo-fetuin, asialo-a1 -acid glycoprotein from human plasma Cohn fraction V, transfer of GlcNAc to a terminal Gal in a b1,3-linkage [5]; <3> best acceptor among the glycoproteins that contain Galb(1-4)GlcNAc [6]) (Reversibility: ? <1-3> [1,5,6,10]) [1, 5, 6, 10] UDP + N-acetylglucosaminylated asialo-a1 -acid glycoprotein <2> [10] UDP-N-acetylglucosamine + asialo-fetuin <1> (<1> much less effective than asialo-a1 -acid glycoprotein, transfer of GlcNAc to a terminal Gal in a b1,3-linkage [5]) (Reversibility: ? <1> [5]) [5] UDP + N-acetylglucosaminylated asialo-fetuin

2.4.1.149

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

S UDP-N-acetylglucosamine + asialo-transferrin <1> (<1> much less effective than asialo-a1 -acid glycoprotein, transfer of GlcNAc to a terminal Gal in a b1,3-linkage [5]) (Reversibility: ? <1> [5]) [5] P UDP + N-acetylglucosaminylated asialo-transferrin S UDP-N-acetylglucosamine + lactoneotetraosylceramide <2> (<2> lactoneotetraosylceramide from bovine erythrocytes, higher rate than with lactonorhexaosylceramide, efficiency decreases with growing acceptor chain length [4]) (Reversibility: ? <2> [4]) [4] P ? S UDP-N-acetylglucosamine + lactonorhexaosylceramide <2> (<2> lactonorhexaosylceramide from bovine erythrocytes, lower rate than with lactoneotetraosylceramide, efficiency decreases with growing acceptor chain length [4]) (Reversibility: ? <2> [4]) [4] P ? S UDP-N-acetylglucosamine + lactose <1-3> (<2> less physiological acceptor than N-acetyllactosamine [2]; <2> less effective acceptor than N-acetyllactosamine [3]; <1> 70% of activity with N-acetyllactosamine [5]; <3> transfer of one GlcNAc to position C-3 of the terminal Gal residue of lactose or N-acetyllactosamine in the b linkage, 51% of activity with N-acetyllactosamine [6]) (Reversibility: ? <1-3> [2,3,5,6,10]) [2, 3, 5, 6] P UDP + GlcNAcb(1-3)Galb(1-4)Glc <1-3> [2, 5, 6, 10] S UDP-N-acetylglucosamine + lactosylceramide <2> (<2> lactosylceramide from human erythrocytes [4]) (Reversibility: ? <2> [4]) [4] P ? S UDP-N-acetylglucosamine + lactotetraosylceramide <2> (<2> lactotetraosylceramide from human meconium, poor substrate [4]) (Reversibility: ? <2> [4]) [4] P ? S UDP-N-acetylglucosamine + porcine submaxillary asialo-afuco-mucin <1> (<1> poor acceptor, Galb(1-3)GalNAc as terminal acceptor structure [1]; <1> very poor substrate [5]) (Reversibility: ? <1> [1,5]) [1, 5] P UDP + N-acetylglucosaminylated porcine submaxillary asialo-afuco-mucin S Additional information <1-3> (<2> no acceptors: melibiose, gentiobiose, galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine [3]; <2> transfer of GlcNAc occurs mainly to type 2 chain nonfucosylated structures, elongation of type 1 chain structure Lc4 is also detectable, no transfer to any fucosylated derivative of either type 1 or 2 chains, transfer of GlcNAc to a terminal Gal residue on a lacto-series core chain [4]; <1> asialo-serum glycoproteins are much more effective than O-glycoproteins as acceptors, overview over oligosaccharide substrates [5]; <3> terminal Galb(1-4)Glc(NAc) sequences, i.e. type II chains, are preferred substrates, no substrates: terminal Galb(1-3)GlcNAc sequences, i.e. type I chains, Lewis X trisaccharides, i.e. Galb(1-4)(Fuca(1-3))GlcNAc, monosaccharides, e.g. galactose, asialo-bovine submaxillary mucin that contains GalNAca1Ser/Thr and Galb(1-3)GalNAca1-Ser/Thr [6]; <2> no acceptor: Galb(13)GlcNAcb(1-3)Galb(1-4)Glc [10]) [3-6, 10] P ? 301

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

2.4.1.149

Inhibitors 2-mercaptoethanol <3> [6] 4-thiouridine diphosphate <3> (<3> inhibits in a concentration-dependent manner [6]) [6] CH3 CN <3> [6] CaCl2 <1> (<1> weak [5]) [1, 5] CdCl2 <1> [5] EDTA <1, 2> (<2> 10 mM, complete inhibition [4]; <1> strong [5]) [1, 4, 5] FeCl2 <1> [5] N-acetyllactosamine <3> (<3> strong product inhibition [6]) [6] NiCl2 <1> [5] SDS <3> [6] UDP <3> (<3> 1 mM, 70% inhibition [6]) [6] ZnCl2 <1> (<1> strong [5]) [1, 5] Additional information <1> (<1> nerve growth factor stimulated PC-12 cells with reduced GnT-i activity compared to unstimulated cells, no effect in PC12D cells [8]) [8] Activating compounds ATP <2> (<2> omitting ATP from reaction mixture decreases activity by 87% [3]) [3] CHAPSO <2> (<2> activates solubilized enzyme [4]) [4] G-3634-A <2> (<2> activates solubilized enzyme [4]) [4] Triton CF-54 <2> (<2> activates solubilized enzyme, activity is optimal when assayed in the presence of a final concentration of 0.3% [4]) [4] Triton X-100 <2> (<2> activates solubilized enzyme [4]) [4] deoxycholate <2> (<2> activates solubilized enzyme [4]) [4] Additional information <2> (<2> not activated by Triton X-100 [3]; <2> CDPcholine stimulates by protecting UDP-GlcNAc from hydrolysis by endogenous enzymes [4]) [3, 4] Metals, ions Cd2+ <1> (<1> CdCl2 slightly stimulates [1]) [1] Co2+ <3> (<3> slightly stimulates, 12.8% as effective as Mn2+ at 10 mM [6]) [6] Mg2+ <1> (<1> MgCl2 slightly stimulates [1]) [1] Mn2+ <1-3> (<1> MnCl2 : 10-25 mM for optimal activity required [1]; <2> requirement [2]; <2> essential for activity, neither Mg2+ nor Ca2+ can substitute for Mn2+ [3]; <2,3> strict requirement [4,6]; <1> MnCl2 stimulates 2.5fold, optimal concentration: 20 mM [5]; <3> optimum concentration: 215 mM, cooperative activating effects by combination of Mn2+ at a low concentration and Mg2+ or Ca2+ [6]) [1-6] Additional information <1-3> (<2> not activated by Mg2+ or Ca2+ [3]; <1> absolute requirement for divalent cations, not stimulated by Mg2+ [5]; <3> enzyme possesses 2 distinct metal binding sites, not activated by Ca2+ , Mg2+ , Zn2+ , Fe2+ , Cu2+ , Ni2+ , K+ , Na+ [6]) [3, 5, 6]

302

2.4.1.149

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

Specific activity (U/mg) 0.00153 <1> [5] 0.118 <3> [6] Additional information <1> [8] Km-Value (mM) 0.129 <3> (UDP-N-acetyl-d-glucosamine) [6] 0.133 <2> (UDP-N-acetyl-d-glucosamine, <2> lactose as acceptor [3]) [3] 0.17 <2> (UDP-N-acetyl-d-glucosamine, <2> lactosylceramide as acceptor [4]) [4] 0.19 <2> (lactoneotetraosylceramide) [4] 0.19 <2> (lactonorhexaosylceramide) [4] 0.44 <1> (Galb(1-4)GlcNAcb(1-4)GlcNAc-2-aminopyridine, <1> PC-12 cells [8]) [8] 0.6 <1> (asialo-a1 -acid glycoprotein) [5] 0.9 <1> (Galb(1-4)GlcNAcb(1-4)GlcNAc-2-aminopyridine, <1> PC-12D cells [8]) [8] 1 <1> (Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAc) [5] 2.2 <1> (N-acetyllactosamine) [5] 5.2 <1> (lactose) [5] 7 <2> (N-acetyllactosamine) [3] 8 <1> (UDP-N-acetyl-d-glucosamine, <1> PC-12 and PC-12D cells [8]) [8] 18.2 <3> (lactose) [6] 19.6 <3> (N-acetyllactosamine) [6] 29.8 <2> (lactose) [3] Additional information <1> [5] pH-Optimum 5.8-7.5 <2> (<2> broad [4]) [4] 6.5-8 <1> [1] 6.8-7.2 <1> (<1> asialo-a1 -acid glycoprotein as acceptor [5]) [5] 6.8-7.8 <2> (<2> broad [2]) [2] 7 <3> (<3> broad pH-optimum around pH 7 [6]) [6] 7-7.2 <2> (<2> highest activity, with Hepes buffer [4]) [4] Additional information <4> (<4> pI: 8.7 [9]) [9] pH-Range 6-7.5 <3> (<3> substantial reaction in the range, 65% of activity at pH 7 [6]) [6] Temperature optimum ( C) 37 <1-3> (<1-3> assay at [1-8,10]) [1-8, 10] 42 <3> (<3> 112% of activity compared with that at 37 C [6]) [6]

4 Enzyme Structure Molecular weight 90000 <3> (<3> gel filtration [6]) [6] 303

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

2.4.1.149

Subunits ? <2, 4> (<4> x * 45000, calculated from the amino acid sequence [9]; <2> x * 47125, calculated from the amino acid sequence, mature N-glycosylated iGnT: may be approximately 53 kDa [10]) [9, 10] monomer <3> (<3> 1 * 70000, SDS-PAGE [6]) [6] Posttranslational modification glycoprotein <1-3> (<2> 2 potential N-glycosylation sites [10]) [5, 6, 10]

5 Isolation/Preparation/Mutation/Application Source/tissue CHO cell <5> (<5> Pro-5 and Lec2 CHO cells [12]) [12] FM3A cell <4> (<4> breast cancer cell line, higher expression of b3GnT7 in cancer cell lines with low degrees of invasiveness [9]) [9] KLN205 cell <4> (<4> KLN205-MUC1, lung cancer cell line, higher expression of b3GnT7 in cancer cell lines with low degrees of invasiveness [9]) [9] Novikoff ascites tumor <1> (<1> strain N1S1-67 [1,5]) [1, 5] PC-12 cell <1> (<1> pheochromacytoma cells, nerve growth factor stimulated cells with reduced GnT-i activity compared to unstimulated cells [8]) [8] PC-12D cell <1> (<1> new subline of pheochromacytoma cells PC-12, low GnT-i activity [8]) [8] SW-1116 cell <2> (<2> colonic carcinoma cell line [10]) [10] SW-40 cell <2> (<2> colonic adenocarcinoma cell line [4]) [4] WM-266-4 cell <2> (<2> human melanoma cell line [10]) [10] brain <2> (<2> highly expressed in fetal and adult brain [10]) [10] cerebellum <4> (<4> very weak expression of b3GnT7 [9]) [9] cerebrum <4> (<4> very weak expression of b3GnT7 [9]) [9] colon <4> (<4> b3GnT7 is most strongly expressed in the placenta and colon [9]) [9] heart <4> (<4> very weak expression of b3GnT7 [9]) [9] kidney <2, 4> (<4> moderate expression of b3GnT7 [9]; <2> highly expressed in fetal kidney [10]) [9, 10] lung <4> (<4> moderate expression of b3GnT7 [9]) [9] placenta <4> (<4> b3GnT7 is most strongly expressed in the placenta at the later stages of gestation and in colon [9]) [9] serum <2, 3> (<2> normal blood group 0 serum [3]; <3> newborn calf serum [6]) [2, 3, 6, 11] small intestine <4> (<4> moderate expression of b3GnT7 [9]) [9] stomach <4> (<4> moderate expression of b3GnT7 [9]) [9] testis <4> (<4> very weak expression of b3GnT7 [9]) [9] Additional information <2> (<2> enzyme is undetectable in normal colonic epithelial cells, but is greatly enhanced both during fetal development and in colonic adenocarcinomas [4]; <2> enzyme is expressed ubiquitously in various adult tissues [10]) [4, 10]

304

2.4.1.149

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

Localization Golgi apparatus <2> (<2> stem region and large catalytic domain reside presumably in the Golgi lumen [10]) [10] membrane <2> (<2> membrane-bound [4]; <2> type II membrane protein topology, longest transmembrane domain among glycosyltransferases cloned so far [10]) [4, 10] soluble <2> [3, 7] Purification <1> (partial, 178fold, from Novikoff tumor cell ascites fluid [5]) [5] <3> (125000fold [6]) [6] Cloning <2> (plasmid cDNA encoding the catalytic domain of i-GlcNAcT, amino acids 53-415, is expressed in COS-1 cells [7]; cDNA from melanoma and colonic cancer cells is cloned and overexpressed in human Burkitt lymphoma Namalwa KJM-1 cells overexpressing the i-antigen, nucleotide and amino acid sequence of the 415-amino acids protein, cDNA fragment encoding the stem region plus putative catalytic domain of iGnT fused to protein A is expressed in COS-1 cells [10]) [7, 10] <4> (cDNA from placenta encoding b3GnT7 is cloned and partially characterized, open reading frame encodes a 397-amino acids protein [9]) [9] <6> (human ortholog to mouse enzyme b3GnT7 has the chromosomal locus 2q37.1 [9]) [9] Application medicine <4> (<4> enzyme may be used as diagnostic marker of human cancer or as target molecule for the development of a new therapy [9]) [9]

6 Stability Temperature stability 4 <3> (<3> 4% of activity compared with that at 37 C [6]) [6] 12 <3> (<3> 9% of activity compared with that at 37 C [6]) [6] 20 <3> (<3> 28% of activity compared with that at 37 C [6]) [6] 30 <3> (<3> 63% of activity compared with that at 37 C [6]) [6] 42 <3> (<3> 112% of activity compared with that at 37 C [6]) [6] 45 <3> (<3> 88% of activity compared with that at 37 C [6]) [6] 50 <3> (<3> 10% of activity compared with that at 37 C [6]) [6] 55 <3> (<3> 2% of activity compared with that at 37 C [6]) [6] Storage stability <1>, 0-4 C, lyophilized enzyme form, over 6 months, stable [5] <3>, 4 C, purified enzyme, at least 2 weeks, stable [6]

305

N-Acetyllactosaminide b-1,3-N-acetylglucosaminyltransferase

2.4.1.149

References [1] van den Eijnden, D.H.; Winterwerp, H.; Smeeman, P.; Schiphorst, W.E.C.M.: Novikoff ascites tumor cells contain N-acetyllactosaminide b 1 leads to 3 and b1!6 N-acetylglucosaminyltransferase activity. J. Biol. Chem., 258, 3435-3437 (1983) [2] Zielenski, J.; Koscielak, J.: The occurrence of two novel N-acetylglucosaminyltransferase activities in human serum. FEBS Lett., 158, 164-168 (1983) [3] Hosomi, O.; Takeya, A.; Kogure, T.: Human serum contains N-acetyllactosamine:b1-3 N-acetylglucosaminyltransferase activity. J. Biochem., 95, 16551659 (1984) [4] Holmes, E.H.: Characterization of a b1!3-N-acetylglucosaminyltransferase associated with synthesis of type 1 and type 2 lacto-series tumor-associated antigens from the human colonic adenocarcinoma cell line SW403. Arch. Biochem. Biophys., 260, 461-468 (1988) [5] van den Eijnden, D.H.; Koenderman, A.H.L.; Schiphorst, W.E.C.M.: Biosynthesis of blood group i-active polylactosaminoglycans. Partial purification and properties of an UDP-GlcNAc:N-acetyllactosaminide b1!3-Nacetylglucosaminyltransferase from Novikoff tumor cell ascites fluid. J. Biol. Chem., 263, 12461-12471 (1988) [6] Kawashima, H.; Yamamoto, K.; Osawa, T.; Irimura, T.: Purification and characterization of UDP-GlcNAc:Galb1-4Glc(NAc)b-1,3-N-acetylglucosaminyltransferase (poly-N-acetyllactosamine extension enzyme) from calf serum. J. Biol. Chem., 268, 27118-27126 (1993) [7] McAuliffe, J.C.; Ujita, M.; Fukuda, M.; Hindsgaul, O.: Synthesis of selectively radiolabeled hexasaccharides for the determination of enzymatic regioselectivity. Glycoconjugate J., 16, 767-772 (1999) [8] Fukuzumi, M.; Maruyama, S.; Sano, M.; Fukui, S.: Comparison of the expression of cell surface poly-N-acetyllactosamine-type oligosaccharides in PC12 cells with those in its variant PC12D. Glycobiology, 11, 481-494 (2001) [9] Kataoka, K.; Huh, N.-h.: A novel b1,3-N-acetylglucosaminyltransferase involved in invasion of cancer cells as assayed in vitro. Biochem. Biophys. Res. Commun., 294, 843-848 (2002) [10] Sasaki, K.; Kurata-Miura, K.; Ujita, M.; Angata, K.; Nakagawa, S.; Sekine, S.; Nishi, T.; Fukuda, M.: Expression cloning of cDNA encoding a human b1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis. Proc. Natl. Acad. Sci. USA, 94, 14294-14299 (1997) [11] Salo, H.; Aitio, O.; Ilves, K.; Bencomo, E.; Toivonen, S.; Penttilä, L.; Niemelä, R.; Salminen, H.; Grabenhorst, E.; Renkonen, R.; Renkonen, O.: Several polylactosamine-modifying glycosyltransferases also use internal GalNAcb1-4GlcNAc units of synthetic saccharides as acceptors. Glycobiology, 12, 217-228 (2002) [12] Hummel, M.; Hedrich, H.C.; Hasilik, A.: Elongation of N-acetyllactosamine repeats in diantennary oligosaccharides. Eur. J. Biochem., 245, 428-433 (1997)

306

N-Acetyllactosaminide b-1,6-N-acetylglucosaminyl-transferase

2.4.1.150

1 Nomenclature EC number 2.4.1.150 Systematic name UDP-N-acetyl-d-glucosamine:b-d-galactosyl-1,4-N-acetyl-d-glucosaminide b-1,6-N-acetyl-d-glucosaminyltransferase Recommended name N-acetyllactosaminide b-1,6-N-acetylglucosaminyl-transferase Synonyms Galb1!4GlcNAc-R b1!6 N-acetylglucosaminyltransferase I N-acetylglucosaminyltransferase IGnT N-acetylglucosaminyltransferase UDP-GlcNAc:Gal-R, b-d-6-N-acetylglucosaminyltransferase acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-acetyllactosaminide b1!6b1!6GnT uridine diphosphoacetylglucosamine-acetyllactosaminide b1!6-acetylglucosaminyltransferase Additional information <2> (<2> C2GnT forms the core 2 O-glycan branch, IGnT forms the I antigen, both are members of a b-1,6-N-acetylglucosaminyltransferase gene family [6]) [6] CAS registry number 85638-40-0

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

Rattus norvegicus (Sprague Dawley rats [1]) [1, 9] Homo sapiens [2, 3, 6, 7, 9] Mus musculus [4, 9] Sus scrofa [8, 9] Mus musculus (<5> IGnT A, 2 isoenzymes: IGnT A and B [5]) [5] Mus musculus (<5> IGnT B, 2 isoenzymes: IGnT A and B [5]) [5]

307

N-Acetyllactosaminide b-1,6-N-acetylglucosaminyl-transferase

2.4.1.150

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-d-glucosaminylR = UDP + N-acetyl-b-d-glucosaminyl-1,6-b-d-galactosyl-1,4-N-acetyl-dglucosaminyl-R Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + oligo-N-acetyllactosaminoglycan <2> (<2> involved in midchain branching of oligo-N-acetyllactosaminoglycans by transferring GlcNAc in b1,6-linkage to internal galactose residues [2]) [2] P ? S UDP-N-acetyl-d-glucosamine + poly-N-acetyllactosamine <2-6> (<3,5,6> forms branches in poly-N-acetyllactosamines, which bear the I blood group antigen [4,5]; <2> responsible for the conversion of linear to branched polylactosamines, cIGnT6 actions at central rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens [7]; <4> responsible for the formation of the b16-branched poly-N-acetyllactosamine structure, involved in generating branches to central positions of preformed as well as growing polylactosamine chains but not in synthesizing the distal branches to growing chains [8]) [4, 5, 7, 8] P ? S UDP-N-acetyl-d-glucosamine + poly-N-acetyllactosaminoglycan <1> (<1> might function in biosynthesis of cell surface polylactosaminoglycans on Novikoff cells and blood group I antigenic structures, formation of the GlcNAcb(1-3)(GlcNAcb(1-6))Gal-R branching points in the branched type of polylactosylaminoglycans [1]) [1] P ? S Additional information <2> (<2> initiates formation of side chains, key enzyme in biosynthesis of I antigen of erythrocytes, N-acetyllactosamine is a more physiological acceptor than lactose [3]; <2> C2GnT forms the core 2 O-glycan branch, which is critical for oligosaccharide-mediated cell-cell interaction, IGnT forms the I antigen, both are members of a b1,6-N-acetylglucosaminyltransferase gene family [6]; <2> expression of I-antigen is entirely dependent on IGnT, expression of IGnT is developmentally regulated [9]) [3, 6, 9] P ? Substrates and products S UDP-N-acetyl-d-glucosamine + Gala(1-3)Galb(1-4)GlcNAcb(1-3)Galb(14)Glc <5, 6> (<6> IGnT B: at 148% of the rate with lacto-N-neotetraose [5]; <5> IGnT A: at 256% of the rate with lacto-N-neotetraose [5]) (Reversibility: ? <5,6> [5]) [5]

308

2.4.1.150

N-Acetyllactosaminide b-1,6-N-acetylglucosaminyl-transferase

P ? S UDP-N-acetyl-d-glucosamine + Gala(1-3)Galb(1-4)GlcNAcb(1-3)Galb(14)GlcNAc <2> (<2> reaction rate is 38% of that with GlcNAcb(13)Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAc [2]) (Reversibility: ? <2> [2]) [2] P ? S UDP-N-acetyl-d-glucosamine + Galb(1-3)GlcNAcb(1-3)Galb(1-4)Glc <5, 6> (<5,6> poor substrate [5]; <6> IGnT B: at 6% of the rate with lactoN-neotetraose [5]; <5> IGnT A: at 4% of the rate with lacto-N-neotetraose [5]) (Reversibility: ? <5,6> [5]) [5] P ? S UDP-N-acetyl-d-glucosamine + Galb(1-3)GlcNAcb(1-3)Galb(1-4) GlcNAIb(1-3)Galb(1-4)Glc <5, 6> (<5,6> poor substrate [5]; <6> IGnT B: at 10% of the rate with lacto-N-neotetraose [5]; <5> IGnT A: at 6% of the rate with lacto-N-neotetraose [5]) (Reversibility: ? <5,6> [5]) [5] P ? S UDP-N-acetyl-d-glucosamine + Galb(1-4)(Fuca(1-3))GlcNAcb(1-3) Galb(1-4)Glc <5, 6> (<5,6> very poor substrate [5]) (Reversibility: ? <5,6> [5]) [5] P ? S UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb(1-3)Galb(1-4)Glc <6> (<6> i.e. lacto-N-neotetraose, IGnT B forms branch in the internal Gal residue [5]) (Reversibility: ? <6> [5]) [5] P UDP + Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)Glc <6> (<6> no transfer to the terminal Gal residue [5]) [5] S UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAc <2, 4> (<2> cIGnT [7]; <4> cIGnT6 [8]) (Reversibility: ? <2,4> [7,8]) [7, 8] P UDP + Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)GlcNAc <2, 4> [7, 8] S UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb(1-3)Galb(1-4) GlcNAcb(1-3)Galb(1-4)Glc <5, 6> (<5,6> i.e. para-lacto-N-neohexaose [5]; <6> IGnT B: at 181% of the rate with lacto-N-neotetraose [5]; <5> IGnT A: at 170% of the rate with lacto-N-neotetraose [5]) (Reversibility: ? <5,6> [5]) [5] P UDP + Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)GlcNAcb(1-3) Galb(1-4)Glc <5, 6> (<6> IGnT B: major product, minor product is Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(14)Glc, IGnT B forms b-1,6 branch in both of the internal galactosyl residues, prolonged incubation results in di-branched oligosaccharide [5]) [5] S UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb(1-3)Galb(1-4) GlcNAcb(1-3)Galb(1-4)GlcNAc <4-6> (<6> IGnT B: at 164% of the rate with lacto-N-neotetraose [5]; <5> IGnT A: at 194% of the rate with lactoN-neotetraose [5]; <4> cIGnT6 [8]) (Reversibility: ? <4-6> [5,8]) [5, 8] P UDP + Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)GlcNAcb(1-3) Galb(1-4)GlcNAc <4> (<4> cIGnT6, 60% of the heptasaccharide product, 40% of the heptasaccharide product is Galb(1-4)GlcNAcb(1-3)Galb(1309

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4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)GlcNAc, 95% heptasaccharide and 5% di-branched octasaccharide product [8]) [8] UDP-N-acetyl-d-glucosamine + Galb(1-4)GlcNAcb(1-3)Galb(1-4) GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAc <2> (<2> cIGnT [7]) (Reversibility: ? <2> [7]) [7] UDP + (GlcNAc)1-Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)Galb(14)GlcNAcb(1-3)Galb(1-4)GlcNAc <2> (<2> also as product: (GlcNAc)2 Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3) Galb(1-4)GlcNAc, cIGnT6 generates in a partial reaction nona- and decasaccharide products, which represent mixtures of isomers carrying one or two GlcNAc-branches on the linear octasaccharide acceptor [7]) [7] UDP-N-acetyl-d-glucosamine + GlcNAcb(1-3)Galb(1-3)GalNAca1-R <4> (<4> dIGnT [9]) (Reversibility: ? <4> [9]) [9] UDP + GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-3)GalNAca1 -R <4> [9] UDP-N-acetyl-d-glucosamine + GlcNAcb(1-3)Galb(1-4)Glc <4> (<4> dIGnT [9]) (Reversibility: ? <4> [9]) [9] UDP + GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)Glc <4> [9] UDP-N-acetyl-d-glucosamine + GlcNAcb(1-3)Galb(1-4)Glc(NAc)b1-R <4> (<4> dIGnT [9]) (Reversibility: ? <4> [9]) [9] UDP + GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)Glc(NAc)b1-R <4> [9] UDP-N-acetyl-d-glucosamine + GlcNAcb(1-3)Galb(1-4)GlcNAc <2> (<2> very poor acceptor, reaction rate is 2% of that with GlcNAcb(1-3)Galb(14)GlcNAcb(1-3)Galb(1-4)GlcNAc [2]) (Reversibility: ? <2> [2]) [2] UDP + GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)GlcNAc <2> [2] UDP-N-acetyl-d-glucosamine + GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)Gal <2> (<2> poor acceptor, reaction rate is 6% of that with GlcNAcb(13)Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAc [2]) (Reversibility: ? <2> [2]) [2] ? UDP-N-acetyl-d-glucosamine + GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3) Galb(1-4)Glc <2, 5, 6> (<2> reaction rate is 41% of that with GlcNAcb(13)Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAc [2]; <6> IGnT B: at 41% of the rate with lacto-N-neotetraose [5]; <5> IGnT A: at 22% of the rate with lacto-N-neotetraose [5]) (Reversibility: ? <2,5,6> [2,5]) [2, 5] UDP + GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4)Glc <2, 5, 6> (<5,6> only product [5]) [2, 5] UDP-N-acetyl-d-glucosamine + GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3) Galb(1-4)GlcNAc <2, 4> (<2> GlcNAc residue at the reducing end side of the branching galactose plays a role in the reaction [2]; <4> cIGnT6 [8]) (Reversibility: ? <2,4> [2,8]) [2, 8] UDP + GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(1-4) GlcNAc <2, 4> [2, 8] UDP-N-acetyl-d-glucosamine + N-acetyllactosamine <2> (Reversibility: ? <2> [3]) [3] UDP + GlcNAcb(1-6)Galb(1-4)GlcNAc UDP-N-acetyl-d-glucosamine + NeuAca(2-3)Galb(1-4)GlcNacb(1-3) Galb(1-4)Glc <5, 6> (<6> IGnT B: at 123% of the rate with lacto-N-neo-

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N-Acetyllactosaminide b-1,6-N-acetylglucosaminyl-transferase

tetraose [5]; <5> IGnT A: at 35% of the rate with lacto-N-neotetraose [5]) (Reversibility: ? <5,6> [5]) [5] ? UDP-N-acetyl-d-glucosamine + NeuAca(2-6)Galb(1-4)GlcNacb(13)Galb(1-4)Glc <5, 6> (<6> IGnT B: at 148% of the rate with lacto-Nneotetraose [5]; <5> IGnT A: at 256% of the rate with lacto-N-neotetraose [5]) (Reversibility: ? <5,6> [5]) [5] ? UDP-N-acetyl-d-glucosamine + asialo-a1 -acid glycoprotein <1, 2> (<1> GlcNAc residues are introduced to position C-6 of the terminal galactose of the glycoprotein, relative high activity towards asialo-a1 -acid glycoprotein in Novikoff ascites tumor cells [1]; <1,2> acts on b-galactosyl-1,4-Nacetylglucosaminyl-termini on asialo-a1 -acid glycoproteins [1,3]) (Reversibility: ? <1,2> [1,3]) [1, 3] UDP + N-acetylglucosaminylated asialo-a1 -acid glycoprotein UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-R <1, 2> (<1,2> acts on b-galactosyl-1,4-N-acetylglucosaminyl-termini on asialo-a1 -acid glycoproteins [1,3]; <1> GlcNAc residues are introduced to position C-6 of the terminal galactose of the glycoprotein [1]) (Reversibility: ? <1,2> [1-3]) [1-3] UDP + N-acetyl-b-d-glucosaminyl-1,6-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-R <1, 2> [1-3] UDP-N-acetyl-d-glucosamine + lactose <2> (Reversibility: ? <2> [3]) [3] UDP + GlcNAcb(1-6)Galb(1-4)Glc <2> [3] UDP-N-acetyl-d-glucosamine + poly-N-acetyllactosamine <2-6> (<2-6> forms branches in poly-N-acetyllactosamines [4-8]; <2> IGnT [6]; <2> cIGnT6 transfers one or multiple GlcNAc branches to midchain galactoses of long linear polylactosamines [7]; <4> cIGnT6 [8]; <2> transfer of GlcNAc to b1,4-linked Gal residue in a linear poly-N-acetyllactosamine with the approximate structure Galb(1-4)GlcNAcb(1-3)Galb(14)Glc(NAc)-R, forming Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(14)Glc(NAc)-R [9]) (Reversibility: ? <2-6> [4-9]) [4-9] ? UDP-N-acetyl-d-glucosamine + porcine submaxillary asialo-afuco-mucin <1> (<1> poor acceptor, Galb(1-3)GalNAc as terminal acceptor structure [1]) (Reversibility: ? <1> [1]) [1] UDP + N-acetylglucosaminylated porcine submaxillary asialo-afuco-mucin UDP-N-acetyl-d-glucosamine + pyridylaminated Galb(1-4)GlcNAcb(13)Galb(1-4)Glc <4> (<4> i.e. pyridylaminated lacto-N-neotetraose, cIGnT6 [8]) (Reversibility: ? <4> [8]) [8] UDP + pyridylaminated Galb(1-4)GlcNAcb(1-3)(GlcNAcb(1-6))Galb(14)Glc <4> [8] Additional information <2-6> (<3,5,6> 2 isoforms IGnT A and IGnT B, Cterminal 1/4 of IGnT B is identical to that of IGnT A, the rest of the sequences shows 63% identity [4,5]; <5,6> generally, oligosaccharides with the Galb(1-4)GlcNAcb(1-3)Galb(1-4)Glc(Nac) sequence serve as good substrates, addition of Gala(1-3) or sialic acid a(2-6) to the non-reducing 311

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end gelactose enhances the acceptor activity, while sialic acid a2-3 linkage shows a repressive effect, no substrate: N-acetyl-b-d-glucosaminyl-1,3-bd-galactosyl-1,4-b-d-glucose [5]; <2,4> 2 types of branching enzyme activities: cIGnT6 generates b-1,6-N-acetylglucosaminyl branches at the central galactose residue, and dIGnT6 acts on peridistal galactose residues [7-9]; <2> no substrate of cIGnT6: GlcNAcb(1-3)Galb(1-4)GlcNAc, Galb(1-4)GlcNAcb(1-3)Galb(1-4)(Fuca(1-3))GlcNAc, no transfer of GlcNAc to substrates with a1-3-fucosyl residues and to midchain galactoses that belongs to Lewis x determinants [7]; <4> absolute requirement of at least a complete Galb(1-4)GlcNAc residue bound to position 3 of the acceptor Gal residues, i.e. it is capable of acting only on the Gal residues of internal Galb(1-4)GlcNAc units, no substrates: pyridylaminated GlcNAcb(1-3)Galb(1-4)Glc and pyridylaminated Galb(1-3)GlcNAcb(13)Galb(1-4)Glc, 4 major groups according to their acceptor specificities: blood group I structure: IGnT6, core 2 in O-glycans: C2GnT6, core 4 in O-glycans: C4GnT6, 2,6-branched N-linked core: GnT V [8]) [4, 5, 7-9] P ? Inhibitors AMP <4> (<4> 10% inhibition [8]) [8] CaCl2 <1> [1] CuCl2 <4> [8] EDTA <1> [1] GDP <4> (<4> 19% inhibition [8]) [8] NiCl2 <4> [8] UDP <4> (<4> most potent inhibitor of donor substrate analogues, complete inhibition [8]) [8] UDP-galactose <4> (<4> 42% inhibition [8]) [8] UDP-glucose <4> (<4> 51% inhibition [8]) [8] UDP-glucronic acid <4> (<4> 36% inhibition [8]) [8] UDP-hexanolamine <4> (<4> 66% inhibition [8]) [8] UMP <4> (<4> 41% inhibition [8]) [8] UTP <4> (<4> most potent inhibitor of donor substrate analogues, complete inhibition [8]) [8] ZnCl2 <1, 4> [1, 8] Metals, ions Cd2+ <1> (<1> CdCl2 , slightly stimulates [1]) [1] Mg2+ <1> (<1> MgCl2 , slightly stimulates [1]) [1] Mn2+ <1, 2> (<1> MnCl2 , 10-25 mM for optimal activity required [1]; <2> requirement [3]) [1, 3] Additional information <4> (<4> no requirement for any divalent metal ions [8]) [8] Specific activity (U/mg) 0.058 <4> [8] Additional information <5, 6> (<5,6> when expressed as proteins IGnT B shows higher specific activity than IGnT A [5]) [5]

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Km-Value (mM) 0.44 <6> (Galb(1-4)GlcNAcb(1-3)Galb(1-4)Glc, <6> IGnT B [5]) [5] 0.52 <6> (NeuAca(2-6)Galb(1-4)GlcNacb(1-3)Galb(1-4)Glc, <6> IGnT B [5]) [5] 0.55 <6> (Galb(1-4)GlcNAcb(1-3)Galb(1-4)GlcNAcb(1-3)Galb(1-4)Glc, <6> IGnT B [5]) [5] 0.96 <4> (pyridylaminated lacto-N-neotetraose) [8] 2.59 <4> (UDP-N-acetyl-d-glucosamine) [8] pH-Optimum 6.5-8 <1> [1] 6.8-7.8 <2> (<2> broad [3]) [3] 7 <4> [8] Temperature optimum ( C) 37 <1, 2, 4-6> (<1,2,4-6> assay at [1-3,5,8]) [1-3, 5, 8]

4 Enzyme Structure Subunits ? <2, 4> (<2> x * 67000-74000, glycosylated protein fused to glutathione Stransferase, size heterogenicity is at least partially due to differences in Nglycosylation, SDS-PAGE [7]; <4> x * 76000, SDS-PAGE under nonreducing conditions [8]) [7, 8] Posttranslational modification glycoprotein <2> (<2> recombinant enzyme, expressed in SF9 insect cells as fusion protein with glutathione S-transferase has 5 potential N-glycosylation sites [7]) [7]

5 Isolation/Preparation/Mutation/Application Source/tissue ES-D3 cell <5, 6> (<5,6> D-3 embryonic stem cells moderately express IGnT A and B [5]) [5] F9 cell <3> (<3> F9 embryonal carcinoma cells moderately express IGnT A and B [4]) [4] HL-60 cell <2> (<2> C2GnT, promyelocytic leukaemia HL-60 cells [6]) [6] Novikoff ascites tumor <1> (<1> strain N1S1-67 [1]) [1] P-19 cell <5, 6> (<5,6> P-19 embryonal carcinoma cells moderately express IGnT A and B [5]) [5] PA-1 cell <2> (<2> IGnT, teratocarcinoma PA-1 cells [6]; <2> tetracarcinoma cells, IGnT mainly acts as cIGnT, but can also act as dIGnT with 6-30% of the activity of cIGnT [9]; <2> cIGnT6, embryonal carcinoma cell line [7]) [6, 7, 9]

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T-lymphocyte <2> (<2> C2GnT is highly expressed in activated T lymphocytes [6]) [6] brain <2> (<2> IGnT is highly expressed in adult cerebellum and the frontal lobe of adult brain, also expression in fetal brain [9]) [9] cerebellum <2> (<2> IGnT is highly expressed in adult cerebellum [9]) [9] colon <2> (<2> IGnT is moderately expressed in adult colon [9]) [9] gastric mucosa <4> (<4> dIGnT [9]) [9] heart <2> (<2> IGnT is moderately expressed in adult heart [9]) [9] intestine <3, 5, 6> (<3,5,6> strong expression of IGnT A and B in adults [4,5]) [4, 5] kidney <2, 3, 5, 6> (<3,5,6> strong expression of IGnT A and B in adults [4,5]; <2> IGnT expression in fetal kidney [9]) [4, 5, 9] liver <1, 3, 5, 6> (<3,5,6> strong expression of IGnT A and B in adults [4,5]; <1> cIGnT [9]) [4, 5, 9] lung <2> (<2> IGnT expression in fetal lung [9]) [9] mammary gland <3, 5, 6> (<3,5,6> moderate expression of IGnT A and B [4,5]) [4, 5] myeloid cell <2> (<2> C2GnT is highly expressed in [6]) [6] prostate gland <2> (<2> IGnT is highly expressed in adult prostata [9]) [9] serum <2> (<2> from freshly drawn human blood of healthy donors [2]) [2, 3] small intestine <2, 4> (<4> highest enzyme activity per tissue protein [8]; <2> IGnT is moderately expressed in adult small intestine [9]; <4> cIGnT is the dominant form [9]) [8, 9] submaxillary gland <3, 5, 6> (<3,5,6> moderate expression of IGnT A and B [4,5]) [4, 5] Additional information <2, 3> (<3> no expression of IGnT A and B in N2a cells [4]; <2> high expression of C2GnT in granulocyte-monocyte cell lineage [6]) [4, 6] Localization membrane <2, 3, 5, 6> (<2,3,5,6> type II transmembrane topology [5,9]) [5, 9] microsome <4> (<4> enzyme is concentrated in the microsome fraction [8]) [8] Purification <2> (purification of recombinant enzyme, expressed in SF9 insect cells as protein fused to glutathione S-transferase [7]; cIGnT, purified from PA-1 cells [9]) [7, 9] <4> (210000fold, cIGnT6 [8]) [8, 9] Cloning <2> (C2GnT and IGnT cDNAs are cloned and sequenced, genes are both localized on chromosome 9, band q2l, genomic structures [6]; IGnT6 is cloned, sequenced and cDNA encoding cIGnT6 in PA-1 cells is expressed in SF9 insect cells as protein fused to glutathione S-transferase [7]; cDNA encoding IGnT is cloned from PA-1 cells, 400-amino acids protein [9]) [6, 7, 9]

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<3, 5, 6> (2 isoforms IGnT A and IGnT B are produced by alternative splicing of the IGnT gene, the C-terminal 1/4 of IGnT B is identical to that of IGnT A [4,5]; expression of each isoform is controlled by a different promoter, characterization of the promoters: GT boxes play crucial roles in transcriptional regulation of the genes [4]; IGnT A is cloned [4,5]; IGnT B is cloned from mouse liver cDNA, nucleotide and amino acid sequence, exon organization of the IGnT gene, expression of IGnT A and B in COS-7 and CHO cells [5]) [4, 5, 9] Engineering Additional information <3> (<3> mutation experiments at the GT boxes of the promoters of IGnT A and B genes [4]) [4] Application synthesis <2> (<2> cIGnT6 transfers multiple GlcNAc branches to long linear polylactosamines, a prerequisite for improving enzyme-assisted in vitro synthesis of a type of multivalent sialyl Lewis x glycans that are high affinity inhibitors of lymphocyte l-selectin, enzyme allows general polylactosamine synthesis [7]) [7]

6 Stability General stability information <4>, bovine serum albumin, 2 mg/ml, preserves enzyme activity at 37 C, especially the highly purified enzyme fraction [8] Storage stability <2>, -20 C, recombinant enzyme, a few days, stable [7]

References [1] van den Eijnden, D.H.; Winterwerp, H.; Smeeman, P.; Schiphorst, W.E.C.M.: Novikoff ascites tumor cells contain N-acetyllactosaminide b1!3 and b1!6 N-acetylglucosaminyltransferase activity. J. Biol. Chem., 258, 3435-3437 (1983) [2] Leppänen, A.; Penttilä, L.; Niemelä, R.; Helin, J.; Seppo, A.; Lusa, S.; Renkonen, O.: Human serum contains a novel b1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo (N-acetyllactosaminoglycans). Biochemistry, 30, 9287-9296 (1991) [3] Zielenski, J.; Koscielak, J.: The occurrence of two novel N-acetylglucosaminyltransferase activities in human serum. FEBS Lett., 158, 164-168 (1983) [4] Chen, G.-Y.; Kurosawa, N.; Muramatsu, T.: Functional analysis of promoter activity of murine b-1,6-N-acetylglucosaminyltransferase. Gene, 275, 253-259 (2001)

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[5] Chen, G.-Y.; Kurosawa, N.; Muramatsu, T.: A novel variant form of murine b1,6-N-acetylglucosaminyltransferase forming branches in poly-N-acetyllactosamines. Glycobiology, 10, 1001-1011 (2000) [6] Bierhuizen, M.F.A.; Maemura, K.; Kudo, S.; Fukuda, M.: Genomic organization of core 2 and I branching b-1,6-N-acetylglucosaminyltransferases. Implication for evolution of the b-1,6-N-acetylglucosaminyltransferase gene family. Glycobiology, 5, 417-425 (1995) [7] Mattila, P.; Salminen, H.; Hirvas, L.; Niittymäki, J.; Salo, H.; Niemelä, R.; Fukuda, M.; Renkonen, O.; Renkonen, R.: The centrally acting b1,6N-acetylglucosaminyltransferase (GlcNAc to Gal). Functional expression, purification, and acceptor specificity of a human enzyme involved in midchain branching of linear poly-N-acetyllactosamines. J. Biol. Chem., 273, 27633-27639 (1998) [8] Sakamoto, Y.; Taguchi, T.; Tano, Y.; Ogawa, T.; Leppänen, A.; Kinnunen, M.; Aitio, O.; Parmanne, P.; Renkonen, O.; Taniguchi, N.: Purification and characterization of UDP-GlcNAc:Galb1-4-GlcNAcb1-6*Galb1-4Glc(NAc)-R(GlcNAc to *Gal) b1,6N-acetylglucosaminyltransferase from hog small intestine. J. Biol. Chem., 273, 27625-27632 (1998) [9] Fukuda, M.: b6 -N-acetylglucosaminyltransferase (IGnT). Handbook of Glycosyltransferases and Related Genes, 2002, 125-132 (2002)

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N-Acetyllactosaminide a-1,3-galactosyltransferase

2.4.1.151

1 Nomenclature EC number 2.4.1.151 (transferred to EC 2.4.1.87) Recommended name N-acetyllactosaminide a-1,3-galactosyltransferase

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4-Galactosyl-N-acetylglucosaminide 3-a-L-fucosyltransferase

2.4.1.152

1 Nomenclature EC number 2.4.1.152 Systematic name GDP-b-l-fucose:1,4-b-d-galactosyl-N-acetyl-d-glucosaminyl-R transferase

3-l-fucosyl-

Recommended name 4-galactosyl-N-acetylglucosaminide 3-a-l-fucosyltransferase Synonyms FUT5 FUT6 Fuc-TV Fuc-Tb FucT-VI GDL-l-fucose:N-acetyl-b-d-glucosaminyl a-3-l-fucosyltransferase GDP-Fuc:Galb1-4GlcNAc (Fuc to GlcNAc) a1-3 fucosyltransferase GDP-l-fucose:1,4-b-d-galactosyl-N-acetyl-d-galactosaminyl-R 3-l-fucosyltransferase GDP-fucose:Galb(1-4)GlcNAc-R a(1-3)fucosyltransferase GDP-fucose:b-d-N-acetylglucosaminide 3-a-fucosyltransferase a(1-3)fucosyltransferase a-3-l-fucosyltransferase a-3-fucosyltransferase a1,3 fucosyltransferase a1,3FT a1-3 fucosyltransferase a1!3fucosyltransferase a3-fucosyltransferase-V a3-fucosyltransferase-VI fucosyltransferase, guanosine diphosphofucose-glucoside a1!3galactoside 3-fucosyltransferase guanosine diphosphofucose glucoside a1!3fucosyltransferase lewis-negative a-3-fucosyltransferase plasma a-3-fucosyltransferase Additional information (cf. EC 2.4.1.65) CAS registry number 111310-38-4

318

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2 Source Organism <1> Homo sapiens (<1>, enzyme is produced by infection of Trichoplusia ni cells with recombinant baculovirus [7]; recombinant enzyme expressed in Sf-9 cells via baculovirus infection [8]; fusion protein: protein A coupled to the catalytic domain of FucT-V [1]; fucosyltransferase V [23]) [1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, 15, 16, 17, 20, 21, 22, 23] <2> Pan troglodytes [28] <3> Bos taurus [5, 24] <4> Cricetulus griseus [6] <5> Lymnaea stagnalis [9] <6> Mus musculus [18] <7> Rattus norvegicus [19] <8> Macaca mulatta [22] <9> Homo sapiens [13, 26] <10> Schistosoma mansoni [27] <11> Pan troglodytes [28] <12> Homo sapiens [25, 26, 29]

3 Reaction and Specificity Catalyzed reaction GDP-b-l-fucose + 1,4-b-d-galactosyl-N-acetyl-d-glucosaminyl-R = GDP + 1,4-b-d-galactosyl-(a-1,3-l-fucosyl)-N-acetyl-d-glucosaminyl-R (i.e. Lewis X determinent) Reaction type hexosyl group transfer Natural substrates and products S Additional information <1, 6, 12> (<1>, enzyme is involved in the expression of the sialyl-LewisX determinant, a ligand for E- and P-selectin [15]; <1>, the hepatocellular enzyme is involved in the synthesis of sialosyl LeX determinants on cirrhotic a1AGP [17]; <6>, Fuc-TVII participates in the generation of a(1,3)fucosylated ligands for l-selectin, evidence for a role for this enzyme in E- and P-selectin ligand expression in leukocytes [18]; <12>, the gene is capable of directing expression of the Lewis x Galb(1,4)(Fuca(1,3))GlcNAc, sialyl Lewis x NeuNAca(2,3)Galb(1,4) (Fuca(1,3))GlcNAc, and difucosyl sialyl Lewis x NeuNAca(2,3)Galb(1,4) epitopes [25]; (Fuca(1,3))GlcNAcb(1,3)Galb(1,4)(Fuca(1,3))GlcNAc <12>, the enzyme is involved in the biosynthesis of the E-selectin ligand, sialyl-Lewis X. Catalyzes the transfer of fucose from GDP-b-fucose to a2,3 sialylated substrates [25,26,29]) [15, 17, 18, 19, 25, 26, 29] P ?

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Substrates and products S GDP-l-fucose + 2'-fucosyllactose <9> (<9>, 42% of the activity with Galb(1,4)GlcNAc [13]) (Reversibility: ? <9> [13]) [13] P GDP + ? S GDP-l-fucose + 3'-sialyl N-acetyl lactosamine <1> (Reversibility: ? <1> [17]) [17] P GDP + ? S GDP-l-fucose + Fuca(1,2)Galb(1,3)GlcNAc-R <1, 8> [22] P GDP + ? S GDP-l-fucose + Fuca(1,2)Galb(1,4)Glc <1, 9> (<1>, i.e. 2'-fucosyllactose, 17% of the activity with Galb(1,4)GlcNAc [2]) (Reversibility: ? <1,9> [1,2,4,13,17]) [1, 2, 4, 13, 17] P GDP + Fuca(1,2)Galb(1,4)(Fuca(1,3))Glc S GDP-l-fucose + Fuca(1,2)Galb(1,4)GlcNAc <1> (<1>, 162% of the activity with Galb(1,4)GlcNAc [2]) (Reversibility: ? <1> [2,11]) [2, 11] P GDP + Fuca(1,2)Galb(1,4)(Fuca(1,3))GlcNAc S GDP-l-fucose + Fuca(1,2)Galb(1,4)GlcNAc <1> (<1>, mutant enzyme A349D shows 8fold higher activity than the wild-type enzyme [23]) (Reversibility: ? <1> [23,24]) [23, 24] P GDP + Fuca(1,2)Galb(1,4)(Fuca(1,3))GlcNAc S GDP-l-fucose + Fuca(1,2)Galb(1,4)GlcNAcb(CH2 )8 COOMe <1> (Reversibility: ? <1> [12]) [12] P GDP + Fuca(1,2)Galb(1,4)(Fuca(1,3))GlcNAcb(CH2 )8 COOMe S GDP-l-fucose + Fuca(1,2)Galb(1,4)GlcNAcb-bovine-serum-albimun <1> (Reversibility: ? <1> [10]) [10] P GDP + Fuca(1,2)Galb(1,4)(Fuca(1,3))GlcNAcb-bovine-serum-albimun S GDP-l-fucose + Gala(1,3)Galb(1,4)GlcNAc-R <1, 8> [22] P GDP + ? S GDP-l-fucose + Galb(1,3)GlcNAc <9> (<9>, i.e. lacto-N-biose I, 10% of the activity with Galb(1,4)GlcNAc [13]) (Reversibility: ? <9> [13]) [13] P GDP + ? S GDP-l-fucose + Galb(1,4)-Glc <1> (<1>, 3% of the activity with Galb(1,4)GlcNAc [2]) (Reversibility: ? <1> [2]) [2] P GDP + Galb(1,4)(Fuca(1,3))Glc S GDP-l-fucose + Galb(1,4)GlcNAc <9> (Reversibility: ? <9> [13]) [13] P GDP + Galb(1,4)(Fuca(1,3))GlcNAc S GDP-l-fucose + Galb(1,4)GlcNAc <1, 5, 9> (i.e. N-acetyllactosamine) (Reversibility: ? <1,5,9> [2,9,11,13,17]) [2, 9, 11, 13, 17] P GDP + Galb(1,4)(Fuca(1,3))GlcNAc <5> [9] S GDP-l-fucose + Galb(1,4)GlcNAc-O(CH2 )8 CO2 CH3 <1> (Reversibility: ? <1> [3]) [3] P GDP + Galb(1,4)(Fuca(1,3))GlcNAc-O(CH2 )8 CO2 CH3 S GDP-l-fucose + Galb(1,4)GlcNAc-R <1, 8> [22] P GDP + Galb(1,4)(Fuca(1,3))GlcNAc-R S GDP-l-fucose + Galb(1,4)GlcNAcb(1,2)Mana(1,6)Manb(1,4)GlcNAc <1> [15] P GDP + Galb(1,4)(Fuca(1,3))GlcNAcb(1,2)Mana(1,6)Manb(1,4)GlcNAc 320

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S GDP-l-fucose + Galb(1,4)GlcNAcb(1,3)Galb(1,4)Glc <1> (Reversibility: ? <1> [2]) [1] P GDP + Galb(1,4)(Fuca(1,3))GlcNAcb(1,3)Galb(1,4)Glc + Galb(1,4)GlcNAcb(1,3)Galb(1,4)(Fuca(1,3))Glc <1> [1] S GDP-l-fucose + Galb(1,4)GlcNAcb(1,3)Galb(1,4)Glc <1> (Reversibility: ? <1> [11]) [11] P GDP + Galb(1,4)(Fuca(1,3))GlcNAcb(1,3)Galb(1,4)Glc S GDP-l-fucose + NeuAca(2,3)Galb(1,4)GlcNAc <1> (<1>, 147% of the activity with Galb(1,4)GlcNAc [2]) (Reversibility: ? <1> [2,11]) [2, 11] P GDP + NeuAca(2,3)Galb(1,4)(Fuca(1,3))GlcNAc S GDP-l-fucose + NeuAca(2,3)Galb(1,4)GlcNAcb(1,2)Mana(1,6)Manb(1,4)GlcNAc <1> (Reversibility: ? <1> [15]) [15] P GDP + NeuAca(2,3)Galb(1,4)(Fuca(1,3))GlcNAcb(1,2)Mana(1,6)Manb(1,4)GlcNAc S GDP-l-fucose + a(2,3)-sialyllactosamine <1> (<1>, 115% of the activity with Galb(1,4)GlcNAc [11]) (Reversibility: ? <1> [11]) [11] P GDP + ? S GDP-l-fucose + a(2,3)sialyllactosamine <9> (Reversibility: ? <9> [13]) [13] P GDP + ? S GDP-l-fucose + a1 acid glycoprotein <1> (<1>, with the major terminal structure on N-linked chains: NeuAc(2,3/6)Galb(1,4)GlcNAcb-R [15]) (Reversibility: ? <1> [15]) [15] P GDP + ? S GDP-l-fucose + asialo-a1 -acid glycoprotein <1> (<1>, with the major terminal structure on N-linked chains: Galb(1,4)GlcNAcb-R [15]) (Reversibility: ? <1> [15]) [15] P GDP + ? S GDP-l-fucose + asialo-fetuin <1> (<1>, with the major terminal structure on N-linked chains: Galb(1,4)GlcNAcb-R [15]) (Reversibility: ? <1> [15]) [15] P GDP + ? S GDP-l-fucose + asialotransferrin <1> (Reversibility: ? <1> [17]) [17] P GDP + ? S GDP-l-fucose + desialylated a1 -acid glycoprotein <7> (Reversibility: ? <7> [19]) [19] P GDP + ? S GDP-l-fucose + fetuin <1, 7> (<1>, with the major terminal structure on N-linked chains: NeuAca(2,3/6)Galb(1,4)GlcNAcb-R [15]) (Reversibility: ? <1,7> [15,19]) [15, 19] P GDP + ? S GDP-l-fucose + lacto-N-biose I <9> (Reversibility: ? <9> [13]) [13] P GDP + ? S GDP-l-fucose + lacto-N-tetraose <1> (Reversibility: ? <1> [4]) [4] P GDP + ? S GDP-l-fucose + lactofucopentaose I <1> (Reversibility: ? <1> [4]) [4] P GDP + ? S GDP-l-fucose + lactofucopentaose II <1> (Reversibility: ? <1> [4]) [4] 321

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P GDP + ? S GDP-l-fucose + lactose <1, 9> (<1>, weak activity [17]; <9>, 11% of the activity with Galb(1,4)GlcNAc [13]) (Reversibility: ? <1,9> [3,4,13,17]) [3, 4, 13, 17] P GDP + ? S Additional information <1, 3, 7, 8> (<3>, native enzyme utilizes only type 2 acceptors. Double mutation R115W/E116D slightly increases H-type 1 activity [24]; <1>, enzyme utilizes only type-2 chain polylactosamines as substrates, sialylated and nonsialylated oligosaccharides are good substrates, the enzyme produces sialyl Lewis X and Lewis X determinants [5]; <1>, the enzyme is active on the polylactosamine substrate having 6-sulfate modification at the GlcNAc moiety and gives rise to sialyl and nonsialyl 6-sulfo Lewis X. The enzyme is also active on a(1,2) fucosylated type 2 chain substrates [5]; <1>, no activity with Galb(1,3)GlcNAc, NeuAca2,8NeuAc and NeuAca2, NeuAca2,6Galb(1,4)GlcNAc, 6Galb(1,4)Glc [2]; <1>, predominantly activity with sialylated or nonsialylated type-2 chain acceptors, only a low a(1,4)-fucosyltransferase activity with type-1 chain acceptors [6]; <1>, fetuin and asialofetuin bearing N-linked carbohydrate chains containing the type II lactosamine core (Galb(1,4)GlcNAc) [7]; <1>, acceptor specificity with glycoprotein and glycolipid acceptors [11]; <1>, acceptor: type 2 H-oligosaccharide 8methoxycarbonyloctyl glycoside [14]; <7>, 11 nonidentical amino acids, found within a hypervariable peptide segment positioned at the NH2 -terminus determines whether or not an a(1,3)-fucosyltransferase can utilize type I acceptor substrates to form Lewis a and sialyl Lewis a moieties [19]; <1>, weak activity with type 1 substrates [22]; <8>, no activity with type 1 substrates [22]) [2, 5, 6, 7, 11, 14, 15, 19, 20, 22, 24] P ? Inhibitors Cu2+ <7> [19] GDP <1> (<1>, 0.191 mM causes 50% inhibition in the scintillation proximity assay and 0.214 mM causes 50% inhibition in the Dowex assay [7]) [7] GDP-choline <1> (<1>, 0.5 mM, 90% inhibition [17]) [17] GDP-hexanolamine <1> [21] N-bromosuccinimide <1> (<1>, moderate inhibition [8]) [8] NEM <1, 4> (<1>, 50% inhibition at 0.1 mM, complete inhibition at 0.5 mM. At 37 C complete inhibition is reached after 3 min. At 23 C more than 10 min are required to reach maximal activity. No inactivation after 10 min at 4 C [8]; <1>, activity in HL-60 cells is essentially unaffected. The RPMI 8226 enzyme activity is significantly inhibited [12]; recombinant fusion protein of protein A coupled to the catalytic domain of FucT-V, irreversible inactivation, effective protection by GDP-fucose and GDP [14]) [5, 6, 8, 12, 14] diethyldicarbonate <1> (<1>, IC50: 0.092. Preincubation at 23 C gives maximal inhibition, 75%, after 180 s. Preincubation at 13 C gives 55% inhibition after 3 min. Preincubation at 4 C results in only 35% inhibition [8]) [5, 8] dithionitrobenzene <1> [8]

322

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Metals, ions Ca2+ <1, 7> (<1>, activates, Km : 16 mM [11]; <7>, Mn2+ , Mg2+ or Ca2+ required [19]) [11, 19] Mg2+ <1, 7> (<1>, activates, Km : 22 mM [11]; <7>, Mn2+ , Mg2+ or Ca2+ required [19]) [11, 19] Mn2+ <1, 5, 7> (<5>, stimulates, optimal concentration is about 20 mM [9]; <1>, required [4,6]; <1>, 20 mM Mn2+ are required for full activity [5]; <1>, activates, Km : 12 mM [11]; <7>, Mn2+ , Mg2+ or Ca2+ required [19]) [4, 5, 6, 9, 11, 19] Turnover number (min±1) 102 <1> (Fuca(1,2)Galb(1,4)GlcNAc, <1>, fucosyltransferase V [23]) [23] 216 <1> (Fuca(1,2)Galb(1,4)GlcNAc, <1>, fucosyltransferase V mutant enzyme A349D [23]) [23] Specific activity (U/mg) 0.000002 <1> [17] 0.002289 <7> [19] 1.4 <1> [11] Additional information <1> (<1>, measurement of a(1-3)fucosyltransferase activity using scintillation proximity [7]) [3, 7] Km-Value (mM) 0.0016 <1> (GDP-fucose, <1>, pH 6.5, 37 C [7]) [7] 0.0026 <1> (N-acetyllactosamine, <1>, pH 7.3, 37 C [11]) [11] 0.0065 <1> (GDP-fucose, <1>, pH 7.2, 37 C [17]) [17] 0.009 <1> (GDP-l-fucose) [6] 0.0188 <1> (GDP-l-fucose, <1>, fucosyltransferase V [23]) [23] 0.0233 <1> (GDP-l-fucose, <1>, fucosyltransferase V mutant enzyme A349D [23]) [23] 0.062 <1> (GDP-l-fucose, pH 7.6, 15 mM MnCl2 [4]) [4] 0.2 <1> (NeuAca(2,3)Galb(1,4)GlcNAc) [2] 0.38 <1> (a(2,3) bisialylated biantennary glycan, <1>, pH 7.2, 37 C [17]) [17] 0.65 <1> (3'-sialyl N-acetyl lactosamine, <1>, pH 7.2, 37 C [17]) [17] 0.7 <1> (Fuca(1,2)Galb(1,4)GlcNAc) [2] 0.74 <1> (unsialylated biantennary glycan, <1>, pH 7.2, 37 C [17]) [17] 0.9 <1> (NeuAca(2,3)Galb(1,4)GlcNAc, <1>, pH 7.3, 37 C [11]) [11] 1.1 <1> (Fuca(1,2)Galb(1,4)GlcNAc, <1>, fucosyltransferase V mutant enzyme A349D [23]) [23] 1.3 <1> (Fuca(1,2)Galb(1,4)GlcNAc) [11] 1.4 <1> (Galb(1,4)GlcNAc) [2] 1.4 <1> (lactofucopentaose I, pH 7.6, 15 mM MnCl2 , 0.095 mM GDP-fucose [4]) [4] 1.5 <1> (Galb(1,4)GlcNAcb(1,3)Galb(1,4)Glc, <1>, pH 7.3, 37 C [11]) [11] 2.6 <1> (N-acetyl lactosamine, <1>, pH 7.2, 37 C [17]) [17] 2.9 <1> (Galb(1,4)GlcNAc, <1>, pH 7.3, 37 C [11]) [11] 3.9 <1> (Fuca(1,2)Galb(1,4)GlcNAc, <1>, fucosyltransferase V [23]) [23] 3.9 <1> (NeuAca(2,3)Galb(1,4)Glc) [2]

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5 <1> (lacto-N-tetraose, pH 7.6, 15 mM MnCl2 , 0.095 mM GDP-fucose [4]) [4] 6.7 <1> (Fuca(1,2)Galb(1,4)Glc) [2] 17 <1> (lactofucopentaose II, pH 7.6, 15 mM MnCl2 , 0.095 mM GDP-fucose [4]) [4] 20 <1> (Fuca(1,2)Galb(1,4)Glc, pH 7.6, 15 mM MnCl2 , 0.095 mM GDP-fucose [4]) [4] 23 <5> (Galb(1,4)GlcNAc, <5>, pH 7.0, 37 C [9]) [9] 25 <1> (Galb(1,4)Glc) [2] 83 <1> (lactose, <1>, pH 7.6, 15 mM MnCl2 , 0.095 mM GDP-fucose [4]) [4] Additional information <1> [5] pH-Optimum 5-7 <4> [6] 6 <7> (<7>, soluble enzyme [19]) [19] 6.2 <7> (<7>, membrane-bound enzyme [19]) [19] 7-8 <1> (<1>, reaction with N-acetyllactosamine [11]) [11] 7.2-8 <1> [5] 7.5 <5> [9] 7.6-8 <1> [4] pH-Range 6-8.8 <1> (<1>, pH 6.0: about 40% of maximal activity, pH 8.8: about 50% of maximal activity, reaction with N-acetyllactosamine [11]) [11]

4 Enzyme Structure Molecular weight 41000 <1> (<1>, gel filtration [11]) [11] Subunits ? <7> (<7>, x * 40680, SDS-PAGE [19]) [19] monomer <1, 2, 9, 10, 12> (<12>, 1 * 41859, calculation from nucleotide sequence [29]; <2>, 1 * 41892, calculation from nucleotide sequence [28]; <9>, 1 * 43008, calculation from nucleotide sequence [13,26]; <11>, 1 * 43034, calculation from nucleotide sequence [28]; <1>, 1 * 44000, SDS-PAGE [11]; <10>, 1 * 50556, calculation from nucleotide sequence [27]) [11, 13, 25, 26, 27, 28, 29]

5 Isolation/Preparation/Mutation/Application Source/tissue CCRF-CEM cell <1> [12] COS-1 cell <4> [6] HL-60 cell <1> [12] Hep-G2 cell <1> [11]

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K-562 cell <1> [12] KG-1 cell <1> [12] MOLT-4 cell <1> [12] RPMI-8226 cell <1> [12] WEGLI 18 cell <1> [15] bladder <1> (<1>, transcript of FUT6 mRNA detected [5]) [5] blood plasma <1> (<1>, in individuals with plasma fucosyltransferase deficiency from Indonesia, either a missense mutation, G739A, in the catalytic domain, or a nonsense mutation, C945A, truncating the COOH-terminus of the enzyme, leads to the total loss of the enzymatic activity of FUT6. 9% of individuals on the island of Java lack the plasma type enzyme. Activity is elevated in patients with a variety of malignant disorders, including liver cancer, elevated in heavy alcohol drinkers. Reduction of enzyme level in patients with schizophrenia [5]; <1>, enzyme activity is significantly higher in patients with chronic liver diseases than in that of normal controls [10]; <1>, the plasma enzyme appears at the last trimester of gestation [16]) [2, 5, 10, 16] colon <1, 7, 9> (<1>, transcript of FUT6 mRNA detected [5]) [5, 19, 26] connective tissue <5> [9] endothelial cell <1> [5] endothelium <6> (<6>, the enzyme is expressed in endothelial cells lining the high endothelial venules of perpheral lymph nodes, mesenteric lymph nodes and Pever`s patches [18]) [18] granulocyte <1> (<1> eosinophilic [15]) [15] granulocyte <1> (<1> neutrophilic [15]) [15] hepatocyte <1> [17] jejunum <7> [19] kidney <1, 9, 12> (<1>, transcript of FUT6 mRNA detected [5]; <1>, during renal organogenesis the myeloid enzyme is progressively replaced by the plasma enzyme in the proximal tubules and later by the Lewis enzyme in Bellini`s ducts and calyce [16]) [5, 16, 26] leukocyte <9> [13] liver <1, 9> (<1>, transcript of FUT6 mRNA detected [5]) [5, 11, 17, 26] milk <1> [3, 4, 5, 15] peripheral blood <9> [13] saliva <1> [1, 5] small intestine <1> [5] uterus <1> (<1>, transcript of FUT6 mRNA detected [5]) [5] Additional information <1> (<1>, enzyme is produced by infection of Trichoplusia ni cells with recombinant baculovirus [7]; <1>, recombinant enzyme expressed in Sf-9 cells via baculovirus infection [8]; <1>, no activity detected in Daudi, Raji and Reh leukemia cells [12]) [8, 12] Localization Golgi trans-face <1> [5] Golgi vesicle <1> [17]

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membrane <1, 2, 7, 9, 11, 12> (<1, 7>, bound to [14, 19]; <2, 9, 11>, type II membrane protein, membrane-bound form in trans cistermae of Golgi [13, 26, 28]) [13, 14, 19, 25, 26, 28, 29] microsome <5> [9] secretory granule <1> (<1>, granules of endothelial cells (Weibel-Parade bodies) [5]) [5] soluble <7> [19] Purification <1> (partial [17]) [3, 4, 11, 17] <7> (soluble enzyme [19]) [19] Cloning <6> (COS-7 cells transiently transfected with Fuc-TVII expression vectors [18]) [18] <9> (expression in COS cells [13]) [13] <10> [27] <12> (expression in either insect cells or yeast [5]; expression in Spodoptera frugiperda [6]; transfection of COS-1 and CHO-T cells [25]) [5, 6, 25, 29] Engineering A349D <1> (<1>, mutant enzyme shows higher activity with a range of acceptor substrates, higher affinity for Fuca(1,2)Galb(1,4)GlcNAc, 8fold higher overall catalytic efficiency than that of wild-type enzyme. The single amino acid site Asp336 of FucT III and Ala349 of FucT V constitutes the only difference in the sequence of FucT III and V over the final 210 COOH-terminal amino acid residues, impacts the acceptor substrate profiles of FucT III and FuvT V [23]) [23] C104S <1> (<1>, mutant enzyme is inactive, mutant enzyme produces a series of lower molecular weight bands when characterized by Wester blot and does not bind GDP [21]) [21] C351S <1> (<1>, mutant enzyme is inactive [21]) [21] C354S <1> (<1>, mutant enzyme is inactive [21]) [21] C94S <1> (<1>, mutant enzyme is inactive [21]) [21] R115W/E116D <3> (<3>, double mutation slightly increases H-type 1 activity [24]) [24]

References [1] Johnson, P.H.; Yates, A.D.; Watkins, W.M.: Human salivary fucosyltransferases: evidence for two distinct a-3-l-fucosyltransferase activities one or which is associated with the Lewis blood group Le gene. Biochem. Biophys. Res. Commun., 100, 1611-1618 (1981) [2] Johnson, P.H.; Watkins, W.M.: Sialyl compounds as acceptor substrates for human a-3- and a-3/4-l-fucosyltransferases. Biochem. Soc. Trans., 13, 1119-1120 (1985)

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[3] Johnson, P.H.; Watkins, W.M.: Separation of an a3-l-fucosyltransferase from the blood-group-Le-gene specified a-3/4-l-fucosyltransferase in human milk. Biochem. Soc. Trans., 10, 445-446 (1982) [4] Prieels, J.-P.; Beyers, T.; Hill, R.L.: Human milk fucosyltransferase. Biochem. Soc. Trans., 5, 838-839 (1977) [5] Kannagi, R.: a3-Fucosyltransferase-VI (FUT6). Handbook of Glycosyltransferases and Related Genes; (Taniguchi, N.; Honk, K.; Fukuda, M., eds.), 237245 (2002) [6] Kannagi, R.: a3-Fucosyltransferase-V (FUT5). Handbook of Glycosyltransferases and Related Genes; (Taniguchi, N.; Honk, K.; Fukuda, M., eds.), 232236 (2002) [7] Hood, C.M.; Kelly, V.A.; Bird, M.I.; Britten, C.J.: Measurement of a(1-3)fucosyltransferase activity using scintillation proximity. Anal. Biochem., 255, 8-12 (1998) [8] Britten, C.J.; Bird, M.I.: Chemical modification of an a3-fucosyltransferase; definition of amino acid residues essential for enzyme activity. Biochim. Biophys. Acta, 1334, 57-64 (1997) [9] Mulder, H.; Schachter, H.; Thomas, J.R.; Halkes, K.M.; Kamberling, J.P.; Vliegenthart, J.F.G.: Identification of a GDP-Fuc:Galb1-3GalNAc-R (Fuc to Gal) a1-2 fucosyltransferase and a GDP-Fuc:Galb1-4GlcNAc (Fuc to GlcNAc) a1-3 fucosyltransferase in connective tissue of the snail Lymnaea stagnalis. Glycoconjugate J., 13, 107-113 (1996) [10] Hada, T.; Fukui, K.; Ohno, M.; Akamatsu, S.; Yazawa, S.; Enomoto, K.; Yamaguchi, K.; Matsuda, Y.; Amuro, Y.; et al.: Increased plasma a-(1!3)-lfucosyltransferase activities in patients with hepatocellular carcinoma. Glycoconjugate J., 12, 627-631 (1995) [11] Johnson, P.H.; Donald, A.S.; Clarke, J.L.; Watkins, W.M.: Purification, properties and possible gene assignment of an a1,3-fucosyltransferase expressed in human liver. Glycoconjugate J., 12, 879-893 (1995) [12] Robinson, N.E.; de Vries, T.; Davis, R.E.; Stults, C.L.M.; Watson, S.R.; van den Eijnden, D.H.; Macher, B.A.: Expression of fucosylated antigens and a1,3 fucosyltransferases in human leukemia cell lines. Glycobiology, 4, 317-326 (1994) [13] Weston, B.W.; Nair, R.P.; Larsen, R.D.; Lowe, J.B.: Isolation of a novel human a (1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group a (1,3/1,4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities. J. Biol. Chem., 267, 4152-4160 (1992) [14] Holmes, E.H.; Xu, Z.; Sherwood, A.L.; Macher, B.A.: Structure-function analysis of human a1!3fucosyltransferases. A GDP-fucose-protected, Nethylmaleimide-sensitive site in FucT-III and FucT-V corresponds to Ser178 in FucT-IV. J. Biol. Chem., 270, 8145-8151 (1995) [15] Easton, E.W.; Schiphorst, W.E.; van Drunen, E.; van der Schoot, C.E.; van den Eijnden, D.H.: Human myeloid a3-fucosyltransferase is involved in the expression of the sialyl-Lewis(x) determinant, a ligand for E- and P-selectin. Blood, 81, 2978-2986 (1993)

327

4-Galactosyl-N-acetylglucosaminide 3-a-L-fucosyltransferase

2.4.1.152

[16] Candelier, J.J.; Mollicone, R.; Mennesson, B.; Bergemer, A.M.; Henry, S.; Coullin, P.; Oriol, R.: a-3-Fucosyltransferases and their glycoconjugate antigen products in the developing human kidney. Lab. Invest., 69, 449-459 (1993) [17] Jezequel-Cuer, M.; N'Guyen-Cong, H.; Biou, D.; Durand, G.: Oligosaccharide specificity of normal human hepatocyte a1-3 fucosyltransferase. Biochim. Biophys. Acta, 1157, 252-258 (1993) [18] Smith, P.L.; Gersten, K.M.; Petryniak, B.; Kelly, R.J.; Rogers, C.; Natsuka, Y.; Alford, J.A., 3rd; Scheidegger, E.P.; Natsuka, S.; Lowe, J.B.: Expression of the a(1,3)fucosyltransferase Fuc-TVII in lymphoid aggregate high endothelial venules correlates with expression of l-selectin ligands. J. Biol. Chem., 271, 8250-8259 (1996) [19] Karaivanova, V.; Mookerjea, S.; Hunt, D.; Nagpurkar, A.: Characterization and purification of fucosyltransferases from the cytosol of rat colon. Int. J. Biochem. Cell Biol., 28, 165-174 (1996) [20] Legault, D.J.; Kelly, R.J.; Natsuka, Y.; Lowe, J.B.: Human a(1,3/1,4)-fucosyltransferases discriminate between different oligosaccharide acceptor substrates through a discrete peptide fragment. J. Biol. Chem., 270, 2098720996 (1995) [21] Holmes, E.H.; Yen, T.-Y.; Thomas, S.; Joshi, R.; Nguyen, A.; Long, T.; Gallet, F.; Maftah, A.; Julien, R.; Macher, B.A.: Human a1,3/4 fucosyltransferases. Characterization of highly conserved cysteine residues and N-linked glycosylation sites. J. Biol. Chem., 275, 24237-24245 (2000) [22] Dupuy, F.; Germot, A.; Marenda, M.; Oriol, R.; Blancher, A.; Julien, R.; Maftah, A.: a1,4-Fucosyltransferase activity: a significant function in the primate lineage has appeared twice independently. Mol. Biol. Evol., 19, 815-824 (2002) [23] Vo, L.; Lee, S.; Marcinko, M.C.; Holmes, E.H.; Macher, B.A.: Human a1,3/4fucosyltransferases II. A single amino acid at the COOH terminus of FucT III and V alters their kinetic properties. J. Biol. Chem., 273, 25250-25255 (1998) [24] Dupuy, F.; Petit, J.-M.; Mollicone, R.; Oriol, R.; Julien, R.; Maftah, A.: A single amino acid in the hypervariable stem domain of vertebrate a1,3/ 1,4-fucosyltransferases determines the type 1/type 2 transfer. Characterization of acceptor substrate specificity of the Lewis enzyme by site-directed mutagenesis. J. Biol. Chem., 274, 12257-12262 (1999) [25] Weston, B.W.; Smith, P.L.; Kelly, R.J.; Lowe, J.B.: Molecular cloning of a fourth member of a human a(1,3) fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes. J. Biol. Chem., 267, 24575-24584 (1992) [26] Cameron, H.S.; Szczepaniak, D.; Weston, B.W.: Expression of human chromosome 19p a(1,3)-fucosyltransferase genes in normal tissues. Alternative splicing, polyadenylation, and isoforms. J. Biol. Chem., 270, 20112-20122 (1995) [27] Trottein, F.; Mollicone, R.; Fontaine, J.; de Mendonca, R.; Piller, F.; Pierce, R.; Oriol, R.; Capron, M.: Molecular cloning of a putative a3-fucosyltrans328

2.4.1.152

4-Galactosyl-N-acetylglucosaminide 3-a-L-fucosyltransferase

ferase from Schistosoma mansoni. Mol. Biochem. Parasitol., 107, 279-287 (2000) [28] Costache, M.; Apoil, P.-A.; Cailleau, A.; Elmgren, A.; Larson, G.; Henry, S.; Blancher, A.; Iordachescu, D.; Oriol, R.; Mollicone, R.: Evolution of fucosyltransferase genes in vertebrates. J. Biol. Chem., 272, 29721-29728 (1997) [29] Koszdin, K.L.; Bowen, B.R.: The cloning and expression of a human a-1,3 fucosyltransferase capable of forming the E-selectin ligand. Biochem. Biophys. Res. Commun., 187, 152-157 (1992)

329

Dolichyl-phosphate a-N-acetylglucosaminyltransferase

2.4.1.153

1 Nomenclature EC number 2.4.1.153 Systematic name UDP-N-acetyl-d-glucosamine:dolichyl-phosphate a-N-acetyl-d-glucosaminyltransferase Recommended name dolichyl-phosphate a-N-acetylglucosaminyltransferase Synonyms UDP-N-acetylglucosamine-dolichol phosphate N-acetylglucosaminyltransferase acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-dolichol phosphate dolichyl phosphate N-acetylglucosaminyltransferase dolichyl phosphate acetylglucosaminyltransferase CAS registry number 63363-73-5

2 Source Organism <1> Homo sapiens (normal controls and patients with cystic fibrosis and diabetes mellitus [2]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + dolichyl phosphate = UDP + dolichyl N-acetyl-a-d-glucosaminyl phosphate Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + dolichyl phosphate <1> (<1>, one of the initial steps of the lipid-mediated glycosylation pathway [2]) (Reversibility: ? <1> [2]) [2] P UDP + dolichyl N-acetyl-a-d-glucosaminyl phosphate 330

2.4.1.153

Dolichyl-phosphate a-N-acetylglucosaminyltransferase

Substrates and products S UDP-N-acetyl-d-glucosamine + dolichyl phosphate <1> (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + dolichyl N-acetyl-a-d-glucosaminyl phosphate Activating compounds Nonidet P-40 <1> (<1>, detergent required [2]) [2] Triton X-100 <1> (<1>, detergent required [2]) [2] detergent <1> (<1>, required at low concentrations [1]) [1] Metals, ions Mn2+ <1> (<1>, divalent cation required, 5 mM Mn2+ is optimal [2]) [2] divalent cation <1> (<1>, required [1]) [1] Specific activity (U/mg) Additional information <1> (<1>, specific activities of dolichyl phosphatemannosyltransferase are comparable in liver homogenates from normal controls and patients with cystic fibrosis and diabetes mellitus [2]) [2] pH-Optimum 7.8 <1> (<1>, broad with maximum near pH 7.8 [1]) [1, 2]

5 Isolation/Preparation/Mutation/Application Source/tissue liver <1> (<1>, normal controls and patients with cystic fibrosis and diabetes mellitus [2]) [1, 2] Localization particle-bound <1> (<1>, 90% of the activity is particulate [1]) [1]

References [1] Alhadeff, J.A., Watkins, P.: Lipid-mediated glycosylation in human liver. Characterization of the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine and mannose from GDP-mannose to dolichyl phosphate. Enzyme, 31, 90-103 (1984) [2] Alhadeff, J.A., Watkins, P.: Dolichyl phosphate-mannosyltransferase and dolichyl phosphate-N-acetylglucosaminyltransferase activities in liver preparations from normal controls and patients with cystic fibrosis and diabetes mellitus. Clin. Chim. Acta, 134, 1-9 (1983)

331

Globotriosylceramide b-1,6-N-acetylgalactosaminyl-transferase

2.4.1.154

1 Nomenclature EC number 2.4.1.154 Systematic name UDP-N-acetyl-d-galactosamine-globotriosylceramide b-1,6-N-acetylgalactosaminyltransferase Recommended name globotriosylceramide b-1,6-N-acetylgalactosaminyl-transferase Synonyms acetylgalactosaminyltransferase, uridine diphosphoacetylgalactosamine-glycosphingolipid GalNAc transferase globoside N-acetylgalactosaminyltransferase glycosphingolipid b-N-acetylgalactosaminyltransferase CAS registry number 83215-90-1

2 Source Organism <1> Mesocricetus auratus [1]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-galactosamine + globotriosylceramide = UDP + globotetraosylceramide Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-galactosamine + globotriosylceramide <1> (<1> glycosphingolipid metabolism [1]) (Reversibility: ? <1> [1]) [1] P UDP + globotetraosylceramide

332

2.4.1.154

Globotriosylceramide b-1,6-N-acetylgalactosaminyl-transferase

Substrates and products S UDP-N-acetyl-d-galactosamine + globotriosylceramide <1> (<1> highly acceptor specific, does not act on globotetraosylceramide or lactosylceramide [1]) (Reversibility: ? <1> [1]) [1] P UDP + globotetraosylceramide <1> (<1> the product has a terminal b-Nacetylgalactosamine residue [1]) [1] Inhibitors II3-a-N-acetylneuraminyl-lactosylceramide <1> [1] globotetraosylceramide <1> [1] Additional information <1> (<1> no effect: lactosylceramide [1]) [1] Activating compounds detergents <1> [1] sodium taurodeoxycholate <1> (<1> greatest activation, 1 mg/ml [1]) [1] Metals, ions Mn2+ <1> (<1> required for maximal activity, 4 mM [1]) [1] Additional information <1> (<1> Mg2+ cannot replace Mn2+ [1]) [1] Km-Value (mM) 0.14 <1> (UDP-N-acetyl-d-galactosamine) [1] 0.42 <1> (globotriosylceramide) [1] pH-Optimum 4.5-8 <1> (<1> maximal activity in 2-(N-morpholino)ethanesulfonic acid [1]) [1] Temperature optimum ( C) 37 <1> (<1> assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue fibroblast <1> (<1> cultured hamster cells, NIL-8 [1]) [1]

References [1] Lockney, M.W.; Sweely, C.S.: Characterization of a glycosphingolipid b-Nacetylgalactosaminyl-transferase activity in cultured hamster (nil) cells. Biochim. Biophys. Acta, 712, 234-241 (1982)

333

a-1,6-Mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

2.4.1.155

1 Nomenclature EC number 2.4.1.155 Systematic name UDP-N-acetyl-d-glucosamine:6-[2-(N-acetyl-b-d-glucosaminyl)-a-d-mannosyl]-glycoprotein 6-b-N-acetyl-d-glucosaminyltransferase Recommended name a-1,6-mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase Synonyms GnT-V acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-a-mannoside b1-6-UDP-N-acetylglucosamine:a-mannoside-b1,6 N-acetylglucosaminyltransferase a-1,3(6)-mannosylglycoprotein b-1,6-N-acetylglucosaminyltransferase a-mannoside b-1,6-N-acetylglucosaminyltransferase Additional information (cf. EC 2.4.1.101 and EC 2.4.1.143-5) CAS registry number 83588-90-3

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

Mesocricetus auratus (hamster [3]) [3-5, 8, 10] Gallus gallus [8] Homo sapiens [2, 8, 12, 13, 14, 15] Mus musculus [1, 8, 9, 11] Rattus norvegicus [6, 7]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + 6-(2-[N-acetyl-b-d-glucosaminyl]-a-d-mannosyl)-b-d-mannosyl-R = UDP + 6-(2,6-bis[N-acetyl-b-d-glucosaminyl]-ad-mannosyl)-b-d-mannosyl-R (<1> mechanism [4]) Reaction type hexosyl group transfer

334

2.4.1.155

a-1,6-Mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

Natural substrates and products S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3(6)-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6(3))-b-dmannosyl-1,4-N-acetyl-d-glucosaminyl-R <1-4> (<1,3,4> involved in biosynthesis of branched N-glycopeptides [1-3]; <1-4> enzyme requires prior action of N-acetylglucosaminyltransferase II which requires prior action of N-acetylglucosaminyltransferase I [8]; <1> biosynthesis of cellsurface ASSN-linked oligosaccharides [10]; <3> promotes angiogenesis in vitro and in vivo at physiological concentrations [13]) [1-3, 8, 10, 13] P UDP + N-acetyl-d-glucosaminyl-1,2-(N-acetyl-d-glucosaminyl-1,6)-1,2-ad-mannosyl-1,3(6)-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6(3))b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R Substrates and products S TDP-glucose + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3(6)-(Nacetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6(3))-b-d-mannosyl-1,4-Nacetyl-d-glucosaminyl-R <3> (Reversibility: ? <3> [15]) [15] P TDP + ? S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,2-a-4-deoxymannosyl-1,6-b-glucosyl-O-(CH2 )7 -CH3 <1> (<1> good substrate [4]) (Reversibility: ? <> []) [4] P UDP + N-acetyl-d-glucosaminyl-1,6-(N-acetyl-d-glucosaminyl-1,2)-a-4deoxy-mannosyl-1,6-b-d-mannosyl-O-(CH2 )7 -CH3 <1> [4] S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3(6)-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6(3))-b-dmannosyl-1,4-N-acetyl-d-glucosaminyl-R <1-5> (<4> transfers N-acetylglucosamine to C-6 of a-linked mannose residue with inversion of configuration at its anomeric center [1]; <3,4> acceptor substrates are only branched mannose glycopeptides with non-reducing N-acetylglucosamine terminal residues [1,2]; <4> acceptors are bi-antennary Asn-linked asialoor agalacto-oligosaccharides containing N-acetylglucosamine at the nonreducing terminal [1]; <3> best substrates are bi- and tri-antennary acceptors [2]; <3> specificity study with various pyridylaminated sugar chains [2]; <1,5> synthetic acceptor substrates [3,7]; <1-4> no substrates are bisected N-glycans [8]; <4> glycopeptides with sialic acid [1]; <3,4> glycopeptides with sialic acid or galactose at non-reducing terminal [1,2]; <3> most active towards biantennary sugars [12]; <3> enzyme also uses bisected oligosaccharides [14]) (Reversibility: ? <1-5> [1-8,12,14,15]) [1-8, 12, 14, 15] P UDP + N-acetyl-d-glucosaminyl-1,2-(N-acetyl-d-glucosaminyl-1,6)-1,2-ad-mannosyl-1,3(6)-(N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6(3))b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R <1> [8] S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6-b-d-glucosyl-O-R <1, 4> (<1> R: hydrophobic group, e.g. (CH2 )7 -CH3 [4, 5]; <1, 4> R: hydrophobic group, e.g. (CH2 )7 -CH3 or (CH2 )8 -COOCH3 [8]; <1> best substrate [4]) (Reversibility: ? <1, 4> [4, 5, 8]) [4, 5, 8]

335

a-1,6-Mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

2.4.1.155

P UDP + N-acetyl-d-glucosaminyl-1,6-(N-acetyl-d-glucosaminyl-1,2)-a-dmannosyl-1,6-b-d-glucosyl-O-R <1, 4> [4, 5, 8] S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6-b-d-mannosyl-O-(CH2 )8 COOCH3 <5> (Reversibility: ? <5> [6]) [6] P UDP + N-acetyl-d-glucosaminyl-1,6-(N-acetyl-d-glucosaminyl-1,2)-amannosyl-1,6-b-d-mannosyl-O-(CH2 )8 COOCH3 S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6-b-d-mannosyl-O-R <1, 4> (<1,4> R: hydrophobic group, e.g. (CH2 )8 -COOCH3 [8]) (Reversibility: ? <1,4> [8]) [8] P UDP + N-acetyl-d-glucosaminyl-1,6-(N-acetyl-d-glucosaminyl-1,2)-a-dmannosyl-1,6-b-d-mannosyl-O-R <1, 4> [8] S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,6-b-glucosaminyl-O-(CH2 )7 -CH3 <1> (Reversibility: ? <1> [10]) [10] P UDP + ? S UDP-glucose + N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3(6)-(Nacetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6(3))-b-d-mannosyl-1,4-Nacetyl-d-glucosaminyl-R <3> (Reversibility: ? <3> [15]) [15] P UDP + ? S Additional information <3> (<3> ADP-glucose, CDP-glucose and GDPglucose do not act as glycosyl donor [15]) [15] P ? Inhibitors ADP-N-acetylglucosamine <3> (<3> 20 mM [12]) [12] CDP-N-acetylglucosamine <3> (<3> 20 mM [12]) [12] Fe3+ <3> (<3> 1 mM inhibits [12]) [12] GDP-N-acetylglucosamine <3> (<3> 20 mM [12]) [12] N-acetyl-d-glucosaminyl-1,2-a-4-O-methyl-mannosyl-1,6-b-d-glucosyl-O(CH2 )7 -CH3 <1> [4] N-acetyl-d-glucosaminyl-1,2-a-6-deoxy-d-mannosyl-1,6-b-d-glucosyl-O(CH2 )7 -CH3 <1> (<1> kinetics [4]; <1> competitive [5]) [3-5] N-acetyl-d-glucosaminyl-1,2-a-6-deoxy-d-mannosyl-1,6-b-d-glucosyl-O(CH2 )8 -COOCH3 <5> [6] N-bromosuccinimide <3> (<3> 1 mM inhibits [12]) [12] TDP-N-acetylglucosamine <3> (<3> 20 mM [12]) [12] Additional information <4, 5> (<4,5> no inhibitor: EDTA [1,6]) [1, 6] Activating compounds NaCl <5> (<5> stabilizes and enhances activity, 0.2 M [6]) [6] Triton X-100 <5> (<5> activation, 1.0-1.5% [6]) [6] glycerol <5> (<5> activation and stabilization, 20% [6]) [6] immunoglobulin G <5> (<5> activation and stabilization, 0.5 mg/ml [6]) [6] Metals, ions Mn2+ <3> (<3> 1 mM stimulates [12]) [12] NaCl <5> (<5> activation, 0.2 M [6]) [6] Additional information <1-5> (<1-5> no requirement for Mn2+ [1-3,6-8]; <5> no requirement for Mn2+ , Mg2+ , Ca2+ [6]) [1-3, 6-8]

336

2.4.1.155

a-1,6-Mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

Specific activity (U/mg) 0.0015-0.031 <4> [11] 0.0036 <3> (<3> BVVD73 mutant [15]) [15] 0.004 <3> (<3> BVVD188 mutant [15]) [15] 0.03-0.06 <4> (<4> Q39-740 mutant [11]) [11] 0.139 <3> [2] 1.22 <5> [6] 110 <3> [12] Additional information <1, 4> [1, 3] Km-Value (mM) 0.023 <1> (N-acetyl-d-glucosaminyl-1,2-a-mannosyl-1,6-b-glucosyl-O(CH2 )7 -CH3 ) [4] 0.036 <1> (N-acetyl-d-glucosaminyl-1,2-a-mannosyl-1,6-b-glucosyl-O(CH2 )7 -CH3 ) [4] 0.074 <1> (N-acetyl-d-glucosaminyl-1,2-a-4-deoxy-mannosyl-1,6-b-d-glucosyl-O-(CH2 )7 -CH3 ) [4] 0.087 <5> (N-acetyl-d-glucosaminyl-1,2-a-mannosyl-1,6-b-mannosyl-O(CH2 )8 COOCH3 ) [6] 0.13 <3> (N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3(6)-(N-acetyl-dglucosaminyl-1,2-a-d-mannosyl-1,6(3))-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R, <3> with TDP-glucose [15]) [15] 0.133 <3> (pyridylaminated biantennary acceptor substrates) [2] 0.15 <3> (N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3(6)-(N-acetyl-dglucosaminyl-1,2-a-d-mannosyl-1,6(3))-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R, <3> with UDP-N-acetylglucosamine [15]) [15] 0.18 <3> (N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3(6)-(N-acetyl-dglucosaminyl-1,2-a-d-mannosyl-1,6(3))-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R) [14] 0.18 <1> (N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6-b-d-mannosyl-O(CH2 )-COOCH3 ) [8] 0.23 <3> (N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,3(6)-(N-acetyl-dglucosaminyl-1,2-a-d-mannosyl-1,6(3))-b-d-mannosyl-1,4-N-acetyl-d-glucosaminyl-R, <3> with UDP-glucose [15]) [15] 0.25 <1> (N-acetyl-d-glucosamine) [8] 0.66-0.7 <1> (UDP-N-acetylgucosamine, <1> cosubstrate: N-acetyl-d-glucosaminyl-1,2-a-6-deoxy- (or-4-O-methyl-) mannosyl-1,6-b-d-glucosyl-O-(CH2 )7 CH3 [4]) [4] 1.1-1.2 <1> (UDP-N-acetylglucosamine, <1> cosubstrate: N-acetyl-d-glucosaminyl-1,2-a-d-mannosyl-1,6-b-d-glucosyl-O-(CH2 )7 -CH3 [4]) [3, 4] 1.4 <1> (UDP-N-acetylgucosamine, <1> cosubstrate: N-acetyl-d-glucosaminyl-1,2-a-4-deoxy-mannosyl-1,6-b-d-glucosyl-O-(CH2 )7 -CH3 [4]) [4] 2.9 <3> (TDP-glucose) [15] 2.9 <3> (UDP-glucose) [15] 3.5 <3> (UDP-N-acetylglucosamine) [2] 4 <3> (UDP-N-acetylglucosamine, <3> BVVD188 mutant [15]) [15] 4.6 <3> (UDP-N-acetylglucosamine, <3> BVVD73 mutant [15]) [15]

337

a-1,6-Mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

2.4.1.155

5.8 <3> (UDP-N-acetylglucosamine) [12] 6 <3> (UDP-N-acetylglucosamine) [14] 7.6 <5> (UDP-N-acetylglucosamine, <5> + pyridylaminated acceptor substrate [7]) [7] 11 <5> (UDP-N-acetylglucosamine) [6] Additional information <1> (<1> kinetic study [4]) [4] Ki-Value (mM) 0.014 <1> (N-acetyl-d-glucosaminyl-1,2-a-4-O-methyl-mannosyl-1,6-b-dglucosyl-O-(CH2 )7 -CH3 ) [4] 0.06-0.077 <1> (N-acetyl-d-glucosaminyl-1,2-a-6-deoxy-mannosyl-1,6-b-dglucosyl-O-(CH2 )7 -CH3 ) [3] 0.063 <1> (N-acetyl-d-glucosaminyl-1,2-a-6-deoxy-mannosyl-1,6-b-d-glucosyl-O-(CH2 )7 -CH3 ) [4, 5] 0.141 <5> (N-acetyl-d-glucosaminyl-1,2-a-6-deoxy-mannosyl-1,6-b-d-glucosyl-O-(CH2 )8 -COOCH3 ) [6] pH-Optimum 6 <1> (<1> around [8]) [8] 6-6.7 <4> [11] 6.3 <5> [7] 6.5 <3> [12] 6.5-7 <5> [6] pH-Range 6.25 <5> [7] Temperature optimum ( C) 37 <1-5> (<1-5> assay at [1,3-5,7,8]) [1, 3-5, 7, 8] 42 <4> (<4> 2fold increase in activity as compared to 37 C [11]) [11] 50 <3> [12]

4 Enzyme Structure Molecular weight 65000 <3> (<3> native PAGE [12]) [12] 67000 <3> (<3> SDS-PAGE [12]) [12] 69000 <5> (<5> SDS-PAGE, 2 bands: 69000 and 75000 [6]) [6] 70000 <3> (<3> SDS-PAGE, BVVD188 [15]) [15] 73000 <3> (<3> HPLC-gel filtration [2]) [2] 75000 <5> (<5> SDS-PAGE, 2 bands: 69000 and 75000 [6]) [6] 85000 <3> (<3> SDS-PAGE, BVVD73 [15]) [15] Subunits monomer <3> (<3> 1 * 73000, SDS-PAGE [2]) [2] Additional information <5> (<5> rat kidney enzyme appears as doublet of MW 69000 and 75000 on SDS-PAGE [6]) [6]

338

2.4.1.155

a-1,6-Mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

Posttranslational modification glycoprotein <3> [12]

5 Isolation/Preparation/Mutation/Application Source/tissue BHK-21 cell <1> (<1> native BHK-21/c13 cells, cells transformed with Rous sarcoma virus, i.e. RS-BHK-cells, and lectin-resistant cell-lines LP 1.6 and 3.3 [3]) [3] BW-5147 cell <4> [1, 8] Hep-3B cell <3> [12] brain <4, 5> [7, 9] central nervous system <4> (<4> of the embryo [9]) [9] colonic cancer cell line <3> [12] embryo <4> [9] gonad <4> (<4> of the adult [9]) [9] kidney <1, 4, 5> (<4> of the embryo [9]) [3-6, 8, 9] lung <3, 5> (<3> QC, small lung cancer cell line [2]) [2, 7] myeloid cell line <3> [8] oviduct <2> [8] respiratory epithelium <4> (<4> of the embryo [9]) [9] skin <4> (<4> of the embryo [9]) [9] small intestine <4, 5> (<4> of the embryo [9]) [7, 9] spleen <5> [7] testis <5> [7] tumor cell line <4> [8] Localization membrane <1, 4, 5> [1, 3-6] microsome <1> [3] soluble <3> (<3> fusion with signal peptide from baculoviral protein to permit secretion into medium [15]) [2, 13, 15] Purification <1> (partial [4,5]) [4, 5] <3> (partial [2]; GnT-VD73, GnT-VD233, GnT-VD436 and GnT-VD188 [13]) [2, 12, 13, 14, 15] <4> [11] <5> [6] Cloning <3> (fusion with signal peptide from baculoviral protein to permit secretion into medium, BVVD73 and BVV D188 [15]) [11, 15] <4> (wild type enzyme and several N- and C-terminal deletions, fusion to protein A from Staphylococcus aurus [11]) [11]

339

a-1,6-Mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

2.4.1.155

Engineering A592G <3> (<3> increased specific activity [12]) [12] T555R <3> (<3> increased specific activity [12]) [12] V375L <3> (<3> increased specific activity [12]) [12]

6 Stability pH-Stability 4.5-10.5 <3> (<3> stable for 24 h at 4 C [12]) [12] Temperature stability 4 <4> (<4> stable for 2 months [11]) [11] General stability information <5>, NaCl, 0.2 M, stabilizes and enhances activity [6] <5>, glycerol, 20%, stabilizes and enhances activity [6] <5>, immunoglobulin G, 0.5 mg/ml, stabilizes and enhances activity [6] Storage stability <4>, -20 C stable for 2 months [11] <5>, 4 C, several months in 20% glycerol [6]

References [1] Cummings, R.D.; Trowbridge, I.S.; Kornfeld, S.: A mouse lymphoma cell line resistant to the leukoagglutinating lectin from Phaseolus vulgaris is deficient in UDP-GlcNAc:a-d-mannoside b 1,6 N-acetylglucosaminyltransferase. J. Biol. Chem., 257, 13421-13427 (1982) [2] Gu, J.; Nishikawa, A.; Tsuruoka, N.; Ohno, M.; Yamaguchi, N.; Kangawa, K.; Taniguchi, N.: Purification and characterization of UDP-N-acetylglucosamine:a-6-d-mannoside b1-6N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase V) from a human lung cancer cell line. J. Biochem., 113, 614-619 (1993) [3] Palcic, M.M.; Ripka, J.; Kaur, K.J.; Shoreibah, M.; Hindsgaul, O.; Pierce, M.: Regulation of N-acetylglucosaminyltransferase V activity. Kinetic comparisons of parental, Rous sarcoma virus-transformed BHK, and l-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog. J. Biol. Chem., 265, 6759-6769 (1990) [4] Khan, S.H.; Crawley, S.C.; Kanie, O.; Hindsgaul, O.: A trisaccharide acceptor analog for N-acetylglucosaminyltransferase V which binds to the enzyme but sterically precludes the transfer reaction. J. Biol. Chem., 268, 24682473 (1993) [5] Hindsgaul, O.; Kaur, K.J.; Srivastava, G.; Blaszcyk-Thurin, M.; Crawley, S.C.; Heerze, L.D.; Palcic, M.M.: Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases. J. Biol. Chem., 266, 17858-17862 (1991)

340

2.4.1.155

a-1,6-Mannosyl-glycoprotein 6-b-N-acetylglucosaminyltransferase

[6] Shoreibah, M.; Hindsgaul, O.; Pierce, M.: Purification and characterization of rat kidney UDP-N-acetylglucosamine: a-6-d-mannoside b-1,6-N-acetylglucosaminyltransferase. J. Biol. Chem., 267, 2920-2927 (1992) [7] Taniguchi, N.; Nishikawa, A.; Fujii, S.; Gu, J.: Glycosyltransferase assays using pyridylaminated acceptors: N-acetylglucosaminyltransferase III, IV, and V. Methods Enzymol., 179, 397-408 (1989) [8] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and O-glycan synthesis. Methods Enzymol., 179, 351-397 (1989) [9] Granovsky, M.; Fode, C.; Warren, C.E.; Campbell, R.M.; Marth, J.D.; Pierce, M.; Fregien, N.; Dennis, J.W.: GlcNAc-transferase V and core 2 GlcNActransferase expression in the developing mouse embryo. Glycobiology, 5, 797-806 (1995) [10] Khan, S.H.; Duus, J.O.; Crawley, S.C.; Palcic, M.M.; Hindsgaul, O.: Acceptorsubstrate recognition by N-acetylglucosaminyltransferase-V: role of the mannose residue in bdGlcNAc(1!2)adMan(1!6)bdGlcOR. Tetrahedron, 5, 2415-2435 (1994) [11] Korczak, B.; Le, T.; Elowe, S.; Datti, A.; Dennis, J.W.: Minimal catalytic domain of N-acetylglucosaminyltransferase V. Glycobiology, 10, 595-599 (2000) [12] Park, C.; Jin, U.-H.; Lee, Y.-C.; Cho, T.-J.; Kim, C.-H.: Characterization of UDP-N-acetylglucosamine:a-6-d-mannoside b-1,6-N-acetylglucosaminyltransferase V from a human hepatoma cell line Hep3B. Arch. Biochem. Biophys., 367, 281-288 (1999) [13] Saito, T.; Miyoshi, E.; Sasai, K.; Nakano, N.; Eguchi, H.; Honke, K.; Taniguchi, N.: A secreted type of b1,6-N-acetylglucosaminyltransferase V (GnT-V) induces tumor angiogenesis without mediation of glycosylation: a novel function of GnT-V distinct from the original glycosyltransferase activity. J. Biol. Chem., 277, 17002-17008 (2002) [14] Sasai, K.; Ikeda, Y.; Eguchi, H.; Tsuda, T.; Honke, K.; Taniguchi, N.: The action of N-acetylglucosaminyltransferase-V is prevented by the bisecting GlcNAc residue at the catalytic step. FEBS Lett., 522, 151-155 (2002) [15] Sasai, K.; Ikeda, Y.; Fujii, T.; Tsuda, T.; Taniguchi, N.: UDP-GlcNAc concentration is an important factor in the biosynthesis of b1,6-branched oligosaccharides: Regulation based on the kinetic properties of N-acetylglucosaminyltransferase V. Glycobiology, 12, 119-127 (2002)

341

Indolylacetyl-myo-inositol galactosyltransferase

2.4.1.156

1 Nomenclature EC number 2.4.1.156 Systematic name UDP-galactose:indol-3-ylacetyl-myo-inositol 5-O-d-galactosyltransferase Recommended name indolylacetyl-myo-inositol galactosyltransferase Synonyms galactosyltransferase, uridine diphosphogalactose-indolylacetylinositol indol-3-ylacetyl-myo-inositol galactoside synthase CAS registry number 85537-80-0

2 Source Organism <1> Zea mays (sweet corn [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + indol-3-ylacetyl-myo-inositol = UDP + 5-O-(indol-3-ylacetyl-myo-inositol) d-galactoside Reaction type hexosyl group transfer Natural substrates and products S UDPgalactose + indol-3-ylacetyl-myo-inositol <1> (<1> involved in metabolism of indol-3-ylacetic acid, a plant growth hormone [1]) (Reversibility: ? <1> [1]) [1] P UDP + 5-O-(indol-3-ylacetyl-myo-inositol) d-galactoside Substrates and products S UDPgalactose + indol-3-ylacetyl-myo-inositol <1> (<1> no substrate: UDPglucose [1]) (Reversibility: ? <1> [1]) [1] P UDP + 5-O-(indol-3-ylacetyl-myo-inositol) d-galactoside <1> [1]

342

2.4.1.156

Indolylacetyl-myo-inositol galactosyltransferase

Inhibitors EDTA <1> [1] Mg2+ <1> (<1> weak [1]) [1] Mn2+ <1> [1] Zn2+ <1> [1] Metals, ions Ca2+ <1> (<1> slight activation [1]) [1] pH-Optimum 7.6 <1> (<1> assay at [1]) [1] Temperature optimum ( C) 37 <1> (<1> assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue kernel <1> (<1> immature [1]) [1] Purification <1> (partial [1]) [1]

6 Stability General stability information <1>, 2-mercaptoethanol stabilizes during ammonium sulfate fractionation and dialysis [1]

References [1] Corcuera, L.J.; Michalczuk, L.; Bandurski, R.S.: Enzymic synthesis of indol-3ylacetyl-myo-inositol galactoside. Biochem. J., 207, 283-290 (1982)

343

1,2-Diacylglycerol 3-glucosyltransferase

2.4.1.157

1 Nomenclature EC number 2.4.1.157 Systematic name UDP-glucose:1,2-diacylglycerol 3-d-glucosyltransferase Recommended name 1,2-diacylglycerol 3-glucosyltransferase Synonyms UDP-glucose-diacylglycerol glucosyltransferase UDP-glucose:1,2-diacylglycerol glucosyltransferase UDPglucose:1,2-diacylglycerol 3-d-glucosyltransferase UDPglucose:diacylglycerol glucosyltransferase glucosyltransferase, uridine diphosphoglucose-diacylglycerol CAS registry number 83744-96-1

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

Anabaena variabilis [1] Anacystis nidulans [2] Acholeplasma laidlawii [3, 4, 5] Staphylococcus aureus [6] Bacillus subtilis (Marburg strain 60015 [7]) [7]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + 1,2-diacylglycerol = UDP + 3-d-glucosyl-1,2-diacylglycerol Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + 1,2-diacylglycerol <3> (<3>, the enzyme synthesizes the major nonbilayer-prone lipid monoglucosyldiacylglycerol in the membrane, which is important for spontaneous curvature, and is an important

344

2.4.1.157

1,2-Diacylglycerol 3-glucosyltransferase

site for the lipid surface charge density [3,4]; <3>, the product monoglucosyldiacylglycerol is the first glucolipid along the glucolipid pathway, and a major nonbilayer-prone lipid in the single membrane [5]) (Reversibility: ? <3> [3, 4, 5]) [3, 4, 5] P UDP + 3-d-glucosyl-1,2-diacylglycerol <3> [3] Substrates and products S UDP-glucose + 1,2-dioleoylglycerol <3> (Reversibility: ? <3> [5]) [5] P UDP + 3-d-glucosyl-1,2-dioleoylglycerol S UDPglucose + 1,2-diacylglycerol <1-5> (<5>, the enzyme successively transfers up to four glucose residues to 1,2-diacylglycerol. The starting activity can be characterized as a UDP-glucose:1,2-diacylglycerol-3-b-dglucosyltransferase, whereas in subsequent steps the glucose acceptors vary and represent the products of previous additions of b-glucosyl residues [7]) (Reversibility: ? <1-5> [1-7]) [1-7] P UDP + 3-d-glucosyl-1,2-diacylglycerol <1> [1] S UDPglucose + 1,2-dipalmitoylglycerol <1, 2, 3> (<3>, twice as high enzyme activity as sn-1,2-dioleoylglycerol at all concentrations up to 2.5 mol% concentration [5]) (Reversibility: ? <1,2,3> [1,2,5]) [1, 2, 5] P UDP + 3-d-glucosyl-1,2-dipalmitoylglycerol S UDPglucose + 1-oleoyl-2-palmitoylglycerol <1> (Reversibility: ? <1> [1]) [1] P UDP + 3-d-glucosyl-1-oleoyl-2-palmitoylglycerol S UDPglucose + 1-stearoyl-2-palmitoylglycerol <1> (Reversibility: ? <1> [1]) [1] P UDP + 3-d-glucosyl-1-stearoyl-2-palmitoylglycerol Inhibitors CaCl2 <2> [2] EDTA <1> [1] Activating compounds 1,2-dioleoyl-sn-glycero-3-phosphoglycerol <3> (<3>, activates [4]) [4] dodecylphosphoglycerol <3> (<3>, anionic amphiphiles are essential for the restoration of a proper conformation. Amphiphilic environment with a critical fraction of negatively charged headgroups induces a catalytic, active site conformation of the enzyme [3]) [3] phosphatidylglycerol <3> (<3>, anionic amphiphiles are essential for the restoration of a proper conformation. Amphiphilic environment with a critical fraction of negatively charged headgroups induces a catalytic, active site conformation of the enzyme [3]) [3] sn-1,2-dioleoylglycerol <3> (<3>, activates [5]) [5] Metals, ions Mg2+ <1, 3> (<1>, required, maximal activity at 20 mM MgCl2 [1]; <3>, strong dependence on, maximal activity at 15-28 mM MgCl2 [5]) [1, 5] Specific activity (U/mg) 12 <3> [5]

345

1,2-Diacylglycerol 3-glucosyltransferase

2.4.1.157

Km-Value (mM) 0.045 <1> (UDPglucose) [1] pH-Optimum 7 <1> [1] pH-Range 5-8.7 <1> (<1>, pH 5.0: about 45% of maximal activity, pH 8.7: about 50% of maximal activity [1]) [1] Temperature optimum ( C) 45 <1> [1] Temperature range ( C) 28-55 <1> (<1>, 28 C: 50% of maximal activity, 55 C: 35% of maximal activity [1]) [1]

4 Enzyme Structure Subunits ? <3, 4, 5> (<3>, 1 * 40000, SDS-PAGE [5]; <5>, x * 43600, SDS-PAGE [7]; <4>, x * 44000, SDS-PAGE [6]) [5, 6, 7]

5 Isolation/Preparation/Mutation/Application Localization membrane <1, 2, 3> (<2>, plasma membranes, thylakoid membranes, not in outer membrane [2]; <3>, bound to [4]) [1, 2, 3, 4, 5] Purification <3> [5] Cloning <3> (expression in Escherichia coli [4]) [4] <4> (expression in Escherichia coli [6]) [6] <5> (expression in Escherichia coli [7]) [7]

6 Stability Temperature stability 4 <3> (<3>, half-life is around 50 h [5]) [5] 20 <3> (<3>, half-life is around 1.8 h [5]) [5] 28 <3> (<3>, half-life in 1,2-dioleoylphosphatidylglycerol-CHAPS micelles is more than 2 h, half-life is less than 0.5 h in 1,2-dioleoylphosphatidylcholineCHAPS micelles [3]) [3]

346

2.4.1.157

1,2-Diacylglycerol 3-glucosyltransferase

General stability information <3>, total digestion by proteinase K, trypsin or chymotrypsin when no lipid is present, the activity is highly restored in micelles containing 1,2-dioleoylphosphatidylglycerol and 1,2-dioleoyl-phosphatidylserine [3] Storage stability <1>, -80 C, stable for at least 2 months, crude enzyme extract [1] <3>, -20 C or -50 C, stable for at least 48 days [5]

References [1] Sato, N.; Murata, N.: Lipid biosynthesis in the blue-green alga (cyanobacterium), Anabaena variabilis III. UDPglucose:diacylglycerol glucosyltransferase activity in vitro. Plant Cell Physiol., 23, 1115-1120 (1982) [2] Omata, T.; Murata, N.: Glucolipid synthesis activities in cytoplasmic and thylakoid membranes from cyanobacterium Anacystis nidulans. Plant Cell Physiol., 27, 485-490 (1986) [3] Li, L.; Karlsson, O.P.; Wieslander, A.: Activating amphiphiles cause a conformational change of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes according to proteolytic digestion. J. Biol. Chem., 272, 29602-29606 (1997) [4] Berg, S.; Edman, M.; Li, L.; Wikstrom, M.; Wieslander, A.: Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes: recognition of a large group of lipid glycosyltransferases in eubacteria and archaea. J. Biol. Chem., 276, 22056-22063 (2001) [5] Karlsson, O.P.; Dahlqvist, A.; Vikstroem, S.; Wieslander, A.: Lipid dependence and basic kinetics of the purified 1,2-diacylglycerol 3-glucosyltransferase from membranes of Acholeplasma laidlawii. J. Biol. Chem., 272, 929936 (1997) [6] Jorasch, P.; Warnecke, D.C.; Lindner, B.; Zahringer, U.; Heinz, E.: Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols. Eur. J. Biochem., 267, 3770-3783 (2000) [7] Jorasch, P.; Wolter, F.P.; Zahringer, U.; Heinz, E.: A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2-diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products. Mol. Microbiol., 29, 419-430 (1998)

347

13-Hydroxydocosanoate 13-b-glucosyltransferase

2.4.1.158

1 Nomenclature EC number 2.4.1.158 Systematic name UDP-glucose:13-hydroxydocosanoate 13-b-d-glucosyltransferase Recommended name 13-hydroxydocosanoate 13-b-glucosyltransferase Synonyms 13-glucosyloxydocosanoate 2'-b-glucosyltransferase UDP-glucose-13-hydroxydocosanoate glucosyltransferase UDP-glucose:13-hydroxydocosanoic acid glucosyltransferase glucosyltransferase, uridine diphosphoglucose-hydroxydocosanoate CAS registry number 70457-13-5

2 Source Organism <1> Candida bogoriensis [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + 13-hydroxydocosanoate = UDP + 13-b-d-glucosyloxydocosanoate Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + 13-(b-d-glucopyranosyl)oxydocosanoic acid methyl ester <1> (<1> glucosyltransferase II [1]) (Reversibility: ? <1> [1]) [1] P UDP + 13-[(2'-O-b-d-glucopyranosyl-b-d-glucopyranosyl)oxy]docosanoic acid methyl ester <1> [1] S UDPglucose + 13-hydroxydocosanoic acid <1> (<1> glucosyltransferase I [1]) (Reversibility: ? <1> [1]) [1] P UDP + 13-(b-d-glucopyranosyl)oxydocosanoic acid

348

2.4.1.158

13-Hydroxydocosanoate 13-b-glucosyltransferase

Substrates and products S UDPglucose + 13-(b-d-glucopyranosyl)oxydocosanoic acid methyl ester <1> (<1> glucosyltransferase II [1]) (Reversibility: ? <1> [1]) [1] P UDP + 13-[(2'-O-b-d-glucopyranosyl-b-d-glucopyranosyl)oxy]docosanoic acid methyl ester <1> [1] S UDPglucose + 13-hydroxydocosanoic acid <1> (<1> glucosyltransferase I [1]) (Reversibility: ? <1> [1]) [1] P UDP + 13-(b-d-glucopyranosyl)oxydocosanoic acid <1> [1] S Additional information <1> (<1> enzyme seems to be a single multifunctional protein catalyzing both transferase reactions [1]) [1] P ? Inhibitors NaCl <1> (<1> 0.2 mM: 50% inhibition of glucosyltransferase I, 0.25 mM: 50% inhibition of glucosyltransferase II [1]) [1] Specific activity (U/mg) 0.0222 <1> (<1> glucosyltransferase I [1]) [1] 0.0242 <1> (<1> glucosyltransferase II [1]) [1] Km-Value (mM) 0.04 <1> (UDPglucose, <1> glucosyltransferase I and II [1]) [1] pH-Optimum 6.5-8 <1> (<1> glucosyltransferase I, within this range activity of glucosyltransferase II increases [1]) [1] Temperature optimum ( C) 25 <1> (<1> assay at [1]) [1] 30-37 <1> [1] Temperature range ( C) 5-37 <1> (<1> 30 C-37 C: 100% of activity, at 5 C: 10% of maximal activity [1]) [1]

4 Enzyme Structure Molecular weight 50000 <1> (<1> gel filtration [1]) [1] Subunits monomer <1> (<1> 1 * 52000, SDS-PAGE [1]) [1]

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

349

13-Hydroxydocosanoate 13-b-glucosyltransferase

2.4.1.158

6 Stability pH-Stability 5.9-8.1 <1> (<1> glucosyltransferase I and II: stable [1]) [1] General stability information <1>, glycerol, 20%, stabilizes [1] Storage stability <1>, 4 C, 20% glycerol: half-life 35 days [1] <1>, 4 C, without glycerol: half-life 5 days [1]

References [1] Breithaupt, T.B.; Light, R.J.: Affinity chromatography and further characterization of the glucosyltransferases involved in hydroxydocosanoic acid sophoroside production in Candida bogoriensis. J. Biol. Chem., 257, 96229628 (1982)

350

Flavonol-3-O-glucoside L-rhamnosyltransferase

2.4.1.159

1 Nomenclature EC number 2.4.1.159 Systematic name UDP-l-rhamnose:flavonol-3-O-d-glucoside l-rhamnosyltransferase Recommended name flavonol-3-O-glucoside l-rhamnosyltransferase Synonyms UDP-rhamnose:flavonol 3-O-glucoside rhamnosyltransferase rhamnosyltransferase, uridine diphosphorhamnose-flavonol 3-O-glucoside CAS registry number 83380-89-6

2 Source Organism <1> Tulipa sp. (tulip, cv. Apeldoorn [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-l-rhamnose + flavonol 3-O-d-glucoside = UDP + flavonol 3-O-l-rhamnosylglucoside Reaction type hexosyl group transfer Natural substrates and products S UDPrhamnose + flavonol 3-O-glucoside <1> (involved in flavonoid metabolism, pathway of flavonol 3-O-triglycoside biosynthesis) (Reversibility: ? <1> [1]) [1] P UDP + flavonol 3-O-rhamnosylglucoside Substrates and products S UDPrhamnose + flavonol 3-O-diglycoside <1> (Reversibility: ? <1> [1]) [1] P UDP + flavonol 3-O-triglycoside <1> [1]

351

Flavonol-3-O-glucoside L-rhamnosyltransferase

2.4.1.159

S UDPrhamnose + flavonol 3-O-glucoside <1> (<1> acceptor substrates are 3-O-glucosides of quercetin, isorhamnetin or kaempferol, rutin, less efficient acceptors: quercetin 3-O-galactoside or quercetin 3-O-rhamnoside, kaempferol 3-O-rhamnoside or myrecetin 3-O-rhamnoside, no substrates: aglycones [1]) (Reversibility: ? <1> [1]) [1] P UDP + flavonol 3-O-rhamnosylglucoside <1> (<1> i.e. 3-O-rutinosides [1]) [1] Inhibitors p-chloromercuribenzoate <1> (<1> weak [1]) [1] Activating compounds 2-mercaptoethanol <1> (<1> slight stimulation [1]) [1] dithioerythritol <1> (<1> slight stimulation [1]) [1] glutathione <1> (<1> slight stimulation [1]) [1] Additional information <1> (<1> no stimulation by sucrose or bovine serum albumin [1]) [1] Metals, ions Mg2+ <1> (<1> slight stimulation [1]) [1] Additional information <1> (<1> no activation by Ca2+ , NH+4 , Mn2+ , Cl- or SO24- [1]) [1] pH-Optimum 8.5-9 <1> [1]

4 Enzyme Structure Molecular weight 40000 <1> (<1> gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue anther <1> [1] Purification <1> (partial [1]) [1]

References [1] Kleinehollenhorst, G.; Behrens, H.; Pegels, G.; Srunk, N.; Wiermann, R.: Formation of flavonol 3O-diglycosides and flavonol 3-O-triglycosides by enzyme extracts from anthers of tulipa cv. Apeldoorn. Z. Naturforsch. C, 37c, 587-599 (1982)

352

Pyridoxine 5'-O-b-D-glucosyltransferase

2.4.1.160

1 Nomenclature EC number 2.4.1.160 Systematic name UDP-glucose:pyridoxine 5'-O-b-d-glucosyltransferase Recommended name pyridoxine 5'-O-b-d-glucosyltransferase Synonyms UDP-glucose-pyridoxine glucosyltransferase UDP-glucose:pyridoxine 5'-O-b-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-pyridoxine 5'-bCAS registry number 83744-97-2

2 Source Organism <1> Pisum sativum (podded pea, L. cv. Kinusaya [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + pyridoxine = UDP + 5'-O-b-d-glucosylpyridoxine Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + pyridoxine <1> (Reversibility: ? <1> [1]) [1] P UDP + 5'-O-b-d-glucosylpyridoxine <1> [1] Substrates and products S UDPglucose + 4'-deoxypyridoxine <1> (Reversibility: ? <1> [1]) [1] P UDP + 5'-O-b-d-glucosyl-4'-deoxypyridoxine S UDPglucose + pyridoxamine <1> (Reversibility: ? <1> [1]) [1] P UDP + 5'-O-b-d-glucosylpyridoxamine

353

Pyridoxine 5'-O-b-D-glucosyltransferase

2.4.1.160

S UDPglucose + pyridoxine <1> (<1> high specificity for UDPglucose [1]) (Reversibility: ? <1> [1]) [1] P UDP + 5'-O-b-d-glucosylpyridoxine <1> [1] Inhibitors 4'-deoxypyridoxine <1> (<1> competitive inhibitor of pyridoxamine and pyridoxine glucosylation [1]) [1] pyridoxamine <1> (<1> competitive inhibitor of pyridoxine glucosylation [1]) [1] pyridoxine <1> (<1> competitive inhibitor of pyridoxamine glucosylation [1]) [1] Metals, ions Mg2+ <1> (<1> required, Km : 1.0 mM [1]) [1] Mn2+ <1> (<1> can replace Mg2+ , 40% as effective as Mg2+ [1]) [1] Km-Value (mM) 0.25 <1> (pyridoxine) [1] 0.26 <1> (pyridoxamine) [1] 0.67 <1> (UDPglucose, <1> cosubstrate pyridoxine [1]) [1] pH-Optimum 7.8-8.8 <1> (<1> pyridoxine + UDPglucose [1]) [1] pH-Range 7-9.5 <1> (<1> about 50% of maximum activity at pH 7 and 9.5 [1]) [1] Temperature optimum ( C) 30 <1> (<1> assay at [1]) [1]

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

References [1] Tadera, K.; Yagi, F.; Kobayashi, A.: Specificity of a particulate glucosyltransferase in seedlings of Pisum sativum L. which catalyzes the formation of 5-O(b-d-glucopyranosyl)pyridoxine. J. Nutr. Sci. Vitaminol., 28, 359-366 (1982)

354

Oligosaccharide 4-a-D-glucosyltransferase

2.4.1.161

1 Nomenclature EC number 2.4.1.161 Systematic name 1,4-a-d-glucan:1,4-a-d-glucan 4-a-d-glucosyltransferase Recommended name oligosaccharide 4-a-d-glucosyltransferase Synonyms 1,4-a-glucan:1,4-a-glucan 4-a-glucosyltransferase amylase III CAS registry number 9000-92-4

2 Source Organism <1> Entamoeba histolytica [1, 2]

3 Reaction and Specificity Catalyzed reaction transfers the non-reducing terminal a-d-glucose residue from a 1,4-a-d-glucan to the 4-position of an a-d-glucan, thus bringing about the hydrolysis of oligosaccharides Reaction type Hexosyl group transfer Natural substrates and products S a-d-glucan + a-d-glucan <1> (<1> transfers the nonreducing terminal a-d-glucose residue from a 1,4-a-d-glucan to the 4-position of an a-dglucan, thus bringing about the hydrolysis of oligosaccharides, acts on amylose, amylopectin, glycogen and maltooligosaccharides [2]) (Reversibility: ? <1> [1, 2]) [1, 2] P ? S Additional information <1> (<1> enzyme may be biologically relevant in the environment of the intestinal tract of man [1]) [1] P ? 355

Oligosaccharide 4-a-D-glucosyltransferase

2.4.1.161

Substrates and products S a-d-glucan + a-d-glucan <1> (<1> transfers the nonreducing terminal a-d-glucose residue from a 1,4-a-d-glucan to the 4-position of an a-dglucan, thus bringing about the hydrolysis of oligosaccharides, acts on amylose, amylopectin, glycogen and maltooligosaccharides and on 4-nitrophenyl-a-glucoside to 4-nitrophenylmaltoheptaoside [1,2]; <1> no substrate: maltose [2]) (Reversibility: ? <1> [1, 2]) [1, 2] P ? <1> (<1> hydrolysis of oligosaccharides, no detectable free glucose is formed [2]) [2] Inhibitors 1-deoxynojirimycin <1> (<1> Bay h 5595 [2]) [2] acarbose <1> (<1> Bay g 5421 [2]) [2] pH-Optimum 6 <1> [1] Temperature optimum ( C) 33 <1> [1]

4 Enzyme Structure Molecular weight 47000 <1> (<1> gel filtration [1]) [1]

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

References [1] Nebinger, P.: Separation and characterization of four different amylases of Entamoeba histolytica. I. Purification and properties. Biol. Chem. HoppeSeyler, 367, 161-167 (1986) [2] Nebinger, P.: Separation and characterization of four different amylases of Entamoeba histolytica. II. Characterization of amylases. Biol. Chem. HoppeSeyler, 367, 169-176 (1986)

356

Aldose b-D-fructosyltransferase

2.4.1.162

1 Nomenclature EC number 2.4.1.162 Systematic name a-d-aldosyl-b-d-fructoside:aldose 1-b-d-fructosyltransferase Recommended name aldose b-d-fructosyltransferase Synonyms Additional information (cf. EC 2.4.1.10) CAS registry number 9031-67-8

2 Source Organism <1> Bacillus subtilis (NCIB 11871, 11872, 11873 [1]) [1] <2> Erwinia sp. (previously Aerobacter levanicum [1]) [1]

3 Reaction and Specificity Catalyzed reaction a-d-aldosyl1 b-d-fructoside + d-aldose2 = d-aldose1 + a-d-aldosyl2 b-dfructoside Reaction type hexosyl group transfer Natural substrates and products S fructose donor + fructose acceptor <1, 2> (Reversibility: ? <1, 2> [1]) [1] P d-aldose + d-aldosyl b-d-fructoside <1, 2> (<1,2> the products can act as acceptors for further reactions, leading to oligosaccharides or polysaccharides [1]) [1] Substrates and products S 6-deoxy-d-glucose + sucrose <1> (Reversibility: ? <1> [1]) [1] P 6-deoxysucrose + d-glucose <1> [1] S d-glucose 6-benzoate + sucrose <1> (Reversibility: ? <1> [1]) [1] P sucrose 6-benzoate + d-glucose <1> [1]

357

Aldose b-D-fructosyltransferase

2.4.1.162

S fructose donor + 1-thio-d-glucose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + 3-O-methyl a-d-glucose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + 3-O-methylglucose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + 4-chloro-4-deoxyglucose 6-acetate <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + 4-chlorogalactose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + 6-O-methylgalactose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + 6-O-methylglucose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + 6-chloro-6-deoxyglucose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + 6-deoxyglucose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + d-arabinose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + d-arabinose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + d-glycero-d-galactoheptose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + l-arabinose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + l-fucose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + cellobiose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + ethanol <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + fructose acceptor <1, 2> (<1,2> the donor must be a bfructosyl ring attached to the anomeric carbon of an aldose by an 1,2 link, acceptors can be monomeric or oligomeric hexoses or pentoses, no acceptors are inosital, 2-deoxyglucose, glucosamine [1]) (Reversibility: ? <1, 2> [1]) [1] P d-aldose + d-aldosyl b-d-fructoside <1, 2> (<1,2> the products can act as acceptors for further reactions, leading to oligosaccharides or polysaccharides [1]) 358

2.4.1.162

Aldose b-D-fructosyltransferase

S fructose donor + galactose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + galactose 6-acetate <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + gluconic acid <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + glucose 6-acetate <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + glucose 6-alkyl ether <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + glucose 6-benzyl ether <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + glucose 6-phosphate <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + glycerol <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + isomaltose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + lactose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + lyxose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + lyxose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + maltohexaose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + maltopentaose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + maltose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + maltotriose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + mannose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + mannose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + mellibiose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + methyl-a-d-glucopyranoside <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside S fructose donor + rhamnose <1, 2> (Reversibility: ? <1, 2> [1]) [1] P corresponding d-aldose + d-aldosyl b-d-fructoside 359

Aldose b-D-fructosyltransferase

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

fructose donor + ribose <1, 2> (Reversibility: ? <1, 2> [1]) [1] corresponding d-aldose + d-aldosyl b-d-fructoside fructose donor + sorbose <1, 2> (Reversibility: ? <1, 2> [1]) [1] corresponding d-aldose + d-aldosyl b-d-fructoside fructose donor + sucrose <1, 2> (Reversibility: ? <1, 2> [1]) [1] corresponding d-aldose + d-aldosyl b-d-fructoside fructose donor + xylitol <1, 2> (Reversibility: ? <1, 2> [1]) [1] corresponding d-aldose + d-aldosyl b-d-fructoside fructose donor + xylose <1, 2> (Reversibility: ? <1, 2> [1]) [1] corresponding d-aldose + d-aldosyl b-d-fructoside glucose 6-acetate + sucrose <1> (Reversibility: ? <1> [1]) [1] sucrose 6-acetate + d-glucose <1> [1] raffinose + fructose acceptor <1, 2> (Reversibility: ? <1, 2> [1]) [1] corresponding d-aldose + d-aldosyl b-d-fructoside raffinose + galactose <1> (Reversibility: ? <1> [1]) [1] b-d-fructofuranosyl-2,1-d-galactopyranoside + a-d-galactopyranosyl-ad-glucopyranoside <1> [1] raffinose + xylose <1> (Reversibility: ? <1> [1]) [1] b-d-fructofuranosyl-2,1-a-d-xylopyranoside + a-d-galactopyranosyl-ad-glucopyranoside <1> [1] stachyose + fructose acceptor <1, 2> (Reversibility: ? <1, 2> [1]) [1] corresponding d-aldose + d-aldosyl b-d-fructoside sucrose + d-galactose <1> (Reversibility: ? <1> [1]) [1] b-d-fructofuranosyl-2,1-d-galactopyranoside + d-glucose <1> [1] sucrose + fructose acceptor <1, 2> (Reversibility: ? <1, 2> [1]) [1] corresponding d-aldose + d-aldosyl b-d-fructoside sucrose + xylose <1> (Reversibility: ? <1> [1]) [1] b-d-fructofuranosyl-2,1-a-d-xylopyranoside + d-glucose <1> [1]

Km-Value (mM) 200 <1> (sucrose, <1> strain NCIB 11871 [1]) [1] pH-Optimum 5.4-6 <1> [1] Temperature optimum ( C) 30 <1> [1]

5 Isolation/Preparation/Mutation/Application Localization extracellular <1, 2> [1] Purification <1> [1]

360

2.4.1.162

2.4.1.162

Aldose b-D-fructosyltransferase

6 Stability Temperature stability 45 <1, 2> (<1,2> stable at least 20 min [1]) [1]

References [1] Rathbone, E.B.; Hacking, A.J.; Cheetham, P.S.J.: Trihalo-trideoxy galactosucrose compounds. UK Patent Application, GB21, 45080 A (1985)

361

b-Galactosyl-N-acetylglucosaminylgalactosylglucosyl-ceramide b-1,3-acetylglucosaminyltransferase

2.4.1.163

1 Nomenclature EC number 2.4.1.163 Systematic name UDP-N-acetyl-d-glucosamine:b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl1,3-b-d-galactosyl-1,4-b-d-glucosylceramide b-1,3-acetylglucosaminyltransferase Recommended name b-galactosyl-N-acetylglucosaminylgalactosylglucosyl-ceramide glucosaminyltransferase

b-1,3-acetyl-

Synonyms acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-acetyllactosaminide b1!3poly-N-acetyllactosamine extension enzyme uridine diphosphoacetylglucosamine-acetyllactosaminide b1!3-acetylglucosaminyltransferase CAS registry number 85638-39-7

2 Source Organism <1> Mus musculus [1]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide = UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl1,4-b-d-glucosylceramide Reaction type hexosyl group transfer

362

Natural substrates and products S Additional information <1> (<1> biosynthesis of Ii core glycosphingolipids [1]) [1] P ? Substrates and products S UDP-N-acetyl-d-glucosamine + asialo a1 -acid glycoprotein <1> (<1> sialic acid depleted a1 -acid glycoprotein, 30% of the activity with neolactotetraosylceramide [1]) (Reversibility: ? <1> [1]) [1] P ? S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <1> (<1> i.e. neolactotetraosylceramide, GlcNAcT-2, biosynthesis in vitro of Ii core glycosphingolipids [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <1> [1] S UDP-N-acetyl-d-glucosamine + lactosylceramide <1> (<1> 15% of the activity with neolactotetraosylceramide [1]) (Reversibility: ? <1> [1]) [1] P ? Inhibitors EDTA <1> [1] Activating compounds Nonidet P-40 <1> (<1> reaction rate is optimal at detergent:protein ratio of 1:3 [1]) [1] Triton CF-54 <1> (<1> reaction rate is optimal at detergent:protein ratio of 1:6 [1]) [1] Triton X-100 <1> (<1> reaction rate is optimal at detergent:protein ratio of 1:6 [1]) [1] detergent <1> (<1> activates, reaction rate is optimal at detergent:protein ratio of 1.6 in Triton CF-54 or X-100, 1:3 in Nonidet P-40 [1]) [1] Metals, ions Ca2+ <1> (<1> activates, 48% as effective as Mn2+ [1]) [1] Co2+ <1> (<1> activates, 52% as effective as Mn2+ [1]) [1] Mn2+ <1> (<1> requirement, Km : 1.25 mM, Mn2+ cannot be replaced by Mg2+ , Zn2+ , Cu2+ or Cd2+ [1]) [1] Additional information <1> (<1> not activated by Mg2+ , Zn2+ , Cu2+ or Cd2+ [1]) [1] Km-Value (mM) 0.09 <1> (neolactotetraosylceramide) [1] 0.33 <1> (UDP-N-acetyl-d-glucosamine, <1> solubilized enzyme [1]) [1] 2.5 <1> (UDP-N-acetyl-d-glucosamine, <1> membrane-bound enzyme [1]) [1] pH-Optimum 7-8 <1> [1]

363

5 Isolation/Preparation/Mutation/Application Source/tissue P-1798 cell <1> (<1> lymphoma cell line [1]) [1] Localization membrane <1> [1]

References [1] Basu, M.; Basu, S.: Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by b1-3- and b1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma. J. Biol. Chem., 259, 12557-12562 (1984)

364

Galactosyl-N-acetylglucosaminylgalactosylglucosyl-ceramide b-1,6-N-acetylglucosaminyltransferase

2.4.1.164

1 Nomenclature EC number 2.4.1.164 Systematic name UDP-N-acetyl-d-glucosamine:d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl1,3-b-d-galactosyl-1,4-b-d-glucosylceramide b-1,6-N-acetylglucosaminyltransferase Recommended name galactosyl-N-acetylglucosaminylgalactosylglucosyl-ceramide b-1,6-N-acetylglucosaminyltransferase Synonyms acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-acetyllactosaminide b1 !6uridine diphosphoacetylglucosamine-acetyllactosaminide b1 !6-acetylglucosaminyltransferase CAS registry number 85638-40-0

2 Source Organism <1> Mus musculus [1]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl1,3-b-d-galactosyl-1,4-b-d-glucosylceramide = UDP + N-acetyl-d-glucosaminyl-1,6-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4b-d-glucosylceramide Reaction type hexosyl group transfer Natural substrates and products S Additional information <1> (<1> biosynthesis of Ii core glycosphingolipids [1]) [1] P ? 365

Substrates and products S UDP-N-acetyl-d-glucosamine + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <1> (<1> i.e. neolactotetraosylceramide, GlcNAcT-3, biosynthesis in vitro of Ii core glycosphingolipids [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-d-glucosaminyl-1,6-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <1> [1] S UDP-N-acetyl-d-glucosamine + lactosylceramide <1> (<1> 15% of the activity with neolactotetraosylceramide [1]) (Reversibility: ? <1> [1]) [1] P ? S UDP-N-acetyl-d-glucosamine + sialic acid-depleted a1 -acid glycoprotein <1> (<1> 30% of the activity with neolactotetraosylceramide [1]) (Reversibility: ? <1> [1]) [1] P ? Inhibitors EDTA <1> (<1> complete inhibition [1]) [1] Activating compounds Nonidet P-40 <1> (<1> reaction rate is optimal at detergent:protein ratio of 1:3 [1]) [1] Triton CF-54 <1> (<1> reaction rate is optimal at detergent:protein ratio of 1:6 [1]) [1] Triton X-100 <1> (<1> reaction rate is optimal at detergent:protein ratio of 1:6 [1]) [1] detergent <1> (<1> activates, reaction rate is optimal at detergent:protein ratio of 1.6 in Triton CF-54 or X-100, 1:3 in Nonidet P-40 [1]) [1] Metals, ions Ca2+ <1> (<1> activates, 48% as effective as Mn2+ [1]) [1] Co2+ <1> (<1> activates, 52% as effective as Mn2+ [1]) [1] Mn2+ <1> (<1> required, Km : 1.25 mM, Mn2+ cannot be replaced by Mg2+ , Zn2+ , Cu2+ or Cd2+ [1]) [1] Additional information <1> (<1> not activated by Mg2+ , Zn2+ , Cu2+ or Cd2+ [1]) [1] Km-Value (mM) 0.09 <1> (neolactotetraosylceramide) [1] 0.33 <1> (UDP-N-acetyl-d-glucosamine, <1> solubilized enzyme [1]) [1] 2.5 <1> (UDP-N-acetyl-d-glucosamine, <1> membrane-bound enzyme [1]) [1] pH-Optimum 7-8 <1> [1]

366

5 Isolation/Preparation/Mutation/Application Source/tissue P-1798 cell <1> (<1> lymphoma cell line [1]) [1] Localization membrane <1> [1]

References [1] Basu, M.; Basu, S.: Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by b1-3- and b1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma. J. Biol. Chem., 259, 12557-12562 (1984)

367

N-Acetylneuraminylgalactosylglucosylceramide b-1,4-N-acetylgalactosaminyltransferase

2.4.1.165

1 Nomenclature EC number 2.4.1.165 Systematic name UDP-N-acetyl-d-galactosamine:N-acetylneuraminyl-2,3-a-d-galactosyl-1,4b-d-glucosylceramide b-1,4-N-acetylgalactosaminyltransferase Recommended name N-acetylneuraminylgalactosylglucosylceramide b-1,4-N-acetylgalactosaminyltransferase Synonyms CT GalNAc transferase Sda-b-1,4-N-acetylgalactosaminyltransferase Sda-b-GalNAc-transferase acetylgalactosaminyltransferase, uridine diphosphoacetylgalactosamine-acetylneuraminyl(a2!3)galactosyl(b1!4)glucosyl b1!4CAS registry number 109136-50-7

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

Homo sapiens [1, 2, 3, 4, 5] Sus srofa [4] Rattus norvegicus [4] Mus musculus (DGS654A transgenic line [7]) [6, 7]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-galactosamine + N-acetylneuraminyl-2,3-a-d-galactosyl1,4-b-d-glucosylceramide = UDP + N-acetyl-d-galactosaminyl-1,4-b-N-acetylneuraminyl-2,3-a-d-galactosyl-1,4-b-d-glucosylceramide Reaction type hexosyl group transfer

368

2.4.1.165

N-Acetylneuraminylgalactosylglucosylceramide b-1,4-N-acetylgalactosaminyltransferase

Natural substrates and products S UDP-N-acetyl-d-galactosamine + N-acetylneuraminyl-2,3-a-d-galactosyl1,4-b-d-glucosylceramide <1, 2, 4> (<1>, responsible for addition of the immunodominant sugar of the Sda-isto-blood-group determinant [2]; <1>, enzymic activity correlates with the degree of enterocytic differentiation [5]; <4>, overexpression of the enzyme in skeletal muscles inhibits muscular dystrophy in mdx mice. The enzyme produces the terminal b1,4GalNAc linkage on the cytotoxic T cell carbohydrate antigen which is also localized to the neuromuscular junction in adult animals [6]; <4>, the enzyme can affect the post-translational processing of dystroglycan and the extent of muscular dystrophy even in muscles where the VIA4-1 antigen is not present [7]) (Reversibility: ? <1, 2, 4> [2, 4, 6, 7]) [2, 4, 5, 6, 7] P UDP + N-acetyl-d-galactosaminyl-1,4-b-N-acetylneuraminyl-2,3-a-d-galactosyl-1,4-b-d-glucosylceramide <1> [2] Substrates and products S UDP-N-acetyl-d-galactosamine + NeuAca(2-3)Galb(1-4)Glc <1, 2> (<1>, i.e. 3'-sialyllactose [1]; <1>, strictly requires the presence of sialic acid in a-2,3-linkage to subterminal galactose [2]) (Reversibility: ? <1, 2> [1, 2, 3, 4, 5]) [1, 2, 3, 4, 5] P UDP + GalNAcb(1-4)(NeuAca(2-3))Galb(1-4)Glc <1, 2> [1, 4] S UDP-N-acetyl-d-galactosamine + NeuAca(2-3)Galb(1-4)GlcNAc <1, 2> (<1>, i.e. 3'-sialyllactosamine [4]) (Reversibility: ? <1, 2> [4, 5]) [4, 5] P UDP + GalNAcb(1-4)(NeuAca(2-3))Galb(1-4)Glc <2> [4] S UDP-N-acetyl-d-galactosamine + a1 -acid glycoprotein <1, 2> (Reversibility: ? <1, 2> [1, 4]) [1, 4] P UDP + N-acetylgalactosaminyl-a1 -acid glycoprotein S UDP-N-acetyl-d-galactosamine + asialofetuin <1, 2> (Reversibility: ? <1, 2> [1, 2, 4, 5]) [1, 2, 4, 5] P UDP + N-acetylgalactosaminylasialofetuin S UDP-N-acetyl-d-galactosamine + di-sialyl-lacto-N-tetraose <1> (Reversibility: ? <1> [5]) [5] P UDP + N-acetylgalactosaminyl-di-sialyl-lacto-N-tetraose S UDP-N-acetyl-d-galactosamine + fetuin <1, 2> (Reversibility: ? <1, 2> [1, 2, 4, 5]) [1, 2, 4, 5] P UDP + N-acetylgalactosaminylfetuin S UDP-N-acetyl-d-galactosamine + glycophorin <1> (<1>, good acceptor [2]) (Reversibility: ? <1> [2]) [2] P UDP + N-acetylgalactosaminyl glycophorin S UDP-N-acetyl-d-galactosamine + glycophorin A <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetylgalactosaminyl glycophorin A S UDP-N-acetyl-d-galactosamine + human chorionic gonadotropin <1, 2> (Reversibility: ? <1, 2> [2, 4]) [2, 4] P UDP + N-acetylgalactosaminyl human chorionic gonadotropin

369

N-Acetylneuraminylgalactosylglucosylceramide b-1,4-N-acetylgalactosaminyltransferase

2.4.1.165

S UDP-N-acetyl-d-galactosamine + sialosylparagloboside <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetylgalactosaminyl sialosylparagloboside S Additional information <1, 2> (<2>, the enzyme strictly recognizes the NeuAca2,3Galb terminal sequence of N- and O-linked oligosaccharides bound to glycoproteins [4]; <1>, the enzyme strictly requires the presence in acceptors of NeuAc in a2,3-linkage to subterminal galactose [5]) [4, 5] P ? Inhibitors ATP <1> [1] Triton X-100 <1, 2> (<1>, weak [1]; <2>, 0.5%, inhibition [4]) [1, 4] Activating compounds Triton X-100 <1> (<1>, activation [2]; <1>, 0.5%, 2fold increment in activity [4]) [1, 2, 4] n-octylglucoside <2> (<2>, 20-30% enhancement of activity with the proteins that behave as good acceptors: fetuin, a1 -glycoprotein and human chorionic gonadotropin. 3fold enhancement of activity of the poor acceptor glycophorin [4]) [4] Metals, ions Mn2+ <1, 2> (<1>, requirement, Km : 16 mM [1]; <1>, optimal concentration is 10 mM [2]; <2>, required [4]) [1, 2, 4] Km-Value (mM) 0.055 <1> (UDP-N-acetylgalactosamine) [1] 0.064 <1> (UDP-N-acetylgalactosamine) [5] 0.222 <1> (fetuin) [5] 1.1 <1> (N-acetylneuraminyl-2,3-a-d-galactosyl-1,4-b-d-glucosylceramide) [1] pH-Optimum 7.4 <2> [4] 7.5 <1> (<1>, with fetuin as acceptor [1]) [1, 2] pH-Range 5.8-9.5 <1> (<1>, about half-maximal activity at pH 5.8 and 9.5 [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue CACO-2 cell <1> (<1>, enzymic activity correlates with the degree of enterocytic differentiation [5]) [2, 5] blood plasma <1> (<1>, group O [1]) [1] cecum <1, 2, 3> [4] kidney <1> [3]

370

2.4.1.165

N-Acetylneuraminylgalactosylglucosylceramide b-1,4-N-acetylgalactosaminyltransferase

large intestine <1, 2> (<1>, colon mucosa or colon carcinoma cell line CaCo2, not in cell lines SW-948, SW-948FL, SW-480, SW-48, SW-1417, COLO-205, LOVO or HT-29 [2]) [2, 4, 5] proximal colon <1, 2> [4] skeletal muscle <4> [6] Localization microsome <2> [4] soluble <2> (<2>, large amounts of the enzyme are released in soluble form, particularly when CaCo-2 cells are maintained in culture for more than 3 weeks in order to ensure an higher degree of enterocyte differentiation [4]) [4] Purification <2> (partial [4]) [4] Cloning <4> (transgenic mice overexpress the enzyme in the skeletal muscle of mdx animals [6]) [6] Application analysis <1> (<1>, the enzyme is a marker of the colonic cell maturation [2]) [2] medicine <4> (<4>, the enzyme can alter a dystroglycan glycosylation, either directly or indirectly, in ways that may be therapeutic even in congenital muscular dystrophies where the glycosylation of dystroglycan is abnormal [7]) [7]

6 Stability Storage stability <2>, -20 C, microsomal fraction, 1 year without significant loss of activity [4]

References [1] Takeya, A.; Hosomi, O.; Kogure, T.: Identification and characterization of UDP-GalNAc:NeuAca2-3Galb1-4Glc(NAc) b1-4(GalNAc to Gal)N-acetylgalactosaminyltransferase in human blood plasma. J. Biochem., 101, 251-259 (1987) [2] Malagolini, N.; Dall'Olio, F.; Serafini-Cessi, F.: UDP-GalNAc:NeuAca2,3Gal bR (GalNAc to Gal) b1,4-N-acetylgalactosaminyltransferase responsible for the Sda specificity in human colon carcinoma CaCo-2 cell line. Biochem. Biophys. Res. Commun., 180, 681-686 (1991) [3] Morton, J.A.; Pickles, M.M.; Vanhegan, R.I.: The Sda antigen in the human kidney and colon. Immunol. Invest., 17, 217-224 (1988)

371

N-Acetylneuraminylgalactosylglucosylceramide b-1,4-N-acetylgalactosaminyltransferase

2.4.1.165

[4] Malagolini, N.; Dall'Olio, F.; Guerrini, S.; Serafini-Cessi, F.: Identification and characterization of the Sda b1,4-N-acetylgalactosaminyltransferase from pig large intestine. Glycoconjugate J., 11, 89-95 (1994) [5] Serafini-Cessi, F.; Malagolini, N.; Guerrini, S.; Turrini, I.: A soluble form of Sda-b1,4-N-acetylgalactosaminyltransferase is released by differentiated human colon carcinoma CaCo-2 cells. Glycoconjugate J., 12, 773-779 (1995) [6] Nguyen, H.H.; Jayasinha, V.; Xia, B.; Hoyte, K.; Martin, P.T.: Overexpression of the cytotoxic T cell GalNAc transferase in skeletal muscle inhibits muscular dystrophy in mdx mice. Proc. Natl. Acad. Sci. USA, 99, 5616-5621 (2002) [7] Jayasinha, V.; Hoyte, K.; Xia, B.; Martin, P.T.: Overexpression of the CT GalNAc transferase inhibits muscular dystrophy in a cleavage-resistant dystroglycan mutant mouse. Biochem. Biophys. Res. Commun., 302, 831-836 (2003)

372

Raffinose-raffinose a-galactotransferase

2.4.1.166

1 Nomenclature EC number 2.4.1.166 Systematic name raffinose:raffinose a-d-galactosyltransferase Recommended name raffinose-raffinose a-galactotransferase Synonyms galactosyltransferase, raffinose (raffinose donor) raffinose:raffinose a-galactosyltransferase CAS registry number 93389-38-9

2 Source Organism <1> Cerastium arvense (enzyme can also be isolated from other species of the Caryophyllaceae [1]) [1]

3 Reaction and Specificity Catalyzed reaction 2 raffinose = 1F-a-d-galactosylraffinose + sucrose Reaction type hexosyl group transfer Natural substrates and products S raffinose + raffinose <1> (Reversibility: ? <1> [1]) [1] P isolychnose + sucrose <1> [1] S raffinose + raffinose <1> (Reversibility: ? <1> [1]) [1] P lychnose + sucrose <1> (<1> i.e. 1F-a-d-galactosylraffinose [1]) [1] S Additional information <1> (<1> involved in the accumulation of the tetrasaccharides lychnose and isolychnose in the leaves of Cerastium arvense and other Caryophyllaceae during late autumn [1]) [1] P ?

373

Raffinose-raffinose a-galactotransferase

2.4.1.166

Substrates and products S raffinose + raffinose <1> (Reversibility: ? <1> [1]) [1] P isolychnose + sucrose <1> (<1> isolychnose is 3F-a-galactosylraffinose, enzyme can also be found in Cerastium arvense leaves, probably another raffinose:raffinose a-d-galactosyltransferase [1]) [1] S raffinose + raffinose <1> (Reversibility: ? <1> [1]) [1] P lychnose + sucrose <1> (<1> i.e. 1F-a-d-galactosylraffinose [1]) [1] Specific activity (U/mg) 0.00025 <1> (<1> lychnose [1]) [1] 0.000316 <1> (<1> isolychnose [1]) [1] pH-Optimum 6 <1> [1] Temperature optimum ( C) 38 <1> (<1> assay at [1]) [1]

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

6 Stability Storage stability <1>, -20 C, 6 months [1]

References [1] Hopf, H.; Gruber, G.; Zinn, A.; Kandler, O.: Physiology and biosynthesis of lychnose in Cerastium arvense. Planta, 162, 283-288 (1984)

374

Sucrose 6F-a-galactotransferase

2.4.1.167

1 Nomenclature EC number 2.4.1.167 Systematic name UDP-galactose:sucrose 6F-a-d-galactosyltransferase Recommended name sucrose 6F-a-galactotransferase Synonyms UDPgalactose:sucrose 6fru-a-galactosyltransferase galactosyltransferase, uridine diphosphogalactose-sucrose 6F-aCAS registry number 92480-04-1

2 Source Organism <1> Sesamum indicum [1]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + sucrose = UDP + 6F-a-d-galactosylsucrose Reaction type hexosyl group transfer Natural substrates and products S UDPgalactose + sucrose <1> (Reversibility: ? <1> [1]) [1] P UDP + 6F-a-galactosylsucrose <1> (<1> i.e. planteose [1]) [1] S Additional information <1> (<1> probably involved in the biosynthesis of planteose in seeds [1]) [1] P ? Substrates and products S UDPgalactose + sucrose <1> (Reversibility: ? <1> [1]) [1] P UDP + 6F-a-galactosylsucrose <1> (<1> i.e. planteose [1]) [1]

375

Sucrose 6F-a-galactotransferase

2.4.1.167

Inhibitors 5'-UMP <1> (<1> 10 mM: 55% inhibition, 1 mM: 34% inhibition [1]) [1] Additional information <1> (<1> UDP, UDPglucose, glucose 1-phosphate, glucose, galactose 1-phosphate, galactose are not inhibitory up to 1 mM [1]) [1] Activating compounds DTT <1> (<1> 10 mM: increase of activity by about 30% [1]) [1] Additional information <1> (<1> no requirement for a sulfhydryl reagent [1]) [1] Metals, ions Additional information <1> (<1> bivalent metal ions, e.g. Ca2+ , Cu2+ , Fe2+ , Mg2+ , Zn2+ have no effect [1]) [1] Km-Value (mM) 0.2-0.5 <1> (UDPgalactose) [1] 3.6-6 <1> (sucrose) [1] pH-Optimum 6.2 <1> [1] pH-Range 5-7 <1> (<1> 30% of maximal activity at pH 5.0, 70% of maximal activity at pH 7.0 [1]) [1] Temperature optimum ( C) 38 <1> (<1> assay at [1]) [1]

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

6 Stability General stability information <1>, freezing and thawing have no effect [1] Storage stability <1>, -20 C, 6 months [1]

References [1] Hopf, H.; Spanfelner, M.; Kandler, O.: Planteose synthesis in seeds of Sesamum indicum L.. Z. Pflanzenphysiol., 114, 485-492 (1984) 376

Xyloglucan 4-glucosyltransferase

2.4.1.168

1 Nomenclature EC number 2.4.1.168 Systematic name UDP-glucose:xyloglucan 1,4-b-d-glucosyltransferase Recommended name xyloglucan 4-glucosyltransferase Synonyms glucosyltransferase, uridine diphosphoglucose-xyloglucan 4bxyloglucan 4b-d-glucosyltransferase xyloglucan glucosyltransferase Additional information (not identical with EC 2.4.1.12) CAS registry number 80237-91-8

2 Source Organism <1> Glycine max (soy bean [1]) [1, 2, 4, 5] <2> Pisum sativum (pea, cv. Alaska [3,6,7]; pea, cv. Caprice [3]) [3, 6, 7] <3> Tamarindus indica (tamarind [8]) [8]

3 Reaction and Specificity Catalyzed reaction transfers a b-d-glucosyl residue from UDP-glucose on to a glucose residue in xyloglucan, forming a b-1,4-d-glucosyl-d-glucose linkage Reaction type hexosyl group transfer Natural substrates and products S UDP-d-glucose + xyloglucan <1> (<1> involved in biosynthesis of xyloglucan in higher plants [2]; <1,2> responsible for the formation of the xyloglucan backbone [1,6]) [1, 2, 6] P UDP + glucosylxyloglucan

377

Xyloglucan 4-glucosyltransferase

2.4.1.168

Substrates and products S UDP-d-glucose + (glucosyl)xyloglucan <1-3> (<1> transfers a b-d-glucosyl residue from UDPglucose on to a glucose residue in xyloglucan, other glucosyl-acceptors are b-1,3-glucan and xylan [1]; <1> no acceptor: b-1,4glucan [4]; <1> no acceptor: cello-oligosaccharides and fragment oligosaccharides from endoglucanase digest [1]; <3> standard oligosaccharides for enzyme assay [8]) (Reversibility: ? <1-3> [1-8]) [1-8] P UDP + (glucosyl-glucosyl)xyloglucan <1, 2> (<1> forms a b-1,4-d-glucosyl-d-glucose linkage [1]) [1-7] Inhibitors GTPglucose <1> (<1> weak [1]) [1, 4] TDPglucose <1> (<1> weak [1]) [1] UTP <1> [1] UTPxylose <1> (<1> above 0.03 mM [2]) [2] Additional information <1, 1> (no inhibition by tunicamycin, ATP or GTP [1]) [1] Activating compounds UDP-xylose <1, 2> (<1> activation, 0.01-0.03 mM [1]) [1, 3-5] Additional information <2> (<2> fusicoccin, indole acetic acid and D2 O activate in vivo, via proton extrusion, in the presence of O2 and sugar substrate, while mannitol, vanadate or nigrecin inhibit this activation, no direct activation in vitro [7]) [7] Metals, ions Mg2+ <2> (<2> activation [3,6,7]; <2> 3-5 mM [6]; <2> can replace Mn2+ [6]; <2> can replace Mn2+ to some extent [3]) [3, 6, 7] Mn2+ <1, 2> (<1,2> activation [3,4,6]; <2> 3-5 mM [6]) [3, 4, 6] Additional information <2> (<2> no activation by Ca2+ [6]) [6] Km-Value (mM) 0.056 <1> (UDPglucose) [4] pH-Optimum 6.5-7 <1> [1] 7-8 <1> [4] Temperature optimum ( C) 35 <1> [1]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture <1> [1, 4, 5] hypocotyl <1> (<1> elongation region [2]) [2] kernel <3> [8] seedling <2> [3, 7]

378

2.4.1.168

Xyloglucan 4-glucosyltransferase

Localization Golgi membrane <1, 2> [2, 3, 7] membrane <1, 2> [1, 4-6]

6 Stability General stability information <1>, DTT, 1 mM, EDTA, 1 mM, sucrose, 0.4 M, bovine serum albumin, 0.1%, stabilize [1] Storage stability <1>, 0 C, crude membrane-bound enzyme preparation, 1 day [4]

References [1] Hayashi, T.; Matsuda, K.: Biosynthesis of xyloglucan in suspension-cultured soybean cells. Occurrence and some properties of xyloglucan 4-b-d-glucosyltransferase and 6-a-d-xylosyltransferase. J. Biol. Chem., 256, 1111711122 (1981) [2] Hayashi, T.; Koyama, T.; Matsuda, K.: Formation of UDP-xylose and xyloglucan in soybean Golgi membranes. Plant Physiol., 87, 341-345 (1988) [3] White, A.R.; Xin, Y.; Pezeshk, V.: Xyloglucan glucosyltransferase in Golgi membranes from Pisum sativum (pea). Biochem. J., 294, 231-238 (1993) [4] Hayashi, T.; Matsuda, K.: Biosynthesis of xyloglucan in suspension-cultured soybean cells. Evidence that the enzyme system of xyloglucan synthesis does not contain b-1,4-glucan 4-b-glucosyltransferase activity (EC 2.4.1.12). Plant Cell Physiol., 22, 1571-1584 (1981) [5] Hayashi, T.; Nakajima, T.; Matsuda, K.: Biosynthesis of xyloglucan in suspension-cultured soybean cells. Processing of the oligosaccharide building blocks. Agric. Biol. Chem., 48, 1023-1027 (1984) [6] Gordon, R.; MacLachlan, G.: Incorporation of UDP-[14 C]glucose into xyloglucan by pea membranes. Plant Physiol., 91, 373-378 (1989) [7] Ray, P.M.: Auxin and fusicoccin enhancement of b-glucan synthase in peas. Plant Physiol., 78, 466-472 (1985) [8] Marry, M.; Cavalier, D.M.; Schnurr, J.K.; Netland, J.; Yang, Z.; Pezeshk, V.; York, W.S.; Pauly, M.; White, A.R.: Structural characterization of chemically and enzymatically derived standard oligosaccharides isolated from partially purified tamarind xyloglucan. Carbohydr. Polym., 51, 347-356 (2002)

379

Xyloglucan 6-xylosyltransferase

1 Nomenclature EC number 2.4.1.169 (transferred to EC 2.4.2.39) Recommended name xyloglucan 6-xylosyltransferase

380

2.4.1.169

Isoflavone 7-O-glucosyltransferase

2.4.1.170

1 Nomenclature EC number 2.4.1.170 Systematic name UDP-glucose:isoflavone 7-O-b-d-glucosyltransferase Recommended name isoflavone 7-O-glucosyltransferase Synonyms UDP-glucose-flavonoid 7-O-glucosyltransferase UDP-glucose:isoflavone 7-O-glucosyltransferase UDPglucose-flavonoid 7-O-glucosyltransferase UDPglucose:isoflavone 7-O-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-isoflavone 7-Ouridine diphosphoglucose-isoflavone 7-O-glucosyltransferase CAS registry number 97089-62-8

2 Source Organism <1> Cicer arietinum [1] <2> Silene latifolia [2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + isoflavone = UDP + isoflavone 7-O-b-d-glucoside Reaction type hexosyl group transfer Substrates and products S UDPglucose + biochanin A <1> (Reversibility: ? <1> [1]) [1] P UDP + biochanin A 7-O-b-glucoside <1> [1] S UDPglucose + daidzein <1> (<1>, poor substrate [1]) (Reversibility: ? <1> [1]) [1] P UDP + daidzein 7-O-b-glucoside

381

Isoflavone 7-O-glucosyltransferase

2.4.1.170

S UDPglucose + formononetin <1> (Reversibility: ? <1> [1]) [1] P UDP + formononetin 7-O-b-glucoside S UDPglucose + genistein <1> (<1>, poor substrate [1]) (Reversibility: ? <1> [1]) [1] P UDP + genistein 7-O-b-glucoside S UDPglucose + isovitexin <2> (Reversibility: ? <2> [2]) [2] P UDP + isovitexin 7-O-b-glucoside Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 0.012 <1> (biochanin A) [1] 0.024 <1> (formononetin) [1] 0.2 <1> (UDPglucose) [1] pH-Optimum 8.5-9 <1> [1]

4 Enzyme Structure Molecular weight 42000 <1> (<1>, sucrose density gradient ultracentrifugation [1]) [1] 50000 <1> (<1>, gel filtration [1]) [1] Subunits ? <2> (<2>, x * 54000, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue petal <2> [2] root <1> [1] Purification <1> [1] <2> [2]

6 Stability pH-Stability 8.5-9 <1> (<1>, optimal stability [1]) [1] General stability information <1>, 2-mercaptoethanol, 40 mM, stabilizes during purification [1] <1>, dithioerythritol, 10 mM, effectively stabilizes during purification [1]

382

2.4.1.170

Isoflavone 7-O-glucosyltransferase

Storage stability <1>, -20 C, 20% v/v glycerol, stable for several months [1]

References [1] Koester, J.; Barz, W.: UDP-glucose:isoflavone 7-O-glucosyltransferase from roots of chick pea (Cicer arietinum L.). Arch. Biochem. Biophys., 212, 98104 (1981) [2] Vellekoop, P.; Lugones, L.; van Brederode, J.: Purification of an UDP-glucose:flavone, 7-O-glucosyltransferase, from Silene latifolia using a specific interaction between the enzyme and phenyl-Sepharose. FEBS Lett., 330, 36-40 (1993)

383

Methyl-ONN-azoxymethanol b-D-glucosyltransferase

2.4.1.171

1 Nomenclature EC number 2.4.1.171 Systematic name UDP-glucose:methyl-ONN-azoxymethanol b-d-glucosyltransferase Recommended name methyl-ONN-azoxymethanol b-d-glucosyltransferase Synonyms UDPglucose-methylazoxymethanol glucosyltransferase cycasin synthase glucosyltransferase, uridine diphosphoglucose-methylazoxymethanol CAS registry number 99283-65-5

2 Source Organism <1> Cycas revoluta (Thunb., Japanese cycad [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + methyl-ONN-azoxymethanol = UDP + cycasin Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + methylazoxymethanol <1> (<1> biosynthesis of the toxic substance cycasin [1]) (Reversibility: ? <1> [1]) [1] P UDP + cycasin Substrates and products S UDPglucose + methylazoxymethanol <1> (Reversibility: ? <1> [1]) [1] P UDP + cycasin <1> [1] Km-Value (mM) 0.3 <1> (methylazoxymethanol) [1] 1.58 <1> (UDPglucose) [1] 384

2.4.1.171

Methyl-ONN-azoxymethanol b-D-glucosyltransferase

pH-Optimum 8.3 <1> [1] Temperature optimum ( C) 30 <1> (<1> assay at [1]) [1]

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

References [1] Tadera, K.; Yagi, F.; Arima, M.; Kobayashi, A.: Formation of cycasin from methylazoxymethanol by UDP-glucosyltransferase from leaves of Japanese cycad. Agric. Biol. Chem., 49, 2827-2828 (1985)

385

Salicyl-alcohol b-D-glucosyltransferase

2.4.1.172

1 Nomenclature EC number 2.4.1.172 Systematic name UDP-glucose:salicyl-alcohol b-d-glucosyltransferase Recommended name salicyl-alcohol b-d-glucosyltransferase Synonyms UDPglucose:salicyl alcohol phenyl-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-salicyl alcohol 2CAS registry number 89400-32-8

2 Source Organism <1> Gardenia jasminoides (Ellis [1]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + salicyl alcohol = UDP + salicin Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + salicyl alcohol <1> (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + salicin Substrates and products S UDPglucose + salicyl alcohol <1> (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + salicin <1> [1, 2] Inhibitors Cu2+ <1> (<1> 2 mM, 80% inhibition [1]) [1] Mn2+ <1> (<1> 2 mM, 20% inhibition [1]) [1] N-ethylmaleimide <1> (<1> 10 mM, 35% inhibition [1]) [1]

386

2.4.1.172

Salicyl-alcohol b-D-glucosyltransferase

Zn2+ <1> (<1> 2 mM, 60% inhibition [1]) [1] p-chloromercuribenzoate <1> (<1> 10 mM, 25% inhibition [1]) [1] Specific activity (U/mg) 0.165 <1> [1] Km-Value (mM) 0.031 <1> (UDPglucose) [1] 0.11 <1> (salicyl alcohol) [1] 0.53 <1> (salicyl alcohol) [2] 0.64 <1> (UDPglucose) [2] pH-Optimum 9 <1> [2] 9-9.5 <1> [1] pH-Range 6-10.5 <1> (<1> 45% of maximal activity at pH 6.0, 10% of maximal activity at pH 10.5 [1]) [1] 7.5-10.2 <1> (<1> half maximal activity at pH 7.5 and 10.2 [2]) [2] Temperature optimum ( C) 30 <1> (<1> assay at [2]) [2] 37 <1> (<1> assay at [1]) [1] 50 <1> [2] Temperature range ( C) 30-55 <1> (<1> 20% of maximal activity at 30 C, 35% of maximal activity at 55 C [2]) [2]

4 Enzyme Structure Molecular weight 51000 <1> (<1> gel filtration [1]) [1]

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

6 Stability pH-Stability 7-9 <1> (<1> stable [1]) [1]

387

Salicyl-alcohol b-D-glucosyltransferase

2.4.1.172

Temperature stability 50 <1> (5 min, 10% remaining activity) [1] Storage stability <1>, -20 C, 3 weeks, 50% loss of activity [2] <1>, 4 C, 2 weeks, 40% loss of activity, or -20 C, 2 weeks, 40% loss of activity [1]

References [1] Mizukami, H.; Terao, T.; Ohashi, H.: Partial purification and characterization of UDP-glucose:salicyl alcohol glucosyltransferase from Gardenia jasminoides cell cultures. Planta Med., 1985, 104-107 (1985) [2] Terao, T.; Ohashi, H.; Mizukami, H.: Position-specific glucosylation of salicyl alcohol with an enzyme preparation from Gardenia jasminoides cultured cells. Plant Sci. Lett., 33, 47-52 (1984)

388

Sterol 3b-glucosyltransferase

2.4.1.173

1 Nomenclature EC number 2.4.1.173 Systematic name UDP-glucose:sterol 3-O-b-d-glucosyltransferase Recommended name sterol 3b-glucosyltransferase Synonyms UDP-glucose-sterol b-glucosyltransferase UDP-glucose-sterol glucosyltransferase UDPG-SGTase UDPG:sterol glucosyltransferase UDPglucose:sterol glucosyltransferase sterol glucosyltransferase sterol-b-d-glucosyltransferase sterol:UDPG glucosyltransferase uridine diphosphoglucose-poriferasterol glucosyltransferase uridine diphosphoglucose-sterol glucosyltransferase uridine diphosphoglucose-sterol glucosyltransferase Additional information (not identical with EC 2.4.1.192 or EC 2.4.1.193) CAS registry number 123940-38-5 9075-00-7

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

Avena sativa [1, 2, 19] Physarum polycephalum [3-4, 8] Zea mays [5, 9, 15-18] Rhodotorula bogoriensis (or Candida bogoriensis [6]) [6] Digitalis purpurea [7] Sinapsis alba (white mustard [8,11]) [8, 11] Asparagus plumosus [10] Pisum sativum (pea [12,13]) [12, 13] Gossypium hirsutum (cotton [14]) [14] Arabidopsis thaliana (ugt80A2 [20]) [20]

389

Sterol 3b-glucosyltransferase

<11> <12> <13> <14> <15> <16>

2.4.1.173

Candida albicans (ugt51c1 [21]) [21] Dictyostelium discoideum (ugt52 [21]) [21] Pichia pastoris (ugt51b1 [21]) [21] Saccharomyces cerevisiae (ugt51=ylr189C [21]) [21] Solanum tuberosum (potato [22]) [22] Avena sativa (ugt80A1 [20,21]) [20, 21]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + a sterol = UDP + a sterol 3-b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + poriferasterol <2> (<2> synthesis of poriferasterol monoglucoside may be involved in differentiation) [4] P UDP + poriferasterol 3-b-d-glucoside Substrates and products S UDP-glucose + 4-demethyl-obtusifoliol <3> (<3> 58% of the activity with sitosterol [17]) (Reversibility: ? <3> [17]) [17] P UDP + 4-demethyl-obtusifoliol 3-b-d-glucoside <3> [17] S UDP-glucose + 5-a-cholestanol <2> (<2> 8% of the activity with sitosterol [3]) (Reversibility: ? <2> [3]) [3] P UDP + 5-a-cholestanol 3-b-d-glucoside <2> [3] S UDP-glucose + 5-cholestenol <2> (<2> 12% of the activity with sitosterol [3]) (Reversibility: ? <2> [3]) [3] P UDP + 5-cholestenol 3-b-d-glucoside <2> [3] S UDP-glucose + 7-dehydrocholesterol <2> (<2> 6% of the activity with sitosterol [3]) (Reversibility: ? <2> [3]) [3] P UDP + 7-dehydrocholesterol 3-b-d-glucoside <2> [3] S UDP-glucose + a-spinasterol <2> (<2> 31% of the activity with sitosterol [3]) (Reversibility: ? <2> [3]) [3] P UDP + a-spinasterol 3-b-d-glucoside <2> [3] S UDP-glucose + campesterol <2-3, 6, 9> (<2> 28% of the activity with sitosterol [3]; <2> 80% of the activity with poriferasterol [4]; <6> best substrate [8]; <3> 68% of the activity with sitosterol [17]) (Reversibility: ? <2-3,6,9> [3-4,8,14,17]) [3-4, 8, 14, 17] P UDP + campesterol 3-b-d-glucoside <2-3, 6, 9> (<9> 4% of the products [14]) [3-4, 8, 14, 17] S UDP-glucose + cholesterol <1-2, 4-6, 10, 12-14, 16> (<2> 23% of the activity with sitosterol [3]; <2> 12% of the activity with poriferasterol [4]; <5> best substrate [7]; <1> with sitosterol, best substrates [19]; <10,16> cloned enzyme exhibits equal activity to the purified protein [20]; <12-14,

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P S P S

P S P S P S

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Sterol 3b-glucosyltransferase

16> cloned enzyme glucosilates cholesterol [21]) (Reversibility: ? <1-2, 46, 10, 12-14, 16> [3-4, 6-7, 11, 19-21]) [3-4, 6-7, 11, 19-21] UDP + cholesterol 3-b-d-glucoside <1-2, 4-6, 10, 12-14, 16> [3-4, 6-7, 11, 19-21] UDP-glucose + epiandrosterone <5> (<5> best substrate [7]) (Reversibility: ? <5> [7]) [7] UDP + epiandrosterone 3-b-glucoside <5> [7] UDP-glucose + ergosterol <1-2, 4, 12-14, 16> (<2> 11% of the activity with sitosterol [3]; 9% of the activity with poriferasterol [4]; <1> 69% of the activity with sitosterol [19]; <12-14, 16> cloned enzyme glucosylates ergosterol [21]) (Reversibility: ? <1-2, 4, 12-14, 16> [3-4, 6, 19, 21]) [3-4, 6, 19, 21] UDP + ergosterol 3-b-d-glucoside <1-2, 4, 12-14, 16> [3-4, 6, 19, 21] UDP-glucose + isofucosterol <3> (<3> 73% of the activity with sitosterol [17]) (Reversibility: ? <3> [17]) [17] UDP + isofucosterol 3-b-d-glucoside <3> [17] UDP-glucose + pregnenolone <5> (<5> 57% of the activity with epiandrosterone [7]) (Reversibility: ? <5> [7]) [7] UDP + pregnenolone 3-b-d-glucoside <5> [7] UDP-glucose + sitosterol <1-3, 5-9, 11-14, 16> (<2> greater activity than with other sterols [3]; <2> maximal activity, the same as poriferasterol [4]; <5> 88% of the activity with epiandrosterone [7]; <2> best substrate [8]; <3> best substrate [17]; <1> with cholesterol, best substrates [19]; <12-14, 16> cloned enzyme glucosylates sitosterol [21]) (Reversibility: ? <1-3, 5-9, 11-14> [1-5, 7-8, 10, 12, 14, 17, 19, 21]) [1-5, 7-8, 10, 12, 14, 17, 19, 21] UDP + sitosterol 3-b-d-glucoside <1-3, 5-9, 11-14, 16> (<9> 75% of the product [14]) [1-5, 7-8, 10, 12, 14, 17, 19, 21] UDP-glucose + stigmasterol <1-2, 5, 9, 12-14, 16> (<2> 50% of the activity with sitosterol [3]; <2> 14% of the activity with poriferasterol [4]; <5> 122% of the activity with epiandrosterone, highest specificity [7]; <1> 63% of the activity with sitosterol [19]; <12-14, 16> cloned enzyme glucosylates stigmasterol [21]) (Reversibility: ? <1-2, 5, 9, 12-14, 16> [3-4, 7, 14, 19, 21]) [3-4, 7, 14, 19, 21] UDP + stigmasterol 3-b-d-glucoside <1-2, 5, 9, 12-14, 16> (<9> 6% of the product [14]) [3-4, 7, 14, 19, 21] UDP-glucose + tomatidine <12-14, 16> (<12-14, 16> cloned enzyme glucosylates tomatidine [21]) (Reversibility: ? <12-14, 16> [21]) [21] UDP + tomatidine 3-b-d-glucoside <12-14, 16> [21] Additional information <1-3, 5-9, 12-14, 16> (<2> D5 -sterols are glucosylated at higher rate than D7 -sterols [3]; <2> stanols and sterols with conjugated double bonds in ring B are poor substrates [3]; <2> sterols possessing an alkyl group at C-24 are better substrates than C27 sterols [3]; <2> the presence of a D22 double bond decreases affinity of the sterol for the enzyme [3]; <2> presence and localization of double bonds in the ring system exert a pronounced effect on the rate of glucosylation [3]; <5> almost no activity with androsterone, digitoxigenin, digitoxin and testoster391

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one [7]; <6> enzyme is absolute specific to UDP-glucose [8]; <2> enzyme can use CDP-glucose and TDP-glucose at 6 times slower rate [8]; <2,6> glucose 1-phosphate, GDP-glucose and ADP-glucose are inactive substrates [8]; <2,6> all 3b-hydroxy sterols containing a double bond in position 5 are glucosilated and a double bond at C24 reduces glucosylation [8]; <2> coprostanol and epicholestanol are not glucosylated [8]; <7> yamogenin is not substrate of this enzyme [10]; <8> UDP-glucose is an effective glucose donor, not so ADP-glucose, CDP-glucose or GDP-glucose [13]; <9> UDP-glucose is an effective glucose donor but not so ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose, UDP-galactose or UDPmannose [14]; <3> p-nitrophenol, estrone and 4-a,10-b-dimethyl-transdecal-3-b-ol are not substrates [17]; <1> UDP-glucose is an effective glucose donor but not so UDP-mannose, UDP-galactose or UMP-glucose [19]; <12-14,16> UDP-glucose best glucose donor, CDP-glucose, GDPglucose and UDP-xylopyranose are used but at significantly lower rates and UDP-mannose, UDP-glucaronic acid, GDP-mannose and TDP-glucose are not effective glucose donor [21]) [3, 7, 8, 10, 13, 14, 17, 19, 21] P ? Inhibitors 4-thio-UDP <1> (<1> 50% of inhibition at 160 uM [19]) [19] ATP <8> (<8> inhibition at 1-3 mM but slight stimulation at low concentration [12]) [12] GDP-glucose <8> (<8> 20% inhibition at high concentration [13]) [13] NH+4 <5> [7] Triton X-100 <2, 8> (<2> strong inhibition at concentrations below 0.05% [3]; <8> moderate inhibition but strong inhibition at low concentrations [13]) [3, 13] Tween 20 <2> (<2> strong inhibition at concentrations below 0.05% [3]) [3] Tween 60 <2> (<2> strong inhibition at concentrations below 0.05% [3]) [3] UDP <1-2, 7-8> (<2> 50% inhibition at 0.06 mM [3]; <7> total inhibition at 1 mM [10]; <8> 60% of inhibition [13]; <1> 50% of inhibition at 70 mM [19]) [3, 10, 13, 19] UDP-2',3'-dialdehyde <3> (<3> inhibits irreversibly [18]) [18] UDP-mannose <1> (<1> 50% of inhibition at 40 mM [19]) [19] UMP <7-8> (<7> inhibition at 1 mM [10]; <8> 20% of inhibition [13]) [10, 13] UTP <2, 7> (<2> 50% inhibition at 0.70 mM [3]; <7> total inhibition at 1 mM [10]) [3, 10] cetyltrimethylammonium bromide <8> (<8> strong inhibition [13]) [13] coprostanol <2> (<2> potent inhibitor [8]; <6> not inhibitory [8]) [8] epicholestanol <2> (<2> potent inhibitor [8]; <6> not inhibitory [8]) [8] maleate buffer <9> (<9> strong inhibition at 50 mM [14]) [14] mersalyl <3> (<3> 50% of inhibition at 40 mM [18]) [18] p-chloromercuribenzoate <2, 7> (<2> 50% inhibition at 0.60 mM [3]; <7> potent inhibitory effect at 0.1 mM indicating that free SH groups are essential for activity [10]) [3, 10]

392

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p-mercuribenzenesulfonate <3> (<3> 50% of irreversible inhibition at 0.5 mM, UDP and UDPG-glucose protect from inhibition of 10 mM [18]) [18] phenylglyoxal <3> (<3> 92% of inhibition at 10 mM [18]) [18] phosphate buffer <9> (<9> strong inhibition at 50 mM [14]) [14] sodium deoxycholate <6, 8> (<6> strong inhibition at 0.1-1% [11]; <8> strong inhibition at 0.2-0.8% [13]) [8, 13] sodium taurocholate <6> (<6> strong inhibition at 0.1-1% [11]) [8] Additional information <8, 15> (<8> not inhibited by ADP, GDP or inorganic phosphate [13]; <15> not affected by tuber slicing or ethephon [22]) [13, 22] Activating compounds 2-mercaptoethanol <2, 5, 7> (<2> 10% stimulation at 1 mM [3]; <5> activated by [7]; <7> slight stimulation [10]) [3, 7, 10] ATP <2, 8> (<2> 15% stimulation at 0.01 mM [3]; <8> slightly stimulation at low concentration [12]) [3, 12] DIECA <5> (<5> diethyldithiocarbonate, activated by [7]) [7] EDTA <2, 5, 8> (<2> 15% stimulation at 0.1 mM [3]; <5> activated by [7]; <8> 70% of inhibition at 13 mM [13]) [3, 7, 13] EGTA <8> (<8> 50% of inhibition at 13 mM [13]) [13] Emulphogen BC-720 <3> (<3> stimulation al low concentrations [15]) [15] KOH <3> (<3> treatment with KOH pH 10.4 increase activity about 65%, hydroxyl ions can be used in place of detergents to reveal latent enzymatic sites [16]) [16] Triton N-101 <3> (<3> stimulation al low concentrations [15]) [15] Triton X-100 <1, 3, 6, 7> (<1> activity stimulated by 0.1-0.5% with highest stimulation at 0.25% [1]; <1> activity stimulated at 0.3% [2]; <7> activity stimulated at 0.1-0.5% [10]; <6> activity stimulated by 0.1-1% with 6.5fold stimulation at 0.1%, highest activity stimulation with respect to others nonionic detergents [11]; <3> stimulation at low concentrations, at intermediate concentration 0.4-0.8% this stimulation is abolished, it appears to solubilize enzyme still membrane-bound [15]; <3> stimulation of plasma membrane vesicles at 0.1-0.2% but not of endoplasmic reticulum vesicles [16]) [1-2, 1011, 16] Triton X-114 <3> (<3> stimulation al low concentrations [15]) [15] Tween-20 <6> (<6> 2fold activity stimulation at 0.1% [11]) [11] Tween-40 <6> (<6> 70% activity stimulation at 0.05% [11]) [11] Tween-60 <6> (<6> 35% activity stimulation at 0.1% [11]) [11] dithiothreitol <2> (<2> 25% stimulation at 1 mM [3]) [3] ethanol <4> (<4> increase of activity at 7-20% [6]) [6] lecithin <2, 9> (<2> 20-25% stimulation [3]; <9> stimulation by cotton and soybean lecithin but not with egg lecithin, appears to increase Vmax with sitosterol and Km of UDP-glucose [14]) [3, 14] p-chloromercuribenzoate <7> (<7> potent inhibitory effect indicating that free SH groups are essential for activity [10]) [10] phosphatidylcholine <3, 8> (<3> increases Vmax [5]; <8> increase 33% of activity [12]) [5, 12]

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phosphatidylethanolamine <8> (<8> increase 125% of activity [12]) [12] phosphatidylglycerol <3> (<3> increases Vmax [5]) [5] phosphatidylserine <8> (<8> increase 33% of activity [12]) [12] phospholipase <8, 3> (<8> treatment with phospholipases A, C and D lowers activity by 21-29%, the effect of phospholipase A is completely reversed by phosphatidylethanolamine but not by phosphatidylcholine or phosphatidylserine, after phospholipase C or D treatment each phospholipid brings about a partial recovery of activity but phosphatidylethanolamine is superior [12]; <3> phospholipase A2 slightly inhibits latent activity but stimulates the patent activity, it can be used in place of detergents to reveal latent enzymatic sites [16]) [12, 16] phospholipids <3> (<3> stimulated by negatively charged phospholipids [5,9]; <3> phosphatidylcholine or phosphatidylglycerol increase Vmax [5]; <3> phosphatidylcholine + phosphatidylglycerol or phosphatidylcholine + phosphatidic acid increase Vmax and Km with respect to phosphatidylcholine alone [9]; <3> the highest Vmax and Km is observed for the ratios 3:1 of phosphatidylcholine + phosphatidylglycerol and 3:1 phosphatidylcholine + phosphatidic acid [9]; stimulation of activity by solubilization of enzyme still membrane-bound [15]; <6> not stimulatory [11]) [5, 9, 15] Metals, ions Ca2+ <2, 6, 8> (<2> slightly inhibition above 0.1 mM [3]; <6> not stimulated between 0.001 mM to 10 mM [8]; <8> maximal stimulation at 13 mM [12]; <8> stimulated at 2-25 mM to a greater extent than Mg 2+ [13]) [3, 8, 12-13] Cu2+ <7> (<7> inhibitory effect at 1 mM [10]) [10] Hg2+ <2, 6-7> (<2> 50% inhibition at 0.07 mM [3]; <6> 50% of inhibition at 0.4 mM [8]; <7> inhibitory effect at 1 mM [10]) [3, 8, 10] Mg2+ <2, 6-8> (<2> slightly inhibition above 0.1 mM [3]; <6> not stimulated between 0.001 mM to 10 mM [8]; <7> not stimulated by 1 mM [10]; <8> stimulated at 2-25 mM [13]) [3, 8, 10, 13] Mn2+ <5, 7> (<5> inhibited by [7]; <7> no effect at 1 mM [10]) [7, 10] Zn2+ <2, 5-7> (<2> 50% inhibition at 0.05 mM [3]; <5> inhibited by [7]; <6> 50% of inhibition at 0.006 mM [8]; <7> inhibitory effect at 1 mM [10]) [3, 7-8, 10] Additional information <7, 8> (<7,8> no metal ion requirement [10,12]) [10, 12] Km-Value (mM) 0.0048 <2> (poriferasterol) [4] 0.005 <6> (sitosterol) [8] 0.0056 <2> (sitosterol) [3] 0.023 <9> (UDP-glucose, <9> lecithin causes a 2fold increase in Km [14]) [14] 0.025 <3> (UDP-glucose) [17] 0.04 <3> (UDP-glucose) [18] 0.04 <3> (UDP-glucose, <3> slightly lowered by phosphatidylcholine and phosphatidylglycerol [5]) [5] 0.08 <6> (UDP-glucose) [8] 394

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0.12 <2> (UDP-glucose) [4] 0.14 <3> (sterol) [17] 0.18 <2> (UDP-glucose) [3] 0.2 <3> (sitosterol) [18] 0.2 <3> (sitosterol, <3> strongly lowered by phosphatidylcholine and phosphatidylglycerol [5]) [5] 0.24-0.54 <1> (sitosterol) [19] 0.34 <1> (UDP-glucose) [19] Ki-Value (mM) 0.02 <3> (5-Hg UDP, <3> reversible inhibitor [18]) [18] 0.047 <1> (UDP) [19] pH-Optimum 7 <2> [4] 7.2 <2> [3] 7.5 <1, 5> (<1> assay at [1-2]) [1-2, 7] 8 <8> [13] 8.3 <7> (<7> Tris-HCl 0.1 M [10]) [10] pH-Range 7-8.7 <7> [10] Temperature optimum ( C) 28 <8> (<8> assay at [13]) [13] 30 <1-8, 10, 16> (<1-8,10,16> assay at [1-12,15-17,19,20]) [1-12, 15-17, 19, 20] 37 <9> (<9> assay at [14]) [14]

4 Enzyme Structure Molecular weight 56000 <1> (<1> purification by Q-Sepharose fast flow column, Hitrap blue column and SDS-PAGE [19]) [19] 66000 <16> (<16> cloned, N-terminal peptide of 133 amino acid could be cleaved resulting in a molecular weight of 52000 in agreement with 56000 of the purified enzyme [20]) [20] 69000 <10> (<10> cloned, calculated molecular mass [20]) [20] 70000 <2> (<2> gel filtration [3]) [3] 72000 <2> (<2> gel filtration [4]) [4] 114000 <12> (<12> cloned, calculated molecular mass [21]) [21] 136000 <13> (<13> cloned, calculated molecular mass [21]) [21] 136000 <14> (<14> cloned, calculated molecular mass [21]) [21] 140000 <6> (<6> by gel filtration [8]) [8] 171000 <11> (<11> cloned, calculated molecular mass [21]) [21]

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5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <2, 4-5> (<2> mycoplasma strain M3C IV [3,8]; <2> strain F and J activity only in diploid cells [4]; <4> crude extracts from culture [6]; <5> leaf cell culture [7]) [3-4, 6-8] coleoptile <3> (<3> etiolated [16-18]) [5, 9, 15-18] cotton fiber <9> [14] flower <10> (<10> cDNA library [20]) [20] leaf <1, 5, 10> (<5> crude extracts have 2-5 times lower activity than cell cultures [7]; <10> cDNA library [20]) [1-2, 7, 20] root <1, 10> (<10> cDNA library [20]) [2, 20] seed <9> [14] seedling <6, 8> (<8> etiolated [12,13]) [8, 11-13] shoot <7, 1, 16> (<1> etiolated [19]; <10> cDNA library [20]; <16> cloned from [20]) [10, 19, 20] tuber <15> (disc <15> [22]) [22] Localization Golgi apparatus <8> (<8> denser class of Golgi apparatus [13]) [12, 13] cytoplasm <2, 7> (<2> 50% of the activity [3]) [3, 10] etioplast <8> [12] membrane <1-2, 4, 6-7, 9> (<1, 7, 9> membrane fraction [1, 10, 14]; <2> membrane bound 50% of the activity [3]; <4> sonicated homogenate [6]; <6> organelles membrane [8,11]) [1-4, 6, 8, 10-11, 14] microsome <1, 3, 5> [5, 7, 9, 17-19] mitochondrion <8> [12] vesicular fraction <3> (<3> plasma membrane vesicles [15]; <3> plasma membrane and endoplasmic reticulum vesicles [16]) [15, 16] Purification <1> (partial purification by differential centrifugation [1-2]; purification about 12500fold by microsomes, extraction with diethyl ether, Q-Sepharose fast flow column, Hitrap blue column and SDS-PAGE [19]) [1-2, 19] <2> (partial purification by differential centrifugation and gel filtration on Sephadex G-100 [3-4]) [3-4] <3> (partial purification by differential centrifugation and DEAE-cellulose column [5,9]; partial purification by DEAE-cellulose column DE-52 [18]) [5, 9, 18] <5> (partial purification by differential centrifugation and Sephadex G-100 column, DEAE-Sephadex A-50 and Sepharose6B [7]) [7] <6> (partial purification by differential centrifugation and Sephadex G-150 column [8]) [8] <6> (partial purification by differential centrifugation and precipitation with Me2 CO [11]) [11] <8> (partial purification by differential centrifugation [12,13]) [12, 13] <9> (partial purification by differential centrifugation and DEAE-cellulose column [14]) [14] 396

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Cloning <10> <11> <12> <13> <14> <16>

(expression (expression (expression (expression (expression (expression

in in in in in in

Escherichia coli [20]) Escherichia coli [21]) Escherichia coli [21]) Escherichia coli [21]) Escherichia coli [21]) Escherichia coli [20])

[20] [21] [21] [21] [21] [20]

Engineering Additional information <14> (<14> null mutant by deletion of ugt52 gene with no enzyme activity [21]) [21] Application synthesis <2, 6> (<2> synthesis of poriferasterol monoglucoside may be involved in differentiation [4]; <2> synthesis of steryl b-d-monoglucosides possible function as glucose carriers through the membrane and intercellular transport of sterols [3]; <6> synthesis of steryl b-d-monoglucosides that are constituents of the cell membrane of higher plants [8]) [3, 4, 8]

6 Stability pH-Stability 7.2 <2, 6> (<2,6> Tris-HCl 0.1 M [3-4,11]) [3-4, 11] 7.3 <6> (<6> Tris-HCl 0.1 M, Triton X-100 0.3% [8]) [8] 7.5 <1, 5, 11-14> (<1> Tris-HCl 0.1 M [1-2]; <5> Tris-HCl 0.1 M, EDTA 10 mM, 2-mercaptoethanol 1 mM, sodium-deoxycholate 0.1% [7]; <11-14> Tris-HCl 100 mM, dithiothreitol 5 mM, pefabloc 200 uM [21]) [1-2, 7, 21] 7.7 <4> (<4> phosphate buffer 0.1 M [6]) [6] 8 <3, 8, 10, 16> (<3> Tris-HCl 50 mM, 1 mM 2-mercaptoethanol, emulphogene BC720 0.36% [5]; <3> Tris-HCl 50 mM, 1 mM 2-mercaptoethanol, Triton X-100 2.1% [9]; <8> Tris-HCl 25 mM [12]; <8> Tris-HCl 50 mM [13]; <3> Tris-HCl 0.1 M, 2-mercaptoethanol 1 mM [15,17]; <3> Tris-HCl 0.1 M, 2mercaptoethanol 5 mM [16]; <16> Tris-HCl 100 mM, dithiothreitol 1 mM, Triton X-100 0.2% [20]) [5, 9, 12-13, 15-17, 20] 8.5 <1, 9> (<9> Tris-HCl 50 mM, Triton X-100 0.25% [14]; <1> Tris-HCl 60 mM, Triton X-100 0.2%, dithiothreitol 2.5 mM [19]) [14, 19] Organic solvent stability ethanol <4> (<4> increases activity between 7-15% but decreases activity above 20% [6]) [6] Storage stability <1>, -20 C, Tris-HCl 0.1 M, pH 7.5, several weeks [1] <6>, -20 C, Tris-HCl 0.1 M, pH 7.2, two weeks [11] <9>, 2 C, Tris-HCl 50 mM, pH 8.5, two weeks 75% of activity [14]

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References [1] Kalinowska, M.; Wojciechowski, Z.A.: Enzymatic synthesis of nuatigenin 3b-d-glucoside in oat (Avena sativa) leaves. Phytochemistry, 25, 2525-2529 (1986) [2] Kalinowska, M.; Wojciechowski, Z.A.: Subcellular localization of UDPG:nuatigenin glucosyltransferase in oats leaves. Phytochemistry, 26, 353-357 (1987) [3] Wojciechowski, J.; Zimowski, J.; Tyski, S.: Enzymatic synthesis of steryl 3-bd-monoglucosides in the slime mold Physarum polycephalum. Phytochemistry, 16, 911-914 (1977) [4] Murakami-Murofushi, K.; Ohta, J.: Expression of UDP-glucose:poriferasterol glucosyltransferase in the process of differentiation of a true slime mold, Physarum polycephalum. Biochim. Biophys. Acta, 992, 412-415 (1989) [5] Ullmann, P.; Bouvier-Nave, P.; Benveniste, P.: Regulation by phospholipids and kinetic studies of plant membrane-bound UDP-glucose sterol b-d-glucosyl transferase. Plant Physiol., 85, 51-55 (1987) [6] Esders, T.W.; Light, R.J.: Occurrence of a uridine diphosphate glucose:sterol glucosyltransferase in Candida bogoriensis. J. Biol. Chem., 247, 7494-7497 (1972) [7] Yoshikawa, T.; Furuya, T.: Purification and properties of sterol:UDPG glucosyltransferase in cell culture of Digitalis purpurea. Phytochemistry, 18, 239-241 (1979) [8] Wojciechowski, Z.A.; Zimowski, J.; Zimowski, J.G.; Lyznik, A.: Specificity of sterol-glucosylating enzymes from Sinapis alba and Physarum polycephalum. Biochim. Biophys. Acta, 570, 363-370 (1979) [9] Ury, A.; Benveniste, P.; Bouvier-Nave, P.: Phospholipid dependence of plant UDP-glucose sterol b-d-glucosyl transferase. Plant Physiol., 91, 567-573 (1989) [10] Paczkowski, C.; Zimowski, J.; Krawczyk, D.; Wojciechowski, Z.A.: Steroidspecific glucosyltransferase in Asparagus plumosus shoots. Phytochemistry, 29, 63-70 (1990) [11] Kalinowska, M.; Wojciechowski, Z.A.: Modulation of activities of steryl glucoside hydrolase and UDPG:sterol glucosyltransferase from Sinapsis alba by detergents and lipids. Phytochemistry, 25, 45-49 (1986) [12] Fang, T.-Y.; Baisted, D.J.: UDPG:sterol glucosyltransferase in etiolated pea seedlings. Phytochemistry, 15, 273-278 (1976) [13] Staver, M.J.; Glick, K.; Baisted, D.J.: Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings. Biochem. J., 169, 297-303 (1978) [14] Forsee, W.T.; Laine, R.A.; Elbein, A.D.: Solubilization of a particulate UDPGlucose:sterol b-glucosyltransferase in developing cotton fibers and seeds and characterization of steryl 6-acyl-d-glucosides. Arch. Biochem. Biophys., 161, 248-259 (1974)

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2.4.1.173

Sterol 3b-glucosyltransferase

[15] Bouvier-Nave, P.; Ullmann, P.; Rimmele, D.; Benveniste, P.: Phospholipiddependence of plant UDP-glucose-sterol-b-d-glucosyltransferase. I. Detergent-mediated delipidatio by selective solubilization. Plant Sci. Lett., 36, 1927 (1984) [16] Quantin-Martenot, E.; Benveniste, P.; Hartmann, M.A.; Bouvier-Nave, P.: Activation of etiolated maize coleoptiles plasma membrane-bound uridine-diphosphate-glucose-sterol-b-d-glucosyltransferase by triton X-100, hydroxyl ions and phospholipase A2 . Plant Sci. Lett., 29, 305-314 (1983) [17] Ullmann, P.; Rimmele, D.; Benveniste, P.; Bouvier-Nave, P.: Phospholipiddependence of plant UDP-glucose-sterol-b-d-glucosyltransferase. II. Acetone-mediated delipidation and kinetic studies. Plant Sci. Lett., 36, 29-36 (1984) [18] Ullmann, P.; Ury, A.; Rimmele, D.; Benveniste, P.; Bouvier-Nave, P.: UDPglucose sterol b-d-glucosyltransferase, a plasma membrane-bound enzyme of plants: Enzymic properties and lipid dependence. Biochimie, 75, 713-723 (1993) [19] Warnecke, D.C.; Heinz, E.: Purification of a membrane-bound UDP-glucose:sterol b-d-glucosyltransferase based on its solubility in diethyl ether. Plant Physiol., 105, 1067-1073 (1994) [20] Warnecke, D.C.; Baltrusch, M.; Buck, F.; Wolter, F.P.; Heinz, E.: UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli. Plant Mol. Biol., 35, 597-603 (1997) [21] Warnecke, D.; Erdmann, R.; Fahl, A.; Hube, B.; Muller, F.; Zank, T.; Zähringer, U.; Heinz, E.: Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum. J. Biol. Chem., 274, 13048-13059 (1999) [22] Bergenstraahle, A.; Tillberg, E.; Jonsson, L.: Effects of ethephon and norbornadiene on sterol and glycoalkaloid biosynthesis in potato tuber disks. Physiol. Plant., 89, 301-308 (1993)

399

Glucuronylgalactosylproteoglycan 4-b-N-acetylgalactosaminyltransferase

2.4.1.174

1 Nomenclature EC number 2.4.1.174 Systematic name UDP-N-acetyl-d-galactosamine:d-glucuronyl-1,3-b-d-galactosyl-proteoglycan b-1,4-N-acetylgalactosaminyltransferase Recommended name glucuronylgalactosylproteoglycan 4-b-N-acetylgalactosaminyltransferase Synonyms N-acetylgalactosaminyltransferase I acetylgalactosaminyltransferase, uridine diphosphoacetylgalactosaminechondroitin, I chondroitin galactosaminyltransferase <3, 4> [3, 4] glucuronylgalactosylproteoglycan b-1,4-N-acetylgalactosaminyltransferase uridine diphosphoacetylgalactosamine-chondroitin acetylgalactosaminyltransferase I Additional information <2-4> (<2,3> the enzyme has also N-acetylgalactosaminyltransferase II activity, EC 2.4.1.175, substrates chondroitin oligo- and polysaccharides [2,3]; <4> the enzyme has also N-acetylgalactosaminyltransferase II activity, EC 2.4.1.175, substrates sulfated and unsulfated chondroitin oligo- and polysaccharides [4]) [2-4] CAS registry number 96189-39-8

2 Source Organism <1> Bos taurus (calf [1]) [1] <2> Homo sapiens (chondroitin sulfate N-acetylgalactosaminyltransferase, SwissProt-ID: Q8TDV6 [2]) [2] <3> Homo sapiens (SwissProt-ID: Q8TDV6) [3, 4] <4> Homo sapiensSwissProt-ID: Q8N6G5); [4]

400

2.4.1.174

Glucuronylgalactosylproteoglycan 4-b-N-acetylgalactosaminyltransferase

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-d-galactosyl-proteoglycan = UDP + N-acetyl-d-galactosaminyl-1,4-b-d-glucuronyl-1,3-b-d-galactosylproteoglycan Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-galactosamine + glucuronylgalactosyl glycosides <1, 2> (<2> involved in chondroitin initiation and elongation [2]; <1> involved in chondroitin sulfate biosynthesis: transfer of the first N-acetylgalactosamine residue [1]; involved in the biosynthesis of chodroitin sulfate. Key enzyme activity for the initiation of chondroitin and dermatan sulfates, transferring GalNAc to the GlcA-Gal-Gal-Xyl-Ser core.) [1, 2] P UDP + N-acetyl-d-galactosaminylglucuronylgalactosyl glycosides Substrates and products S UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-b-d-galactose <1> (<1> minimum requirement for acceptor structure [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-d-galactosamine-1,4-b-d-glucuronyl-1,3-b-d-galactose S UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-b-d-galactosyl-1,3b-d-galactose <1> (<1> best substrate [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-d-galactosaminyl-1,4-b-d-glucuronyl-1,3-b-d-galactosyl1,3-b-d-galactose <1> [1] S UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-b-d-galactosyl-1,3b-d-galactosyl-1,4-b-d-xylosyl-1-O-(Gly)Ser-(Gly-Glu) <4> (Reversibility: ? <4> [4]) [4] P UDP + N-acetyl-d-galactosamine-1,4-b-d-glucuronyl-1,3-b-d-galactosyl1,3-b-d-galactosyl-1,4-b-d-xylosyl-1-O-(Gly)Ser-(Gly-Glu) S UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-b-d-galactosyl-1,3b-d-galactosyl-1,4-b-d-xylosyl-1-O-Ser <4> (Reversibility: ? <4> [4]) [4] P UDP + N-acetyl-d-galactosamine-1,4-b-d-glucuronyl-1,3-b-d-galactosyl1,3-b-d-galactosyl-1,4-b-d-xylosyl-1-O-Ser S UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-b-d-galactosyl-1,3b-d-galactosyl-1,4-b-d-xylosyl-1-O-Ser-peptide <4> (Reversibility: ? <4> [4]) [4] P UDP + N-acetyl-d-galactosamine-1,4-b-d-glucuronyl-1,3-b-d-galactosyl1,3-b-d-galactosyl-1,4-b-d-xylosyl-1-O-Ser-peptide S UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-b-d-galactosyl-1,3b-d-galactosyl-b-d-xylose-O-methoxyphenyl <2> (<2> i.e. linkage tetrasaccharide, synthetic substrate [2]; <2> transfers GalNAc to GlcA at the non-reducing terminus of the linkage tetrasacharide [2]; <2> best acceptor substrate together with chondroitin polysaccharide [2]) (Reversibility: ? <2> [2]) [2]

401

Glucuronylgalactosylproteoglycan 4-b-N-acetylgalactosaminyltransferase

2.4.1.174

P UDP + N-acetyl-d-galactosamine-1,4-b-d-glucuronyl-1,3-b-d-galactosyl1,3-b-d-galactosyl-b-d-xylose-O-methoxyphenyl S UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-b-d-galactosyl-1,4b-d-glucose <1> (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-d-galactosamine-1,4-b-d-glucuronyl-1,3-b-d-galactosyl1,4-b-d-glucose S UDP-N-acetyl-d-galactosamine + b-d-glucuronyl-1,3-b-d-galactosyl-1-OC2 H4 -NH-benzyloxycarbonyl <3, 4> (Reversibility: ? <3,4> [3,4]) [3, 4] P UDP + N-acetyl-d-galactosamine-1,4-b-d-glucuronyl-1,3-b-d-galactosyl1-O-C2 H4 -NH-benzyloxycarbonyl S UDP-N-acetyl-d-galactosamine + glucuronylgalactosyl glycosides <1-4> (<1> acceptors are tri- or disaccharides with terminal glucuronyl residue and a galactosyl residue in penultimate position, transfers acetylgalactosamine in b-linkage, no substrates are glucuronic acid or disaccharides with terminal galactosyl residues [1]) (Reversibility: ? <1-4> [1,2]) [1-4] P UDP + N-acetyl-d-galactosaminyl-1,4-b-d-glucuronylgalactosyl glycosides <1-4> [1-4] Metals, ions Mn2+ <1, 3, 4> (<1> requirement [1]) [1, 3, 4] Specific activity (U/mg) Additional information <1, 2> (<1> 13.1 cpm/min/mg protein [1]) [1, 2] pH-Optimum 6.4 <1> [1] 6.5 <2, 3> (<2,3> assay at [2,3]) [2, 3] Temperature optimum ( C) 30 <1> (<1> assay at [1]) [1] 37 <2, 4> (<2,4> assay at [2,4]) [2, 4]

4 Enzyme Structure Subunits ? <3, 4> (<3,4> x * 95000, recombinant enzyme, SDS-PAGE [3,4]) [3, 4] Posttranslational modification glycoprotein <3, 4> (<3,4> 2 potential N-glycosylation sites, either one or both of which are used in COS-1 cells during expression of the recombinant fusion protein [3,4]) [3, 4]

5 Isolation/Preparation/Mutation/Application Source/tissue G-361 cell <2> (<2> melanoma cell line [2]) [2] SW-1736 cell <2> (<2> thyroid cancer cell line [2]) [2]

402

2.4.1.174

Glucuronylgalactosylproteoglycan 4-b-N-acetylgalactosaminyltransferase

U266 cell <2> (<2> myeloma cell line [2]) [2] adrenal gland <2> (<2> low content [2]) [2] aorta thoracica <1> [1] bone marrow <2, 3> [2, 3] brain <2, 3> [2, 3] cerebral cortex <2> [2] colon <2, 3> (<2,3> low content [2,3]) [2, 3] heart <3> [3] kidney <2, 3> (<2> very low content [2]) [2, 3] leukocyte <2-4> (<2> low content [2]; <3,4> peripheral blood [3,4]) [2-4] liver <2, 3> (<2> very low content [2]) [2, 3] lung <2-4> (<2> low content [2]) [2-4] ovary <2> [2] pancreas <2> [2] peripheral blood <3, 4> [3, 4] placenta <2, 3> (<2,3> very high content [2,3]) [2, 3] skeletal muscle <3, 4> [3, 4] small intestine <2, 3> (<2> high content [2]) [2, 3] spleen <2, 3> [2, 3] testis <2> (<2> low content [2]) [2] thymus <2, 3> (<2,3> low content [2,3]) [2, 3] thyroid gland <2, 3> (<3> high content [3]; <2> very high content [2]) [2, 3] uterus <2> [2] Localization membrane <3, 4> (<3,4> type II membrane protein topology [3,4]) [3, 4] microsome <1> [1] Purification <1> (partially solubilized with a buffer containing 2% Triton X-100, 20% glycerol and phospholipase A2 [1]) [1] Cloning <2> (DNA sequence determination and analysis, expression of a truncated form, consisting of the putative catalytic domain, in Spodoptera frugiperda Sf21 insect cells as a soluble FLAG-tagged protein [2]) [2] <3> (DNA sequence determination, genomic organization and chromosome localization, functional expression as soluble protein fused to protein A IgGbinding domain in COS-1 cells, secretion into the medium [3]) [3] <4> (DNA sequence determination, genomic organization and chromosome localization, functional expression as soluble protein fused to protein A IgGbinding domain in COS-1 cells, secretion into the medium [4]) [4]

6 Stability Temperature stability 50 <1> (<1> 1 h, 10% loss of activity [1]) [1]

403

Glucuronylgalactosylproteoglycan 4-b-N-acetylgalactosaminyltransferase

2.4.1.174

References [1] Rohrmann, K.; Niemann, R.; Buddecke, E.: Two N-acetylgalactosaminyltransferases are involved in the biosynthesis of chondroitin sulfate. Eur. J. Biochem., 148, 463-469 (1985) [2] Gotoh, M.; Sato, T.; Akashima, T.; Iwasaki, H.; Kameyama, A.; Mochizuki, H.; Yada, T.; Inaba, N.; Zhang, Y.; Kikuchi, N.; Kwon, Y.-D.; Togayachi, A.; Kudo, T.; Nishihara, S.; Watanabe, H.; Kimata, K.; Narimatsu, H.: Enzymatic synthesis of chondroitin with a novel chondroitin sulfate N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine to glucuronic acid in initiation and elongation of chondroitin sulfate synthesis. J. Biol. Chem., 277, 38189-38196 (2002) [3] Uyama, T.; Kitagawa, H.; Tamura, J.-I.; Sugahara, K.: Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase. The key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate. J. Biol. Chem., 277, 8841-8846 (2002) [4] Uyama, T.; Kitagawa, H.; Tanaka, J.; Tamura, J.-i.; Ogawa, T.; Sugahara, K.: Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/ dermatan sulfate. J. Biol. Chem., 278, 3072-3078 (2003)

404

Glucuronosyl-N-acetylgalactosaminylproteoglycan 4-b-N-acetylgalactosaminyltransferase

2.4.1.175

1 Nomenclature EC number 2.4.1.175 Systematic name UDP-N-acetyl-d-galactosamine:b-d-glucuronosyl-(1,3)-N-acetyl-b-d-galactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase Recommended name glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase Synonyms N-acetylgalactosaminyltransferase II UDP-N-acetyl-d-galactosamine:d-glucuronyl-N-acetyl-1,3-b-d-galactosaminylproteoglycan b-1,4-N-acetylgalactosaminyltransferase acetylgalactosaminyltransferase, uridine diphosphoacetylgalactosaminechondroitin, II chondroitin galactosaminyltransferase <4, 5> [3, 4] chondroitin polymerase <6> [5] chondroitin synthase glucuronyl-N-acetylgalactosaminylproteoglycan b1,4-N-acetylgalactosaminyltransferase uridine diphosphoacetylgalactosamine-chondroitin acetylgalactosaminyltransferasec II Additional information <1, 4-8> (<4, 5, 7> the enzyme also has N-acetylgalactosaminyltransferase I activity, EC 2.4.1.174 [3, 4, 7]; <4> the enzyme shows no 3-b-glucuronosyltransferase activity, EC 2.4.1.226 [3]; <1, 3, 6, 8> the enzyme also has the 3-b-glucuronosyltransferase, EC 2.4.1.226, activity [2, 5, 8-10]; the human form of this enzyme is a bifunctional glycosyltransferase, which also has the 3-b-glucuronosyltransferase, EC 2.4.1.226, activity required for the synthesis of the chondroitin sulfate disaccharide repeats. Similar chondroitin synthase co-polymerases can be found in Pasteurella multocida and Escherichia coli.) [2-5, 7-10] CAS registry number 96189-40-1

405

Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase 2.4.1.175

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

Bos taurus (fetal [6,9]; calf [1,6]; adult [6]) [1, 6, 9] Rattus norvegicus [6] Homo sapiens (SwissProt-ID: Q8TDX6) [2] Homo sapiens (SwissProt-ID: Q8TDX6, chondroitin N-acetylgalactosaminyltransferase I, not N-acetylgalactosaminyltransferase I EC 2.4.1.174 [3,4]) [3, 4] Homo sapiens (SwissProt-ID: Q8N6G5, chondroitin N-acetylgalactosaminyltransferase II [4]) [4] Escherichia coli (strain K4 [5]) [5] Homo sapiens (SwissProt-ID: Q86X52, chondroitin sulfate N-acetylgalactosaminyltransferase [7]) [7] Pasteurella multocida (type F [8,10]) [8, 10] Escherichia coli (strain K4 [10]) [10]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-galactosamine + b-d-glucuronosyl-(1!3)-N-acetyl-b-d-galactosaminyl-proteoglycan = UDP + N-acetyl-b-d-galactosaminyl-(1!4)-bd-glucuronosyl-(1!3)-N-acetyl-b-d-galactosaminyl-proteoglycan (<8> enzyme contains 2 distinct catalytic sites for GalNAc and GlcNAc transferase activity [10]) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-b-d-galactosamine + chondroitin oligosaccharide <1, 4-6, 9> (<1, 4, 5> involved in chondroitin biosynthesis [1, 3, 4, 6, 9]; <6, 9> biosynthesis of K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone to which b-fructose is linked at position C-3 of the GlcNAc residue [5,10]) [1, 3-6, 9, 10] P UDP + N-acetyl-b-d-galactosaminyl chondroitin oligosaccharide S UDP-N-acetyl-b-d-galactosamine + chondroitin sulfate oligosaccharide <1, 5, 6> (<1, 4, 5> involved in chondroitin sulfate biosynthesis: elongation of growing chondroitin sulfate chain [1,3,4,6,9]) [1, 3-6, 9] P UDP + N-acetyl-b-d-galactosaminyl chondroitin sulfate oligosaccharide Substrates and products S UDP-N-acetyl-d-galactosamine + chondroitin oligosaccharide <1-9> (<8, 9> in vitro: addition of single sugars to a nonreducing terminal GlcNAc residue of a chondroitin-derived oligosaccharide acceptor [10]; <9> long polymers are not formed [10]; <8> high specificity for sugar donor substrates [8, 10]; <7> low activity [7]; <1, 3, 4> best acceptor substrate [2, 3, 6]; <6> acceptor substrates longer than a tetrasaccharide are absolutely required [5]; <1, 6, 7> acceptors are even-numbered chondroitin oligosac-

406

2.4.1.175 Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase

P S P S P S P S

P S

P S

P S

P

charides with terminal glucuronyl-N-acetyl-1,3-b-d-galactosamine sequence at non-reducing end, transfers N-acetyl-galactosamine to non-reducing glucuronic acid residues in b-glycosidic linkage [1, 5, 7, 9]; <1> no substrates are glucuronyl-N-acetyl-1,3-b-galactosamine or glucuronyl-Nacetyl-1,3-b-d-galactosaminyl-N-acetyl-1,3-b-d-galactosaminyl-N-acetylgalactosaminol [1]) (Reversibility: ? <1-9> [1-10]) [1-10] UDP + N-acetyl-d-galactosaminyl chondroitin oligosaccharide <1-9> (<3, 4, 6, 8> transfer in b-1,4-linkage [2,3,5,8,10]) [1-10] UDP-N-acetyl-d-galactosamine + chondroitin polysaccharide <7> (<7> best acceptor substrate [7]) (Reversibility: ? <7> [7]) [7] UDP + N-acetyl-d-galactosaminyl chondroitin polysaccharide <7> [7] UDP-N-acetyl-d-galactosamine + chondroitin sulfate C <3> (<8,9> no activity [10]; <3> 11% activity compared to chondroitin [2]) (Reversibility: ? <3> [2]) [2] UDP + N-acetyl-d-galactosaminyl chondroitin sulfate C <3> [2] UDP-N-acetyl-d-galactosamine + chondroitin sulfate oligosaccharide <6, 7> (<8,9> no activity [10]; <6> acceptor substrates longer than a tetrasaccharide are absolutely required [5]) (Reversibility: ? <6,7> [5,7]) [5, 7] UDP + N-acetyl-d-galactosaminyl chondroitin sulfate oligosaccharide <6, 7> [5, 7] UDP-N-acetyl-d-galactosamine + defructosylated K4 oligosaccharide <9> (<9> the native K4 oligosaccharide, containing a fructose residue branch at C-3 of each GlcNAc residue of the chondroitin backbone, is no substrate [10]; <9> in vitro: addition of single sugars to a nonreducing terminal GlcNAc residue of a chondroitin-derived oligosaccharide acceptor, but long polymers are not formed [10]) (Reversibility: ? <9> [10]) [10] UDP + N-acetyl-d-galactosaminyl defructosylated K4 oligosaccharide <9> [10] UDP-N-acetyl-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-d-galactosamine 4-sulfate)4 <1> (<1> i.e. chondroitin sulfate octasaccharide, glycosylated at 32% the rate of chondroitin octasaccharide [1]) (Reversibility: ? <1> [1]) [1] UDP + N-acetyl-b-d-galactosaminyl-1,4-glucuronyl-N-acetyl-1,3-b-d-galactosaminyl-(glucuronyl-N-acetyl-1,3-b-d-galactosamine)3 UDP-N-acetyl-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-dgalactosamine)2 <1, 6> (<6> minimal required acceptor substrate chain length [5]; <1> i.e. chondroitin tetrasaccharide, glycosylated at 40% the rate of decamer glycosylation [1]) (Reversibility: ? <1,6> [1,5]) [1, 5] UDP + N-acetyl-b-d-galactosaminyl-1,4-glucuronyl-N-acetyl-1,3-b-d-galactosaminyl-glucuronyl-N-acetyl-1,3-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-dUDP-N-acetyl-b-d-galactosamine galactosamine)3 <1, 3, 6, 7> (<1> minimal chain length required for activity [9]; <3> low activity [2]; <1> i.e. chondroitin hexasaccharide, glycosylated at 60% the rate of decamer glycosylation [1]) (Reversibility: ? <1, 3, 6, 7> [1,2,5,7-9]) [1, 2, 5, 7-9] UDP + N-acetyl-b-d-galactosaminyl-1,4-glucuronyl-N-acetyl-1,3-b-d-galactosaminyl-(glucuronyl-N-acetyl-1,3-b-d-galactosamine)2 407

Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase 2.4.1.175

S UDP-N-acetyl-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-dgalactosamine)4 <1, 3, 7> (<3> low activity [2]; <1> i.e. chondroitin octasaccharide, glycosylated at 75% the rate of decamer glycosylation [1]) (Reversibility: ? <1,3,7> [1,2,7]) [1, 2, 7] P UDP + N-acetyl-b-d-galactosaminyl-1,4-glucuronyl-N-acetyl-1,3-b-d-galactosaminyl-(glucuronyl-N-acetyl-1,3-b-d-galactosamine)3 S UDP-N-acetyl-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-dgalactosamine)5 <1, 7> (<1> i.e. chondroitin decasaccharide, best substrate [1]) (Reversibility: ? <1,7> [1,7]) [1, 7] P UDP + N-acetyl-b-d-galactosaminyl-1,4-glucuronyl-N-acetyl-1,3-b-d-galactosaminyl-(glucuronyl-N-acetyl-1,3-b-d-galactosamine)4 <1> [1] S UDP-N-acetyl-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-d-galactosamine)6 <1, 7> (<1> i.e. chondroitin dodecasaccharide, glycosylated at about 60% the rate of decamer glycosylation [1]) (Reversibility: ? <1, 7> [1, 7]) [1, 7] P UDP + N-acetyl-b-d-galactosaminyl-1,4-glucuronyl-N-acetyl-1,3-b-d-galactosaminyl-(glucuronyl-N-acetyl-1,3-b-d-galactosamine)5 S UDP-N-acetyl-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-d-galactosamine)7 <1, 7> (<1> i.e. chondroitin tetradecasaccharide, glycosylated at about 45% the rate of decamer glycosylation [1]) (Reversibility: ? <1, 7> [1, 7]) [1, 7] P UDP + N-acetyl-b-d-galactosaminyl-1,4-glucuronyl-N-acetyl-1,3-b-d-galactosaminyl-(glucuronyl-N-acetyl-1,3-b-d-galactosamine)6 S UDP-N-acetyl-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-d-galactosamine)8 <1> (<1> i.e. chondroitin hexadecasaccharide, glycosylated at 15% the rate of decamer glycosylation [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-b-d-galactosaminyl-1,4-glucuronyl-N-acetyl-1,3-b-d-galactosaminyl-(glucuronyl-N-acetyl-1,3-b-d-galactosamine)7 S UDP-N-acetyl-b-d-galactosamine + (glucuronyl-N-acetyl-1,3-b-d-glucosamine)4 <1> (<1> i.e. hyaluronate octasaccharide, glycosylated at 25% the rate of chondroitin octasaccharide [1]) (Reversibility: ? <1> [1]) [1] P UDP + ? S Additional information <1, 3-7> (<7> low activity with sulfated oligosaccharide substrates [7]; <4,5> the enzyme also has N-acetylgalactosaminyltransferase I activity, EC 2.4.1.174 [3, 4]; <4> the enzyme shows no 3-bglucuronosyltransferase activity, EC 2.4.1.226 [4]; <1, 3, 6> the enzyme also has the 3-b-glucuronosyltransferase, EC 2.4.1.226, activity [2, 5, 9]; <3> chondroitin sulfate A and D are very poor acceptor substrates [2]; <1, 7> chondroitin sulfates A, C, D, and E, and dermatan sulfate are poor acceptor substrates [6, 7]) [2-7, 9] P ? Inhibitors Ca2+ <6> (<6> complete inhibition [5]) [5] Cu2+ <6> (<6> complete inhibition [5]) [5] EDTA <6> (<6> complete inhibition [5]) [5]

408

2.4.1.175 Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase

Metals, ions Fe2+ <6> (<6> can partly substitute for Mn2+ , 30.6% activity compared to Mn2+ [5]) [5] Mg2+ <6> (<6> can partly substitute for Mn2+ , 30.7% activity compared to Mn2+ [5]) [5] Mn2+ <1, 3-6> (<6> required, 20 mM [5]; <1> required, maximal at about 10 mM [6]; <6> can partly be substituted by Fe2+ and Mg2+ [5]) [2, 3-6] Additional information <1> (<1> Mg2+ , Cu2+ , Ca2+ , Zn2+ , Ba2+ do not effect the enzyme activity [6]) [6] Specific activity (U/mg) 0.00053 <6> (<6> purified recombinant enzyme, substrate chondroitin polysaccharide with MW 10 kDa [5]) [5] 0.00124 <6> (<6> purified recombinant enzyme, substrate chondroitin hexasaccharide [5]) [5] 0.00331 <6> (<6> purified recombinant enzyme, substrate chondroitin sulfate polysaccharide with MW 20 kDa [5]) [5] 0.00334 <6> (<6> purified rcombinant enzyme, substrate chondroitin sulfate hexasaccharide [5]) [5] 0.0425 <1> (<1> purified enzyme [9]) [9] Additional information <1-4, 7> (<1> assay method development [6]; <1> 34.6 cpm/min/mg protein [1]) [1-3, 6, 7] Km-Value (mM) 0.036 <6> (UDP-N-acetyl-b-d-galactosamine) [5] 0.05 <1> (UDP-N-acetyl-b-d-galactosamine) [6] pH-Optimum 6.5 <1, 3-5> (<1,3-5> assay at [2-4,9]) [2-4, 6, 9] 7-7.5 <6> [5] 7.4 <1> [1] Temperature optimum ( C) 25 <6> (<6> highest product chain length [5]) [5] 30 <1, 6> (<6> highest reaction rate [5]; <1> assay at [1]) [1, 5] 37 <1, 5> (<1,5> assay at [4,6]) [4, 6]

4 Enzyme Structure Molecular weight 160000 <1> (<1> gel filtration [9]) [9] Subunits ? <4, 5> (<4,5> x * 95000, recombinant enzyme, SDS-PAGE [3,4]) [3, 4] Posttranslational modification glycoprotein <4, 5> (<4,5> 2 potential N-glycosylation sites, either one or both of which are used in COS-1 cells during expression of the recombinant fusion protein [3,4]) [3, 4] 409

Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase 2.4.1.175

5 Isolation/Preparation/Mutation/Application Source/tissue G-361 cell <7> (<7> melanoma cell line [7]) [7] SW-1736 cell <7> (<7> thyroid cancer cell line [7]) [7] U266 cell <7> (<7> myeloma cell line [7]) [7] adrenal gland <7> (<7> low content [7]) [7] aorta thoracica <1> [1] bone marrow <4, 7> [3, 7] brain <3, 4, 7> [2, 3, 7] cerebral cortex <7> [7] colon <4, 7> (<4,7> low content [3,7]) [3, 7] heart <3, 4> [2, 3] kidney <3, 4, 7> (<7> very low content [7]) [2, 3, 7] leukocyte <3-5, 7> (<7> low content [7]; <3> high content [2]; <3,4> peripheral blood [2,3]) [2-4, 7] liver <3, 4, 7> (<7> very low content [7]) [2, 3, 7] lung <3-5, 7> (<7> low content [7]; <3> high content [2]) [2-4, 7] ovary <7> [7] pancreas <7> [7] peripheral blood <3-5> [2-4] placenta <3, 4, 7> (<3,4,7> very high content [2,3,7]) [2, 3, 7] serum <1, 2> [6, 9] skeletal muscle <3-5> [2-4] small intestine <4, 7> (<7> high content [7]) [3, 7] spleen <3, 4, 7> (<3> high content [2]) [2, 3, 7] testis <7> (<7> low content [7]) [7] thymus <3, 4, 7> (<4,7> low content [3,7]) [2, 3, 7] thyroid gland <4, 7> (<7> very high content [7]; <4> high content [3]) [3, 7] uterus <7> [7] Localization cytosol <1> [6] membrane <3-5> (<3-5> type II membrane protein topology [2-4]) [2-4] microsome <1> [1] Additional information <9> (<9> transport of the nascent polymer chain out of the cell through the lipid membranes requires at least seven distinct polypeptide species, possible transport mechanism [10]; <8> the carboxy-terminal tail is likely to contain a docking segment that interacts with the transport mechanism [10]) [10] Purification <1> (partially solubilized with a buffer containing 2% Triton X-100, 20% glycerol and phospholipase A2 [1]) [1, 9] <6> (recombinant His-tagged enzyme expressed in E. coli strain TOP10 [5]) [5]

410

2.4.1.175 Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase

Cloning <3> (genomic organization and chromosome localization, functional expression as soluble protein fused to protein A IgG-binding domain in COS-1 cells, secretion into the medium [2]) [2] <4> (DNA sequence determination, genomic organization and chromosome localization, functional expression as soluble protein fused to protein A IgGbinding domain in COS-1 cells, secretion into the medium [3]) [3] <5> (DNA sequence determination, genomic organization and chromosome localization, functional expression as soluble protein fused to protein A IgGbinding domain in COS-1 cells, secretion into the medium [4]) [4] <6> (DNA and amino acid sequence determination and analysis, expression as soluble His-tagged protein in Escherichia coli strain TOP10 [5]) [5] <7> (DNA sequence determination and analysis, expression of a truncated form, consisting of the putative catalytic domain, in Spodoptera frugiperda Sf21 insect cells as a soluble FLAG-tagged protein [7]) [7] <8> (DNA sequence determination, expression in Escherichia coli of amino acid residues 1-704 of total 965 as soluble functional protein [8]) [8, 10] <9> [10] Application medicine <8, 9> (<8,9> antimicrobial approach, understanding microbial capsule biosynthesis may assist the uncloaking of bacterial camouflage without disrupting host GAG synthesis [10]) [10]

6 Stability Temperature stability 50 <1> (<1> t1=2 : 5 min, 90% loss of activity within 20 min [1]) [1] General stability information <1>, ion-exchange chromatography on DEAE-trisacryl inactivates [1] <1>, rapid thermodenaturation [1] <1>, stable against freeze-thawing in liquid N2 several times [9] <1>, unstable upon purification [1] Storage stability <1>, -80 C, MES/NaOH, 50 mM, pH 6.5, 1 mM dithiothreitol, stable for at least a few months [9]

References [1] Rohrmann, K.; Niemann, R.; Buddecke, E.: Two N-acetylgalactosaminyltransferases are involved in the biosynthesis of chondroitin sulfate. Eur. J. Biochem., 148, 463-469 (1985) [2] Kitagawa, H.; Uyama, T.; Sugahara, K.: Molecular cloning and expression of a human chondroitin synthase. J. Biol. Chem., 276, 38721-38726 (2001)

411

Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-b-N-acetylgalactosaminyltransferase 2.4.1.175

[3] Uyama, T.; Kitagawa, H.; Tamura, J.-I.; Sugahara, K.: Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase. The key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate. J. Biol. Chem., 277, 8841-8846 (2002) [4] Uyama, T.; Kitagawa, H.; Tanaka, J.; Tamura, J.-i.; Ogawa, T.; Sugahara, K.: Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/ dermatan sulfate. J. Biol. Chem., 278, 3072-3078 (2003) [5] Ninomiya, T.; Sugiura, N.; Tawada, A.; Sugimoto, K.; Watanabe, H.; Kimata, K.: Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4. J. Biol. Chem., 277, 21567-21575 (2002) [6] Kitagawa, H.; Tsuchida, K.; Ujikawa, M.; Sugahara, K.: Detection and characterization of UDP-GalNAc: chondroitin N-acetylgalactosaminyltransferase in bovine serum using a simple assay method. J. Biochem., 117, 10831087 (1995) [7] Gotoh, M.; Sato, T.; Akashima, T.; Iwasaki, H.; Kameyama, A.; Mochizuki, H.; Yada, T.; Inaba, N.; Zhang, Y.; Kikuchi, N.; Kwon, Y.-D.; Togayachi, A.; Kudo, T.; Nishihara, S.; Watanabe, H.; Kimata, K.; Narimatsu, H.: Enzymatic synthesis of chondroitin with a novel chondroitin sulfate N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine to glucuronic acid in initiation and elongation of chondroitin sulfate synthesis. J. Biol. Chem., 277, 38189-38196 (2002) [8] DeAngelis, P.L.; Padgett-McCue, A.J.: Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F. J. Biol. Chem., 275, 24124-24129 (2000) [9] Tsuchida, K.; Lind, T.; Kitagawa, H.; Lindahl, U.; Sugahara, K.; Lidholt, K.: Purification and characterization of fetal bovine serum b-N-acetyl-d-galactosaminyltransferase and b-d-glucuronyltransferase involved in chondroitin sulfate biosynthesis. Eur. J. Biochem., 264, 461-467 (1999) [10] DeAngelis, P.L.: Microbial glycosaminoglycan glycosyltransferases. Glycobiology, 12, 9R-16R (2002)

412

Gibberellin b-D-glucosyltransferase

2.4.1.176

1 Nomenclature EC number 2.4.1.176 Systematic name UDP-glucose:gibberellin 2-O-b-d-glucosyltransferase Recommended name gibberellin b-d-glucosyltransferase Synonyms gibberellin b-d-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-gibberellate 3-Oglucosyltransferase, uridine diphosphoglucose-gibberellate 7CAS registry number 94489-97-1 99775-14-1

2 Source Organism <1> Phaseolus coccineus [1] <2> Lycopersicon peruvianum [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + gibberellin = UDP + gibberellin 2-O-b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + gibberellin GA3 <1> (<1> acts on the plant hormone gibberellin GA3 and related compounds [1]) [1] P UDP + gibberellin GA3 -O(3)-b-d-glucopyranoside Substrates and products S UDPglucose + gibberellin GA3 <1> (<2> no substrate [1]) (Reversibility: ? <1> [1]) [1] P UDP + gibberellin GA3 -O(3)-b-d-glucopyranoside <1> [1]

413

Gibberellin b-D-glucosyltransferase

2.4.1.176

S P S P S P S

UDPglucose + gibberellin GA30 <1> [1] UDP + gibberellin GA30 -O(3)-b-d-glucopyranoside UDPglucose + gibberellin GA7 <1, 2> [1] UDP + gibberellin GA7 -O(3)b-d-glucopyranoside UDPglucose + gibberellin GA9 <2> [1] UDP + gibberellin GA9 -O(3)-b-d-glucopyranoside <2> [1] Additional information <1, 2> (<1> gibberellins not accepted as substrates: GA1 , GA4 , GA5 , GA8 , 3-epi-GA3 , iso-GA3 , 3-dehydro-GA3 , 3OH-epi-allogibberic acid, 1b-OH-GA5 [1]; <2> gibberellins not accepted as substrates: GA1 , GA3 , GA4 , GA5 , GA6 , GA8 , GA20 , GA7 -methylester, GA8 -methylester [1]) [1] P ? pH-Optimum 8.2 <1> (<1> assay at [1]) [1] 8.4 <2> (<2> assay at [1]) [1] Temperature optimum ( C) 25 <1> (<1> assay at [1]) [1] 37 <2> (<2> assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture <2> [1] fruit <1> (<1> maturing [1]) [1] Localization soluble <1, 2> [1]

6 Stability Storage stability <1, 2>, deep-frozen, stable for months [1]

References [1] Sembdner, G.; Knöfel, H.D.; Schwarzkopf, E.; Liebisch, H.W.: In vitro glucosylation of gibberellins. Biol. Plant., 27, 231-236 (1985)

414

Cinnamate b-D-glucosyltransferase

2.4.1.177

1 Nomenclature EC number 2.4.1.177 Systematic name UDP-glucose:trans-cinnamate b-d-glucosyltransferase Recommended name cinnamate b-d-glucosyltransferase Synonyms UDPG:t-cinnamate glucosyltransferase uridine diphosphoglucose-cinnamate glucosyltransferase CAS registry number 83744-95-0

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

Ipomoea batatas (sweet potato, Lam cv. Norin 1 [5]) [5] Medicago sativa (alfalfa, cv. Apollo [3]) [3] Phaseolus vulgaris (bean, cv. Canadian Wonder [3]) [3] Vanilla planifolia (Andr. [2,4]) [2, 4] Verbesina carcasana [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + trans-cinnamate = UDP + trans-cinnamoyl b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + cinnamate <1, 4> (<1> involved in biosynthesis of chlorogenic acid in root of Ipomoea batatas [5]; <4> enzyme of benzoate metabolism [4,2]) [5, 4, 2] P UDP + cinnamoyl b-d-glucoside

415

Cinnamate b-D-glucosyltransferase

2.4.1.177

Substrates and products S UDP-glucose + 2,3-dimethoxybenzyl alcohol <5> (Reversibility: ? <5> [1]) [1] P UDP + 2,3-dimethoxybenzyl b-d-glucose <5> [1] S UDP-glucose + 2,4-dimethoxybenzyl alcohol <5> (Reversibility: ? <5> [1]) [1] P UDP + 2,4-dimethoxybenzyl b-d-glucose <5> [1] S UDP-glucose + 2,4-dimethoxycinnamate <5> (Reversibility: ? <5> [1]) [1] P UDP + 2,4-dimethoxycinnamoyl b-d-glucose <5> [1] S UDP-glucose + 2,5-dimethoxybenzyl alcohol <5> (Reversibility: ? <5> [1]) [1] P UDP + 2,5-dimethoxybenzyl b-d-glucose <5> [1] S UDP-glucose + 2,5-dimethoxycinnamate <5> (Reversibility: ? <5> [1]) [1] P UDP + 2,5-dimethoxycinnamoyl b-d-glucose <5> [1] S UDP-glucose + 2,6-dimethoxycinnamate <5> (Reversibility: ? <5> [1]) [1] P UDP + 2,6-dimethoxycinnamoyl b-d-glucose <5> [1] S UDP-glucose + 2-dimethoxycinnamate <5> (Reversibility: ? <5> [1]) [1] P UDP + 2-dimethoxycinnamoyl b-d-glucose <5> [1] S UDP-glucose + 3,4,5-trimethoxycinnamate <4> (<4> glucosylation at 84% the rate of 4-methoxycinnamate [4]) (Reversibility: ? <4> [4]) [4] P UDP + 3,4,5-trimethoxycinnamoyl b-d-glucose S UDP-glucose + 3,4-dimethoxybenzyl alcohol <5> (Reversibility: ? <5> [1]) [1] P UDP + 3,4-dimethoxybenzyl b-d-glucose <5> [1] S UDP-glucose + 3,4-dimethoxycinnamate <4, 5> (<4> glucosylation at 78% the rate of 4-methoxycinnamate [4]) (Reversibility: ? <4,5> [1,4]) [1, 4] P UDP + 3,4-dimethoxycinnamoyl b-d-glucose (<5> (E)-isomer [1]) [1] S UDP-glucose + 3,5-dimethoxycinnamate <5> (Reversibility: ? <5> [1]) [1] P UDP + 3,5-dimethoxycinnamoyl b-d-glucose <5> [1] S UDP-glucose + 3-methoxycinnamate <4> (<4> glucosylation at 52% the rate of 4-methoxycinnamate [4]) (Reversibility: ? <4> [4]) [4] P UDP + 3-methoxycinnamoyl b-d-glucose S UDP-glucose + 4-dimethoxycinnamate <5> (Reversibility: ? <5> [1]) [1] P UDP + 4-dimethoxycinnamoyl b-d-glucose <5> [1] S UDP-glucose + 4-hydroxy-3-methoxycinnamate <1> (<1> glucosylation at 35% the rate of cinnamate [5]) (Reversibility: ? <1> [5]) [5] P UDP + 4-hydroxy-3-methoxycinnamoyl b-d-glucose S UDP-glucose + 4-methoxycinnamate <4> (<4> best substrate [2,4]) (Reversibility: ? <4> [2,4]) [2, 4] P UDP + 4-methoxycinnamoyl b-d-glucose S UDP-glucose + benzoate <1> (<1> glucosylation at 71% the rate of cinnamate [5]) (Reversibility: ? <1> [5]) [5] P UDP + benzoyl b-d-glucose S UDP-glucose + caffeate <1> (<1> glucosylation at 15% the rate of cinnamate [5]) (Reversibility: ? <1> [5]) [5] P UDP + caffeoyl b-d-glucose 416

2.4.1.177

Cinnamate b-D-glucosyltransferase

S UDP-glucose + cinnamate <1, 2, 3, 4, 5> (<1> trans-cinnamate [5]; <1> best substrate [5]; <4> glucosylation at 8% the rate of 4-methoxycinnamate [4]) (Reversibility: ? <1,2,3,4,5> [1,2,3,4,5]) [1, 2, 3, 4, 5] P UDP + cinnamoyl b-d-glucose <1, 2, 3> (<1> trans-isomer [5]) [3, 5] S UDP-glucose + feruloate <1, 4> (<1> glucosylation at 27% the rate of cinnamate [5]; <4> glucosylation 26% the rate of 4-methoxycinnamate [4]) (Reversibility: ? <1,4> [5,4]) [5, 4] P UDP + feruloyl b-d-glucose S UDP-glucose + isoferulic acid <4> (<4> glucosylation at 46% the rate of 4-methoxycinnamate [4]) (Reversibility: ? <4> [4]) [4] P UDP + isoferuloyl b-d-glucose S UDP-glucose + o-coumarate <1> (<1> glucosylation at 52% the rate of cinnamate [5]) (Reversibility: ? <1> [5]) [5] P UDP + o-coumaroyl b-d-glucose S UDP-glucose + p-coumarate <1, 4> (<1> glucosylation at 57% the rate of cinnamate [5]; <4> glucosylation at 43% the rate of 4-methoxycinnamate [4]; <1> the glucosyl-group is introduced exclusively into the carboxylgroup [5]) (Reversibility: ? <1,4> [4,5]) [4, 5] P UDP + p-coumaroyl-d-glucose <1> [5] S UDP-glucose + sinapic acid <4> (<4> glucosylation at 6.5% the rate of 4methoxycinnamate [4]) (Reversibility: ? <4> [4]) [4] P UDP + sinapoyl b-d-glucose Inhibitors HgCl2 <1> (<1> strong [5]) [5] p-chloromercuribenzoate <1> [5] Additional information <1, 2, 3> (<1> no inhibition by MgCl2 , CaCl2 , MnCl2 , chlorogenic acid (1 mM each) [5]; <2,3> no inhibition by hydroxycinnamic acid [3]) [3, 5] Metals, ions Additional information <1> (<1> no activation by MgCl2 , CaCl2 or MnCl2 , 1 mM each [5]) [5] Specific activity (U/mg) 0.0279 <4> [4] 70.1 <1> [5] Km-Value (mM) 0.1 <1> (UDP-glucose) [5] 0.2 <1> (trans-cinnamic acid) [5] 0.4 <2, 3> (cinnamic acid) [3] pH-Optimum 5.8 <1> [5] Temperature optimum ( C) 30 <1, 4> (<1,4> assay at [5,4]) [5, 4]

417

Cinnamate b-D-glucosyltransferase

2.4.1.177

4 Enzyme Structure Molecular weight 45000 <1> (<1> gel filtration [5]) [5]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture <2, 3, 4, 5> [1, 2, 3, 4] root <1> [5] Localization cytosol <2, 3> [3] soluble <1> [5] Purification <1> (partial [5]) [5] <2> (partial [3]) [3] <4> (partial [4]) [4]

6 Stability General stability information <1>, PMSF and 2-mercaptoethanol stabilize during purification [5] <1>, freezing inactivates, sorbitol stabilizes [5] <1>, sorbitol, 10%, stabilizes during storage [5] Storage stability <1>, -20 C, 10% sorbitol, about 10% loss of activity within 30 days [5] <1>, sorbitol, 10%, stabilizes during storage [5]

References [1] Vitali, A.; Monache, G.; Zappia, G.; Misiti, D.; Gacs-Baitz, E.; Botta, B.: bglucosyltransferase in cell cultures of Verbesina caracasana. Heterocycles, 50, 721-730 (1999) [2] Funk, C.; Brodelius, P.E.: Phenylpropanoid metabolism in suspension cultures of Vanilla planifolia Andr. IV. Induction of vanillic acid formation. Plant Physiol., 99, 256-262 (1992) [3] Edwards, R.; Mavandad, M.; Dixon, R.A.: Metabolic fate of cinnamic acid in elicitor treated cell suspension cultures of Phaseolus vulgaris. Phytochemistry, 29, 1867-1873 (1990)

418

2.4.1.177

Cinnamate b-D-glucosyltransferase

[4] Funk, C.; Brodelius, P.E.: Phenylpropanoid metabolism in suspension cultures of Vanilla planifolia Andr. III. Conversion of 4-methoxycinnamic acids into 4-hydroxybenzoic acids. Plant Physiol., 94, 102-108 (1990) [5] Shimizu, T.; Kojima, M.: Partial purification and characterization of UDPG:tcinnamate glucosyltransferase in the root of sweet potato, Ipomoea batatas Lam. J. Biochem., 95, 205-212 (1984)

419

Hydroxymandelonitrile glucosyltransferase

2.4.1.178

1 Nomenclature EC number 2.4.1.178 Systematic name UDP-glucose:4-hydroxymandelonitrile glucosyltransferase Recommended name hydroxymandelonitrile glucosyltransferase Synonyms UDPglucose:4-hydroxymandelonitrile glucosyltransferase cyanohydrin glucosyltransferase glucosyltransferase, uridine diphosphoglucose-cyanohydrin CAS registry number 89287-39-8

2 Source Organism <1> Triglochin maritima [1] <2> Prunus serotina (wild black cherry [2]) [2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + 4-hydroxymandelonitrile = UDP + taxiphyllin Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + (R,S)-mandelonitrile <2> (Reversibility: ? <2> [2]) [2] P UDP + (R)-prunasin <2> [2] S UDP-glucose + 4-hydroxymandelonitrile <1> (<1> last step in biosynthesis of cyanogenic glucosides [1]) (Reversibility: ? <1> [1]) [1] P UDP + taxiphyllin <1> [1] Substrates and products S UDP-glucose + (R,S)-mandelonitrile <2> (Reversibility: ? <2> [2]) [2] P UDP + (R)-prunasin <2> [2]

420

2.4.1.178

Hydroxymandelonitrile glucosyltransferase

S UDP-glucose + 3,4-dihydroxymandelonitrile <1> (Reversibility: ? <1> [1]) [1] P UDP + 3,4-dihydroxymandelonitrile glucoside <1> [1] S UDP-glucose + 4-hydroxymandelonitrile <1> (Reversibility: ? <1> [1]) [1] P UDP + taxiphyllin <1> [1] S UDP-glucose + benzoic acid <2> (Reversibility: ? <2> [2]) [2] P UDP + benzoyl glucoside S UDP-glucose + benzyl alcohol <2> (Reversibility: ? <2> [2]) [2] P UDP + benzoyl glucoside S UDP-glucose + mandelamide <2> (Reversibility: ? <2> [2]) [2] P ? S UDP-glucose + mandelic acid <2> (Reversibility: ? <2> [2]) [2] P ? Metals, ions high salt concentrations <1> (<1> 160% activity observed at 0.6 M concentration of NaCl, KCl or ammonium sulfate with 4-hydroxymandelonitrile, and 20% activity observed at 0.1-0.2 M concentration with 3,4-dihydroxymandelonitrile [1]) [1] Specific activity (U/mg) 0.001 <2> (<2> mandelic acid [2]) [2] 0.002 <2> (<2> benzyl alcohol [2]) [2] 0.0025 <2> (<2> mandelamide [2]) [2] 0.012 <2> (<2> (R,S)-mandelonitrile [2]) [2] 0.018 <2> (<2> benzoic acid [2]) [2] 0.31 <1> (<1> 3,4-dihydroxymandelonitrile [1]) [1] 0.94 <1> (<1> 4-hydroxymandelonitrile [1]) [1] Km-Value (mM) 0.32 <2> (UDP-glucose, <2> assay at pH 7.3 in the presence of 20 mM of (R,S)-mandelonitrile. The enzyme exclusively utilizes the (R)-enantiomer [2]) [2] 0.5 <1> (UDP-glucose, <1> 4-hydroxymandelonitrile or 3,4-dihydroxymandelonitrile [1]) [1] pH-Optimum 6.5-8.5 <1> [1] 7-8 <2> (<2> highest rates are realized in Tris-HCl buffer at pH 7.5 [2]) [2] 8 <1> (<1> assay at [1]) [1] pH-Range 5.8-9.3 <2> (<2> about 50% of activity maximum [2]) [2] 6-9.5 <1> (<1> about 50% of activity maximum at pH 6 and pH 9.5 [1]) [1] Temperature optimum ( C) 30 <1> [1]

421

Hydroxymandelonitrile glucosyltransferase

2.4.1.178

4 Enzyme Structure Molecular weight 50000 <1> (<1> gel filtration [1]) [1] Subunits monomer <1> (<1> 1 * 47000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue seedling <1> [1] Localization soluble <1> [1] Purification <1> (method that includes chromatography on Sephadex PD10 and DEAESephadex [1]) [1] <2> (method that includes chromatography on Sephadex G-25 [2]) [2]

6 Stability pH-Stability 7.5 <1> (<1> unstable below, crude enzyme extract [1]) [1] Storage stability <1>, -20 C, 10% glycerol, crude extract stable for several weeks [1] <2>, -20 C, 10% glycerol, 72% of 2 days crude extract stable for several weeks [2]

References [1] Hösel, W.; Schiel, O.: Biosynthesis of cyanogenic glucosides: in vitro analysis of the glucosylation step. Arch. Biochem. Biophys., 229, 177-186 (1984) [2] Poulton, J.E.; Shin, S.I.: Prunasin biosynthesis by cell-free extracts from black cherry (Prunus serotina Ehrh.) fruits and leaves. Z. Naturforsch. C, 38, 369-374 (1983)

422

Lactosylceramide b-1,3-galactosyltransferase

2.4.1.179

1 Nomenclature EC number 2.4.1.179 Systematic name UDP-galactose:d-galactosyl-1,4-b-d-glucosyl-R b-1,3-galactosyltransferase Recommended name lactosylceramide b-1,3-galactosyltransferase Synonyms UDP-galactose:lactose b1,3-galactosyltransferase <2> [2] galactosyltransferase, uridine diphosphogalactose-lactosylceramide b1-3uridine diphosphogalactose-lactosylceramide b1-3-galactosyltransferase CAS registry number 106769-64-6

2 Source Organism <-1> no activity in Mus musculus (lactating mammary gland [2]) [2] <1> Homo sapiens [1, 3-6] <2> Macropus eugenii (female tammar wallaby [2]) [2]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + d-galactosyl-1,4-b-d-glucosyl-R = UDP + d-galactosyl-1,3b-d-galactosyl-1,4-b-d-glucosyl-R (R may be an oligosaccharide or a glycolipid; lactose can also act as acceptor, but more slowly. Involved in the elongation of oligosaccharide chains, especially in glycolipids.) Reaction type hexosyl group transfer Natural substrates and products S UDP-galactose + d-galactosyl-1,4-b-d-glucosyl-glycolipid <1> (<1> involved in elongation of oligosaccharide chains in glycolipids [1, 6]; <1> associated with biosynthesis of type 1 lactoseries core chain carbohydrate structure [4-6]) [1, 4-6] P UDP + d-galactosyl-1,3-b-d-galactosyl-1,4-b-d-glucosyl-glycolipid 423

Lactosylceramide b-1,3-galactosyltransferase

2.4.1.179

Substrates and products S UDP-galactose + d-galactosyl-1,3-b-d-galactosyl-1,4-b-d-glucose <2> (Reversibility: ? <2> [2]) [2] P UDP + d-galactosyl-1,3-b-d-galactosyl-1,3-b-d-galactosyl-1,4-b-d-glucose S UDP-galactose + d-galactosyl-1,4-b-d-glucose <1, 2> (<1,2> i.e. lactose [1-3]; <2> best substrate [2]) (Reversibility: ? <1> [1-3]) [1-3] P UDP + d-galactosyl-1,3-b-d-galactosyl-1,4-b-d-glucose <1> [1] S UDP-galactose + d-galactosyl-1,4-b-d-glucosyl-R <1, 2> (<1> R may be an oligosaccharide or a glycolipid [1, 2]; <1> p-nitrophenyl-b-glycosides of galactose or lactose are by far the best substrates among oligosaccharides [1]; <1> substrate specificity [1, 5]; <1> hydrophobic substrates preferred [1]; <1> poor substrates are a-galactosides [1]) (Reversibility: ? <1, 2> [1-6]) [1-6] P UDP + d-galactosyl-1,3-b-d-galactosyl-1,4-b-d-glucosyl-R <1> [1, 4-6] S UDP-galactose + N-acetylgalactosamine <2> (Reversibility: ? <2> [2]) [2] P UDP + b-d-galactosyl-1,3-N-acetylgalactosamine S UDP-galactose + N-acetyllactosamine <2> (Reversibility: ? <2> [2]) [2] P UDP + b-d-galactosyl-1,3-N-acetyllactosamine S UDP-galactose + asialo-fetuin <1> (Reversibility: ? <1> [1]) [1] P UDP + b-d-galactosyl-1,3-asialo-fetuin S UDP-galactose + asialo-glycophorin A <1> (Reversibility: ? <1> [1]) [1] P UDP + b-d-galactosyl-1,3-asialo-glycophorin A S UDP-galactose + asialo-orosomucoid <1> (Reversibility: ? <1> [1]) [1] P UDP + b-d-galactosyl-1,3-asialo-orosomucoid S UDP-galactose + b-d-galactose <2> (Reversibility: ? <2> [2]) [2] P UDP + b-d-galactosyl-1,3-b-d-galactose S UDP-galactose + b-d-galactosyl-1,3-N-acetyl-d-galactosamine <1> (Reversibility: ? <1> [1]) [1] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-1,3-N-acetyl-d-galactosamine S UDP-galactose + b-d-galactosyl-1,6-N-acetylglucosylamine <2> (Reversibility: ? <2> [2]) [2] P UDP + b-d-galactosyl-1,3-b-d-galactosyl-1,6-N-acetylglucosylamine S UDP-galactose + b-d-galactosyl-O-4-nitrophenyl <1> (Reversibility: ? <1> [1]) [1] P UDP + b-d-galactosyl-1,3-b-d-galactose-O-4-nitrophenyl S UDP-galactose + ganglioside GM2 <1> (Reversibility: ? <1> [1]) [1] P UDP + b-d-galactosyl-1,3-ganglioside GM2 S UDP-galactose + lacto-N-tetraose <2> (Reversibility: ? <2> [2]) [2] P UDP + b-d-galactosyl-1,3-lacto-N-tetraose S UDP-galactose + lactosylceramide <1> (<1> best glycolipid substrate [1]) (Reversibility: ? <1> [1,3]) [1, 3] P ? S UDP-galactose + lactotriaosylceramide <1> (<1> best substrate [5]) (Reversibility: ? <1> [4-6]) [4-6] P UDP + lactotetraosylceramide <1> [4-6] S UDP-galactose + lactulose <2> (Reversibility: ? <2> [2]) [2] P UDP + b-d-galactosyl-1,3-lactulose S UDP-galactose + melibiose <2> (Reversibility: ? <2> [2]) [2] 424

2.4.1.179

P S P S P S P S P S P

Lactosylceramide b-1,3-galactosyltransferase

UDP + b-d-galactosyl-1,3-melibiose UDP-galactose + methyl-a-galactoside <2> (Reversibility: ? <2> [2]) [2] UDP + b-d-galactosyl-1,3-methyl-a-galactoside UDP-galactose + methyl-b-galactoside <1, 2> (Reversibility: ? <1,2> [1,2]) [1, 2] UDP + b-d-galactosyl-1,3-methyl-b-galactoside UDP-galactose + p-nitrophenyl-a-N-acetylgalactosylamine <2> (Reversibility: ? <2> [2]) [2] UDP + b-d-galactosyl-1,3-N-acetyl-a-galactosylaminyl-4-nitrophenyl UDP-galactose + stachyose <2> (Reversibility: ? <2> [2]) [2] UDP + b-d-galactosyl-1,3-stachyose Additional information <2> (<2> glucose is a very poor acceptor substrate [2]) [2] ?

Inhibitors EDTA <1, 2> (<2> 97% inhibition at 30 mM [2]) [2, 3] Additional information <1, 2> (<1> inhibition by salt-concentrations above 0.1 M [1]; <1> no inhibition by N-acetylgalactosamine [1]; <2> bovine or tammar a-lactalbumin [2]) [1, 2] Activating compounds CHAPSO <1> (<1> 5 to 7fold less effective compared to Triton CF-54 [5]) [5] Triton CF-54 <1> (<1> activation, optimal at 0.1% [5]) [5, 6] Triton X-100 <1> (<1> activation, can replace Triton CF-54 with 75% less efficiency [5]) [5] bovine liver non-specific transfer proteins <1> (<1> activation [5]) [5] deoxycholate <1> (<1> 5 to 7fold less effective compared to Triton CF-54 [5]) [5] glycerol <1> (<1> requirement, above 20% w/v [1]) [1] phosphatidylethanolamine <1> (<1> activation, less efficient than Triton CF-54 [5]) [5] taurodeoxycholate <1> (<1> 5 to 7fold less effective compared to Triton CF-54 [5]) [5] Additional information <1> (<1> little or no activation by phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin, Brij-58, Empigen BB or detergent G-3634-A [5]) [5] Metals, ions Ca2+ <1> (<1> activation, can replace Mn2+ with 26% efficiency [5]) [5] Cd2+ <1> [1] Co2+ <1> (<1> activation, can replace Mn2+ with 51% efficiency [5]) [5] Cu2+ <1> (<1> activation, can replace Mn2+ with 15% efficiency [5]) [5] Mg2+ <1> (<1> activation, can replace Mn2+ with 13% efficiency [5]) [5] Mn2+ <1, 2> (<2> absolute requirement [2]; <1> requirement, 25 mM [5]) [2, 3, 5] Ni2+ <1> (<1> activation, can replace Mn2+ with 16% efficiency [5]) [5] Additional information <1> (<1> no activation by Zn2+ or Cd2+ [5]) [5]

425

Lactosylceramide b-1,3-galactosyltransferase

2.4.1.179

Specific activity (U/mg) 0.00013 <1> (<1> recombinant in HCT-15 cells [6]) [6] 0.00077 <1> [5] 0.02-0.032 <2> (<2> from day 90 to day 225 post partum [2]) [2] Km-Value (mM) 0.013 <1> (lactotriaosylceramide) [5] 0.048 <1> (UDP-galactose) [5] 0.17 <1> (UDP-galactose) [1] 2.5 <1> (lactosylceramide) [1] 46 <2> (lactose) [2] 242 <1> (lactose) [1] pH-Optimum 6 <1> [1] 6.5 <2> [2] 6.8 <1> (<1> assay at [3]) [3] 7 <1> (<1> HEPES buffer [5,6]; <1> assay at [6]) [5, 6] pH-Range 5-8 <2> (<2> about 75% of maximal activity at pH 5 and about half-maximal activity at pH 8.0 [2]) [2] 5.3-7.5 <1> (<1> about 80% of maximal activity at pH 5.3 and about 55% of maximal activity at pH 7.5 [1]) [1] 5.8-8.2 <1> (<1> about half-maximal activity at pH 5.8 and pH 8.2, cacodylate or Tris-HCl buffer [5]) [5] Temperature optimum ( C) 37 <1, 2> (<1> assay at [1-6]) [1-6]

5 Isolation/Preparation/Mutation/Application Source/tissue Colo 205 cell <1> (<1> colonic adenocarcino cell line [4-6]) [4-6] SW-403 cell <1> (<1> colonic adenocarcino cell line [5]) [5] cell suspension culture <1> [5] colonic adenocarcinoma cell <1> [4, 5] kidney <1> [1, 3] mammary gland <2> [2] Additional information <1, 2> (<1> no activity in normal HCT-15 cells, human colonic adenocarcinoma cell line [6]; <2> dramatic changes during lactation [2]) [2, 6] Localization Golgi apparatus <1> [5] endoplasmic reticulum <1> [5] membrane <1> [1, 3, 5] microsome <1> [1, 3]

426

2.4.1.179

Lactosylceramide b-1,3-galactosyltransferase

Purification <1> (partial [3,5]; from Colo 205 cell line [5]) [3, 5] Cloning <1> (functional expression in HCT-15 cells [6]) [6]

6 Stability Temperature stability 36 <2> (<2> t1=2 : 20 min [2]) [2] 39 <2> (<2> 30 min, complete inactivation, in the presence of Mn2+ 11% loss of activity within 60 min [2]) [2] General stability information <1>, glycerol, 20% w/v and above, stabilizes [1] <2>, Mn2+ enhances thermal stability [2] Storage stability <1>, 4 C, in 20% glycerol, several weeks [3]

References [1] Bailly, P.; Piller, F.; Cartron, J.-P.: Characterization and specific assay for a galactoside b-3-galactosyltransferase of human kidney. Eur. J. Biochem., 173, 417-422 (1988) [2] Messer, M.; Nicholas, K.R.: Biosynthesis of marsupial milk oligosaccharides: characterization and developmental changes of two galactosyltransferases in lactating mammary glands of the tammar wallaby, Macropus eugenii. Biochim. Biophys. Acta, 1077, 79-85 (1991) [3] Bailly, P.; Piller, F.; Cartron, J.-P.: Identification of UDP-galactose: lactose (lactosylceramide) a-4 and b-3 galactosyltransferases in human kidney. Biochem. Biophys. Res. Commun., 141, 84-91 (1986) [4] Holmes, E.H.; Levery, S.B.: Preparative in vitro generation of lacto-series type 1 chain glycolipids catalyzed by b1-3-galactosyltransferase from human colonic adenocarcinoma Colo 205 cells. Arch. Biochem. Biophys., 274, 14-25 (1989) [5] Holmes, E.H.: Characterization and membrane organization of b 1-3- and b 1-4-galactosyltransferases from human colonic adenocarcinoma cell lines Colo 205 and SW403: basis for preferential synthesis of type 1 chain lactoseries carbohydrate structures. Arch. Biochem. Biophys., 270, 630-646 (1989) [6] Sherwood, A.L.; Greene, T.G.; Holmes, E.H.: Stable expression of a cDNA encoding a human b1!3galactosyltransferase responsible for lacto-series type 1 core chain synthesis in non-expressing cells: variation in the nature of cell surface antigens expressed. J. Cell. Biochem., 50, 165-177 (1992)

427

Lipopolysaccharide N-acetylmannosaminouronosyltransferase

2.4.1.180

1 Nomenclature EC number 2.4.1.180 Systematic name UDP-N-acetyl-b-d-mannosaminouronate:lipopolysaccharide N-acetyl-b-dmannosaminouronosyltransferase Recommended name lipopolysaccharide N-acetylmannosaminouronosyltransferase Synonyms ManNAcA transferase uridine diphosphoacetylmannosaminuronate-acetylglucosaminylpyrophosphorylundecaprenol acetylmannosaminuronosyltransferase CAS registry number 113478-30-1

2 Source Organism <1> Salmonella typhimurium (PR122 [1]) [1] <2> Escherichia coli (F1467, lacking enzyme activity and 21259, wild-type [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-b-d-mannosaminouronate + lipopolysaccharide = UDP + Nacetyl-b-d-mannosaminouronosyl-1,4-lipopolysaccharide Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-mannosaminuronic acid + N-acetyl-d-glucosamine-diphosphorylundecaprenol <1, 2> (<1, 2> i.e. lipid I [1]) (Reversibility: r <1, 2> [1]) [1] P UDP + ManNAcA-GlcNAc-diphosphorylundecaprenol <1, 2> (<1, 2> i.e. lipid II [1]) [1]

428

2.4.1.180

Lipopolysaccharide N-acetylmannosaminouronosyltransferase

S Additional information <1> (<1> involved in the biosynthesis of common antigen in Enterobacteriaceae [1]) [1] P ? Substrates and products S UDP-N-acetyl-d-mannosaminuronic acid + N-acetyl-d-glucosamine-diphosphorylundecaprenol <1, 2> (<1,2> i.e. lipid I [1]) (Reversibility: r <1, 2> [1]) [1] P UDP + ManNAcA-GlcNAc-diphosphorylundecaprenol <1, 2> (<1,2> i.e. lipid II [1]) [1] Temperature optimum ( C) 37 <1> (assay at) [1]

5 Isolation/Preparation/Mutation/Application Localization membrane <1> [1] Purification <1> [1] Cloning <2> (transfection of wild-type enzyme code to mutant [1]) [1]

References [1] Barr, K.; Ward, S.; Meier-Dieter, U.; Mayer, H.; Rick, P.D.: Characterization of an Escherichia coli rff mutant defective in transfer of N-acetylmannosaminuronic acid (ManNAcA) from UDP-ManNAcA to a lipid-linked intermediate involved in enterobacterial common antigen synthesis. J. Bacteriol., 170, 228-233 (1988)

429

Hydroxyanthraquinone glucosyltransferase

2.4.1.181

1 Nomenclature EC number 2.4.1.181 Systematic name UDP-glucose:hydroxyanthraquinone O-glucosyltransferase Recommended name hydroxyanthraquinone glucosyltransferase Synonyms anthraquinone-specific glucosyltransferase uridine diphosphoglucose-anthraquinone glucosyltransferase CAS registry number 112198-78-4

2 Source Organism <1> Cinchona succirubra (5 distinct glucosylating activities with different pI values and substrate specificities, named isoforms I-V [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + an hydroxyanthraquinone = UDP + a glucosyloxyanthraquinone Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + a hydroxyanthraquinone <1> (<1> a range of anthraquinones and some flavones can act as acceptors [1]) (Reversibility: ? <1> [1]) [1] P UDP + a glucosyloxyanthraquinone <1> [1] Substrates and products S UDP-glucose + 1,8-dihydroanthraquinone <1> (Reversibility: ? <1> [1]) [1] P ?

430

2.4.1.181

Hydroxyanthraquinone glucosyltransferase

S UDP-glucose + 2,6-dihydroanthraquinone <1> (Reversibility: ? <1> [1]) [1] P ? S UDP-glucose + a hydroxyanthraquinone <1> (<1> a range of anthraquinones and some flavones can act as acceptors [1]) (Reversibility: ? <1> [1]) [1] P UDP + a glucosyloxyanthraquinone <1> [1] S UDP-glucose + anthrapurpurin <1> (Reversibility: ? <1> [1]) [1] P UDP + a glucosyloxyanthrapurpurin S UDP-glucose + emodin <1> (Reversibility: ? <1> [1]) [1] P UDP + a glucosyloxyemodin S UDP-glucose + quinizarin <1> (Reversibility: ? <1> [1]) [1] P UDP + a glucosyloxyquinizarin Inhibitors Co2+ <1> (<1> 60% inhibition at 1 mM [1]) [1] Cu2+ <1> (<1> 50% inhibition at 1 mM [1]) [1] Mn2+ <1> (<1> 45% inhibition at 10 mM [1]) [1] N-ethylmaleimide <1> (<1> 92% inhibition at 1 mM [1]) [1] Zn2+ <1> (<1> 77% inhibition at 1 mM [1]) [1] iodoacetamide <1> (<1> 60% inhibition at 1 mM [1]) [1] iodoacetate <1> (<1> 30% inhibition at 1 mM [1]) [1] p-chloromercuribenzoate <1> (<1> 47% inhibition at 1 mM, 10 mM dithiothreitol protects [1]) [1] phenylmercuric acetate <1> (<1> 80% inhibition at 1 mM, 10 mM dithiothreitol protects [1]) [1] Activating compounds EDTA <1> (<1> 30% activation at 1 mM [1]) [1] dithiothreitol <1> (<1> 63% activation at 10 mM [1]) [1] Metals, ions Additional information <1> (<1> no cation requirement [1]) [1] Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 0.01 <1> (1,8-dihydroanthraquinone, <1> isoform V [1]) [1] 0.01 <1> (2,6-dihydroanthraquinone, <1> isoform IV [1]) [1] 0.01 <1> (anthrapurpurin, <1> isoform II [1]) [1] 0.01 <1> (emodin, <1> isoform I [1]) [1] 0.01 <1> (quinizarin, <1> isoform III [1]) [1] pH-Optimum 7 <1> (<1> for isoforms I-V [1]) [1] Temperature optimum ( C) 30 <1> (<1> assay at [1]) [1]

431

Hydroxyanthraquinone glucosyltransferase

2.4.1.181

4 Enzyme Structure Molecular weight 50000 <1> (<1> gel filtration [1]) [1]

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

6 Stability General stability information <1>, in absence of SH-group protectors, 50% loss of activity after 48 h, in presence of 14 mM 2-mercaptoethanol, 50% loss of activity after 2 weeks [1] Storage stability <1>, -20 C, 25 mM histidine/HCl buffer, pH 7.0, 10% glycerol, 10 mM dithioerythritol, stable for 2 months [1]

References [1] Khouri, H.E.; Ibrahim, R.K.: Purification and some properties of five anthraquinone-specific glucosyltransferases from Cinchona succirubra cell suspension culture. Phytochemistry, 26, 2531-2535 (1987)

432

Lipid-A-disaccharide synthase

2.4.1.182

1 Nomenclature EC number 2.4.1.182 Systematic name UDP-2,3-bis(3-hydroxytetradecanoyl)glucosamine:2,3-bis(3-hydroxyte tradecanoyl)-b-d-glucosaminyl-1-phosphate 2,3-bis(3-hydroxytetradecanoyl)-glucosaminyltransferase Recommended name lipid-A-disaccharide synthase Synonyms LpxB <1> [2, 7] lipid A disaccharide synthase synthase, lipid A disaccharide CAS registry number 105843-81-0

2 Source Organism <1> Escherichia coli (gene lpxB gene [2,3,7]; MC1061/pSR8, overvproducing strain [4,5]) [1-7] <2> Rhizobium leguminosarum (biovars phaseoli CE3 and viciae 8401 [8]) [8]

3 Reaction and Specificity Catalyzed reaction UDP-2,3-bis(3-hydroxytetradecanoyl)glucosamine + 2,3-bis(3-hydroxytetradecanoyl)-b-d-glucosaminyl 1-phosphate = UDP + 2,3-bis(3-hydroxytetradecanoyl)-d-glucosaminyl-1,6-b-d-2,3-bis(3-hydroxytetradecanoyl)-b-d-glucosaminyl 1-phosphate Reaction type hexosyl group transfer Natural substrates and products S 2,3-diacylglucosamine 1-phosphate + UDP-2,3-diacylglucosamine <1> (<1> involved in lipid A biosynthesis [2, 3]; <1> involved with EC

433

Lipid-A-disaccharide synthase

2.4.1.182

2.3.1.129 and EC 2.7.1.130 in the biosynthesis of the phosphorylated glycolipid, Lipid A, in the outer membrane of E. coli and other gram-negative bacteria [1,5]) [1-3, 5, 7] P 2,3-diacylglucosamine-(b-1,6)-2,3-diacylglucosamine 1-phosphate + UDP Substrates and products S 2,3-diacylglucosamine 1-phosphate + ADP-2,3-diacylglucosamine <1> (<1> no substitutes for UDP-2,3-diacylglucosamine [1]; <1> 2.66% of the activity with UDP-2,3-diacylglucosamine [4]) (Reversibility: ? <1> [4]) [4] P 2,3-diacylglucosamine-(b-1,6)-2,3-diacylglucosamine 1-phosphate + ADP S 2,3-diacylglucosamine 1-phosphate + CDP-2,3-diacylglucosamine <1> (<1> no substitutes for UDP-2,3-diacylglucosamine [1]; <1> 0.70% of the activity with UDP-2,3-diacylglucosamine [4]) (Reversibility: ? <1> [4]) [4] P 2,3-diacylglucosamine-(b-1,6)-2,3-diacylglucosamine 1-phosphate + CDP S 2,3-diacylglucosamine 1-phosphate + GDP-2,3-diacylglucosamine <1> (<1> no substitutes for UDP-2,3-diacylglucosamine [1]; <1> 0.11% of the activity with UDP-2,3-diacylglucosamine [4]) (Reversibility: ? <1> [4]) [4] P 2,3-diacylglucosamine-(b-1,6)-2,3-diacylglucosamine 1-phosphate + GDP S 2,3-diacylglucosamine 1-phosphate + TDP-2,3-diacylglucosamine <1> (<1> no substitutes for UDP-2,3-diacylglucosamine [1]; <1> 33.3% of the activity with UDP-2,3-diacylglucosamine [4]) (Reversibility: ? <1> [4]) [4] P 2,3-diacylglucosamine-(b-1,6)-2,3-diacylglucosamine 1-phosphate + TDP S 2,3-diacylglucosamine 1-phosphate + UDP-2,3-diacylglucosamine <1, 2> (<2> lipid X isolated from E. coli [8]; <1> strong preference for 2,3-diacylated substrates, but the (R)-3-hydroxy substituent is not required [5]; <1,2> 2,3-diacylglucosamine 1-phosphate is lipid X [4-6,8]) (Reversibility: ir <1> [2,5,7]; ? <1,2> [1,3,4,6,8]) [1-8] P 2,3-diacylglucosamine-(b-1,6)-2,3-diacylglucosamine 1-phosphate + UDP <1> (<1> product analysis [1]) [1-8] S 2,3-diacylglucosamine 1-phosphate + UDP-3-O-((R)-3-hydroxymyristoyl)GlcNAc <1> (<1> 0.44% of the activity with UDP-2,3-diacylglucosamine [4]) (Reversibility: ? <1> [4]) [4] P ? Inhibitors octyl-b-d-glucoside <1> [1] Activating compounds Additional information <1> (<1> no requirement for detergent [1,4,5]) [1, 4, 5] Metals, ions Additional information <1> (<1> no requirement for divalent cations [1]) [1]

434

2.4.1.182

Lipid-A-disaccharide synthase

Specific activity (U/mg) 9 <1> (<1> purified enzyme [7]) [7] 15.9 <1> (<1> purified enzyme [4,5]) [4, 5] Additional information <1, 2> (<2> coupled assay, first 3 steps of lipid A biosynthesis [8]; <1> specific activity in different recombinant strains [3]) [3, 8] Km-Value (mM) 0.11 <1> (UDP-2,3-diacylglucosamine) [4] 0.27 <1> (2,3-diacylglucosamine 1-phosphate) [4] pH-Optimum 8 <1, 2> (<1,2> assay at [4,5,8]) [1, 4, 5, 8] Temperature optimum ( C) 25 <1> (<1> assay at [4]) [4] 30 <2> (<2> assay at [8]) [8] 37 <1> (<1> assay at [4]) [4]

4 Enzyme Structure Molecular weight 86000 <1> (<1> gel filtration [4]) [4] Additional information <1> (<1> glycerol-3-phosphate dehydrogenase specifically binds to purified His-tagged enzyme [7]; <1> nucleotide sequence [1]) [1, 7] Subunits ? <1> (<1> x * 42000, SDS-PAGE [3]; <1> x * 42339, DNA sequence determination [2,5]) [2, 3, 5] dimer <1> (<1> 2 * 42000, SDS-PAGE [4]) [4]

5 Isolation/Preparation/Mutation/Application Localization cytosol <1> [1, 5] Additional information <1> (<1> enzyme possibly interacts with membranes due to its extreme hydrophobic reaction product [1]) [1] Purification <1> (purification as recombinant His-tagged protein from overproducing strain in presence of Triton X-100, copurification of glycerol-3-phosphate dehydrogenase specifically bound to the enzyme [7]; purification of lpxB-lacZ gene product [2]) [2, 4, 5, 7] Cloning <1> (overexpression from plasmid as His-tagged protein [7]; overexpression in Escherichia coli from plasmid, alone and together with gene lpxA, UDP435

Lipid-A-disaccharide synthase

2.4.1.182

GlcNAc acyltransferase [3]; expression from plasmid, DNA sequence analysis, lpxB gene is cotranscribed with the lpxA gene located in the same operon [2]) [2, 3, 7] Application synthesis <1> (<1> synthesis of a variety of artificial fluorinated lipid A precursor analogues on a preparative scale for investigation of structure-function relationships of lipid A derivatives [6]) [6]

6 Stability Temperature stability 60 <1> (<1> 30 min, inactivation [1]) [1] Storage stability <1>, -80 C, frozen in liquid N2 , stable for several years [5]

References [1] Ray, B.L.; Painter, G.; Raetz, C.R.H.: The biosynthesis of gram-negative endotoxin. Formation of lipid A disaccharides from monosaccharide precursors in extracts of Escherichia coli. J. Biol. Chem., 259, 4852-4859 (1984) [2] Crowell, D.N.; Reznikoff, W.S.; Raetz, C.R.H.: Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase. J. Bacteriol., 169, 5727-5734 (1987) [3] Crowell, D.N.; Anderson, M.S.; Raetz, C.R.H.: Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli. J. Bacteriol., 168, 152-159 (1986) [4] Radika, K.; Raetz, C.R.H.: Purification and properties of lipid A disaccharide synthase of Escherichia coli. J. Biol. Chem., 263, 14859-14867 (1988) [5] Raetz, C.R.H.: Lipid A disaccharide synthase from Escherichia coli. Methods Enzymol., 209, 455-466 (1992) [6] Vyplel, H.; Scholz¹ D.; Loibner¹ H.; Kern, M.; Bednarik, K.; Schaller, H.: Synthesis of fluorinated analogues of lipid A. Tetrahedron Lett., 33, 1261-1264 (1992) [7] Milla, M.E.; Raetz, C.R.H.: Association of lipid A disaccharide synthase with aerobic glycerol-3-phosphate dehydrogenase in extracts of Escherichia coli. Biochim. Biophys. Acta, 1304, 245-253 (1996) [8] Price, N.P.J.; Kelly, T.M.; Raetz, C.R.H.; Carlson, R.W.: Biosynthesis of a structurally novel lipid A in Rhizobium leguminosarum: Identification and characterization of six metabolic steps leading from UDP-GlcNAc to 3deoxy-d-manno-2-octulosonic acid2 -lipid IVA. J. Bacteriol., 176, 4646-4655 (1994)

436

a-1,3-Glucan synthase

2.4.1.183

1 Nomenclature EC number 2.4.1.183 Systematic name UDP-glucose:a-d-(1-3)-glucan 3-a-d-glucosyltransferase Recommended name a-1,3-glucan synthase Synonyms 1,3-a-d-glucan synthase glucosyltransferase, uridine diphosphoglucose-1,3-a-glucan uridine diphosphoglucose-1,3-a-glucan glucosyltransferase CAS registry number 113478-38-9

2 Source Organism <1> Streptococcus mutans (AHT strain [1]) [1] <2> Lactobacillus helveticus (strain NCC2745 [2]; gene epsF [2]) [2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + [a-d-glucosyl-(1!3)]n = UDP + [a-d-glucosyl-(1!3)]n+1 (a glucan primer is needed to start the reaction, which brings about elongation of the glucan chains) Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + [a-d-glucosyl-(1-3)]n <1> [1] P UDP + [a-d-glucosyl-(1-3)]n+1

437

a-1,3-Glucan synthase

2.4.1.183

Substrates and products S UDP-glucose + 2-(octadecylthio)-ethyl-b-d-glucose <2> (<2> synthetic acceptor composed of a monosaccharide linked to a long hydrophobic aliphatic chain, -CH2 CH2 SC18 H37 [2]) (Reversibility: ? <2> [2]) [2] P UDP + 2-(octadecylthio)-ethyl-b-d-glucose-1,3-a-d-glucose <2> [2] S UDP-glucose + [a-d-glucosyl-(1-3)]n <1, 2> (<2> substrate specificity [2]; <2> enzyme forms a-1,3- and a-1,6-linkages onto the initial b-d-glucose-phosphate [2]; <2> enzyme EpsF: introduction of an a-1,3-linkage branch at the first b-d-glucose-phosphate of the side-chain [2]; <1> introduction of an a-1,3-linkage branch at the C3-position to the a-1,6chain [1]; <1> enzyme catalyzes both the hydrolysis of sucrose to glucose and fructose and the glucosyl transfer to glucosyl polymers to yield waterinsoluble glucan involving 2 different active sites, the enzyme catalyzes only sucrose hydrolysis however in the absence of 1,6-a-d-glucan as acceptor [1]; <1> a glucan primer is needed to begin the reaction which brings about elongation of the glucan chains [1]) (Reversibility: ? <1,2> [1,2]) [1, 2] P UDP + [a-d-glucosyl-(1-3)]n+1 <1, 2> (<2> product analysis [2]) [1, 2] S Additional information <2> (<2> no activity with p-aminophenyl, p-nitrophenyl, and 6-(fluorescein-5-carboxamido)-hexanoic acid succimidyl ester [2]) [2] P ? Inhibitors antiserum against the isolated 1,3-a-d-glucan synthase <1> (<1> glucosyl transfer activity is completely inhibited, not sucrose hydrolysis activity [1]) [1] Specific activity (U/mg) Additional information <2> (<2> assay development [2]) [2] pH-Optimum 6 <1> (<1> assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification <1> [1] <2> (recombinant as His-tagged or GST-tagged proteins from Escherichia coli [2]) [2] Cloning <2> (cloning of eps gene cluster from genomic library and expression in Escherichia coli as His-tagges or GST-tagged proteins, DNA sequence analysis [2]) [2]

438

2.4.1.183

a-1,3-Glucan synthase

References [1] Yamashita, Y.; Hanada, N.; Itoh-Andoh, M.; Takehara, T.: Evidence for the presence of two distinct sites of sucrose hydrolysis and glucosyl transfer activities on 1,3-a-d-glucan synthase of Streptococcus mutans. FEBS Lett., 243, 343-346 (1989) [2] Jolly, L.; Newell, J.; Porcelli, I.; Vincent, S.J.F.; Stingele, F.: Lactobacillus helveticus glycosyltransferases: from genes to carbohydrate synthesis. Glycobiology, 12, 319-327 (2002)

439

Galactolipid galactosyltransferase

2.4.1.184

1 Nomenclature EC number 2.4.1.184 Systematic name mono-b-d-galactosyldiacylglycerol:mono-b-d-galactosyldiacylglycerol b-dgalactosyltransferase Recommended name galactolipid galactosyltransferase Synonyms GGGT galactolipid:galactolipid galactosyltransferase galactosyltransferase, galactolipid-galactolipid interlipid galactosyltransferase CAS registry number 66676-74-2

2 Source Organism <1> Spinacia oleracea (cv. New Asia [6]) [1-6]

3 Reaction and Specificity Catalyzed reaction 2 mono-b-d-galactosyldiacylglycerol = a-d-galactosyl-b-d-galactosyldiacylglycerol + diacylglycerol Reaction type hexosyl group transfer Natural substrates and products S mono-b-d-galactosyldiacylglycerol + mono-b-d-galactosyldiacylglycerol <1> (Reversibility: ? <1> [2]) [2] P a-d-galactosyl-b-d-galactosyldiacylglycerol + diacylglycerol <1> (<1> monogalactosyldiacylglycerol synthesis, maintaining of a low concentration of diacylglycerol in the chloroplast outer envelope membrane [5]) [5]

440

2.4.1.184

Galactolipid galactosyltransferase

Substrates and products S mono-b-d-galactosyldiacylglycerol + mono-b-d-galactosyldiacylglycerol <1> (<1> digalactosyldiacylglycerol synthesis proceeds most rapidly by galactosyl transfer to hexaene species of monogalactosyldiacylglycerol [4]) (Reversibility: r <1> [5]) [1-6] P a-d-galactosyl-b-d-galactosyldiacylglycerol + diacylglycerol <1> (<1> by further transfer of galactosyl residues to the digalactosyldiacylglycerol, trigalactosyldiacylglycerol and tetragalactosyldiacylglycerol are also formed [2-4]) [1-6] Inhibitors CdCl2 <1> (<1> 2 mM, 50% inhibition [4]) [4] N-ethylmaleimide <1> [4] UDP <1> (<1> no effect [5]; <1> 8 mM, 50% inhibition [4]) [4] UMP <1> (<1> 10 mM, 50% inhibition [4]) [4] Zn2+ <1> (<1> 2 mM, complete inhibition [3]; <1> 1 mM, 75% inhibition [4]) [3, 4] p-hydroxymercuribenzoate <1> [4] thermolysin <1> [5] Activating compounds NH+4 <1> (<1> 50 mM, 5fold stimulation [4]) [4] a-linolenic acid <1> (<1> 0.3 mM, 28fold stimulation in the presence of 2 mM MgCl2 , MgCl2 can be replaced by MnCl2 , CaCl2 or high concetrations of KCl [6]) [6] hexadecatrienoic acid <1> (<1> approx. 3fold activation [6]) [6] linoleic acid <1> (<1> approx. 4fold activiation [6]) [6] oleic acid <1> (<1> approx. 4fold activiation [6]) [6] palmitic acid <1> (<1> approx. 2fold activiation [6]) [6] palmitoleic acid <1> (<1> approx. 4fold activiation [6]) [6] stearic acid <1> (<1> approx. 2fold activiation [6]) [6] Metals, ions Ba2+ <1> (<1> 10 mM, 23fold stimulation [4]) [4] Ca2+ <1> (<1> 10 mM, 23fold stimulation [4]; <1> a-linolenic acid stimulates activity in presence of CaCl2 [6]) [4, 6] Co2+ <1> (<1> 10 mM, 3fold simulation [4]) [4] Fe2+ <1> (<1> 10 mM, 6fold simulation [4]) [4] K+ <1> (<1> 50 mM, weak stimulation [3]; <1> a-linolenic acid stimulates in presence of high concentrations of KCl [6]; <1> 50 mM, 5fold stimulation [4]) [3, 4, 6] Li+ <1> (<1> 50 mM, weak stimulation [3]) [3] Mg2+ <1> (<1> stimulates forward reaction, no effect on reverse reaction [5]; <1> 10 mM, 20fold stimulation [4]; <1> EDTA inhibits stimulation [3,6]; <1> unsaturated 16- and 18-carbon fatty acids stimulate in presence of MgCl2 [6]; <1> a-linolenic acid causes drastic increase in activity under limiting concentrations of MgCl2 , without affecting its maximum activity at higher MgCl2

441

Galactolipid galactosyltransferase

2.4.1.184

concentration, free a-linolenic acid alone does not affect the activity [6]) [36] Mn2+ <1> (<1> a-linolenic acid stimulates activity in presence of MnCl2 [6]; <1> 10 mM, 23fold stimulation [4]) [3, 4, 6] Na+ <1> (<1> 50 mM, weak stimulation [3]) [3] pH-Optimum 5.9-7 <1> [3] 6-7 <1> (<1> forward reaction [5]) [5] 6.5 <1> (<1> reverse reaction [5]) [5] 8 <1> (<1> assay at [2]) [2] pH-Range 4.7-8.9 <1> (<1> approx. 50% of maximal activity at pH 5.3 and pH 8.0 [3]) [3] Temperature optimum ( C) 30 <1> (<1> assay at [4]) [4] Temperature range ( C) 4-30 <1> (<1> still active at 4 C [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [3, 6] Localization chloroplast envelope <1> (<1> envelope [3-6]; <1> outer envelope membrane [1,2,5]; <1> outer surface of [2]) [1-6] chloroplast outer membrane <1> [1]

References [1] Heemskerk, J.W.M.; Wintermans, J.F.G.M.; Joyard, J.; Block, M.A.; Dorne, A.J.; Douce, R.: Localization of galactolipid:galactolipid galactosyltransferase and acyltransferase in outer envelope membrane of spinach chloroplasts. Biochim. Biophys. Acta, 877, 281-289 (1986) [2] Dorne, A.J.; Block, M.A.; Joyard, J.; Douce, R.: The galactolipid:galactolipid galactosyltransferase is located on the outer surface of the outer membrane of the chloroplast envelope. FEBS Lett., 145, 30-34 (1982) [3] Heemskerk, J.W.; Bögemann, G.; Wintermans, J.F.G.M.: Turnover of galactolipids incorporated into chloroplast envelopes an assay for galactolipid:galactolipid galactosyltransferase. Biochim. Biophys. Acta, 754, 181-189 (1983) [4] Heemskerk, J.W.M.; Jacobs, F.H.H.; Scheijen, M.A.M.; Helsper, J.P.F.G.; Wintermans, J.F.G.M.: Characterization of galactosyltransferases in spinach chloroplast envelopes. Biochim. Biophys. Acta, 918, 189-203 (1987)

442

2.4.1.184

Galactolipid galactosyltransferase

[5] Heemskerk, J.W.M.; Jacobs, F.H.H.; Wintermans, J.F.G.M.: UDPgalactose-independent synthesis of monogalactosyldiacylglycerol. An enzymatic activity of the spinach chloroplast envelope. Biochim. Biophys. Acta, 961, 38-47 (1988) [6] Sakaki, T.; Kondo, N.; Yamada, M.: Free fatty acids regulate two galactosyltransferases in chloroplast envelope membranes isolated from spinach leaves. Plant Physiol., 94, 781-787 (1990)

443

Flavanone 7-O-b-glucosyltransferase

2.4.1.185

1 Nomenclature EC number 2.4.1.185 Systematic name UDP-glucose:flavanone 7-O-b-d-glucosyltransferase Recommended name flavanone 7-O-b-glucosyltransferase Synonyms UDPglucose:flavanone 7-O-b-d-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-flavanone 7-Ohesperetin 7-O-glucosyl-transferase naringenin 7-O-glucosyltransferase CAS registry number 125752-73-0

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

Citrus paradisi (grapefruit [1]) [1, 2] Citrus mitis [3] Citrus maxima [3] Petunia hybrida [4]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + a flavanone = UDP + a flavanone 7-O-b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + 4',5',7-trihydroxyflavanone <1-4> (<1> trivial name naringenin [1,2]; <1> enzyme does not accept other flavanone or flavonol aglycones [1]; <4> peak I-III [4]) (Reversibility: ? <1-4> [1, 3, 4]) [1-4] P UDP + naringenin 7-O-glucoside <1-4> (<1> trivial name prunin [1]) [14]

444

2.4.1.185

Flavanone 7-O-b-glucosyltransferase

Substrates and products S UDPglucose + 2',4',6',4-tetrahydroxychalcone <1> (<1> 64% of activity with naringenin [2]) (Reversibility: ? <1> [2]) [2] P UDP + 2',4',6',4-tetrahydroxychalcone 4'-O-glucoside <1> [2] S UDPglucose + 3',5,7-trihydroxy-4-methoxyflavanone <1-4> (<1> trivial name hesperetin, 83% of activity with naringenin [1]; <4> peak I, 70% of activity with naringenin) (Reversibility: ? <1-4> [1-4]) [1-4] P UDP + hesperetin 7-O-glucoside <1-4> [1-4] S UDPglucose + 4',5',7-trihydroxyflavanone <1-4> (<1> trivial name naringenin [1,2]; <1> enzyme does not accept other flavanone or flavonol aglycones [1]; <4> peak I-III [4]) (Reversibility: ? <1-4> [1, 3, 4]) [1-4] P UDP + naringenin 7-O-glucoside <1-4> (<1> trivial name prunin [1]) [14] S UDPglucose + apigenin <4> (<4> peak I-III, 49%, 45% and 95% of activity with naringenin respectively [4]) (Reversibility: ? <4> [4]) [4] P UDP + apigenin 7-O-glucoside <4> [4] S UDPglucose + kaempferol <1, 4> (<1> 90% of activity with naringenin [2]; <4> peak I-III, 50%, 46% and 70% of activity with naringenin respectively [4]) (Reversibility: ? <1, 4> [2, 4]) [2, 4] P UDP + kaempferol 7-O-glucoside <1, 4> [2, 4] S UDPglucose + luteolin <4> (<4> peak I, 52% of activity with naringenin [4]) (Reversibility: ? <4> [4]) [4] P UDP + luteolin 7-O-glucoside <4> [4] S UDPglucose + quercetin <1, 4> (<1> 164% of activity with naringenin [2]; <4> peak I, 42% of activity with naringenin [4]) (Reversibility: ? <1, 4> [2, 4]) [2, 4] P UDP + quercetin 7-O-glucoside <1, 4> [2, 4] Inhibitors ADP <1> (<1> 10 mM, strong [2]) [2] ATP <1> (<1> 10 mM, strong [2]) [2] NADPH <1> (<1> 10 mM, weak [2]) [2] TTP <1> (<1> 10 mM, strong [2]) [2] UDP <1, 4> (<1> 10 mM, weak [2]; <4> competitive inhibition [4]) [2, 4] UTP <1> (<1> 10 mM, strong [2]) [2] Metals, ions Additional information <1, 4> (<1> CaCl2 , MnCl2 , NaCl, MgCl2 at 0.1 mM, 1.0 mM and 10 mM have no effect [2]; <4> activity is not affected by CaCl2 or MgCl2 [4]) [2, 4] Specific activity (U/mg) 0.0017 <1> [1] Km-Value (mM) 0.008 <4> (naringenin, <4> peak III [4]) [4] 0.01 <4> (naringenin, <4> peak I [4]) [4] 0.027 <4> (naringenin, <4> peak II [4]) [4] 0.063 <1> (naringenin) [1] 445

Flavanone 7-O-b-glucosyltransferase

2.4.1.185

0.074 <4> (UDPglucose, <4> peak II [4]) [4] 0.08 <1> (naringenin) [2] 0.11 <1> (hesperetin) [2] 0.124 <1> (hesperetin) [1] 0.13 <1> (2',4',6',4-tetrahydroxychalcone) [2] 0.243 <1> (UDPglucose) [1] 0.269 <4> (UDPglucose, <4> peak I [4]) [4] 0.37 <1> (UDPglucose) [2] Ki-Value (mM) 0.00089 <4> (UDP, <4> peak I [4]) [4] 0.074 <4> (UDP, <4> peak II [4]) [4] pH-Optimum 6.5-7.5 <1> [2] 7.5 <1, 4> (<1> naringenin [1]; <4> 50% of maximal activity at pH 6.5, 62% of maximal activity at pH 9.0 [4]) [1, 4] 7.5-8 <1> (<1> hesperetin [1]) [1] pH-Range 5.5-7.5 <1> (<1> 60% of maximal activity at pH 5.5 and pH 7.5 [1]) [1] Temperature optimum ( C) 30 <1> (<1> assay at [1]) [1] 37 <1> [2]

4 Enzyme Structure Molecular weight 54400 <4> (<4> gel filtration [4]) [4] 54900 <1> (<1> gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue flower bud <4> [4] fruit <2, 3> [3] leaf <1, 2, 3, 4> (<3> highest actiity in young leaves [3]) [1, 3, 4] seedling <1> (<1> highest activity in leaves, more than in stem and roots [2]) [2] sepal <4> [4] Purification <1> (ammonium sulfate, Sephadex G-100, hydroxyapatite, UDP-glucuronic acid agarose, Mono Q, Mono P [1]) [1, 2] <4> (ammonium sulfate, Superose 12, Mono Q, 3 peaks with 7-O-glucosyltransferase activity [4]) [4]

446

2.4.1.185

Flavanone 7-O-b-glucosyltransferase

6 Stability Storage stability <1>, 4 C, 1% bovine serum albumin, 2-3 days, no loss of activity [2]

References [1] McIntosh, C.A.; Latchinian, L.; Mansell, R.L.: Flavanone-specific 7-O-glucosyltransferase activity in Citrus paradisi seedlings: purification and characterization. Arch. Biochem. Biophys., 282, 50-57 (1990) [2] McIntosh, C.A.; Mansell R.L.: Biosynthesis of naringin in Citrus paradisi: UDP-glucosyl-transferase activity in grapefruit seedlings. Phytochemistry, 29, 1533-1538 (1990) [3] Lewinsohn, E.; Britsch, L.; Mazur, Y.; Gressel, J.: Flavanone glycoside biosynthesis in Citrus. Chalcone synthase, UDP-glucose: flavanone-7-O-glucosyltransferase and -rhamnosyltransferase activities in cell-free extracts. Plant Physiol., 91, 1323-1328 (1989) [4] Durren, R.L.; McIntosh, C.A.: Flavanone-7-O-glucosyltransferase activity from Petunia hybrida. Phytochemistry, 52, 793-798 (1999)

447

Glycogenin glucosyltransferase

2.4.1.186

1 Nomenclature EC number 2.4.1.186 Systematic name UDP-glucose:glycogenin glucosyltransferase Recommended name glycogenin glucosyltransferase Synonyms M-glycogenin <2, 3> [3] glycogenin glycosyltransferase <1> [1] glyogenin priming glucosyltransferase CAS registry number 117590-73-5

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

Oryctolagus cuniculus [1, 2, 4-6] Neurospora crassa [3] Escherichia coli [3] Rattus norvegicus [5, 6] Gallus gallus (hen [5]) [5] Mus musculus [5] Bos taurus [5, 6] Coturnix sp. (quail [5]) [5]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + glycogenin = UDP + glucosylglycogenin (The glycogenin subunit of glycogen synthase (EC 2.4.1.11, glycogen(starch) synthase) catalyses this reaction, i.e. the enzyme catalyses its own autoglycosylation. Five molecules of glucose can be transferred to one molecule of glycogenin. The product acts as a primer for the reaction catalysed by glycogen synthase.; <1, 4, 7> stereochemistry and mechanism [5]; <1-4, 7> highly conserved protein

448

2.4.1.186

Glycogenin glucosyltransferase

[3, 5, 6]; <1> initiation of glycogen biosynthesis is a 2-step mechanism, requiring first the covalent attachment of a glucose residue to Tyr-194 of glycogenin and then elongation to form an oligosaccharide chain [6]; <2> independent active sites for glucosylation of exogenous and self-acceptors [3]) Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + glycogenin <1-7> (<1,4> regulation [5]; <1-4> enzyme forms the protein part of proteoglycogen [3-5]; <1,4> the glycogenin subunit of glycogen synthase, EC 2.4.1.11, catalyzes this reaction, i.e. the enzyme catalyzes its own glucosylation [1,2,5]) [1-5] P UDP + glucosylated glycogenin Substrates and products S CDP-glucose + glycogenin <4> (<4> recombinant enzyme expressed in E. coli, 71% activity compared to UDP-glucose [5]) (Reversibility: ? <4> [5]) [5] P CDP + glucosylated glycogenin <4> [5] S CDP-glucose + p-nitrophenyl-a-maltoside <4> (<4> recombinant enzyme expressed in E. coli [5]) (Reversibility: ? <4> [5]) [5] P CDP + glucosylated p-nitrophenyl-a-maltoside S TDP-glucose + glycogenin <4> (<4> recombinant enzyme expressed in E. coli, 33% activity compared to UDP-glucose [5]) (Reversibility: ? <4> [5]) [5] P TDP + glucosylated glycogenin <4> [5] S TDP-glucose + p-nitrophenyl-a-maltoside <4> (<4> recombinant enzyme expressed in E. coli [5]) (Reversibility: ? <4> [5]) [5] P TDP + glucosylated p-nitrophenyl-a-maltoside S UDP-galactose + glycogenin <1> (<1> autoglycosylation reaction [4]) (Reversibility: ? <1> [4]) [4] P UDP + galactosylated glycogenin <1> [4] S UDP-glucose + N-(maltosyl-a-1,4-(1-deoxyglucitol))-peptide <1> (<1> peptide sequence: SISIYSYLP [4]; <1> simultaneously and independently of the autoglycosylation reaction [4]) (Reversibility: ? <1> [4]) [4] P UDP + glucosylated N-(maltosyl-a-1,4-(1-deoxyglucitol))-peptide S UDP-glucose + glycogenin <1-7> (<1-7> autoglycosylation reaction [1-6]; <4> one attached glucose molecule is needed for intramolecular self-glucosylation [5]; <1> i.e. unprimed glycogenin [1]; <1> no activity with UDP-N-acetylglucosamine and GDP-mannose [4]; <4> no activity with CDP-glucose [5]; <2,4> UDP-glucose can not be replaced by ADP- or GDP-glucose [3,5]; <1-7> enzyme forms the protein part of proteoglycogen [3-5]) (Reversibility: ? <1-7> [1-6]) [1-6] P UDP + glucosylated glycogenin <1-7> (<1> glucose molecule is attached to Tyr-194 [6]; <1-7> forms glucosyl-a1,4-glucosyl linkage [3,5]; <1> i.e. primed glycogenin [1]; <1> glucosylation reaches a plateau, when 5 additional glucose residues have been added to glycogenin [1]) [1-6]

449

Glycogenin glucosyltransferase

2.4.1.186

S UDP-glucose + n-dodecyl-b-d-maltoside <1-4, 7> (<4,7> hydrophobic nature of the aglycon is required for binding to the active site [5]; <7> renal enzyme [5]; <2-4,7> transglucosylation reaction [3,5]; <1-3> simultaneously and independently of the autoglycosylation reaction [3,4]) (Reversibility: ? <1-4,7> [3-5]) [3-5] P UDP + n-dodecyl-b-d-maltotriose <2> [3] S UDP-glucose + n-octyl-a-d-maltoside <4, 7> (<4,7> hydrophobic nature of the aglycon is required for binding to the active site [5]; <4,7> transglucosylation reaction [5]) (Reversibility: ? <4,7> [5]) [5] P ? S UDP-glucose + n-octyl-b-d-maltoside <4, 7> (<4,7> hydrophobic nature of the aglycon is required for binding to the active site [5]; <4,7> transglucosylation reaction [5]) (Reversibility: ? <4,7> [5]) [5] P ? S UDP-glucose + n-tetradecyl-b-d-maltoside <4, 7> (<4,7> hydrophobic nature of the aglycon is required for binding to the active site [5]; <4,7> transglucosylation reaction [5]) (Reversibility: ? <4,7> [5]) [5] P ? S UDP-xylose + glycogenin <4-6> (<4> renal and skeletal muscle glycogenin, lower activity compared to UDP-glucose [5]; <4-6> autoglycosylation reaction [5]) (Reversibility: ? <4-6> [5]) [5] P UDP + xylosylated glycogenin <4-6> [5] S UDP-xylose + n-dodecyl-b-d-maltoside <4> (<4> transglucosylation reaction [5]) (Reversibility: ? <4> [5]) [5] P ? S Additional information <1> (<1> no activity with nonapeptide SISIYSYLP and N-lactosylated peptide [4]) [4] P ? Inhibitors ADP <1> (<1> skeletal muscle enzyme, allosteric inhibition of autoglucosylation [5]) [5] ATP <1, 4> (<4> slight inhibition, renal enzyme [5]; <1> skeletal muscle enzyme, allosteric inhibition of autoglucosylation, complete inhibition at 5 mM, possible role as natural regulator [5]) [5] CDP <4> (<4> renal enzyme, 90% inhibition at 0.025 mM [5]) [5] CDP-choline <4> (<4> renal enzyme, 75% inhibition at 0.1 mM [5]) [5] CDP-glucose <4> [5] EDTA <2> [3] TDP-glucose <4> [5] UDP <1, 2> (<1,2> inhibits both autoglucosylation and glucosylation of exogenous acceptor [3,4]) [3, 4] UDP-xylose <4> (<4> competitive inhibitor to glucosylation of glycogenin by UDP-glucose [5]) [5] maltose <4, 7> (<4,7> very poor, 50% inhibition at 40 mM [5]) [5] phosphate <2> (<2> slight inhibition at 100 mM [3]) [3]

450

2.4.1.186

Glycogenin glucosyltransferase

Additional information <1, 8> (<8> enzyme is phosphorylated in embryonic muscle [5]; <1> no inhibition by phosphorylation of catalytic subunit of the glycogen synthase complex [1]; <1> not inhibitory: UDP-pyridoxal, LiBr [1]) [1, 5] Activating compounds Additional information <8> (<8> enzyme is phosphorylated in embryonic muscle [5]) [5] Metals, ions Mg2+ <1> (<2> can not substitute Mn2+ [3]; <1> less effective than Mn2+ [1]) [1] Mn2+ <1, 2> (<1,2> dependent on, also required for transglucosylation [3,4]; <1> absolutely dependent on divalent cations, Mn2+ can be replaced by Mg2+ with less effectivity [1]) [1, 3, 4, 6] Specific activity (U/mg) Additional information <1> [4] Km-Value (mM) 0.002 <1> (UDP-glucose) [1] 0.1 <4> (n-dodecyl-b-d-maltoside) [5] 3 <4> (p-nitrophenyl-a-maltoside) [5] Additional information <1, 4-7> (<1,4-7> Km -values for UDP-glucose in different tissues [5]) [5] pH-Optimum 7 <1, 2> (<1,2> assay at [1-4]) [1-4] Temperature optimum ( C) 30 <1, 2> (<1,2> assay at [1-4]) [1-4]

4 Enzyme Structure Molecular weight 31000 <2> (<2> SDS-PAGE [3]) [3] 32000 <4> (<4> kidney enzyme [5]) [5] 37280 <1> (<1> amino acid sequence determination [5]) [5] 38000 <1> (<1> gel filtration, SDS-PAGE, glycogenin glucosyltransferase represents the smaller subunit of glycogen synthase [1,2]) [1, 2] 124000 <1> (<1> gel filtration, heterodimeric glycogen synthase complex [1]) [1] 200000 <1> (<1> gel filtration, active proteoglycogen [4]) [4] Subunits ? <2> (<2> x * 31000, SDS-PAGE [3]) [3] Additional information <1-4, 7> (<1,4,7> in muscle a glycogen b-particle is bound to glycogenin in a 1:1 ratio, the enzyme/glycogen ratio in liver is lower [5]; <1-4,7> enzyme forms the protein part of proteoglycogen [3-5]; <1> pur-

451

Glycogenin glucosyltransferase

2.4.1.186

ified glycogenin associates as a dimer in absence of SDS, MW 64 kDa, glycerol density gradient centrifugation [2]; <1,4> glycogenin glucosyltransferase, MW 38 kDa, represents the smaller subunit of glycogen synthase, both enzyme form a heterodimeric complex of molar ratio 1:1 [1,2,5]) [1-5] Posttranslational modification glycoprotein <1-6> (<1-6> autoglycosylation [1-6]; <1> 1 single glucose attachment site at tyrosine-194 [5,6]; <4> one attached glucose molecule is needed for intramolecular self-glucosylation [5]; <1> five molecules of glucose can be transferred to one molecule of glycogenin, which contains already 1 glucose molecule prior to autoglycosylation [1]) [1-6]

5 Isolation/Preparation/Mutation/Application Source/tissue embryo <8> [5] kidney <1, 4, 7> (<1> low content [6]) [5, 6] liver <1, 4> (<1> very low content [6]) [5, 6] mastocytoma <6> [5] mycelium <2> [3] oviduct <5> [5] skeletal muscle <1, 4, 7, 8> [1, 2, 4-6] Additional information <1, 4, 7> (<7> a glycogenin-like protein has also been found in retina [5]; <4> a glycogenin-like protein has also been found in thymus, brain, heart [5]; <1,4> enzyme activity does not depend on physiological state of the organism [5]) [5] Localization cytosol <4> [5] Additional information <1, 4, 7> (<1,4,7> not membrane-bound [5]) [5] Purification <1> (separation from proteoglycogen [4]; separation from glycogen synthase, EC 2.4.1.11, by LiBr [1,2,5]) [1, 2, 4, 5] <2> (separation from proteoglycogen [3]) [3] Cloning <1> (expression in COS cells [5]; functional expression of the wild-type enzyme in Escherichia coli [6]; expression of mutant Y194F in Escherichia coli [5,6]) [5, 6] <4> (expression of glucose-free apo-glycogenin in an Escherichia coli mutant lacking UDP-glucose, enzyme is active towards itself and other substrates [5]) [5] Engineering Y194F <1, 4> (<4> mutant glycosylates other substrates with nearly the same activity as the wild-type [5]; <1,4> exchange of glucose attachment site, no autoglucosylation activity [5,6]) [5, 6]

452

2.4.1.186

Glycogenin glucosyltransferase

Y194T <4> (<4> exchange of glucose attachment site, no autoglucosylation activity, mutant glycosylates other substrates but with less activity compared to the wild-type [5]) [5] Application analysis <1, 4-7> (<1,4-7> development of assay method with n-dodecyl-bd-maltoside as substrate [5]) [5]

6 Stability General stability information <2>, loss of activity on Q-Sepharose during ion exchange chromatography [3] <4-6>, alkali-stable [5] Storage stability <2>, -20 C, amylolyzed M-glycogenin preparation, at least 1 month without appreciable loss of activity [3] <2>, 4 C, amylolyzed M-glycogenin preparation, at least 1 week without appreciable loss of activity [3]

References [1] Pitcher, J.; Smythe, C.; Cohen, P.: Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle. Eur. J. Biochem., 176, 391-395 (1988) [2] Pitcher, J.; Smythe, C.; Campell, D.G.; Cohen, P.: Identification of the 38-kDa subunit of rabbit skeletal muscle glycogen synthase as glycogenin. Eur. J. Biochem., 169, 497-502 (1987) [3] Goldraij, A.; Curtino, J.A.: M-glycogenin, the protein moiety of Neurospora crassa proteoglycogen, is an auto- and transglucosylating enzyme. Biochem. Biophys. Res. Commun., 227, 909-914 (1996) [4] Carrizo, M.E.; Miozzo¹ M.C.; Goldraij, A.; Curtino, J.A.: Purification of rabbit skeletal muscle proteoglycogen: studies on the glucosyltransferase activity of polysaccharide-free and -bound glycogenin. Glycobiology, 7, 571-578 (1997) [5] Meezan, E.; Manzella, S.; Roden, L: Menage a trois: glycogenin, proteoglycan core protein xylosyltransferase and UDP-xylose. Trends Glycosci. Glycotechnol., 7, 303-332 (1995) [6] Viskupic, E.; Cao, Y.; Zhang, W.; Cheng, C.; DePaoli-Roach, A.A.; Roach, P.J.: Rabbit skeletal muscle glycogenin. Molecular cloning and production of fully functional protein in Escherichia coli. J. Biol. Chem., 267, 25759-25763 (1992)

453

N-Acetylglucosaminyldiphosphoundecaprenol N-acetyl-b-D-mannosaminyltransferase

2.4.1.187

1 Nomenclature EC number 2.4.1.187 Systematic name UDP-N-acetyl-d-mannosamine:N-acetyl-b-d-glucosaminyldiphosphoundecaprenol b-1,4-N-acetylmannosaminyltransferase Recommended name N-acetylglucosaminyldiphosphoundecaprenol N-acetyl-b-d-mannosaminyltransferase Synonyms N-acetylmannosaminyltransferase UDP-N-acetylmannosamine:N-acetylglucosaminyl diphosphorylundecaprenol N-acetylmannosaminyltransferase acetylmannosaminyltransferase, uridine diphosphoacetyl-mannosamineacetylglucosaminylpyrophosphorylundecaprenol CAS registry number 118731-82-1

2 Source Organism <1> Bacillus subtilis (AHU 1035) [1]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-mannosamine + N-acetyl-d-glucosaminyldiphosphoundecaprenol = UDP + N-acetyl-b-d-mannosaminyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenol Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetylmannosamine + N-acetylglucosaminyl diphosphoryl-undecaprenol <1> (Reversibility: ? <1> [1]) [1]

454

2.4.1.187

N-Acetylglucosaminyldiphosphoundecaprenol N-acetyl-b-D-mannosaminyltransferase

P UDP + N-acetyl-b-d-mannosaminyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenol <1> [1] S Additional information <1> (<1> involved in the biosynthesis of teichoic acid linkage units in bacterial cell walls [1]) [1] P ? Substrates and products S UDP-N-acetylmannosamine + N-acetylglucosaminyl diphosphoryl-undecaprenol <1> (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-b-d-mannosaminyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenol <1> [1] Inhibitors Triton X-100 <1> (<1> strong [1]) [1] UDP <1> (<1> 2 mM, strong [1]) [1] UMP <1> (<1> weak [1]) [1] Activating compounds Nonidet P-40 <1> (<1> 0.1%: small stimulatory effect [1]) [1] glycerol <1> (<1> 25%: twofold increase of activity [1]) [1] Metals, ions (NH4 )2 SO4 <1> (<1> 0.3-0.5 M: stimulation [1]) [1] KCl <1> (<1> 0.3 M: stimulation [1]) [1] MgCl2 <1> (<1> 10 mM, highest stimulatory effect [1]) [1] NH4 Cl <1> (<1> 0.3-0.5 M: stimulation [1]) [1] NaCl <1> (<1> 0.3-0.5 M: stimulation [1]) [1] Specific activity (U/mg) 0.0000022 <1> [1] Km-Value (mM) 0.0044 <1> (UDP-N-acetylmannosamine) [1] pH-Optimum 7.3 <1> [1] pH-Range 6-9 <1> (<1> 40% of maximal activity at pH 6, 45% of maximal activity at pH 9 [1]) [1] Temperature optimum ( C) 25 <1> (<1> assay at [1]) [1]

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

455

N-Acetylglucosaminyldiphosphoundecaprenol N-acetyl-b-D-mannosaminyltransferase

2.4.1.187

6 Stability General stability information <1>, Nonidet P-40, 0.1-0.2%, stabilizes [1] <1>, glycerol, 25%, stabilizes [1] Storage stability <1>, -18 C, several months, no significant loss of activity [1]

References [1] Murazumi, N.; Kumita, K.; Araki, Y.; Ito, E.: Partial purification and properties of UDP-N-acetylmannosamine:N-acetylglucosaminyl pyrophosphorylundecaprenol N-acetylmannosaminyltransferase from Bacillus subtilis. J. Biochem., 104, 980-984 (1988)

456

N-Acetylglucosaminyldiphosphoundecaprenol glucosyltransferase

2.4.1.188

1 Nomenclature EC number 2.4.1.188 Systematic name UDP-glucose:N-acetyl-d-glucosaminyldiphosphoundecaprenol 4-b-d-glucosyltransferase Recommended name N-acetylglucosaminyldiphosphoundecaprenol glucosyltransferase Synonyms UDP-d-glucose:N-acetylglucosaminyl pyrophosphorylundecaprenol glucosyltransferase uridine diphosphoglucose-acetylglucosaminylpyrophosphorylundecaprenol glucosyltransferase CAS registry number 118731-83-2

2 Source Organism <1> Bacillus coagulans (AHU 1366 [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + N-acetyl-d-glucosaminyldiphosphoundecaprenol = UDP + bd-glucosyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenol Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + N-acetyl-d-glucosaminyldiphosphoundecaprenol <1> (Reversibility: ? <1> [1]) [1] P UDP + b-d-glucosyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenol <1> [1]

457

N-Acetylglucosaminyldiphosphoundecaprenol glucosyltransferase

2.4.1.188

Substrates and products S UDP-glucose + N-acetyl-d-glucosaminyldiphosphoundecaprenol <1> (Reversibility: ? <1> [1]) [1] P UDP + b-d-glucosyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenol <1> [1] Activating compounds (NH4 )2 SO4 <1> (<1> stimulation [1]) [1] KCl <1> (<1> at 0.6 M stimulation [1]) [1] MgCl2 <1> (<1> at 40 mM stimulation [1]) [1] NH4 Cl <1> (<1> stimulation [1]) [1] NaCl <1> (<1> stimulation [1]) [1] Nonidet P-40 <1> (<1> at 0.1% slight stimulatory [1]) [1] glycerol <1> (<1> at 20% stimulates 3fold [1]) [1] Km-Value (mM) 0.021 <1> (UDP-glucose) [1] pH-Optimum 6.6-8 <1> [1] Temperature optimum ( C) 25 <1> (<1> assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Localization membrane <1> [1] Purification <1> (partial [1]) [1]

6 Stability General stability information <1>, stabilization of the solubilized enzyme by cations and glycerol [1] Storage stability <1>, -18 C, several months without significant loss of activity [1]

References [1] Kumita, K.; Murazumi, N.; Araki, Y.; Ito, E.: Solubilization and properties of UDP-d-glucose:N-acetylglucosaminyl pyrophosphorylundecaprenol glucosyltransferase from Bacillus coagulans AHU 1366 membranes. J. Biochem., 104, 985-988 (1988)

458

Luteolin 7-O-glucuronosyltransferase

2.4.1.189

1 Nomenclature EC number 2.4.1.189 Systematic name UDP-glucuronate:luteolin 7-O-glucuronosyltransferase Recommended name luteolin 7-O-glucuronosyltransferase Synonyms LGT glucuronosyltransferase, uridine diphosphoglucuronate-luteolin 7-OCAS registry number 115490-49-8

2 Source Organism <1> Secale cereale (rye [1,2]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucuronate + luteolin = UDP + luteolin 7-O-b-d-glucuronide Reaction type hexosyl group transfer Natural substrates and products S UDPglucuronic acid + luteolin <1> (<1> first enzyme in the biosynthesis of luteolin triglucuronide [1]) (Reversibility: ir <1> [1]) [1] P UDP + luteolin 7-O-b-d-glucuronide <1> [1] Substrates and products S UDPgalactose + luteolin <1> (<1> 10% of activity with UDPglucuronic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + luteolin 7-O-b-d-galactoside <1> S UDPglucose + luteolin <1> (<1> 10% of activity with UDPglucuronic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + luteolin 7-O-b-d-glucoside <1>

459

Luteolin 7-O-glucuronosyltransferase

2.4.1.189

S UDPglucuronic acid + apigenin <1> (<1> 38% of activity compared to luteolin [1]) (Reversibility: ? <1> [1]) [1] P UDP + apigenin 7-O-glucuronide <1> [1] S UDPglucuronic acid + luteolin <1> (Reversibility: ir <1> [1]) [1] P UDP + luteolin 7-O-glucuronide <1> [1] S UDPxylose + luteolin <1> (<1> 10% of activity with UDPglucuronic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + luteolin 7-O-xyloside <1> Inhibitors UDP <1> (<1> strong inhibition [1]) [1] luteolin <1> (<1> 0.14 mM, 60% inhibition [1]) [1] Metals, ions Ca2+ <1> (<1> 0.25 mM, 25% stimulation [1]) [1] Mg2+ <1> (<1> up to 1 mM, 20% stimulation [1]) [1] Specific activity (U/mg) 1.16 <1> [1] Km-Value (mM) 0.008 <1> (luteolin) [1] 0.012 <1> (UDPglucuronic acid) [1] Ki-Value (mM) 0.26 <1> (UDP) [1] pH-Optimum 6.5 <1> (<1> 50% of maximal activity at pH 5.6 [1]) [1] pH-Range 5.6-9 <1> (<1> 50% of maximal activity at pH 5.6 and pH 9.0 [1]) [1] Temperature optimum ( C) 50 <1> [1]

4 Enzyme Structure Molecular weight 34500 <1> (<1> gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [1] Localization cytosol <1> (<1> probably [1]; <1> no activity in vacuoles [2]) [1, 2]

460

2.4.1.189

Luteolin 7-O-glucuronosyltransferase

Purification <1> (ultrogel AcA 44, DEAE-cellulose, partial purification [1]) [1]

6 Stability General stability information <1>, freezing without glycerol, complete inactivation [1] Storage stability <1>, -20 C, 50% glycerol, 3 months, 30-50% loss of activity [1]

References [1] Schulz, M.; Weissenböck, G.: Three specific UDP-glucuronate: flavone-glucuronosyl-transferases from primary leaves of Secale cereale. Phytochemistry, 27, 1261-1267 (1988) [2] Anhalt, S.; Weissenböck, G.: Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll. Planta, 187, 83-88 (1992)

461

Luteolin-7-O-glucuronide 7-Oglucuronosyltransferase

2.4.1.190

1 Nomenclature EC number 2.4.1.190 Systematic name UDP-glucuronate:luteolin-7-O-b-d-glucuronide 7-O-glucuronosyltransferase Recommended name luteolin-7-O-glucuronide 7-O-glucuronosyltransferase Synonyms LMT UDP-glucuronate:luteolin 7-O-glucuronide-glucuronosyltransferase UDPglucuronate:luteolin-7-O-b-d-glucuronide 7-O-glucuronosyltransferase glucuronosyltransferase, uridine diphosphoglucuronate-luteolin 7-O-glucuronide CAS registry number 115490-51-2

2 Source Organism <1> Secale cereale (rye [1,2]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucuronate + luteolin 7-O-glucuronide = UDP + luteolin 7-O-b-d-diglucuronide Reaction type hexosyl group transfer Natural substrates and products S UDPglucuronic acid + luteolin 7-O-b-d-glucuronide <1> (<1> involved in the sequential biosynthesis of luteolin triglucuronide [1]) (Reversibility: ? <1> [1]) [1] P UDP + UDP + luteolin-7-O-b-d-diglucuronide <1> [1, 2]

462

2.4.1.190

Luteolin-7-O-glucuronide 7-O-glucuronosyltransferase

Substrates and products S UDPglucuronic acid + apigenin 7-O-glucoside <1> (<1> 51.5% of activity with luteolin 7-O-glucuronide [1]) (Reversibility: ? <1> [1]) [1] P UDP + apigenin-7-O-b-d-glucosyl-1,4-b-d-glucuronoside <1> [1] S UDPglucuronic acid + apigenin 7-O-glucuronide <1> (<1> 88% of activity with luteolin 7-O-glucuronide [1]) (Reversibility: ? <1> [1]) [1] P UDP + apigenin-7-O-b-d-diglucuronide <1> [1] S UDPglucuronic acid + chrysoeriol 7-O-glucuronide <1> (<1> 65% of activity with luteolin-7-O-glucuronide [1]) (Reversibility: ? <1> [1]) [1] P UDP + chrysoeriol-7-O-b-d-glucuronosyl-1,4-b-d-glucuronoside S UDPglucuronic acid + luteolin 7-O-glucuronide <1> (Reversibility: ir <1> [1, 2]) [1, 2] P UDP + luteolin-7-O-b-d-diglucuronide <1> [1, 2] Inhibitors Ag+ <1> (<1> 0.75 mM, 40% inhibition [1]) [1] Cu2+ <1> (<1> 0.75 mM, 40% inhibition [1]) [1] Pb2+ <1> (<1> 0.75 mM, 40% inhibition [1]) [1] UDP <1> [1] luteolin <1> (<1> 0.1 mM, 50% inhibition [1]) [1] Specific activity (U/mg) 0.96 <1> [1] Km-Value (mM) 0.012 <1> (luteolin 7-O-glucuronide) [1] 0.04 <1> (UDPglucuronic acid) [1] Ki-Value (mM) 0.12 <1> (UDP) [1] pH-Optimum 6.5 <1> [1] 8.5 <1> (<1> pH-optimum at pH 8.5 is absent in crude enzyme preparations [1]) [1] pH-Range 6-9 <1> (<1> 50% of maximal activity at pH 6.0 and pH 9.0 [1]) [1] Temperature optimum ( C) 52 <1> [1]

4 Enzyme Structure Molecular weight 37000 <1> (<1> gel filtration [1]) [1]

463

Luteolin-7-O-glucuronide 7-O-glucuronosyltransferase

2.4.1.190

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [1, 2] Localization cytosol <1> (<1> no activity in vacuoles [2]) [2] Purification <1> (Ultrogel AcA 44, ultrafiltration, hydroxylapatite, partial purification [1]) [1, 2]

6 Stability General stability information <1>, freezing without glycerol, complete inactivation [1] Storage stability <1>, -20 C, 50% glycerol, 3 months, 30-50% loss of activity [1]

References [1] Schulz, M.; Weissenböck, G.: Three specific UDP-glucuronate: flavone-glucuronosyl-transferases from primary leaves of Secale cereale. Phytochemistry, 27, 1261-1267 (1988) [2] Anhalt, S.; Weissenböck, G.: Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll. Planta, 187, 83-88 (1992)

464

Luteolin-7-O-diglucuronide 4'-Oglucuronosyltransferase

2.4.1.191

1 Nomenclature EC number 2.4.1.191 Systematic name UDP-glucuronate:luteolin-7-O-b-d-diglucuronide 4'-O-glucuronosyltransferase Recommended name luteolin-7-O-diglucuronide 4'-O-glucuronosyltransferase Synonyms LDT UDP-glucuronate:luteolin 7-O-diglucuronide-glucuronosyltransferase UDPglucuronate:luteolin 7-O-diglucuronide-4'-O-glucuronosyl-transferase glucuronosyltransferase, uridine diphosphoglucuronate-luteolin 7-O-diglucuronide CAS registry number 115490-50-1

2 Source Organism <1> Secale cereale (rye [1,2]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucuronate + luteolin 7-O-b-d-diglucuronide = UDP + luteolin 7-O-[bd-glucuronosyl-(1!2)-b-d-glucuronide]-4'-O-b-d-glucuronide Reaction type hexosyl group transfer Natural substrates and products S UDPglucuronic acid + luteolin 7-O-diglucuronide <1> (<1> involved in the sequential biosynthesis of luteolin triglucuronide [1]) (Reversibility: ir <1> [1]) [1, 2] P UDP + luteolin 7-O-[b-d-glucuronosyl-1,2-b-d-glucuronide]-4'-O-b-dglucuronide <1> [1]

465

Luteolin-7-O-diglucuronide 4'-O-glucuronosyltransferase

2.4.1.191

Substrates and products S UDPgalactose + luteolin 7-O-b-d-diglucuronide <1> (<1> 7.5% of activity with UDPglucuronic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + luteolin 7-O-[b-d-glucuronosyl-1,2-b-d-glucuronide]-4'-O-b-dgalactoside <1> [1] S UDPglucose + luteolin 7-O-b-d-diglucuronide <1> (<1> 10% of activity with UDPglucuronic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + luteolin 7-O-[b-d-glucuronosyl-1,2-b-d-glucuronide]-4'-O-b-dglucoside <1> [1] S UDPglucuronic acid + luteolin 7-O-b-d-diglucuronide <1> (Reversibility: ir <1> [1]) [1, 2] P UDP + luteolin 7-O-[b-d-glucuronosyl-1,2-b-d-glucuronide]-4'-O-b-dglucuronide <1> [1] S UDPglucuronic acid + luteolin-7-O-b-d-glucuronide <1> (<1> 34% of activity with luteolin 7-O-diglucuronide [1]) (Reversibility: ? <1> [1]) [1] P UDP + luteolin 7-O-b-d-glucuronosyl-4'-O-glucuronide <1> [1] S UDPxylose + luteolin 7-O-b-d-diglucuronide <1> (<1> 10% of activity with UDPglucuronic acid [1]) (Reversibility: ? <1> [1]) [1] P UDP + luteolin 7-O-[b-d-glucuronosyl-1,2-b-d-glucuronide]-4'-O-b-dxyloside <1> [1] Inhibitors UDP <1> (<1> strong [1]) [1] luteolin <1> (<1> 0.02 mM, 50% inhibition [1]) [1] Metals, ions Additional information <1> (<1> not affected by Ca2+ or Mg2+ up to 0.5 and 0.75 mM [1]) [1] Specific activity (U/mg) 0.14 <1> [1] Km-Value (mM) 0.009 <1> (luteolin 7-O-b-d-diglucuronide) [1] 0.09 <1> (UDPglucuronic acid) [1] pH-Optimum 7 <1> [1] pH-Range 6-9 <1> (<1> 50% of maximal activity at pH 6 and pH 9 [1]) [1] Temperature optimum ( C) 40 <1> [1]

4 Enzyme Structure Molecular weight 29000 <1> (<1> gel filtration [1]) [1]

466

2.4.1.191

Luteolin-7-O-diglucuronide 4'-O-glucuronosyltransferase

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [1] mesophyll <1> (<1> mesophyll protoplasts [2]) [2] Localization vacuole <1> (<1> probably [2]) [2] Purification <1> (Ultrogel AcA 44, Sephadex G-25, ultrafiltration, partial purification [1]) [1, 2]

6 Stability General stability information <1>, freezing without glycerol, complete loss of activity [1] Storage stability <1>, -20 C, 50% glycerol, 3 months, 30-50% loss of activity [1]

References [1] Schulz, M.; Weissenböck, G.: Three specific UDP-glucuronate:flavone-glucuronosyl-transferases from primary leaves of Secale cereale. Phytochemistry, 27, 1261-1267 (1988) [2] Anhalt, S.; Weissenböck, G.: Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll. Planta, 187, 83-88 (1992)

467

Nuatigenin 3b-glucosyltransferase

2.4.1.192

1 Nomenclature EC number 2.4.1.192 Systematic name UDP-glucose:(20S,22S,25S)-22,25-epoxyfurost-5-ene-3b,26-diol 3-O-b-d-glucosyltransferase Recommended name nuatigenin 3b-glucosyltransferase Synonyms uridine diphosphoglucose-nuatigenin glucosyltransferase Additional information (not identical with EC 2.4.1.173 or EC 2.4.1.193) CAS registry number 108891-57-2

2 Source Organism <1> Avena sativa (oat [1,2,3]) [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + (20S,22S,25S)-22,25-epoxyfurost-5-ene-3b,26-diol = UDP + (20S,22S,25S)-22,25-epoxyfurost-5-ene-3b,26-diol 3-O-b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + (20S,22S,25S)-22,25-epoxyfurost-5-ene-3b,26-diol <1> (<1> participates in the initiation of sugar chain formation during the biosynthesis of oat saponins: avenacosides A and B [1]) (Reversibility: ? <1> [1]) [1] P UDP + (20S,22S,25S)-22,25-epoxyfurost-5-ene-3b,26-diol 3-O-b-d-glucoside

468

2.4.1.192

Nuatigenin 3b-glucosyltransferase

Substrates and products S 20S,22S,25S-spirost-5-ene-3b,25-diol + UDP-glucose <1> (<1> i.e. isonuatigenin, at 57% of the activity with nuatigenin [1]) (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + 20S,22S,25S-spirost-5-ene-3b,25-diol 3-O-b-d-glucoside S 22S,25S-5a-tomatanin-3b-ol + UDP-glucose <1> (<1> i.e. tomatidine, at 57% of the activity with nuatigenin [1]) (Reversibility: ? <1> [1]) [1] P UDP + 22S,25S-5a-tomatanin-3b-ol 3-O-b-d-glucoside S 22S,25S-solanid-5-enin-3b-ol + UDP-glucose <1> (<1> i.e. solanidine, at 39% of the activity with nuatigenin [1]) (Reversibility: ? <1> [1]) [1] P UDP + g-chaconine <1> [1] S 25R-5a-spirostan-3b-ol + UDP-glucose <1> (<1> i.e. tigogenin, at 12% of the activity with nuatigenin [1]) (Reversibility: ? <1> [1]) [1] P UDP + 25R-5a-spirostan-3b-ol 3-O-b-d-glucoside S 25R-5a-spirostane-3b,6a-diol + UDP-glucose <1> (<1> i.e. chlorogenin, at 30% of the activity with nuatigenin [1]) (Reversibility: ? <1> [1]) [1] P UDP + 25R-5a-spirostane-3b,6a-diol 3-O-b-d-glucoside S 25R-spirost-5-en-3b-ol + UDP-glucose <1> (<1> i.e. diosgenin, at 17% of the activity with nuatigenin [1]) (Reversibility: ? <1> [1, 2]) [1, 2] P UDP + 25R-spirost-5-en-3b-ol 3-O-b-d-glucoside S UDP-glucose + (20S,22S,25S)-22,25-epoxyfurost-5-ene-3b,26-diol <1> (<1> i.e. nuatigenin, TDPglucose at 8%, ADPglucose at 11%, UDPgalactose at 40% the rate of UDP-glucose [1]) (Reversibility: ? <1> [1, 2, 3]) [1, 2, 3] P UDP + (20S,22S,25S)-22,25-epoxyfurost-5-ene-3b,26-diol 3-O-b-d-glucoside <1> (<1> i.e. nuatigenin 3b-d-monoglucoside [1,2,3]) [1, 2, 3] S androst-5-en-3b-ol-17-one + UDP-glucose <1> (<1> i.e. androsterone, at 15% of the activity with nuatigenin [1]) (Reversibility: ? <1> [3]) [1] P UDP + androst-5-en-3b-ol-17-one 3-O-b-d-glucoside S pregn-5-en-3b-ol-20-one + UDP-glucose <1> (<1> i.e. pregnenolone, at 26% of the activity with nuatigenin) (Reversibility: ? <1> [1]) [1] P UDP + pregn-5-en-3b-ol-20-one 3-O-b-d-glucoside Inhibitors Hg2+ <1> [1, 2] Triton X-100 <1> [1, 2, 3] UDP <1> [1] UMP <1> [1] Zn2+ <1> [1, 2] p-chloromercuribenzoate <1> [1] Metals, ions Additional information <1> (<1> no stimulation by divalent cations: Ca2+ , Mn2+ or Mg2+ [2,3]) [2, 3] pH-Optimum 6.5-8.5 <1> [1, 2] 7.5-8.5 <1> [3]

469

Nuatigenin 3b-glucosyltransferase

2.4.1.192

pH-Range 5.8-8.5 <1> (<1> at pH 5.8 about 50% of activity maximum, at pH 6.5-8.5 activity maximum [2]) [2] Temperature optimum ( C) 30 <1> [1, 2] Temperature range ( C) 30-39 <1> (at 30 C activity maximum, at 39 C activity is 7times lower than at 30 C [2]) [2]

4 Enzyme Structure Molecular weight 58000-62000 <1> (<1> gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf <1> [1, 2, 3] Localization cytosol <1> (<1> 70% of the activity [2]) [1, 2] Purification <1> (partial [1]) [1]

6 Stability Temperature stability 45 <1> (<1> unstable [1]) [1] General stability information <1>, 2-mercaptoethanol, 10 mM, improves stability in solution, 35% loss of activity after 24 h at 4 C [1] <1>, rather unstable when dissolved in buffer, 65% loss of activity after 24 h at 4 C [1] Storage stability <1>, -20 C, cytosolic fraction stable for several months [2] <1>, -20 C, in the form of dry acetone powder, stable for several weeks [1]

470

2.4.1.192

Nuatigenin 3b-glucosyltransferase

References [1] Kalinowska, M.; Wojciechowski, Z.A.: Substrate specificity of partially purified UDP-glucose:nuatigenin glucosyltransferase from oat leaves. Plant Sci., 55, 239-245 (1988) [2] Kalinowska, M.; Wojciechowski, Z.A.: Subcellular localization of UDPG:nuatigenin glucosyltransferase in oat leaves. Phytochemistry, 26, 353-357 (1987) [3] Kalinowska, M.; Wojciechowski, Z.A.: Enzymatic synthesis of nuatigenin 3b-d-glucoside in oat (Avena sativa) leaves. Phytochemistry, 25, 2525-2529 (1986)

471

Sarsapogenin 3b-glucosyltransferase

2.4.1.193

1 Nomenclature EC number 2.4.1.193 Systematic name UDP-glucose:(25S)-5b-spirostan-3b-ol 3-O-b-d-glucosyltransferase Recommended name sarsapogenin 3b-glucosyltransferase Synonyms UDPglucose:(25S)-5b-spirostan-3b-ol 3-O-b-d-glucosyltransferase glucosyltransferase, uridine diphosphoglucose-sarsapogenin Additional information (not identical with EC 2.4.1.173 and 2.4.1.192) CAS registry number 117698-14-3

2 Source Organism <1> Asparagus officinalis [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + (25S)-5b-spirostan-3b-ol = UDP + (25S)-5b-spirostan-3b-ol 3-O-b-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + (25S)-5b-spirostan-3b-ol <1> (<1>, involved in the biosynthesis of plant saponins [1]) (Reversibility: ? <1> [1]) [1] P UDP + (25S)-5b-spirostan-3b-ol 3-O-b-d-glucoside <1> [1] Substrates and products S UDPglucose + (25R)-5a-spirostan-3b-ol <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + (25R)-5a-spirostan-3b-ol 3-O-b-d-glucoside

472

2.4.1.193

Sarsapogenin 3b-glucosyltransferase

S UDPglucose + (25R)-5a-spirostan-3b-ol-12-one <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + (25R)-5a-spirostan-3b-ol-12-one 3-O-b-d-glucoside S UDPglucose + (25R)-5a-spirostane-3b,6a-diol <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + (25R)-5a-spirostane-3b,6a-diol 3-O-b-d-glucoside S UDPglucose + (25R)-5b-spirostan-3b-ol <1> (<1>, i.e. smilagenin [1]) (Reversibility: ? <1> [1]) [1] P UDP + (25R)-5b-spirostan-3b-ol 3-O-b-d-glucoside S UDPglucose + (25R)-spirost-5-en-3b-ol <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + (25R)-spirost-5-en-3b-ol 3-O-b-d-glucoside S UDPglucose + (25S)-5b-spirostan-3b-ol <1> (<1>, i.e. sarsapogenin [1]) (Reversibility: ? <1> [1]) [1] P UDP + (25S)-5b-spirostan-3b-ol 3-O-b-d-glucoside <1> [1] S UDPglucose + 5a-cholestan-3b-ol <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + 5a-cholestan-3b-ol 3-O-b-d-glucoside S UDPglucose + 5b-cholestan-3b-ol <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + 5b-cholestan-3b-ol 3-O-b-d-glucoside S UDPglucose + stigmast-5-en-3b-ol <1> (<1>, weak activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + stigmast-5-en-3b-ol 3-O-b-d-glucoside Inhibitors EDTA <1> (<1>, 1 mM, 43% inhibition [1]) [1] EGTA <1> (<1>, 10 mM, 50% inhibition [1]) [1] PCMB <1> (<1>, 0.01 mM, 94% inhibition [1]) [1] Triton X-100 <1> [1] UDP <1> (<1>, 1 mM, 89% inhibition [1]) [1] UMP <1> (<1>, 1 mM, 51% inhibition [1]) [1] Metals, ions Mg2+ <1> (<1>, 1 mM, stimulates [1]) [1] Mn2+ <1> (<1>, 1 mM, stimulates [1]) [1] pH-Optimum 9.6 <1> [1] pH-Range 7.2-9.9 <1> (<1>, pH 7.2: about 40% of maximal activity, pH 9.9: about 60% of maximal activity [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue shoot <1> (<1>, of 3-week-old plants [1]) [1] 473

Sarsapogenin 3b-glucosyltransferase

2.4.1.193

Localization cytosol <1> [1]

References [1] Paczkowski, C.; Wojciechowski, Z.A.: The occurence of UDPG-dependent glucosyltransferase specific for sarsasapogenin in Asparagus officinalis. Phytochemistry, 27, 2743-2747 (1988)

474

4-Hydroxybenzoate 4-O-b-Dglucosyltransferase

2.4.1.194

1 Nomenclature EC number 2.4.1.194 Systematic name UDP-glucose:4-hydroxybenzoate 4-O-b-d-glucosyltransferase Recommended name 4-hydroxybenzoate 4-O-b-d-glucosyltransferase Synonyms HBA glucosyltransferase PHB glucosyltransferase PHB-O-glucosyltransferase UDP-glucose:4-(b-d-glucopyranosyloxy)benzoic acid glucosyltransferase p-hydroxybenzoate glucosyltransferase uridine diphosphoglucose-4-hydroxybenzoate glucosyltransferase CAS registry number 120860-68-6

2 Source Organism <1> Lithospermum erythrorhizon [1, 3-5] <2> Pinus densiflora [2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + 4-hydroxybenzoate = UDP + 4-(b-d-glucosyloxy)benzoate Reaction type hexosyl group transfer Natural substrates and products S Additional information <1, 2> (<2> probably control of activity during pine pollen germination [2]; <1> key step in the shikonin biosynthesis [3]) [2, 3] P ?

475

4-Hydroxybenzoate 4-O-b-D-glucosyltransferase

2.4.1.194

Substrates and products S UDPglucose + 4-hydroxybenzoate <1, 2> (Reversibility: ir <1, 2> [1, 2]) [1, 2] P UDP + 4-(b-d-glucosyloxy)benzoate <1, 2> [1, 2] S UDPglucose + 4-nitrophenol <1> (Reversibility: ? <1> [1]) [1] P UDP + 4-nitrophenyl O-b-d-glucoside <1> [1] Inhibitors 2-hydroxybenzoic acid <2> [2] 3-hydroxybenzoic acid <2> (<2> competitive inhibition [2]) [2] 4-(b-d-glucosyloxy)benzoate <1> (<1> non competitive inhibition with respect to both 4-hidroxybenzoic acid and UDP [1]) [1] 4-coumaric acid <2> [2] 4-hydroxybenzoic acid <2> [2] Ca2+ <1> (<1> weak effect [1]) [1] Co2+ <1> (<1> strong inhibition at 1 mM and 10 mM [1]) [1] Cu2+ <1> (<1> strong inhibition at 1 mM and 10 mM [1]) [1] EDTA <1> (<1> negative influence on activity at 10 mM, but reversible [1]) [1] Fe2+ <1> (<1> strong inhibition at 1 mM and 10 mM [1]) [1] Mg2+ <1> (<1> weak effect [1]) [1] Mn2+ <1> (<1> weak effect [1]) [1] Ni2+ <1> (<1> strong inhibition at 1 mM and 10 mM [1]) [1] UDP <1> (<1> non competitive inhibition with respect to 4-hydroxybenzoic acid, competitive inhibitor with respect to UDPglucose [1]) [1] Zn2+ <1> (<1> strong inhibition at 1 mM and 10 mM [1]) [1] gallic acid <2> (<2> competitive inhibition [2]) [2] vanillic acid <2> (<2> competitive inhibition [2]) [2] Activating compounds EDTA <2> (<2> 60% increase of activity at 0.1 mM, probably due to removal of heavy metal ions [2]) [2] Metals, ions Ca2+ <2> (<2> 50% increase of activity at 0.3 mM [2]) [2] Additional information <1, 2> (<1,2> no metal ions required [1,2]; <2> Mg2+ has no effect [2]) [1, 2] Specific activity (U/mg) 0.00192 <1> [1] 2.94 <1> [5] Additional information <1> (<1> activity is regulated by white light in cell suspension cultures [4]) [4] Km-Value (mM) 0.24 <2> (UDPglucose) [2] 0.264 <1> (4-hydroxybenzoate) [1] 0.268 <1> (UDPglucose) [1] 0.6 <1> (4-nitrophenol) [1] 2.9 <2> (4-hydroxybenzoate) [2]

476

2.4.1.194

4-Hydroxybenzoate 4-O-b-D-glucosyltransferase

Ki-Value (mM) 0.4 <2> (3-hydroxybenzoic acid) [2] 0.7 <2> (gallic acid) [2] 1.3 <2> (vanillic acid) [2] 3.1 <2> (2-hydroxybenzoic acid) [2] 5 <2> (4-coumaric acid) [2] pH-Optimum 7.5 <2> [2] 7.8 <1> [1] pH-Range 6.6-8.6 <1> (<1> half-maximal activity at pH 6.6 and 8.6 [1]) [1] Temperature optimum ( C) 30 <2> (<2> assay at [2]) [2] 37 <1> (<1> assay at [5]) [5] 45 <1> [1]

4 Enzyme Structure Molecular weight 33000 <2> (<2> apparent, from gel filtration [2]) [2] 47500 <1> (<1> apparent, from gel filtration [1]) [1] Subunits monomer <1> (<1> 1 * 51000, SDS-PAGE [5]) [5]

5 Isolation/Preparation/Mutation/Application Source/tissue callus <1> (<1> cell culture [1]) [1] pollen <2> [2] Localization cytoplasm <1> [3] Purification <1> (partial, using ammonium sulfate precipitation and chromatography on DEAE-Sepharose and phenyl-Sepharose [1]; using gel filtration followed by hydroxyapatite and HiTrap blue affinity chromatography [5]) [1, 5] <2> (partial [2]) [2]

477

4-Hydroxybenzoate 4-O-b-D-glucosyltransferase

2.4.1.194

6 Stability General stability information <1>, dithiothreitol stabilizes at 1-10 mM [1] <1>, phenylmethylsulfonyl fluoride stabilizes at 0.02-1 mM [1] Storage stability <1>, -20 C, 0.05 M Tris-HCl, 1 mM DTT, 0.02 mM phenylmethylsulfonyl fluoride, pH 7.6, 3 months, 10% loss of activity [1] <1>, 4-8 C, 0.01 M Tris-HCl, pH 7.2, 15 days, 76% loss of activity [1]

References [1] Bechthold, A.; Berger, U.; Heide, L.: Partial purification, properties, and kinetic studies of UDP-glucose:p-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon. Arch. Biochem. Biophys., 288, 39-47 (1991) [2] Katsumata, T.; Shige, H.; Ejiri, S.: Biochemical studies on pollen. Part XXXIV. UDP-glucose:4-(b-d-glucopyranosyloxy)benzoic acid glucosyltransferase from the pollen of Pinus densiflora. Phytochemistry, 28, 359-362 (1989) [3] Yazaki, K.; Inushima, K.; Kataoka, M.; Tabata, M.: Intracellular localization of UDPG:p-hydroxybenzoate glucosyltransferase and its reaction product in Lithospermum cell cultures. Phytochemistry, 38, 1127-1130 (1995) [4] Gaisser, S.; Heide, L.: Inhibition and regulation of shikonin biosynthesis in suspension cultures of Lithospermum. Phytochemistry, 41, 1065-1072 (1996) [5] Li, S.-M.; Wang, Z.-X.; Heide, L.: Purification of UDP-glucose:4-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon. Phytochemistry, 46, 27-32 (1997)

478

Thiohydroximate b-D-glucosyltransferase

2.4.1.195

1 Nomenclature EC number 2.4.1.195 Systematic name UDP-glucose:thiohydroximate S-b-d-glucosyltransferase Recommended name thiohydroximate b-d-glucosyltransferase Synonyms desulfoglucosinolate-uridine diphosphate glucosyltransferase uridine diphosphoglucose (UDPglucose):thiohydroximate glucosyltransferase <7> [3] uridine diphosphoglucose-thiohydroximate glucosyltransferase CAS registry number 9068-14-8

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

Arabidopsis thaliana [7] Armoracia lapathifolia [1] Brassica campestris (cv R-500 [3]) [3, 6] Brassica carinata [6] Brassica juncea (cv. Cutlass [2-4]; cv Domo [3]; Coss [2]) [2-4, 6] Brassica napus (cv Westar [3,5]) [3, 5, 6, 8] Brassica nigra (cv 526 [3]) [3, 6] Brassica oleracea (savoy cabbage [3]; cauliflower, ssp. botrytis cv Snowball, self-blanching type white and open type brown [6]) [3, 6] <9> Nasturtium officinale (R.Br. [1]) [1] <10> Sinapis alba [1] <11> Tropaeolum majus [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + phenylacetothiohydroximate = UDP + desulfoglucotropeolin (involved with EC 2.8.2.24 (desulfoglucosinolate sulfotransferase) in the biosynthesis of thioglucosides in cruciferous plants) 479

Thiohydroximate b-D-glucosyltransferase

2.4.1.195

Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + phenylacetothiohydroximate <1-11> (<5,7> biosynthetic pathway for glucosinolates [2,3]; <1> catalyses the penultimate reaction in glucosinolate biosynthesis [7]; <11> integral step in the biosynthesis of benzylglucosinolate [1]; <6> second to last step in glucosinolate biosynthesis [5]) (Reversibility: ? <1-11> [1-7]) [1-7] P UDP + desulfoglucotropeolin Substrates and products S UDP-glucose + 2-(3-indolyl)acetothiohydroximate <6> (Reversibility: ? <6> [5]) [5] P UDP + S-(a-d-glucopyranosyl)-2-(3-indoyl)acetothiohydroximate S UDP-glucose + 3-phenylpropanothiohydroximate <6> (Reversibility: ? <6> [5]) [5] P UDP + S-(a-d-glucopyranosyl)-3-phenylpropanothiohydroximate S UDP-glucose + 4-methylthiobutyrothiohydroximate <11> (<11> relative reaction rate 77% [1]) (Reversibility: ? <11> [1]) [1] P UDP + S-(a-d-glucopyranosyl)-4-methylthiobutyrothiohydroximate S UDP-glucose + benzothiohydroximate <11> (<11> relative reaction rate 10% [1]) (Reversibility: ? <11> [1]) [1] P UDP + S-(a-d-glucopyranosyl)-benzothiohydroximate S UDPglucose + butyrothiohydroximate <11> (<11> relative reaction rate 70% [1]) (Reversibility: ? <11> [1]) [1] P UDP + S-(a-d-glucopyranosyl)-butyrothiohydroximate S UDPglucose + isobutyrothiohydroximate <11> (<11> relative reaction rate 41% [1]) (Reversibility: ? <11> [1]) [1] P UDP + S-(a-d-glucopyranosyl)-isobutyrothiohydroximate S UDPglucose + phenylacetothiohydroximate <1-11> (Reversibility: ? <111> [1-7]) [1-7] P UDP + desulfoglucotropeolin S UDPglucose + propiothiohydroximate <11> (<11> relative reaction rate 50% [1]) (Reversibility: ? <11> [1]) [1] P UDP + S-(a-d-glucopyranosyl)-propiothiohydroximate S Additional information <6, 11> (<11> acetothiohydroximate, phenylacetohydroximic acid, ethanol, 2-mercaptoethanol and cysteine hydrochloride are no substrates [1]; <6> the oxygen analog phenylacetohydroximate and the hydroxylated phenolic substances quercetin and caffeic acid are not glucosylated [5]) [1, 5] P ? Inhibitors 1,10-phenanthroline <1> (<1> causes 50% inhibition [7]) [7] Co2+ <6> [5] Cu2+ <6> [5] CuCl2 <1> [7]

480

2.4.1.195

Thiohydroximate b-D-glucosyltransferase

HgCl2 <11> [1] Zn2+ <6> [5] ZnCl2 <1> [7] p-chloromercuribenzoate <11> [1] Additional information <1, 11> (<11> no inhibtion by KCN or iodoacetic acid, only moderately inhibited by N-ethylmaleimide [1]; <1> iodoacetic acid and 5,5'-dithio-bis-(2-nitrobenzoic acid) are less inhibitory [7]) [1, 7] Activating compounds 1,10-phenanthroline <11> (<11> 0.1 mM, activation [1]) [1] 2,2'-dipyridyl <11> (<11> 1.0 mM, activation [1]) [1] 2-mercaptoethanol <1, 6, 11> (<11> activity dissappears in absence [1]; <11> requirement [1]; <6> 1.0 mM, relative activity 250%, 10 mM, relative activity 260%, in absence enzyme solutions will lose up to 35% activity in 1 week, activity is not recoverable by addition of 2-mercaptoethanol [5]) [1, 5, 7] EDTA <11> (<11> 0.01 mM, activation [1]) [1] l-cysteine <1> [7] cysteine hydrochloride <11> (<11> 1.0 mM, activation [1]) [1] dithiothreitol <6, 11> (<11> 1.0 mM, activation [1]; <6> 5.0 mM, relative activity 230% [5]) [1, 5] glutathione <11> (<11> 1.0 mM, activation [1]) [1] Metals, ions Ca2+ <6> (<6> 10 mM, relative activity 130% [5]) [5] CaCl2 <1> (<1> activity is stimulated by thiol reducing agents [7]) [7] Mg2+ <6> (<6> 10 mM, relative activity 140% [5]) [5] MgCl2 <1> (<1> activity is stimulated by thiol reducing agents [7]) [7] MnCl2 <1> (<1> activity is stimulated by thiol reducing agents [7]) [7] Additional information <1> (<1> purified enzyme lacks an absolute requirement for metal ions [7]) [7] Specific activity (U/mg) 0.561 <1> [7] 2.8 <6> [5] 3.2 <8> [6] 45 <11> [1] Km-Value (mM) 0.05 <6> (phenylacetothiohydroximate, <6> pH 6.0, 30 C [5]) [5] 0.27 <1> (UDPglucose, <1> pH 6.0, 30 C [7]) [7] 0.46 <6> (UDPglucose, <6> pH 6.0, 30 C [5]) [5] 1.05 <11> (4-methylthiobutyrothiohydroximate, <11> pH 7.4, 30 C [1]) [1] 1.47 <11> (UDPglucose, <11> pH 7.4, 30 C, 4-methylthiobutyrothiohydroximate [1]) [1] 1.54 <11> (UDPglucose, <11> pH 7.4, 30 C, phenylacetothiohydroximate [1]) [1]

481

Thiohydroximate b-D-glucosyltransferase

2.4.1.195

pH-Optimum 6 <1, 6> [5, 7] 6.5-7.5 <11> (<11> greater activity in phosphate and Tris-HCl buffers than in Tris-maleate [1]) [1]

4 Enzyme Structure Molecular weight 44000 <5> (<5> gel filtration [4]) [4] 50000 <11> (<11> gel filtration [1]) [1] 51000 <6> (<6> calculated from cDNA open reading frame [8]) [8] 55500 <3, 5, 8> (<8> SDS-PAGE [6]) [6] 57000 <7> [6] 57600 <8> (<8> gel filtration [6]) [6] 57800 <1> [7] Subunits monomer <6, 8> (<6> 1 * 46000, SDS-PAGE [5]; <8> 1 * 55500, SDS-PAGE [6]) [5, 6]

5 Isolation/Preparation/Mutation/Application Source/tissue cotyledon <5> [2] floret <8> [6] hypocotyl <5> [2] inflorescene <1> [7] leaf <1, 2, 8-11> [1, 6, 7] root <5> [2] seedling <1, 5, 6, 8> [2, 3, 5-7] Localization cytoplasm <5> (<5> located outside the plastids and mitochondria, not excluded association with endoplasmic reticulum and or vacuoles [2]) [2, 4] Purification <1> (partial [7]) [7] <5> (partially [2]; co-purified with EC 2.8.2.24 [4]) [2, 4] <6> [3, 5] <11> [1] Cloning <6> (cDNA cloned and expressed in Escherichia coli [8]) [8] Application agriculture <6, 8> (<6,8> glucosinolates have antinutritional properties and causes acute and chronic diseases, particularly monogastrics, in domestic

482

2.4.1.195

Thiohydroximate b-D-glucosyltransferase

animals, great nutritional and therefore economic concern, since the meal fraction is directed to animal feed markets as a protein source, presence of glucosinolates in the meal precludes its use as a feed for nonruminants, this results in a worldwide effort to breed low glucosinolate varieties of rapeseed, beside traditional plant breeding there are molecular genetic studies and modification of these pathways [5,6]) [5, 6] nutrition <6, 8> (<6,8> glucosinolates have antinutritional properties and causes acute and chronic diseases, particularly monogastrics, in domestic animals, great nutritional and therefore economic concern, since the meal fraction is directed to animal feed markets as a protein source, presence of glucosinolates in the meal precludes its use as a feed for nonruminants, this results in a worldwide effort to breed low glucosinolate varieties of rapeseed, beside traditional plant breeding there are molecular genetic studies and modification of these pathways [5,6]) [5, 6]

6 Stability pH-Stability 4-10 <5> (<5> stable below pH 7.5 [4]) [4] 7.5 <6> (<6> most stable in Tris buffer [5]) [5] Temperature stability 5-30 <5> (<5> stable up to 30 C [4]) [4] 30-40 <6> (<6> stable up to 30 C for at least 1 h, activity is lost quickly at 40 C or greater [5]) [5] General stability information <6>, enzyme stability decreases in buffers at pH optimum 6.0 [5] Storage stability <6>, 0 C, storage at temperatures below with or without glycerol results in substantial loss of activity [5] <6>, 4 C, only a 5% loss of activity can be detected after 10 days [5] <8>, -20 , crude extract stored frozen without loss of activity for 1 year [6] <11>, -15 C, freeze-dried protein, specific activity remains unchanged for more than 3 months [1]

References [1] Matsuo, M.; Underhill, E.W.: Purification and properties of a UDP glucose:thiohydroximate glucosyltransferase from higher plants. Phytochemistry, 10, 2279-2288 (1971) [2] Jain, J.C.; Michayluk, M.R.; Groot Wassink, J.W.D.; Underhill, E.W.: Distribution of enzymes catalyzing the glucosylation and sulfation steps of glucosinolate biosynthesis in Brassica juncea seedlings and cultured cells. Plant Sci., 64, 25-29 (1989)

483

Thiohydroximate b-D-glucosyltransferase

2.4.1.195

[3] Jain, J.C.; Reed, D.W.; Groot Wassink, J.W.D.; Underhill, E.W.: A radioassay of enzymes catalyzing the glucosylation and sulfation steps of glucosinolate biosynthesis in Brassica species. Anal. Biochem., 178, 137-140 (1989) [4] Jain, J.C.; Groot Wassink, J.W.D.; Reed, D.W.; Underhill, E.W.: Persistent copurification of enzymes catalyzing the sequential glucosylation and sulfation steps in glucosinolate biosynthesis. J. Plant Physiol., 136, 356-361 (1990) [5] Reed, D.W.; Davin, L.; Jain, J.C.; Deluca, V.; Nelson, L.; Underhill, E.W.: Purification and properties of UDP-glucose: thiohydroximate glucosyltransferase from Brassica napus L. seedlings. Arch. Biochem. Biophys., 305, 526-532 (1993) [6] Groot Wassink, J.W.D.; Reed, D.W.; Kolenovsky, A.D.: Immunopurification and immunocharacterization of the glucosinolate biosynthetic enzyme thiohydroximate S-glucosyltransferase. Plant Physiol., 105, 425-433 (1994) [7] Guo, L.; Poulton, E.: Partial purification and characterization of Arabidopsis thaliana UDPG:thiohydroximate glucosyltransferase. Phytochemistry, 36, 1133-1138 (1994) [8] Marillia, E.-F.; MacPherson, J.M.; Tsang, E.W.T.; Van Audenhove, K.; Keller, W.A.; Groot Wassink, J.W.D.: Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli. Physiol. Plant., 113, 176 (2001)

484

Nicotinate glucosyltransferase

2.4.1.196

1 Nomenclature EC number 2.4.1.196 Systematic name UDP-glucose:nicotinate N-glucosyltransferase Recommended name nicotinate glucosyltransferase Synonyms UDP-glucose:nicotinic acid-N-glucosyltransferase uridine diphosphoglucose-nicotinate N-glucosyltransferase CAS registry number 120858-56-2

2 Source Organism <1> Petroselinum hortense (Hoffm., parsley [3]) [3] <2> Solanum tuberosum (HH258 and F81 [2]) [2] <3> Nicotiana tabacum (XD-6, inducible enzyme by culture with niacin [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + nicotinate = UDP + N-glucosylnicotinate Reaction type hexosyl group transfer Natural substrates and products S UDPglucose + nicotinate <1> (<1> the reversible glucosyltransferase reaction provides free nicotinate for the synthesis of NAD+ and NADP+ in pyridine nucleotide cycle [3]) [3] P ? Substrates and products S UDP-glucose + catechol <1> (Reversibility: ? <1> [3]) [3] P ?

485

Nicotinate glucosyltransferase

2.4.1.196

S UDP-glucose + nicotinate <1, 2, 3> (<1> high specificity for UDPglucose as glucosyl donor [3]) (Reversibility: r <1> [3]; ? <2,3> [1,2]) [1, 2, 3] P UDP + N-glucosylnicotinate <1, 2> [3, 2] S UDP-glucose + nicotinic acid methyl ester <1> (Reversibility: ? <1> [3]) [3] P UDP + N-glucosylnicotinic acid methyl ester S UDP-glucose + vanillic acid <1> (Reversibility: ? <1> [3]) [3] P ? S UDP-glucose + vanillin <1> (Reversibility: ? <1> [3]) [3] P ? Inhibitors 6-hydroxynicotinic acid <3> (<3> 73% inhibition at 1 mM [1]) [1] Cd2+ <3> (<3> at 1mM [1]) [1] Cu2+ <3> (<3> at 1mM [1]) [1] Hg2+ <3> (<3> at 1mM [1]) [1] N1 -methylnicotinamide <3> (<3> 33% inhibition at 1 mM [1]) [1] Pb2+ <3> (<3> at 1mM [1]) [1] UTP <3> (<3> 33% inhibition at 1 mM [1]) [1] Zn2+ <1, 3> (<3> at 1mM [1]) [1, 3] benzoic acid <3> (<3> 88% inhibition at 1 mM [1]) [1] isonicotinic acid <3> (<3> 98% inhibition at 1 mM [1]) [1] nicotinic acid N-oxide <3> (<3> 31% inhibition at 1 mM [1]) [1] p-chloromercuribenzoate <1> [3] salicylamide <3> (<3> 30% inhibition at 1 mM [1]) [1] Metals, ions Additional information <1> (<1> no requirement for divalent cations [3]) [3] Specific activity (U/mg) Additional information <1> [3] Km-Value (mM) 0.17 <1> (nicotinic acid) [3] 0.22 <1> (nicotinic acid) [3] 1.2 <1> (UDPglucose) [3] 4.3 <1> (UDPglucose) [3] pH-Optimum 6.1 <3> [1] 7.8-8.2 <1> [3] pH-Range 5-11 <1> (<1> at pH 5 nearly inactive, at pH 11 about 50% of activity maximum [3]) [3] Temperature optimum ( C) 25 <3> [1] 30 <1> [3]

486

2.4.1.196

Nicotinate glucosyltransferase

4 Enzyme Structure Molecular weight 46000 <1> (<1> gel filtration [3]) [3] Subunits monomer <1> (1 * 46000, SDS-PAGE [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue callus <2> [2] cell suspension culture <1, 2> (<1> heterotrophic [3]) [3, 2] leaf <2> [2] tuber <2> [2] Localization soluble <1> [3] Purification <1> (partial [3]) [3] <3> (partial [1]) [1]

References [1] Taguchi, H.; Sasatani, K.; Nishitani, H.; Okumura, K.: Finding of UDP-glucose:nicotinic acid-N-glucosyltransferase activity in cultured tobacco cells and its properties. Biosci. Biotechnol. Biochem., 61, 720-722 (1997) [2] Köster, S.; Upmeier, B.; Komossa, D.; Barz, W.: Nicotinic acid conjugation in plants and plant cell cultures of potato (Solanum tuberosum). Z. Naturforsch. C, 44c, 623-628 (1989) [3] Upmeier, B.; Thomzik, J.E.; Barz, W.: Enzymatic studies on the reversible synthesis of nicotinic acid-N-glucoside in heterotrophic parsley cell suspension cultures. Z. Naturforsch. C, 43c, 835-842 (1988)

487

High-mannose-oligosaccharide b-1,4-N-acetylglucosaminyltransferase

2.4.1.197

1 Nomenclature EC number 2.4.1.197 Systematic name UDP-N-acetyl-d-glucosamine:high-mannose-oligosaccharide b-1,4-N-acetylglucosaminyltransferase Recommended name high-mannose-oligosaccharide b-1,4-N-acetylglucosaminyltransferase Synonyms UDP-GlcNAc:oligosaccharide b-N-acetylglucosaminyltransferase acetylglucosamine-oligosaccharide acetylglucosaminyltransferase acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-oligosaccharide CAS registry number 123425-54-7

2 Source Organism <1> Dictyostelium discoideum [1, 2]

3 Reaction and Specificity Catalyzed reaction transfers an N-acetyl-d-glucosamine residue from UDP-N-acetyl-d-glucosamine to the 4-position of a mannose linked a-1,6 to the core mannose of high-mannose oligosaccharides produced by Dictyostelium discoideum Reaction type hexosyl group transfer Substrates and products S UDP-N-acetyl-d-glucosamine + O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,3-[O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,3-(O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,6)O-mannopyranosyl-a-1,6]-O-mannopyranosyl-b-1,4-N-acetylglucosamine <1> (<1> i.e. Man9 GlcNAc oligosaccharide, enzyme catalyzes the

488

2.4.1.197

P

S P S

P

S P S P S P

High-mannose-oligosaccharide b-1,4-N-acetylglucosaminyltransferase

transfer of N-acetyl-glucosamine to both a- and b linked mannoses [1]) (Reversibility: ? <1> [1]) [1] UDP + O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,3-[O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,3-(Omannopyranosyl-a-1,2-O-mannopyranosyl-a-1,6)-(N-acetyl-d-glucoseamine-b-1,4)-O-mannopyranosyl-a-1,6]-O-mannopyranosyl-b-1,4-N-acetyl-d-glucosamine <1> (<1> i.e. Man9 GlcNAc oligosaccharide with an additional N-acetyl-d-glucosamine residue attached b-1,4 to the mannose linked a-1,6 to the core mannose in an intersecting position [1]) [1] UDP-N-acetyl-d-glucosamine + (O-mannopyranosyl-a-1,3)-mannopyranosyl-b-1,4-N-acetyl-glucosamine <1> (<1> i.e. Man2 GlcNAc, very low activity [1]) (Reversibility: ? <1> [1]) [1] UDP + O-mannopyranosyl-a-1,3-[N-acetyl-glucoseamine-b-1,4]-mannopyranosyl-b-1,4-N-acetyl-d-glucosamine <1> [1] UDP-N-acetyl-d-glucosamine + O-mannopyranosyl-a-1,3-[O-mannopyranosyl-a-1,3-(O-mannopyranosyl-a-1,6)-O-mannopyranosyl-a-1,6]-Omannopyranosyl-b-1,4-N-acetyl-d-glucosamine <1> (<1> i.e. Man5 GlcNAc oligosaccharide, 20% of activity compared to Man9 GlcNAc [1]) (Reversibility: ? <1> [1]) [1] UDP + O-mannopyranosyl-a-1,3-O-mannopyranosyl-a-1,3-(O-mannopyranosyl-a-1,6)-[N-acetylglucoseamine-b-1,4-O-mannopyranosyl-a1,6]-O-mannopyranosyl-b-1,4-N-acetyl-d-glucosamine <1> (<1> i.e. Man5 GlcNAc oligosaccharide with an additional N-acetyl-d-glucosamine residue in an intersected and/or bisected position, so that either the core b-mannose or the branching a-mannose residue accepts the transferred N-acetyl-d-glucosamine residue [1]) [1] UDP-N-acetyl-d-glucosamine + O-mannopyranosyl-a-1,3-mannopyranosyl-a-methyl <1> (<1> i.e. Man2 Me [1]) (Reversibility: ? <1> [1]) [1] ? UDP-N-acetyl-d-glucosamine + O-mannopyranosyl-a-1,3[O-mannopyranosyl-a-1,6]-O-mannopyranosyl-b-1,4-N-acetylglucosamine <1> (<1> i.e. Man3 GlcNAc [1]) (Reversibility: ? <1> [1]) [1] UDP + O-mannopyranosyl-a-1,3[O-mannopyranosyl-a-1,6-(N-acetyl-dglucoseamine b-1,4)]-O-mannopyranosyl-b-1,4-N-acetylglucosamine <1> [1] UDP-N-acetyl-d-glucosamine + methyl O-a-mannopyranosyl-1,6-[O-ad-mannopyranosyl-1,3]-a-d-mannopyranoside <1> (<1> trivial name Man3 Me [1]) (Reversibility: ? <1> [1]) [1, 2] UDP + methyl O-a-mannopyranosyl-1,6-[O-a-d-mannopyranosyl-1,3]a-d-mannopyranosyl-1,4-N-acetyl-d-glucosamine <1> [1, 2]

Inhibitors EDTA <1> (<1> strong [1]) [1] EGTA <1> (<1> strong [1]) [1] Mg2+ <1> (<1> strong [1]) [1] dithiothreitol <1> (<1> strong inhibition in the presence of Mg2+ and Mn2+ [1]) [1]

489

High-mannose-oligosaccharide b-1,4-N-acetylglucosaminyltransferase

2.4.1.197

Metals, ions Mn2+ <1> (<1> stimulation [1]) [1] Additional information <1> (<1> neither Mg2+ nor Ca2+ can substitute for Mn2+ [2]) [2] Specific activity (U/mg) 0.0000077 <1> (<1> Man5 GlcNAc, intersected [1]) [1] 0.0000129 <1> (<1> Man9 GlcNAc, intersected [1]) [1] 0.0000133 <1> (<1> Man5 GlcNAc, bisected [1]) [1] Km-Value (mM) 0.3-0.43 <1> (O-mannopyranosyl-a-1,3-[O-mannopyranosyl-a-1,3-(O-mannopyranosyl-a-1,6)-O-mannopyranosyl-a-1,6]-O-mannopyranosyl-b-1,4-Nacetyl-d-glucosamine, <1> i.e. Man5 GlcNAc, depending whether N-acetyl-dglucosamine residue is intersected or bisected [1]) [1] 0.65 <1> (O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,3-[O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,3-(O-mannopyranosyl-a-1,2-O-mannopyranosyl-a-1,6)-O-mannopyranosyl-a-1,6]-Omannopyranosyl-b-1,4-N-acetyl-d-glucosamine, <1> i.e. Man9 GlcNAc, intersected product [1]) [1] 1.2 <1> (UDP-N-acetyl-d-glucosamine, <1> cosubstrate Man9 GlcNAc [1]) [1] 1.42 <1> (methyl O-a-mannopyranosyl-1,6-[O-a-d-mannopyranosyl-1,3]-ad-mannopyranoside, <1> intersected product [1]) [1] 2.54 <1> (O-mannopyranosyl-a-1,3[O-mannopyranosyl-a-1,6]-O-mannopyranosyl-b-1,4-N-acetylglucosamine, <1> bisected product [1]) [1] 18.52 <1> (O-mannopyranosyl-a-1,3-mannopyranosyl-a-methyl, <1> intersected product [1]) [1] 19.48 <1> (O-mannopyranosyl-a-1,3-mannopyranosyl-b-1,4-N-acetyl-glucoseamine, <1> bisected product [1]) [1] pH-Optimum 6-6.9 <1> [1] pH-Range 5.5-7.2 <1> (<1> 20% of maximal activity at pH 5.5, 70% of maximal activity at pH 7.2 [1]) [1] Temperature optimum ( C) 37 <1> (<1> assay at [1]) [1, 2]

5 Isolation/Preparation/Mutation/Application Localization Golgi membrane <1> (<1> integral membrane protein, at least the catalytic center is on the luminal site of the vesicles [2]) [2] membrane <1> (<1> membrane-associated [1]) [1, 2]

490

2.4.1.197

High-mannose-oligosaccharide b-1,4-N-acetylglucosaminyltransferase

Purification <1> (strain M4, partial [1]) [1]

6 Stability Temperature stability 45 <1> (<1> 75% decrease of activity after 2 min [1]) [1]

References [1] Sharkey, D.J.; Kornfeld, R.: Identification of an N-acetylglucosaminyltransferase in Dictyostelium discoideum that transfers an intersecting N-acetylglucosamine residue to high mannose oligosaccharides. J. Biol. Chem., 264, 10411-10419 (1989) [2] Capasso, J.M.; Capasso, A.; Kaplan, A.: Subcellular distribution of intersecting b-N-acetylglucosaminyltransferase in Dictyostelium discoideum. A likely marker for the Golgi apparatus. Biochim. Biophys. Acta, 1281, 15-22 (1996)

491

Phosphatidylinositol N-acetylglucosaminyltransferase

2.4.1.198

1 Nomenclature EC number 2.4.1.198 Systematic name UDP-N-acetyl-d-glucosamine:1-phosphatidyl-1d-myo-inositol 6-(N-acetyl-ad-glucosaminyl)transferase Recommended name phosphatidylinositol N-acetylglucosaminyltransferase Synonyms GPI-GlcNAc transferase <4, 5> [5] GPI-GnT <4, 5> [5] UDP-N-acetyl-d-glucosamine:phosphatidylinositol N-acetyl-d-glucosaminyltransferase acetyl-d-glucosaminyltransferase, uridine diphosphoacetylglucosamine a1,6phosphatidylinositol/UDP-GlcNAc:GlcNAc transferase <3> [4] uridine diphosphoacetylglucosamine a1,6-acetyl-d-glucosaminyltransferase CAS registry number 144388-35-2

2 Source Organism <1> Trypanosoma brucei (cloned variant ILTat 1.3, isolated from rat blood [1]; variant MITat 1.4 [2]) [1, 2] <2> Homo sapiens [3, 5, 6, 8, 10] <3> Bombyx mori [4] <4> Saccharomyces cerevisiae (strain XM37-10c [9]; at least 3 components forming the enzyme complex: GPI1, GPI2 or PIG-C, GPI3 or PIG-A [9]) [5, 9] <5> Homo sapiens (gene hGPI1 [5]) [5, 8] <6> Mus musculus (gene mGPI1 clone [6]) [6] <7> Mus musculus (gene mGPI1 clone [6]) [6] <8> Cricetulus griseus [7] <9> Mus musculus [7] <10> Homo sapiens (gene PIG-P [8]) [8] <11> Mus musculus (gene PIG-P [8]) [8]

492

2.4.1.198

Phosphatidylinositol N-acetylglucosaminyltransferase

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + 1-phosphatidyl-1d-myo-inositol = UDP + 6(N-acetyl-a-d-glucosaminyl)-1-phosphatidyl-1d-myo-inositol (In some species, the long-chain acyl groups of the phosphatidyl group are partly replaced by long-chain alkyl or alk-1-enyl groups.) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + phosphatidylinositol <1, 2, 4-11> (<1, 2, 4-11> involved in the first step of glycosylphosphatidylinositol, i.e. GPI, membrane anchor formation of surface glycoproteins [1-3, 5-9]) [1-3, 5-9] P UDP + N-acetyl-d-glucosaminyl-phosphatidylinositol S Additional information <2, 4-11> (<4> enzyme exists as a complex of at least 3 proteins PIG-A or GPI3, PIG-C or GPI2 and GPI1, in which PIG-A, not PIG-C, is the substrate binding component [9]; <8,9> biosynthetic pathway and subcellular localization, overview [7]; <2,10,11> enzyme is active in a complex of at least 5 proteins, termed GPI1, PIG-P, PIG-A, PIG-C and PIG-H, PIG-P is absolutely required [8]; <2,5-7> enzyme is active in a complex of at least 4 proteins, termed GPI1, PIG-A, PIG-C and PIG-H, in which GPI1 is absolutely essential for stabilization [5,6]; <4> glycosylphosphatidylinositol anchoring is essential for transport of cell surface proteins and enzyme deficieny leads to defective cell wall synthesis and cell death [5]; <2> enzyme defect in paroxysmal nocturnal hemoglobinuria [3]) [3, 5-9] P ? Substrates and products S UDP-N-acetyl-d-glucosamine + phosphatidylinositol <1-11> (<2, 4-7, 10, 11> enzyme complex [5, 6, 8, 9]; <5> substrate specificity [5]) (Reversibility: ? <1-11> [1-9]) [1-9] P UDP + N-acetyl-d-glucosaminyl-phosphatidylinositol <1-11> (<1, 2> in a1-6 linkage [2,3]) [1-9] Inhibitors N-ethylmaleimide <1> (<1> strong [2]; <1> binding site is close or at the active site, UDP-N-acetyl-d-glucosamine protects [2]) [2] SH-alkylating reagents <1> [2] iodoacetic acid <1> [2] p-chloromercuriphenylsulfonic acid <1> [2] Activating compounds component 2 of dolichyl-phosphate-mannose synthase <2> (<2> i.e. DPM2 [8]; <2> DPM2 protein binds directly to the enzyme complex and enhances the enzyme activity, regulatory role [8]) [8] Additional information <3> (<3> no activation by Triton X-100 or Triton X114 [4]) [4] 493

Phosphatidylinositol N-acetylglucosaminyltransferase

2.4.1.198

Metals, ions Ca2+ <3> (<3> activates [4]) [4] K+ <3> (<3> activates [4]) [4] Mg2+ <3> (<3> activates [4]) [4] Mn2+ <3> (<3> activates [4]) [4] Additional information <3> (<3> requires cations for activity, such as Mn2+ , Mg2+ , K+ or Ca2+ [4]) [4] pH-Optimum 7.4 <1, 2, 5-7, 10, 11> (<1,2,5-7,10,11> assay at [1-3,5,6,8]) [1-3, 5, 6, 8] Temperature optimum ( C) 37 <1, 2, 6-11> (<1,2,6-11> assay at [2,3,6-8]) [2, 3, 6-8]

4 Enzyme Structure Molecular weight Additional information <2, 4-7, 10, 11> (<4> enzyme exists as a complex of at least 3 proteins PIG-A or GPI3, PIG-C or GPI2 and GPI1, in which PIG-A, not PIG-C, is the substrate binding component [9]; <2,10,11> enzyme is active in a complex of at least 5 proteins, termed GPI1, PIG-P, PIG-A, PIG-C and PIG-H [8]; <2,5-7> enzyme is active in a complex of at least 4 proteins, termed GPI1, PIG-A, PIG-C and PIG-H, in which GPI1 is absolutely essential for stabilization [5,6]) [5, 6, 8] Subunits Additional information <2, 10, 11> (<2> topological model of the enzyme complex [10]; <2,10,11> structure of enzyme complex, overview [8]) [8, 10]

5 Isolation/Preparation/Mutation/Application Source/tissue CHO cell <8> (<8> CHO-K1 cell lines G9PLAP and mutant G9PLAP.85 [7]) [7] larva <3> (<3> 5th instar larvae [4]) [4] lymphoblastoid cell line <2> (<2> with paroxysmal nocturnal hemoglobinuria phenotype, i.e. PNH, obtained by Epstein-Barr immortalization of lymphocytes from PNH-patients [3]) [3] midgut <3> (<3> of the 5th instar larvae [4]) [4] thymus lymphoma cell line <9> (<9> BW514.3 thymoma cell line [7]) [7] Localization endoplasmic reticulum <2, 4-11> (<2> major part at the cytoplasmic side of perinuclear and mitochondria-associated lamellae [10]) [5-10] membrane <1, 2, 4, 5> [1-3, 5] microsome <2, 3, 6, 7> [3, 4, 6] Additional information <2> (<2> type II membrane protein [10]) [10] 494

2.4.1.198

Phosphatidylinositol N-acetylglucosaminyltransferase

Purification <2> (recombinant FLAG-tagged and His-tagged PIG-A from human B lymphoblastoid JY5 cells and from Escherichia coli [10]) [10] <2, 5> (purification of GPI1 by overexpression of PIG-A in JY5 cells and isolation of the complex by affinity chromatography [8]) [8] Cloning <2> (expression of FLAG-tagged and His-tagged PIG-A in PIG-A-deficient human B lymphoblastoid JY5 cells and in Escherichia coli, complementation in JY5 cells [10]; overexpression of FLAG- and GST-double tagged PIG-A in human B lymphoblastoid JY5 cells [8]) [8, 10] <4> (expression of PIG-A/GPI3 and PIG-C/GPI2 as FLAG-tagged fusion proteins in yeast, expression of FLAG-tagged PIG-A/GPI3 in Escherichia coli [9]) [9] <5> (hGPI1, DNA sequence determination, coexpression as GST-tagged protein in human B lymphoblastoid JY25 or JY5 cells with the other three GSTor FLAG-tagged protein of the active protein complex [5]) [5] <6> (coexpression of FLAG-tagged PIG-C, GST-tagged PIG-A and PIG-H with and without mGPI1 in the mGPI1-deficient F9 cells [6]; mGPI1, DNA sequence determination, expression in embryonal carcinoma F9 cells [6]) [6] <7> (coexpression of FLAG-tagged PIG-C, GST-tagged PIG-A and PIG-H with and without mGPI1 in the mGPI1-deficient F9 cells [6]; mGPI1, DNA sequence determination, expression in embryonal carcinoma F9 cells [6]) [6] <10> (expression of PIG-P in enzyme deficient mouse mutant T cell line can complement and restore the enzyme activity [8]) [8] Engineering Additional information <6, 7> (<6,7> disruption of the genes in F9 cells via homologous recombination, causing a severe but not complete defect in the generation of glycosylphosphatidylinositol-anchored proteins, highly reduced activity in vivo, no activity in vitro, decrease in GPI1 also caused decrease of PIG-C and PIG-H [6]) [6]

References [1] Doering, T.L.; Masterson, W.J.; Englund, P.T.; Hart, G.W.: Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine. J. Biol. Chem., 264, 11168-11173 (1989) [2] Milne, K.G.; Ferguson, M.A.J.; Masterson, W.J.: Inhibition of the GlcNAc transferase of the glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes. Eur. J. Biochem., 208, 309-314 (1992) [3] Hillmen, P.; Bessler, M.; Mason, P.J.; Watkins, W.M.; Luzzatto, L.: Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria. Proc. Natl. Acad. Sci. USA, 90, 5272-5276 (1993) 495

Phosphatidylinositol N-acetylglucosaminyltransferase

2.4.1.198

[4] Takano, D.; Kishimoto, T.; Hayakawa, T.; Mitsui, T.; Hori, H.: Activity of phosphatidylinositol/UDP-GlcNAc:GlcNAc transferase in midgut tissue of Bombyx mori. Niigata Daigaku Nogakubu Kenkyu Hokoku, 55, 123-131 (2003) [5] Watanabe, R.; Inoue, N.; Westfall, B.; Taron, C.H.; Orlean, P.; Takeda, J.; Kinoshita, T.: The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1. EMBO J., 17, 877-885 (1998) [6] Hong, Y.; Oshihi, K.; Watanabe, R.; Endo, Y.; Maeda¹ Y.; Kinoshita, T.: GPI1 stabilizes an enzyme essential in the first step of glycosylphosphatidylinositol biosynthesis. J. Biol. Chem., 274, 18582-18588 (1999) [7] Vidugiriene, J.; Sharma, D.K.; Smith, T.K., Baumann, N.A.; Menon, A.K.: Segregation of glycosylphosphatidylinositol biosynthetic reactions in a subcompartment of the endoplasmic reticulum. J. Biol. Chem., 274, 1520215212 (1999) [8] Watanabe, R.; Murakami, Y.; Marmor, M.; Inoue, N.; Maeda, Y.; Hino, J.; Kangawa, K.; Julius, M.; Kinoshita, T.: Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2. EMBO J., 19, 4402-4411 (2000) [9] Kostova, Z.; Rancour, D.M.; Menon, A.K.; Orlean, P.: Photoaffinity labelling with P3-(4-azidoanilido)uridine 5'-triphosphate identifies Gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyses formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis. Biochem. J., 350, 815-822 (2000) [10] Tiede, A.; Nischan, C.; Schubert, J.; Schmidt, R.: Characterization of the enzymatic complex for the first step in glycosylphosphatidylinositol biosynthesis. Int. J. Biochem. Cell Biol., 32, 339-250 (2000)

496

b-Mannosylphosphodecaprenolmannooligosaccharide 6-mannosyltransferase

2.4.1.199

1 Nomenclature EC number 2.4.1.199 Systematic name b-d-mannosylphosphodecaprenol:1,6-a-d-mannosyloligosaccharide 1,6-a-dmannosyltransferase Recommended name b-mannosylphosphodecaprenol-mannooligosaccharide 6-mannosyltransferase Synonyms mannosylphospholipid-methylmannoside a-1,6-mannosyltransferase mannosyltransferase, mannosylphospholipid-methylmannoside a-1,6CAS registry number 125008-27-7

2 Source Organism <1> Mycobacterium smegmatis [1]

3 Reaction and Specificity Catalyzed reaction b-d-mannosylphosphodecaprenol + 1,6-a-d-mannosyloligosaccharide = decaprenol phosphate + 1,6-a-d-mannosyl-1,6-a-d-mannosyl-oligosaccharide Reaction type hexosyl group transfer Natural substrates and products S mannosylphosphodecaprenol + 1,6-a-d-mannooligosaccharides <1> (<1> involved in the formation of mannooligosaccharides in membrane of Mycobacterium smegmatis [1]) [1] P decaprenol phosphate + 1,6-a-d-mannosyl-1,6-a-d-mannooligosaccharides

497

b-Mannosylphosphodecaprenol-mannooligosaccharide 6-mannosyltransferase

2.4.1.199

Substrates and products S mannosylphosphodecaprenol + 1,6-a-d-mannooligosaccharides <1> (<1> poor substrates are mannose and 1,6-a-mannobiose, no substrates are 1,2-, 1,3-a-mannooligosaccharides or myo-inositol-2,6-dimannoside [1]) (Reversibility: ? <1> [1]) [1] P decaprenol phosphate + 1,6-a-d-mannosyl-1,6-a-d-mannooligosaccharides <1> [1] S mannosylphosphodecaprenol + 1,6-a-d-mannopentaose <1> (Reversibility: ? <1> [1]) [1] P decaprenol phosphate + 1,6-a-d-mannohexaose S mannosylphosphodecaprenol + 1,6-a-d-mannotetraose <1> (Reversibility: ? <1> [1]) [1] P decaprenol phosphate + 1,6-a-d-mannosylpentaose S mannosylphosphodecaprenol + 1,6-a-d-mannotriose <1> (Reversibility: ? <1> [1]) [1] P decaprenol phosphate + 1,6-a-d-mannosyltetraose S mannosylphosphodecaprenol + methyl-a-d-mannoside <1> (Reversibility: ? <1> [1]) [1] P decaprenol phosphate + ? S mannosylphosphodecaprenol + myo-inositol-trimannoside <1> (<1> with an 1,6-a-mannobiose unit as part of its structure [1]) (Reversibility: ? <1> [1]) [1] P decaprenol phosphate + ? S Additional information <1> (<1> substrate specificity [1]) [1] P ? Metals, ions Mg2+ <1> [1] Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 2 <1> (1,6-a-d-mannotriose) [1] 2.4 <1> (1,6-a-d-mannotetraose) [1] 2.6 <1> (1,6-a-d-mannopentaose) [1] 28.6 <1> (methyl-a-d-mannoside) [1] 35.7 <1> (1,6-a-mannobiose) [1] 86.9 <1> (mannose) [1] pH-Optimum 8 <1> (<1> assay at [1]) [1] Temperature optimum ( C) 37 <1> (<1> assay at [1]) [1]

498

2.4.1.199

b-Mannosylphosphodecaprenol-mannooligosaccharide 6-mannosyltransferase

5 Isolation/Preparation/Mutation/Application Localization membrane <1> [1]

References [1] Yokoyama, K.; Ballou, C.E.: Synthesis of a 1-6-mannooligosaccharides in Mycobacterium smegmatis. Function of b-mannosylphosphoryldecaprenol as the mannosyl donor. J. Biol. Chem., 264, 21621-21628 (1989)

499

Inulin fructotransferase (depolymerizing, difructofuranose-1,2':2',1-dianhydrideforming)

2.4.1.200

1 Nomenclature EC number 2.4.1.200 (transferred to EC 4.2.2.17) Recommended name inulin fructotransferase (depolymerizing, difructofuranose-1,2':2',1-dianhydride-forming)

500

a-1,6-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

2.4.1.201

1 Nomenclature EC number 2.4.1.201 Systematic name UDP-N-acetyl-d-glucosamine:2,6-bis(N-acetyl-b-d-glucosaminyl)-a-d-mannosyl-glycoprotein 4-b-N-acetyl-d-glucosaminyltransferase Recommended name a-1,6-mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase Synonyms GnT VI <4, 5> [3, 4] N-acetylglucosaminyltransferase VI N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase VI UDP-GlcNAc:GlcNAcb1-6(GlcNAcb1-2)Mana-R (GlcNAc to Man) b4-GlcNActransferase VI <1-3> [1] UDP-GlcNAc:GlcNAcb1-6(GlcNAcb1-2)Mana1-R [GlcNAc to Man]b1-4N-acetylglucosaminyltransferase VI <1, 4> [3, 6] UDP-N-acetyl-d-glucosamine (GlcNAc):GlcNAcb1-6(GlcNAcb1-2)-Mana1R[GlcNAc to Man]b1,4N-acetylglucosaminyltransferase VI <5> [4] acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-glycopeptide b-1,4-, VI acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-glycopeptide b-1-4-, VI mannosyl-glycoprotein b-1,4-N-acetylglucosaminyltransferase uridine diphosphoacetylglucosamine-glycopeptide b-1-4-acetylglucosaminyltransferase VI Additional information (cf. EC 2.4.1.101, EC 2.4.1.143, EC 2.4.1.144, EC 2.4.1.145, EC 2.4.1.146) CAS registry number 119699-68-2

2 Source Organism <-5> no <-4> no <-3> no <-2> no

activity activity activity activity

in in in in

Bos taurus (kidney [1]) [1] Homo sapiens (colon [1]) [1] Sus scrofa (colon and stomach [1]) [1] african green monkey (COS-1 cells [4]) [4]

501

a-1,6-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

2.4.1.201

<-1> no activity in Rattus norvegicus (colon and liver [1]) [1] <1> Gallus gallus (hen [2,5,6]) [1, 2, 5, 6] <2> Anas sp. (duck [1]) [1] <3> Meleagris gallopavo (turkey [1]) [1] <4> Oryzias latipes (Medaka fish [3]) [3] <5> Gallus gallus (SwissProt-ID: Q9DGD1) [4]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + 2,6-bis(N-acetyl-b-d-glucosaminyl)-a-dmannosyl-R = UDP + 2,4,6-tris(N-acetyl-b-d-glucosaminyl)-a-d-mannosylR (R represents the remainder of the N-linked oligosaccharide in the glycoprotein acceptor) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2)-b-d-mannosyl-R <1-3, 5> (<1,5> biosynthesis of pentaantennary asparagine-linked N-glycans [4,5]; <1> involved in N-glycan biosynthesis [2]; <1> reaction in biosynthesis of penta-antennary oligosaccharides of hen ovomucoid [1]) [1, 2, 4, 5] P UDP + N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2-b)(N-acetyl-d-glucosaminyl-1,4)-b-d-mannosyl-R Substrates and products S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,6-b-d-(Nacetylglucosaminyl-1,2)-a-mannosyl-1,6-b-d-glucosyl-octyl <4> (<4> synthetic acceptor substrate [3]) (Reversibility: ? <4> [3]) [3] P UDP + N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2-b)(N-acetyl-d-glucosaminyl-1,4)-b-d-glucosyl-octyl <4> (<4> identification and large scale preparation of the product [3]) [3] S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,6-b-d-(Nacetylglucosaminyl-1,2)-a-mannosyl-1,6-b-d-mannosyl-(CH2 )8 -CH3 <1> (<1> synthetic acceptor substrate [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2-b)(N-acetyl-d-glucosaminyl-1,4)-b-d-mannosyl-(CH2 )8 -CH3 S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,6-b-d-(Nacetylglucosaminyl-1,2)-a-mannosyl-1,6-b-d-mannosyl-(CH2 )8 -COOH <1> (<1> synthetic acceptor substrate [1]) (Reversibility: ? <1> [1]) [1] P UDP + N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2-b)(N-acetyl-d-glucosaminyl-1,4)-b-d-mannosyl-(CH2 )8 -COOH S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,6-b-d-(Nacetylglucosaminyl-1,2)-b-d-mannosyl-R <1-5> (<1,5> R represents the remainder of the N-oligosaccharide core of asparagine-linked glycopep-

502

2.4.1.201

a-1,6-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

tides, (+/-)-fucose, minimum structure requirement: N-acetyl-d-glucosaminyl-b-1,6-(N-acetylglucosaminyl-b-1,2)-a-mannosyl-R [2,4,5]; <1> biantennary oligosaccharides, i.e. Gn T I and II reaction products, are no substrates [5]; <1> acts on bisected and non-bisected substrates [1,2]; <1> acceptor substrate specificity [1,5]; <1> compounds lacking b-2linked N-acetylglucosamine residues are no acceptor substrates [2]) (Reversibility: ? <1-5> [1-5]) [1-5] P UDP + N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2-b)(N-acetyl-d-glucosaminyl-1,4)-b-d-mannosyl-R <1-5> [1-5] S UDP-N-acetyl-d-glucosamine + N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2)-b-d-mannosyl-pyridylamine <1> (<1> fluorescent synthetic substrate [6]) (Reversibility: ? <1> [6]) [6] P UDP + N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2-b)(N-acetyl-d-glucosaminyl-1,4)-b-d-mannosyl-pyridylamine <1> [6] Inhibitors AMP <1> (<1> 0.0125 mM, in the presence of 0.125 M N-acetylglucosamine [1]) [1] ATP <1> (<1> 0.0125 mM, in the presence of 0.125 M N-acetylglucosamine [1]) [1] EDTA <1> (<1> complete inhibition at 1 mM [6]) [1, 6] N-acetylglucosamine <1> (<1> weak, 0.125 M [1]) [1] UDP <1> (<1> 0.0125 mM, in the presence of 0.125 M N-acetylglucosamine [1]) [1] UDP-hexanolamine <1> (<1> 0.0125 mM, in the presence of 0.125 M N-acetylglucosamine [1]) [1] Activating compounds Triton X-100 <1-3> (<1-3> activation, 0.125-1.25% [1,2]) [1, 2, 6] Metals, ions Ca2+ <1> (<1> 8.5% activity compared to Mn2+ [5]; <1> activation, 12.5 mM, can substitute partially for Mn2+ [1,2]) [1, 2] Co2+ <1, 4> (<1> activation, 12.5 mM [1, 2]; <1> can substitute partially for Mn2+ [1, 2, 5, 6]; <1> 86% activity compared to Mn2+ [6]; <1> 80% activity compared to Mn2+ [5]; <4> 70% activity compared to Mn2+ [3]) [1-3, 5, 6] Mg2+ <1, 4> (<1> activation, 12.5 mM [1, 2]; <1> can fully substitute for Mn2+ , 25 mM [6]; <1> can substitute partially for Mn2+ [1, 2, 5]; <1> 60% activity compared to Mn2+ [5]; <4> 8.6% activity compared to Mn2+ [3]) [13, 5, 6] Mn2+ <1-4> (<1-4> absolute requirement [1-3,5,6]; <1> maximal activity at 5 mM [5]; <1,4> maximal activity at 25 mM [3,6]; <1-3> maximal activity at 0.05-0.1 M [1,2]) [1-3, 5, 6] Ni2+ <1, 4> (<1> can substitute partially for Mn2+ [5,6]; <1> 14% activity compared to Mn2+ [6]; <1> 27% activity compared to Mn2+ [5]; <4> 23% activity compared to Mn2+ [3]) [3, 5, 6]

503

a-1,6-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

2.4.1.201

Additional information <1, 4> (<1> requirement for divalent cations, no effect of Zn2+ , Fe2+ , Cu2+ [5]; <4> absolute requirement for divalent cations, Cu2+ cannot substitute Mn2+ [3]; <1> no activation by 12.5 mM Cd2+ , Ni2+ or Zn2+ [1,2]) [1-3, 5] Specific activity (U/mg) 0.00000045 <5> (<5> recombinant enzyme in COS-1 cells [4]) [4] 0.032 <1> (<1> purified enzyme [5]) [5] Additional information <1> (<1> assay method development [6]) [6] Km-Value (mM) 0.09 <1> (N-acetyl-d-glucosaminyl-1,6-b-d-(N-acetylglucosaminyl-1,2)-b-dmannosyl-R) [1] 0.6 <1> (UDP-N-acetyl-d-glucosamine) [1] pH-Optimum 7 <4> [3] 7-8.4 <1> [1, 2] 7.8 <1> [5] 8 <1> (<1> HEPES buffer [6]) [6] pH-Range 5-8.4 <1> (<1> about half-maximal activity at pH 5.0 and about 80% of maximal activity at pH 8.4 [1]) [1] Additional information <1> (<1> broad range [5]) [5] Temperature optimum ( C) 37 <1-4> (<1-4> assay at [1-3,5,6]) [1-3, 5, 6]

4 Enzyme Structure Molecular weight Additional information <5> (<5> type II transmembrane protein, primary structure, amino acid sequence [4]) [4] Subunits ? <1, 5> (<1> x * 72000, non-reducing SDS-PAGE [5]; <1,5> x * 60000, reducing SDS-PAGE [4,5]) [4, 5] Posttranslational modification glycoprotein <1, 5> (<5> 2 potential N-glycosylation sites [4]) [4, 5]

5 Isolation/Preparation/Mutation/Application Source/tissue colon <1, 2, 5> [1, 4] intestine <3> [1] liver <1> [1]

504

2.4.1.201

a-1,6-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

lung <5> [4] ovary <4> [3] oviduct <1, 5> [1, 2, 4-6] spleen <5> [4] Additional information <1> (<1> tissue distribution [1]) [1] Localization microsome <1> [1, 2, 5, 6] Purification <1> [5] Cloning <5> (cloning from cDNA library, DNA sequence determination, functional expression in COS-1 cells [4]) [4]

6 Stability General stability information <1>, dithiothreitol is required for stabilization of the purified enzyme [5] <1>, enzyme becomes more stable after elution from a Ni2+ -chelating column during purification [5]

References [1] Brockhausen, I.; Hull, E.; Hindsgaul, O.; Schachter, H.; Shah, R.N.; Michnick, S.W.; Carver, J.P.: Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc b 1-6 (GlcNAc b 1-2)Man a-R (GlcNAc to Man) b-4-N-acetylglucosaminyltransferase VI. J. Biol. Chem., 264, 11211-11221 (1989) [2] Schachter, H.; Brockhausen, I.; Hull, E.: High-performance liquid chromatography assays for N-acetylglucosaminyltransferases involved in N- and Oglycan synthesis. Methods Enzymol., 179, 351-397 (1989) [3] Taguchi, T.; Kitajima, K.; Inoue, S.; Inoue, Y.; Yang, J.-M.; Schachter, H.; Brockhausen, I.: Activity of UDP-GlcNAc:GlcNAcb1-6(GlcNAcb1-2)Mana1R[GlcNAc to Man]b1-4N-acetylglucosaminyltransferase VI (GnT VI) from the ovaries of Oryzias latipes (Medaka fish). Biochem. Biophys. Res. Commun., 230, 533-536 (1997) [4] Sakamoto, Y.; Taguchi, T.; Honke, K.; Korekane, H.; Watanabe, H.; Tano, Y.; Dohmae, N.; Takio, K.; Horii, A.; Taniguchi, N.: Molecular cloning and expression of cDNA encoding chicken UDP-N-acetyl-d-glucosamine (GlcNAc):GlcNAcb1-6(GlcNAcb1-2)-Mana1-R[GlcNAc to Man]b1,4N-acetylglucosaminyltransferase VI. J. Biol. Chem., 275, 36029-36034 (2000) [5] Taguchi, T.; Ogawa, T.; Inoue, S.; Inoue, Y.; Sakamoto, Y.; Korkane, H.; Taniguchi, N.: Purification and characterization of UDP-GlcNAc:GlcNAcb1-

505

a-1,6-Mannosyl-glycoprotein 4-b-N-acetylglucosaminyltransferase

2.4.1.201

6(GlcNAcb1-2)Mana1-R [GlcNAc to Man]-b1,4-N-acetylglucosaminyltransferase VI from hen oviduct. J. Biol. Chem., 175, 32598-32602 (2000) [6] Taguchi, T.; Ogawa, T.; Kitajima, K.; Inoue, S.; Inoue, Y.; Ihara, Y.; Sakamoto, Y.; Nagai, K.; Taniguchi, N.: A method for determination of UDPGlcNAc:GlcNAcb1-6(GlcNAcb1-2)Mana1-R [GlcNAc to Man]b1-4N-acetylglucosaminyltransferase VI activity using a pyridylaminated tetraantennary oligosaccharide as an acceptor substrate. Anal. Biochem., 255, 155-157 (1998)

506

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin3(4H)-one 2-D-glucosyltransferase

2.4.1.202

1 Nomenclature EC number 2.4.1.202 Systematic name UDP-glucose:2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-d-glucosyltransferase Recommended name 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-d-glucosyltransferase Synonyms uridine diphosphoglucose-2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)one 2-glucosyltransferase CAS registry number 122544-56-3

2 Source Organism <1> Zea mays (two different enzymes with different substrate specificities, genes Bx8 and Bx9 [1,2]) [1, 2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one = UDP + 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-d-glucoside Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one <1> (<1> involved in detoxification of benzoxazinoids [1]) [1, 2] P UDP + 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-d-glucoside

507

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-D-glucosyltransferase

2.4.1.202

Substrates and products S UDP-glucose + 2,4-dihydroxy-1,4-benzoxazin-3-one <1> (<1> peak 1 glucosyltransferase has 3.6% of activity compared to activity towards 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, peak 2 glucosyltransferase has 57% of activity compared to activity towards 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one [2]) (Reversibility: ? <1> [2]) [2] P UDP + 2,4-dihydroxy-2H-1,4-benzoxazin-3-one 2-d-glucoside S UDP-glucose + 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one <1> (Reversibility: ? <1> [1]) [1] P UDP + 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one 2-d-glucoside <1> [1, 2] S UDP-glucose + 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one <1> (Reversibility: ? <1> [1,2]) [1, 2] P UDP + 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-d-glucoside <1> [2] S UDP-glucose + 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one <1> (<1> low glucosylation efficiency [1]) (Reversibility: ? <1> [1]) [1] P UDP + 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one 2-d-glucoside S UDP-glucose + 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one <1> (Reversibility: ? <1> [1]) [1] P UDP + 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-d-glucoside Inhibitors Cu2+ <1> (<1> 5 mM, 3% remaining activity [2]) [2] EDTA <1> (<1> 16% decrease of activity [2]) [2] Fe2+ <1> (<1> 5 mM, 5% remaining activity [2]) [2] N-ethylmaleimide <1> (<1> at 1 mM, 96% decrease of activity, after addition of dithioerythritol, full activity [2]) [2] Cofactors/prosthetic groups ascorbate <1> (<1> at 5 mM, stimulation of 42% [2]) [2] Activating compounds 2-mercaptoethanol <1> (<1> 5 mM, stimulation of 79% [2]) [2] ascorbate <1> (<1> at 5 mM, stimulation of 42% [2]) [2] dithioerythritol <1> (<1> 5 mM, stimulation of 96% [2]) [2] glutathione <1> (<1> 5 mM, stimulation of 92% [2]) [2] Metals, ions CaCl2 <1> (<1> 5 mM, stimulation [2]) [2] MgCl2 <1> (<1> 5 mM, stimulation [2]) [2] Specific activity (U/mg) 0.5 <1> (<1> 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one, BX8-gene encoded protein [1]) [1] 0.5 <1> (<1> 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one, BX9-gene encoded protein [1]) [1]

508

2.4.1.202

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-D-glucosyltransferase

1.218 <1> (<1> enzyme in peak 1 and peak 2 [2]) [2] 1.4 <1> (<1> 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, BX8-gene encoded protein [1]) [1] 2.6 <1> (<1> 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one as substrate, BX9gene encoded protein [1]) [1] 6 <1> (<1> 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, BX9-gene encoded protein [1]) [1] 13 <1> (<1> 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one as substrate, BX9-gene encoded protein [1]) [1] 13.4 <1> (<1> 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one as substrate, BX8-gene encoded protein [1]) [1] 24 <1> (<1> 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one as substrate, BX8-gene encoded protein [1]) [1] Km-Value (mM) 0.061 <1> (2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one, <1> BX8-gene encoded protein [1]) [1] 0.071 <1> (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, <1> BX9-gene encoded protein [1]) [1] 0.081 <1> (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, <1> BX8-gene encoded protein [1]) [1] 0.081 <1> (UDP-glucose, <1> BX8-gene encoded protein [1]) [1] 0.096 <1> (UDP-glucose, <1> BX9-gene encoded protein [1]) [1] 0.174 <1> (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, <1> enzyme in peak 2 [2]) [2] 0.217 <1> (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, <1> enzyme in peak 1 [2]) [2] 0.28 <1> (UDPglucose, <1> reaction with 2,4-dihydroxy-1,4-benzoxazin-3one, enzyme in peak 2 [2]) [2] 0.286 <1> (UDPglucose, <1> reaction with 2,4-dihydroxy-7-methoxy-2H-1,4benzoxazin-3(4H)-one, enzyme in peak 1 [2]) [2] 0.638 <1> (2,4-dihydroxy-1,4-benzoxazin-3-one, <1> enzyme in peak 2 [2]) [2] 1.3 <1> (2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one, <1> BX9-gene encoded protein [1]) [1] pH-Optimum 8.5 <1> [2] pH-Range 6.5-10 <1> (<1> 10% of maximal activity at pH 6.5 and 30% of maximal activity at pH 10.0 [2]) [2] Temperature optimum ( C) 37 <1> (<1> assay at [2]) [2] 45 <1> [2]

509

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-D-glucosyltransferase

2.4.1.202

Temperature range ( C) 25-55 <1> (<1> 88% of maximal activity at 55 C, 60% of maximal activity at 25 C [2]) [2]

4 Enzyme Structure Molecular weight 48000 <1> (<1> gel filtration [1]) [1] 50000 <1> (<1> gel filtration, both peaks [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue seedling <1> [2] Purification <1> (partial, activity elutes from Q-Sepharose column in two peaks, named peak 1 and peak 2, exhibiting different activities towards substrates [2]) [2] Cloning <1> (expressed in Escherichia coli [1]; transfected into Arabidopsis [1]) [1]

References [1] von Rad, U.; Huttl, R.; Lottspeich, F.; Gierl, A.; Frey, M.: Two glucosyltransferases are involved in detoxification of benzoxazinoids in maize. Plant J., 28, 633-642 (2001) [2] Bailey, B.A.; Larson, R.L.: Hydroxamic acid glucosyltransferases from maize seedlings. Plant Physiol., 90, 1071-1076 (1989)

510

trans-Zeatin O-b-D-glucosyltransferase

2.4.1.203

1 Nomenclature EC number 2.4.1.203 Systematic name UDP-glucose:trans-zeatin O-b-d-glucosyltransferase Recommended name trans-zeatin O-b-d-glucosyltransferase Synonyms glucosyltransferase, uridine diphosphoglucose-zeatin Ouridine diphosphoglucose-zeatin O-glucosyltransferase zeatin O-b-d-glucosyltransferase zeatin O-glucosyltransferase Additional information (cf. EC 2.4.2.40, formerly EC 2.4.1.204) CAS registry number 123644-76-8

2 Source Organism <-1> no activity in Phaseolus vulgaris [1] <1> Phaseolus lunatus (cv. Kingston [1,2]; gene ZOG1 [3]) [1-3] <2> Zea mays [3]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + trans-zeatin = UDP + O-b-d-glucosyl-trans-zeatin (unlike EC 2.4.1.215, cis-zeatin O-b-d-glucosyltransferase, UDP-xylose can also act as donor; <1> localisation of site determining the substrate specificity for the UDP-sugar donors [3]) Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + zeatin <1> (<1> important in regulating the level of active cytokinin, i.e. zeatin, in plant tissues [1,2]) [1, 2] P UDP + O-b-d-glucosylzeatin 511

trans-Zeatin O-b-D-glucosyltransferase

2.4.1.203

Substrates and products S UDP-galactose + trans-zeatin <1> (Reversibility: ? <1> [1,2]) [1, 2] P UDP + O-galactosylzeatin <1> [1, 2] S UDP-glucose + trans-zeatin <1> (<1> UDP-glucose is the best donor substrate [3]; <1> specific for trans-zeatin [1]; <1> cis-zeatin, ribosylzeatin, and dihydrozeatin are no substrates [1]) (Reversibility: ? <1> [1-3]) [1-3] P UDP + O-b-d-glucosylzeatin <1> [1, 2] S UDP-xylose + dihydrozeatin <1> (<1> converts dihydrozeatin exclusively with UDP-d-xylose as donor substrates [3]) (Reversibility: ? <1> [2,3]) [2, 3] P UDP + O-b-d-xylosyldihydrozeatin <1> [2, 3] S UDP-xylose + trans-zeatin <1> (Reversibility: ? <1> [1,2]) [1, 2] P UDP + O-xylosylzeatin <1> [1, 2] Specific activity (U/mg) 0.57 <1> (<1> purified enzyme [1]) [1] Km-Value (mM) 0.028 <1> (trans-zeatin) [1] 0.216 <1> (UDP-glucose) [1] 2.7 <1> (UDP-xylose) [1] pH-Optimum 8 <1> (<1> assay at [3]) [1] Temperature optimum ( C) 27 <1> (<1> assay at [1]) [1]

4 Enzyme Structure Molecular weight 44000 <1> (<1> gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue embryo <1> (<1> immature [1]) [1] Additional information <1> (<1> very low expression level in vegetative tissues [2]) [2] Purification <1> [1] Cloning <1> (DNA and amino acid sequence determination, expression in Escherichia coli BL21(DE3) cells [2]) [2, 3]

512

2.4.1.203

trans-Zeatin O-b-D-glucosyltransferase

Engineering Additional information <1> (<1> construction of chimeric recombinant enzyme mutants, exchange of the C-terminus with zeatin O-b-d-xylosyltransferase, EC 2.4.2.40, gene ZOX1, determination of the site determining the substrate specificity [3]) [3]

References [1] Dixon, S.C.; Martin, R.C.; Mok, M.C.; Shaw, G.; Mok, D.W.S.: Zeatin glycosylation enzymes in Phaseolus - isolation of O-glucosyltransferase from Phaseolus lunatus and comparison to O-xylosyltransferase from P. vulgaris. Plant Physiol., 90, 1316-1321 (1989) [2] Martin, R.C.; Mok, M.C.; Mok, D.W.S.: Isolation of a cytokinin gene, ZOG1, encoding zeatin O-glucosyltransferase from Phaseolus lunatus. Proc. Natl. Acad. Sci. USA, 96, 284-289 (1999) [3] Martin, R.C.; Cloud, K.A.; Mok, M.C.; Mok, D.W.S.: Substrate specificity and domain analyses of zeatin O-glycosyltransferases. Plant Growth Regul., 32, 289-293 (2000)

513

Zeatin O-b-D-xylosyltransferase

1 Nomenclature EC number 2.4.1.204 (transferred to EC 2.4.2.40) Recommended name zeatin O-b-d-xylosyltransferase

514

2.4.1.204

Galactogen 6b-galactosyltransferase

2.4.1.205

1 Nomenclature EC number 2.4.1.205 Systematic name UDP-galactose:galactogen b-1,6-d-galactosyltransferase Recommended name galactogen 6b-galactosyltransferase Synonyms 1,6-d-galactosyltransferase UDPgalactose:galactogen b-1,6-d-galactosyltransferase b-(1-6)-d-galactosyltransferase galactosyltransferase, uridine diphosphogalactose-galactogen CAS registry number 88273-54-5

2 Source Organism <1> Helix pomatia [1]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + galactogen = UDP + 1,6-b-d-galactosylgalactogen Reaction type hexosyl group transfer Natural substrates and products S UDPgalactose + galactogen <1> (<1>, biosynthesis of galactogen [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1,6-b-d-galactosylgalactogen Substrates and products S UDP-d-galactose + arabinogalactan <1> (<1>, arabinogalactan from larch wood, 22% as effective as galactogen from Helix pomatia [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1,6-b-d-galactosylarabinogalactan

515

Galactogen 6b-galactosyltransferase

2.4.1.205

S UDP-d-galactose + galactan <1> (<1>, galactan from bovine lung 64% as effective as galactogen from Helix pomatia [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1,6-b-d-galactosylgalactan S UDP-d-galactose + galactogen <1> (<1>, galactogen from Helix pomatia best acceptor, galactogen from Lymnaea stagnalis, 22% as effective as galactogen from Helix pomatia [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1,6-b-d-galactosylgalactogen Metals, ions MgCl2 <1> (<1>, 10 mM, 30% stimulation [1]) [1] Specific activity (U/mg) 3.466 <1> [1] Km-Value (mM) 0.36 <1> (UDP-d-galactose) [1] pH-Optimum 6.8-7.5 <1> [1]

4 Enzyme Structure Molecular weight 600000 <1> (<1>, greater than 600000 Da, gel filtration [1]) [1] Subunits ? <1> (<1>, x * 70000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue albumen gland <1> [1] Localization membrane <1> (<1>, endomembrane-associated, probably in Golgi complex [1]) [1] Purification <1> (partial [1]) [1]

6 Stability Storage stability <1>, -80 C, crude homogenate, 6 months [1] <1>, 0 C, 24 h, 50% loss of activity [1] <1>, 4 C, purified enzyme, stable for 1 week [1]

516

2.4.1.205

Galactogen 6b-galactosyltransferase

References [1] Goudsmit, E.M.; Ketchum, P.A.; Grossens, M.K.; Blake, D.A.: Biosynthesis of galactogen: identification of a b-(1!6)-d-galactosyltransferase in Helix pomatia albumen glands. Biochim. Biophys. Acta, 992, 289-297 (1989)

517

Lactosylceramide 1,3-N-acetyl-b-D-glucosaminyltransferase

2.4.1.206

1 Nomenclature EC number 2.4.1.206 Systematic name UDP-N-acetyl-d-glucosamine:d-galactosyl-1,4-b-d-glucosylceramide b-1,3acetylglucosaminyltransferase Recommended name lactosylceramide 1,3-N-acetyl-b-d-glucosaminyltransferase Synonyms LA2 synthase LacCer(b1 ,3)N-acetylglucosaminyltransferase <3> [4] UDP-GlcNAc:lactosylceramide b1,3-N-acetylglucosaminyltransferase <5, 6> [6] acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-lactosylceramide bb1,3 N-acetylglucosaminyltransferase GlcNAc(b1,3)Gal(b1,4)Glc-ceramide synthase <7, 8> [7] b1-3-N-acetylglucosaminyltransferase b3Gn-T5 <5, 6> [6] lactosylceramide b-acetylglucosaminyltransferase lactosylceramide: N-acetylglucosaminyltransferase <2, 4> [5] lactotriaosylceramide synthase <5, 6> [6] uridine diphosphoacetylglucosamine-lactosylceramide b-acetylglucosaminyltransferase CAS registry number 83682-80-8

2 Source Organism <-1> no activity in Spodoptera frugiperda (Sf9 cells [7]) [7] <1> Homo sapiens [1, 2] <2> Rattus norvegicus (Sprague-Dawley albino [3,5]) [3, 5] <3> Xenopus sp. [4] <4> Mus musculus (enzyme-deficient mutant and normal littermate mice [5]) [5] <5> Homo sapiens [6]

518

2.4.1.206

Lactosylceramide 1,3-N-acetyl-b-D-glucosaminyltransferase

<6> Rattus norvegicus (not a full length clone [6]) [6] <7> Mus musculus [7] <8> Homo sapiens [7]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + d-galactosyl-1,4-b-d-glucosylceramide = UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + lactosylceramide <1-4> (<3> enzyme is active at the branching point of lactosylceramide metabolism, directs lactosylceramide into the glycolipids of the lacto-series [4]; <1> ganglioside metabolism and pattern [1]; <2-4> glycolipid biosynthesis [4,5]; <1> may play a key role in regulating the level of various types of lactoseries tumor-associated antigens with the lacto type 1 or 2 chain [2]; <1,2> biosynthetic pathway of lactoseries and ganglioseries [1,3]) [1-5] P UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide Substrates and products S UDP-N-acetyl-d-glucosamine + d-galactosyl-1,4-b-d-glucosylceramide <1-8> (<1-5> i.e. lactosylceramide [1-6]; <7,8> best substrate [7]; <7,8> acceptor substrate specificity, overview [7]; <5> acceptor substrate is fluorescently labeled with 2-aminobenzamid or 2-pyridylamine [6]) (Reversibility: ? <1-8> [1-7]) [1-7] P UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <1-8> [1-7] S UDP-N-acetyl-d-glucosamine + d-galactosyl-1,4-b-N-acetyl-d-galactosaminyl-1,3-b-(sialyl-a-2,3)-d-galactosyl-1,4-b-d-glucosylceramide <7, 8> (Reversibility: ? <7,8> [7]) [7] P UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-N-acetyl-d-galactosaminyl-1,3-b-(sialyl-a-2,3)-d-galactosyl-1,4-b-d-glucosylceramide <7, 8> [7] S UDP-N-acetyl-d-glucosamine + d-galactosyl-1,4-b-N-acetyl-d-galactosaminyl-1,3-b-(sialyl-a-2,8-sialyl-a-2,3)-d-galactosyl-1,4-b-d-glucosylceramide <7, 8> (Reversibility: ? <7,8> [7]) [7] P UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-N-acetyl-d-galactosaminyl-1,3-b-(sialyl-a-2,8-sialyl-a-2,3)-d-galactosyl-1,4-b-d-glucosylceramide <7, 8> [7]

519

Lactosylceramide 1,3-N-acetyl-b-D-glucosaminyltransferase

2.4.1.206

S UDP-N-acetyl-d-glucosamine + d-galactosyl-1,4-b-N-acetyl-d-galactosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <7, 8> (Reversibility: ? <7,8> [7]) [7] P UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-N-acetyl-d-galactosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <7, 8> [7] S UDP-N-acetyl-d-glucosamine + d-galactosyl-1,4-b-N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosyl-phenyl-C14 H29 <2> (<2> synthetic substrate, 75% specific activity compared to lactosylceramide [3]) (Reversibility: ? <2> [3]) [3] P UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosyl-phenyl-C14 H29 <2> [3] S UDP-N-acetyl-d-glucosamine + d-galactosyl-1,4-b-N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <2, 5, 7, 8> (<5> acceptor substrate is fluorescently labeled with 2-aminobenzamide or 2-pyridylamine [6]) (Reversibility: ? <2,5,7,8> [3,6,7]) [3, 6, 7] P UDP + N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide <2, 5, 7, 8> [3, 6, 7] Inhibitors 12-O-tetradecanoylphorbol-13-acetate <5> (<5> down-regulation of enzyme expression [6]) [6] Activating compounds CHAPS <2> (<2> 41% stimulation compared to Triton CF-54 [3]) [3] Triton CF-54 <2, 3> (<2> stimulation, maximal at 0.4% [3]) [3, 4] Triton X-100 <2> (<2> 89% stimulation compared to Triton CF-54 [3]) [3] Tween 20 <2> (<2> 38% stimulation compared to Triton CF-54 [3]) [3] deoxycholate <2> (<2> 8% stimulation compared to Triton CF-54 [3]) [3] octyl-b-d-glucoside <2> (<2> 46% stimulation compared to Triton CF-54 [3]) [3] retinoic acid <3, 5> (<5> up-regulation of enzyme expression [6]; <3> activates enzyme activity, leads to developmental malformations of the embryos [4]) [4, 6] sodium taurocholate <2> (<2> 25% stimulation compared to Triton CF-54 [3]) [3] taurodeoxycholate <2> (<2> 11% stimulation compared to Triton CF-54 [3]) [3] Metals, ions Ca2+ <2> (<2> 78% activity compared to Mn2+ [3]) [3] Cu2+ <2> (<2> 4% activity compared to Mn2+ [3]) [3] Hg2+ <2> (<2> 4% activity compared to Mn2+ [3]) [3] Mg2+ <2> (<2> 40% activity compared to Mn2+ [3]) [3] Mn2+ <2, 3> (<2> absolute requirement [3]) [3, 4] Zn2+ <2> (<2> 11% activity compared to Mn2+ [3]) [3] Specific activity (U/mg) 0.00000009 <2> (<2> granule cells, 14 days old [5]) [5] 0.0000001 <2> (<2> cerebellum homogenate of 3 days old organisms [5]) [5] 520

2.4.1.206

Lactosylceramide 1,3-N-acetyl-b-D-glucosaminyltransferase

0.00000015 <2> (<2> granule cells, 3 days old [5]) [5] 0.00000034 <1> (<1> colonic mucosa [2]) [2] 0.00000042 <2> (<2> cerebellum homogenate of 14 days old organisms [5]) [5] 0.00000065 <3> (<3> embryo [4]) [4] 0.0000046 <1> (<1> colonic adenocarcinoma tissue sample [2]) [2] 0.00121 <7, 8> (<7,8> recombinant enzyme, substrate lactosylceramide [7]) [7] 0.00123 <7, 8> (<7,8> recombinant enzyme, substrate d-galactosyl-1,4-b-Nacetyl-d-glucosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide [7]) [7] 0.00174 <7, 8> (<7,8> recombinant enzyme, substrate d-galactosyl-1,4-b-Nacetyl-d-galactosaminyl-1,3-b-(sialyl-a-2,3)-d-galactosyl-1,4-b-d-glucosylceramide [7]) [7] 0.0021 <7, 8> (<7,8> recombinant enzyme, substrate d-galactosyl-1,4-b-Nacetyl-d-galactosaminyl-1,3-b-(sialyl-a-2,8-sialyl-a-2,3)-d-galactosyl-1,4-bd-glucosylceramide [7]) [7] 0.00254 <7, 8> (<7,8> recombinant enzyme, substrate d-galactosyl-1,4-b-Nacetyl-d-galactosaminyl-1,3-b-d-galactosyl-1,4-b-d-glucosylceramide [7]) [7] Additional information <1, 2, 5, 6> (<5> activity in diverse tumour cell lines, overview [6]; <6> activity during brain development [6]; <1> activity in different cell lines of normal colonic mucosa and colonic adenocarcinoma [2]) [1-3, 6] Km-Value (mM) 0.021 <2> (d-galactosyl-1,4-b-d-glucosylceramide, <2> cerebellum microsomes [3]) [3] 0.035 <2> (d-galactosyl-1,4-b-N-acetyl-d-glucosaminyl-1,3-b-d-galactosyl1,4-b-d-glucosylceramide, <2> cerebellum microsomes [3]) [3] pH-Optimum 7 <2> [3] 7.2 <1, 5> (<1,5> assay at [2,6]) [2, 6] 7.3 <1> (<1> assay at [1]) [1] 7.6 <3> (<3> assay at [4]) [4] pH-Range 6.8-7.4 <2> (<2> cacodylate buffer [3]) [3] Temperature optimum ( C) 37 <1-3, 5> (<1-3,5> assay at [1-4,6]) [1-4, 6]

5 Isolation/Preparation/Mutation/Application Source/tissue B-lymphocyte <7> (<7> splenic [7]) [7] brain <1-8> (<7> expression during embryonic brain development [7]; <5,7> low content [6,7]) [1, 3-7] cerebellar Purkinje cell <2, 7> (<2> and dendrites [5]) [5, 7] 521

Lactosylceramide 1,3-N-acetyl-b-D-glucosaminyltransferase

2.4.1.206

cerebellum <2, 4-7> (<2,7> distinct cellular layers: Purkinje cell layer, molecular cell layer, granule cell layer + white matter [5,7]; <2> neonatal [3]) [3, 5-7] cerebral cortex <2, 6> (<2,6> fetal [3,6]; <6> not adult [6]) [3, 6] colon <5> [6] colonic adenocarcinoma cell <1, 5> [2, 6] colonic mucosa <1> (<1> not epithelial cells [2]) [2] embryo <3, 7> (<7> expression during mouse development [7]) [4, 7] granulocyte <2> [5] hypophysis <5> [6] liver <5> (<5> very low content [6]) [6] lung <5, 7> (<7> low content [7]) [6, 7] lymph node <5> (<5> low content [6]) [6] medulloblastoma cell line <1> (<1> Daoy and D341 Med, cultured both in vitro and as xenografts in nude mice [1]) [1] neuron <2> [3] placenta <5, 7> [6, 7] spleen <5, 7> (<5> low content [6]) [6, 7] testis <5> [6] thymus <5, 7> (<5,7> low content [6,7]) [6, 7] Additional information <1, 3, 5, 6> (<5> transcript content and activity in diverse tumour cell lines, overview, nearly undetectable content in leukemic cell lines [6]; <6> enzyme levels in brain regions during pre- and postnatal brain development [6]; <3> retinoic acid activates the enzyme and leads to developmental malformations of the embryos, overview [4]; <1> not in colonic epithelial cells [2]) [2, 4, 6] Localization Golgi apparatus <1> [1] membrane <2, 7> [3, 7] microsome <2> [3] Purification <5> (recombinant FLAG-tagged fusion protein from Sf21 insect cells [6]) [6] Cloning <5> (DNA sequence determination and analysis [6]; expression in Nawalma cells, membrane-bound [6]; soluble expression as FLAG-tagged protein in Spodoptera frugiperda Sf21 cells via baculovirus infection [6]) [6] <6> (partial sequence, not a full length clone, DNA sequence determination [6]) [6] <7> (DNA sequence determination, functional expression in Spodoptera frugiperda Sf9 cells via baculovirus infection [7]) [7] <8> (DNA and amino acid sequence determination, functional expression in Spodoptera frugiperda Sf9 cells via baculovirus infection [7]) [7]

522

2.4.1.206

Lactosylceramide 1,3-N-acetyl-b-D-glucosaminyltransferase

References [1] Gottfries, J.; Percy, A.K.; Mansson, J.-E.; Fredman, P.; Wikstrand, C.J.; Friedman, H.S.; Bigner, D.D.; Svennerholm, L.: Glycolipids and glycosyltransferases in permanent cell lines established from human medulloblastomas. Biochim. Biophys. Acta, 1081, 253-261 (1991) [2] Holmes, E.H.; Hakomori, S.-i.; Ostrander, G.K.: Synthesis of type 1 and 2 lacto series glycolipid antigens in human colonic adenocarcinoma and derived cell lines is due to activation of a normally unexpressed b 1-3N-acetylglucosaminyltransferase. J. Biol. Chem., 262, 15649-15658 (1987) [3] Chou, D.K.H.; Jungalwala, F.B.: N-acetylglucosaminyltransferase regulates the expression of neolactoglycolipids including sulfoglucuronylglycolipids in the developing nervous system. J. Biol. Chem., 268, 21727-21733 (1993) [4] Rossi, F.; Gornati, R.; Rizzo, A.M.; Venturini, L.; Bernadini, G.; Berra, B.: Glycolipid glycosyltransferase activities during early development of Xenopus: effect of retinoic acid. Cell Biol. Int., 23, 91-95 (1999) [5] Chou, D.K.H.; Jungalwala, F.B.: N-acetylglucosaminyl Transferase regulates the expression of sulfoglucuronyl glycolipids in specific cell types in cerebellum during development. J. Biol. Chem., 271, 28868-28874 (1996) [6] Togayachi, A.; Akashima, T.; Ookubo, R.; Kudo, T.; Nishihara, S.; Iwasaki, H.; Natsume, A.; Mio, H.; Inokuchi, J.-i.; Irimura, T.; Sasaki, K.; Narimatsu, H.: Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide b1,3-N-acetylglucosaminyltransferase (b3Gn-T5), an essential enzyme for the expression of HNK-1 and lewis X epitopes on glycolipids. J. Biol. Chem., 276, 22032-22040 (2001) [7] Henion, T.R.; Zhou, D.; Wolfer, D.P.; Jungalwala, F.B.; Hennet, T.: Cloning of a mouse b1,3 N-acetylglucosaminyltransferase GlcNAc(b1,3)Gal(b1,4)Glc-ceramide synthase gene encoding the key regulator of lacto-series glycolipid biosynthesis. J. Biol. Chem., 276, 30261-30269 (2001)

523

Xyloglucan:xyloglucosyl transferase

2.4.1.207

1 Nomenclature EC number 2.4.1.207 Systematic name xyloglucan:xyloglucan xyloglucanotransferase Recommended name xyloglucan:xyloglucosyl transferase Synonyms EXGT EXT GenBank L46792-derived protein GI 950299 GenBank X93173-derived protein GI 1890573 GenBank X93174-derived protein GI 1890575 GenBank Z97335-derived protein GI Genbank X93175-derived protein GI 1890577 NXET XET XTH (<20> xyloglucan endotransglucosylase/hydrolase, new unifying nomenclature [28]) [28] endo-xyloglucan transferase endoxyloglucan transferase xyloglucan X93174-derived protein GI 1890575 xyloglucan endotransglycosylase xyloglucan endotransglycosylase (Actinia deliciosa strain Hayward pericarp clone AdXET-5 gene XET precursor) xyloglucan endotransglycosylase (Arabidopsis thaliana gene TCH4 precursor reduced) xyloglucan endotransglycosylase (barley clone PM5 gene HVPM5) xyloglucan endotransglycosylase (barley clone XEA gene HVXEA) xyloglucan endotransglycosylase (barley clone XEB gene HVXEB) xyloglucan endotransglycosylase-related protein XTR-7 (Arabidopsis thaliana) xyloglucan recombinase xyloglucan-specific endo-(1!4)-b-d-glucanase xyloglucan:xyloglucanotransferase xyloglucanotransferase, xyloglucan (xyloglucan donor) xyloglucanotransferase, xyloglucan (xyloglucan donor) (Actinia chinensis clone AdXET-5 gene XET precursor)

524

2.4.1.207

Xyloglucan:xyloglucosyl transferase

xyloglucanotransferase, xyloglucan (xyloglucan donor) (Arabidopsis thaliana gene TCH4 precursor reduced) xyloglucanotransferase, xyloglucan (xyloglucan donor) (barley clone PM5 gene HVPM5) xyloglucanotransferase, xyloglucan (xyloglucan donor) (barley clone XEA gene HVXEA) xyloglucanotransferase, xyloglucan (xyloglucan donor) (barley clone XEB gene HVXEB) CAS registry number 141588-40-1 172279-31-1 (xyloglucanotransferase, xyloglucan (xyloglucan donor) (Arabidopsis thaliana gene TCH4 precursor reduced) /xyloglucan endotransglycosylase (Arabidopsis thaliana gene TCH4 precursor reduced)) 202012-34-8 (xyloglucan endotransglycosylase-related protein XTR-7 (Arabidopsis thaliana) /GenBank Z97335-derived protein GI 2244769) 203402-40-8 (xyloglucanotransferase, xyloglucan (xyloglucan donor) (barley clone PM5 gene HVPM5) /GenBank X93173-derived protein GI 1890573 /xyloglucan endotransglycosylase (barley clone PM5 gene HVPM5)) 203402-41-9 (xyloglucanotransferase, xyloglucan (xyloglucan donor) (barley clone XEA gene HVXEA) /GenBank X93174-derived protein GI 1890575 /xyloglucan X93174-derived protein GI 1890575 /xyloglucan endotransglycosylase (barley clone XEA gene HVXEA)) 203402-42-0 (xyloglucanotransferase, xyloglucan (xyloglucan donor) (barley clone XEB gene HVXEB) /Genbank X93175-derived protein GI 1890577 /xyloglucan endotransglycosylase (barley clone XEB gene HVXEB)) 205513-08-2 (xyloglucanotransferase, xyloglucan (xyloglucan donor) (Actinia chinensis clone AdXET-5 gene XET precursor) /GenBank L46792-derived protein GI 950299 /Xyloglucan endotransglycosylase (Actinia deliciosa strain Hayward pericarp clone AdXET-5 gene XET precursor))

2 Source Organism <1> Pisum sativum (cv. Alaska [3]; var. Feltham First and Pilot [12]; cv. Tyrkys [22]) [1, 3, 12, 14, 22, 28] <2> Marchantia polymorpha [1] <3> Mnium hornum [1] <4> Allium schoenoprasum [1] <5> Zea mays [1, 29] <6> Holcus lanatus [1] <7> Bromus erectus [1] <8> Lupinus polyphyllus [1] <9> Anthriscus sylvestris [1] <10> Acer pseudoplatanus [1] <11> Populus alba [16] <12> Taraxacum officinale [1]

525

Xyloglucan:xyloglucosyl transferase

2.4.1.207

<13> Vigna angularis (Ohwi and Ohashi, cv. Takara [2]; azuki bean [28]) [2, 28] <14> Phaseolus vulgaris (cv. Canadian Wonder [3]) [3, 14] <15> Tamarindus indica [14] <16> Arabidopsis sp. [5] <17> Lycopersicon esculentum (cv. Ailsa Craig [1]; cv. Murrieta [13]) [1, 13, 28] <18> Tropaeolum majus (var. Fiery Festival, 2 enzyme forms encoded by the genes XET1 and NXG1 [11]; 2 isoenzymes: sXET and eXET [21]; cv. Goldshine orange [22]; NXG1 is a member of XET class III [24]) [4, 7, 11, 14, 15, 20-22, 24, 28] <19> Actinidia deliciosa (ripe kiwifruit, [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward, 6 isoenzymes: AdXET1-6 gene family [8]) [8] <20> Arabidopsis thaliana (TCH4, member of the XET-related gene family [9, 10]; ecotype Columbia [10,28]; 3 insect-cell-produced XETs: EXGT, TCH4 and MERI-5, EXGT belongs to XET class I, TCH4 and MERI-5 to XET class II [24]; XETs are encoded by a gene family, 4 isoenzymes: TCH4, Meri-5, EXGT and XTR9 [26]; systematic nomenclature of the XTH gene family with 3 subfamilies [28]) [9, 10, 24, 26, 28, 29] <21> Lens culinaris [14, 24] <22> Brassica oleracea (var. botrytis, 2 isoenzymes: C30, C45 [18]; cauliflower [5,18,23,24]; distinct isoforms [23]; isoenzyme C45a [24]) [5, 18, 23, 24] <23> Apium sp. (celery [5]) [5, 28] <24> Sinapis sp. (mustard [5]) [5] <25> Phaseolus aureus (mung bean [5,18,23]; Vigna radiata [18]; 4 isoenzymes: M35, M45, M55a, M55b [18,24]; distinct isoforms [23]) [5, 18, 23, 24] <26> Lycopersicon esculentum (cv. T5 [6]) [6] <27> Populus tremula (tremuloides [17]) [17] <28> Nicotiana tabacum (tobacco bright yellow-2 [19]; SR-1 [29]) [19, 28, 29] <29> Nicotiana tabacum (cv. Samsun NN, NtXET-1, belongs to group I of XETrelated subfamilies [25]) [25] <30> Arabidopsis thaliana (XTR9, one of 4 isoenzymes: TCH4, Meri-5, EXGT and XTR9 [26]) [26] <31> Populus tremula x Populus tremuloides (hybrid aspen, poplar, major isoenzyme PttXET16A, a member of XET subfamily I [27]) [27] <32> Spinacia oleraceae (spinach [28]) [28] <33> Sellaginella sp. [29] <34> Adianthum capillus-veneris [29] <35> Pteris cretica [29] <36> Microsorum pteropus [29] <37> Azolla sp. [29] <38> Picea abies [29] <39> Pinus parviflora (Glauca [29]) [29] <40> Pinus radiata [29] <41> Cedrus atlantica (Carr. [29]) [29] <42> Chamaecyparis thyloides (Top Point [29]) [29] <43> Chamaecyparis sp. (Snow White [29]) [29] <44> Taxus baccata [29] 526

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<45> <46> <47> <48> <49> <50> <51> <52> <53> <54> <55> <56> <57> <58> <59> <60> <61> <62> <63> <64> <65> <66> <67> <68> <69> <70> <71> <72> <73> <74> <75> <76> <77> <78> <79> <80> <81> <82> <83> <84> <85> <86> <87>

Xyloglucan:xyloglucosyl transferase

Pothomorphe petalta [29] Peperomia rotundifolia [29] Calandrinia landiflora [29] Euphorbia helioscopa [29] Linum usitatissimum [29] Cochlearia officinalis [29] Blumenbachia hieronymi [29] Elsholtzia ciliata [29] Chaenorrhimum minus [29] Pharbitis purpurea (Bojer [29]) [29] Helianthus annuus [29] Cnicus benedictus [29] Callistephus chinensis (Nees [29]) [29] Trigonella caerulea (Ser. [29]) [29] Tragopodon pratensis (subs. Pratensis [29]) [29] Lactuca perennis [29] Scabosia atropurpurea [29] Anthurium upalaense [29] Anthurium willdenowii [29] Lemna minor [29] Crocus vernus (Hill [29]) [29] Iris pseudacorus [29] Dracaena drago [29] Convallaria majalis [29] Aloe sp. [29] Asphodelus fistulosus [29] Chamaedorea elegans [29] Triticum dicoccum [29] Briza maxima [29] Cenchrus ciliaris [29] Vulpia myurus (C.C. Gmel [29]) [29] Beckmannia syzigachne (Fernald [29]) [29] Juncus effusus [29] Luzula luzuloides [29] Typha latifolia [29] Typha angustifolia [29] Cyperus prolifer [29] Carex flacca (subsp. Flacca [29]) [29] Tradescanthia zebrina [29] Commelina nilagirica [29] Pitcairnia imbricata (Regel [29]) [29] Canna indica [29] Musa sapientum [29]

527

Xyloglucan:xyloglucosyl transferase

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3 Reaction and Specificity Catalyzed reaction breaks a b-(1!4) bond in the backbone of a xyloglucan and transfers the xyloglucanyl segment on to O-4 of the non-reducing terminal glucose residue of an acceptor, which can be a xyloglucan or an oligosaccharide of xyloglucan (<1,14,15,18,21> transglycosylic mechanism, i.e. random splitting of the b1,4-linked polyglucose backbone of xyloglucan molecules and rejoining the newly created reducing ends by b-1,4 glycosidic bonds to nonreducing ends of other xyloglucan molecules or xyloglucan subunit oligosaccharides [14,20]; <11> sequential mechanism [16]; <18> ping-pong bi bi reaction mechanism [20]; <1,18> reaction mechanism [22]; <18> double-displacement reaction mechanism [28]) Reaction type transglycosylation (<1, 11, 13-26, 29, 30> endotransglycosylation, a xyloglucan chain is cleaved and transferred to another acceptor chain [1-3, 5-9, 1116, 18, 20, 23, 25, 26, 28]; <1,14> idling reaction [3]) Natural substrates and products S donor xyloglucan + xyloglucan acceptor <1, 5, 13, 14, 17-22, 25, 26, 28-31, 33-87> (<1> enzyme is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and thus causes the wall-loosening required for plant cell expansion and plant growth, in vivo the usual acceptor is polymeric wall-bound xyloglucan [1]; <13> reconnecting enzyme for xyloglucans, involved in the interweaving or reconstruction of cell wall matrix, which is responsible for chemical creepage that leads to morphological changes in the cell wall, responsible for cell wall loosening and integration of cell wall architecture [2]; <1,14> enzyme depolymerises xyloglucan [3]; <18> enzyme is involved in the post-germinative mobilization of xyloglucan storage reserves, involved in cell wall loosening, activity is primarily regulated at the level of gene expression [4]; <26> enzyme mediates cell wall disassembly associated with expansive growth [6]; <18> role in elongation-growth and other processes involving xyloglucan metabolism [7]; <19> XET and xyloglucan may play a role in the cell wall changes that accompany fruit softening during ripening, substrates for XET action are located in the cell wall [8]; <20> enzyme is unlikely to play a role in acidinduced wall loosening but may function in cold acclimation or cold-tolerant growth, TCH4 expression is rapidly regulated by mechanical stimuli, temperature shifts, light and hormones, it may be involved in assembling nascent cell walls or reinforcing existing and expanding walls [9]; <20> the xyloglucan:xyloglucosyl transferase gene TCH4 is strongly upregulated by environmental stimuli, enzyme may function in modifying cell walls to allow growth, airspace formation, the development of vasculature, and reinforcement of regions under mechanical strain, TCH4 protein may contribute to the adaptive changes in morphogenesis that occurs in the organism following exposure to mechanical stimuli [10]; <18,20,22,25> ex-

528

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Xyloglucan:xyloglucosyl transferase

istence of different classes of XET with differing roles in vivo [9,11,18,28]; <18> XET1 may play a role in cell-wall xyloglucan metabolism, such as the incorporation of newly synthesized xyloglucan into the expanding primary cell wall or the modification of xyloglucan polymers forming crosslinks between cellulose microfibrils, NXG1 in vivo predominantly exhibits xyloglucanase activity and mobilizes nonfucosylated xyloglucan seed storage reserves [11]; <1> involved in xyloglucan metabolism [12]; <17> important component of cell wall metabolism, particularly expanding tissue and ripening fruits, study on the role in tomato fruit ripening and vegetative growth, tXET-B1 may incorporate new xyloglucan into the existing network [13]; <1, 14, 15, 18, 21> one of the key enzymes involved in the breakdown of reserve xyloglucan in seeds of some dicotyledonous plants during germination [14]; <18> one of four enzymes, which act in concert to catalyze the mobilisation of storage xyloglucan from the cell wall [15]; <11> activity increases markedly at the exponential growth and decreases immediately at the stationary phase of cells in presence of 2,4-dichlorophenoxyacetic acid, the activity is developmentally regulated during growth but is not directly induced by plant hormones [16]; <22> major role is the integration of new xyloglucan into the cell walls of the densely cytoplasmic florets [18]; <25> associated with primary cell wall metabolism, rather than mobilisation of any seed storage xyloglucan, major role is the re-structuring of existing wall material in the rapidly vacuolating shoots [18]; <28> key enzyme responsible for forming and rearranging the cellulose/xyloglucan network of the cell wall, commitment to the construction of both cell plate and cell wall [19]; <18> 2 isoenzymes: sXET from seeds plays a role in degrading xyloglucan reserves in seeds during germination, eXET from epicotyls is engaged in cell wall rearrangement and integration of new xyloglucan molecules into the preexisting cell wall structure during growth [21]; <1,18> regulation of XET activity: enzyme exists in plant cell walls in a transiently latent state as covalent glycosyl-enzyme complex and is active only when suitable glycosyl acceptors become available [22]; <20,22> Arabidopsis MERI-5 and TCH4 and cauliflower isoenzymes expression occur in dense cytoplasmic tissues predominantly involved in the assembly of new cell walls and thus in the integration of newly secreted xyloglucan into the cell wall [24]; <20,25> Arabidopsis EXGT and mung bean isoenzymes expression in tissues involved in the loosening of existing cell wall material necessary for rapid cell expansion, e.g. vacuolation [24]; <18,21> lentil and nasturtium, NXG1, seed enzymes are involved in the mobilization of cotyledonary xyloglucan reserves after germination [24]; <29> NtXET-1 is involved in the incorporation of small xyloglucan molecules into the cell wall by transglycosylation, role during differentation and growth of the vascular tissue, reduced NtXET-1 expression and increase in the MW of xyloglucans in older leaves might be associated with strengthening of cell walls by reduced turnover and hydrolysis of xyloglucan, control of NtXET-1 expression in leaves [25]; <20,30> XETs encoded by a gene family may influence plant growth and development, low pH would limit XET function in vivo 529

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[26]; <31> multifunctional role in cell wall construction, role in restructuring primary cell walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers [27]; <20> enzyme is capable of splitting and reconnecting xyloglucan molecules in rapidly growing plant tissues, expression and presumed physiological roles of At-XTH22 and 24 [28]; <5,20,28,33-87> acts in root development, necessary for root hair growth [29]; <20> role in cell elongation [29]; <5> role in wall assembly as well as loosening [29]) [1-4, 6-16, 18, 19, 21, 22, 24-29] P ? S Additional information <18, 20-22, 25> (<18,20-22,25> free oligosaccharides are probably not the usual acceptor substrates in vivo [24]) [24] P ? Substrates and products S 2 Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glcb(1-4) (Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glc <18> (<18> acceptor and donor: Glc8-based XXXGXXXG [28]) (Reversibility: ? <18> [28]) [28] P Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glcb(14)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glcb(14)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glc + Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glc <18> (<18> products: XXXGXXXGXXXG + XXXG [28]) [28] S donor xyloglucan + Xyl(1-6)Glc(1-4)(Gal-Xyl(1-6))Glc(1-4)(Gal-Xyl(16))Glc(1-4)Glc-ol-sulphorhodamine conjugate <16, 22-25> (<16,22-25> XLLGol-SR [5]) (Reversibility: ? <16> [5]) [5] P ? S donor xyloglucan + Xyl(1-6)Glc(1-4)(Xyl(1-6))Glc(1-4)(Fuc-Gal-Xyl(16))Glc(1-4)Glc <1, 20, 32> (<1> acceptor: xyloglucan-derived nonasaccharide XG9, Glc4 Xyl3 GalFuc, highly specific for xyloglucan as the glycosyl donor, xyloglucan from Tropaeolum seed is somewhat better than from Rosa cultures, enzyme transfers part of a large xyloglucan molecule to the nonasaccharide forming a polymer with a b-1,4-linkage to the product and the acceptor group of the nonasaccharide is O-4 of the Glc residue furthest from the reducing terminus [1]; <1, 32> XXFG [28]; <1> preference of XLLG over XXXG over XXFG over XXG [28]) (Reversibility: ? <1, 20, 32> [1, 9, 28]) [1, 9, 28] P ? S donor xyloglucan + Xyl(1-6)Glc(1-4)(Xyl(1-6))Glc(1-4)(Fuc-Gal-Xyl(16))Glc(1-4)Glc-ol <20, 30> (<20, 30> XXFG-ol [9,26]; <20> TCH4 protein, 50% as effective as XLLG-ol [9]; <20,30> TCH4, Meri-5, EXGT and XTR9 show a marked preference for XLLG-ol over XXFG-ol or XXXG-ol as acceptor oligosaccharide, TCH4, Meri-5 and EXGT: 25-30% as effective as XLLG-ol, less effective than XXXG-ol, XTR9: more effective than XXXGol, donor: tamarind or pea xyloglucan polymer [26]) (Reversibility: ? <20, 30> [9,26]) [9, 26]

530

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Xyloglucan:xyloglucosyl transferase

P ? S donor xyloglucan + Xyl(1-6)Glc(1-4)(Xyl(1-6))Glc(1-4)Glc <1, 14, 18> (<1,14> Xyl2 Glc3 is less effective than heptasaccharide XG7: Xyl3 Glc4 , minimal structure required for acceptor activity [3]; <18> Glc3 Xyl2 [14]; <1> XXG, preference of XLLG over XXXG over XXFG over XXG [28]) (Reversibility: ? <1,14,18> [3,14,28]) [3, 14, 28] P ? S donor xyloglucan + Xyl(1-6)Glc(1-4)(Xyl(1-6))Glc(1-4)Glc-ol <22, 25> (<22,25> pentamer XXG-ol, all isoenzymes have low but significant activity, tamarind xyloglucan as donor [18]) (Reversibility: ? <22,25> [18]) [18] P ? S donor xyloglucan + Xyl(1-6)Glc(1-4)Glc <25> (<25> trimer XG-ol, only isomer M55a shows a slight activity, tamarind xyloglucan as donor [18]) (Reversibility: ? <25> [18]) [18] P ? S donor xyloglucan + Xyla(1-6)Glcb(1-4)(Gal-Xyla(1-6))Glcb(1-4)(Xyla(16))Glcb(1-4)Glc <18> (<18> XLXG [15]) (Reversibility: ? <18> [15]) [15] P ? S donor xyloglucan + Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(14)(Galb(1-2)Xyla(1-6))Glcb(1-4)Glc-ol <1, 18, 20-22, 25, 30> (<1, 18, 2022, 25, 30> XLLG-ol [9, 18, 22-24, 26, 28]; <20> TCH4 protein, better substrate than XXFG-ol, lower affinity than for polymer xyloglucan [9]; <1, 18, 22, 25> Gal2 Xyl3 Glc3 glucitol prepared from tamarind flour/seed xyloglucan [18, 22, 23]; <18, 20-22, 25> donor: tamarind xyloglucan [18, 23, 24]; <20, 30> donor: tamarind or pea xyloglucan polymer [26]; <22, 25> 1.7-3.6fold preference of XLLG-ol over XXXG-ol, mung bean isoenzymes have higher affinities for XLLG-ol than cauliflower isoenzymes [18]; <22> isoenzyme C45a dissociates from the reaction products after each polysaccharide-to-oligosaccharide transglycosylation event [24]; <18, 20-22, 25> comparison of the size distributions of the different isoenzymes' transglycosylation products [24]; <20, 30> TCH4, Meri-5, EXGT and XTR9 show a marked preference for XLLG-ol over XXFG-ol or XXXG-ol as acceptor oligosaccharide [26]; <20> reaction results in a hybrid product and a leaving group [28]) (Reversibility: ? <1, 18, 20-22, 25, 30> [9, 18, 22-24, 26, 28]) [9, 18, 22-24, 26, 28] P ? S donor xyloglucan + Xyla(1-6)Glcb(1-4)(Galb(1-2)Xyla(1-6))Glcb(14)(Galb(1-2)Xyla(1-6))Glcb(1-4)Glc <1, 16, 18> (<1> XG9n, better substrate than Glc4 Xyl3 GalFuc [1]; <1, 16, 18> XLLG [5, 11, 15, 28]; <18> XLLG from Tropaeolum majus seed, donor xyloglucan from tamarind cotyledons or pea stems, XET1 uses nonfucosylated tamarind seed amyloid xyloglucan or fucosylated pea stem xyloglucan as substrate with equal facility, NXG1 has a significantly higher activity against nonfucosylated tamarind xyloglucan [11]; <18> Glc4 Xyl3 Gal2 [14]; <1> preference of XLLG over XXXG over XXFG over XXG [28]) (Reversibility: ? <1, 16, 18> [1, 5, 11, 14, 15, 28]) [1, 5, 11, 14, 15, 28] 531

Xyloglucan:xyloglucosyl transferase

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P ? S donor xyloglucan + Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Gal-Xyla(16))Glcb(1-4)Glc <18> (<18> XXLG [15]) (Reversibility: ? <18> [15]) [15] P ? S donor xyloglucan + Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(16))Glcb(1-4)Glc <1, 14, 18> (<1> XG7, better substrate than Glc4 Xyl3 GalFuc [1]; <1,14> heptasaccharide XG7, Xyl3 Glc4 , is more effective than Xyl2 Glc3 [3]; <1,18> XXXG [15, 28]; <1> preference of XLLG over XXXG over XXFG over XXG [28]) (Reversibility: ? <1, 14, 18> [1, 3, 15, 28]) [1, 3, 15, 28] P ? S donor xyloglucan + Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(16))Glcb(1-4)Glc-ol <1, 11, 14, 19, 20, 22, 25, 29, 30> (<14> XG7-ol is approximately as effective as heptasaccharide XG7, efficient substrate, reducing group is not required for acceptor activity [3]; <1, 11, 19, 20, 22, 25, 29, 30> XXXG-ol [8, 12, 16, 18, 25, 26]; <1, 22, 25> tamarind seed xyloglucan as donor [12, 18]; <11> pea xyloglucan as donor, specific for xyloglucan as donor [16]; <29> pine or tobacco xyloglucan as donor [25]; <11> transfers a part of xyloglucan to the reduced xyloglucan heptasaccharide XXXG-ol, the acceptor acts by combining with the enzyme independently of the donor, the velocity of the reaction decreases gradually as the heptasaccharide unit is increased from two to four, the affinity for XXXG-ol is increased at a higher concentration of donor xyloglucan [16]; <22, 25> isoenzymes have 9-19times greater activities than on XXG-ol, less activity than with XLLG-ol [18]; <20, 30> TCH4, Meri-5, EXGT and XTR9 show a marked preference for XLLG-ol over XXFG-ol or XXXG-ol as acceptor oligosaccharide, TCH4, Meri-5 and EXGT: more effective than XXFG-ol, XTR9: less effective than XXFG-ol, donor: tamarind or pea xyloglucan polymer [26]) (Reversibility: ? <1, 11, 14, 19, 20, 22, 25, 29, 30> [3, 8, 12, 16, 18, 25, 26]) [3, 8, 12, 16, 18, 25, 26] P ? S donor xyloglucan + Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(16))Glcb(1-4)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(16))Glcb(1-4)Glc-ol <11> (<11> XXXGXXXG-ol, dimer of XXXG-ol, most effective acceptor, pea xyloglucan as donor [16]) (Reversibility: ? <11> [16]) [16] P ? S donor xyloglucan + Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(16))Glcb(1-4)Glcb1-PA <13> (<13> pyridylamino heptasaccharide is a good substrate [2]; <13> pyridylamino XXXG [28]) (Reversibility: ? <13> [2,28]) [2, 28] P ? S donor xyloglucan + acceptor xyloglucan <1-87> (<1, 13, 22, 25> acceptor specificity [1, 2, 18]; <1, 13, 20> donor specificity [1, 2, 9]; <18, 20> specific for xyloglucan [4, 7, 9]; <1> acceptors: xyloglucan-derived nonasaccharide Glc4 Xyl3 GalFuc and certain other xyloglucan oligosaccharides, non-reducing terminal Xyl-Glc group is essential [1]; <1,14> minimum 532

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Xyloglucan:xyloglucosyl transferase

acceptor structure is Xyl2 Glc3 [1, 3]; <1, 11, 13> highly specific for xyloglucan as the glycosyl donor [1, 2, 16]; <1, 13, 14, 16, 18, 20> xyloglucan from Tropaeolum majus seed [1-3, 5, 9, 15]; <1> xyloglucan from Rosa cultures [1]; <13> xyloglucan from Vigna angularis [2]; <1, 13-15, 18, 20-22, 25> xyloglucan from Tamarindus indica [2, 7, 9, 12, 14, 15, 18, 23, 24]; <11> xyloglucan from pea [16]; <20> donors: TCH4 protein is more active against tamarind xyloglucan than nasturtium xyloglucan as donor, pea stem xyloglucan, acceptors: XLLG-ol, XXFG-ol [9]; <19> kiwifruit xyloglucan donor, acceptor: XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan [8]; <29> donor: pine or tobacco xyloglucan [25]; <1, 13, 14> enzyme transfers a large segment of a xyloglucan molecule to another one generating chimeric polymers [1-3]; <13> enzyme requires a basic xyloglucan structure, i.e. a b-1,4-glucosyl backbone with xylosyl side chains, for both acceptor and donor activity, higher activity when xyloglucans with higher MW are used as donor substrates, xyloglucans smaller than 10 kDa are no donor substrates, pyridylamino heptasaccharide, xyloglucan oligomers or polymers are good acceptors [2]; <1, 13, 14> galactosyl or fucosyl side chains are not required for acceptor activity [2, 3]; <1, 14> acceptors: high MW xyloglucan, xyloglucan-derived oligosaccharides with at least two a-d-xylose residues, reducing group is not required for acceptor activity [3]; <1, 11, 13-18, 20-25> acceptors: polymer xyloglucan or its derived oligosaccharides [5, 7, 12-16, 18, 20, 22-24, 28]; <18> donor: Glc8-based XXXGXXXG [28]; <17> preference for solanaceous xyloglucan without Fuc over xyloglucan from other sources as donor [28]; <20> TCH4 protein specifically transglycosylates only xyloglucan, cleavage of both fucosylated and nonfucosylated xyloglucans as donor substrates, but fucosyl content of the substrates lowers reaction rate, preferred acceptor substrate is the nonreducing terminus of high-MW xyloglucan, xyloglucan derived oligosaccharides are also utilized [9]; <18> 2 distinct XETs with different substrate prefences, XET from epicotyl, XET1, uses nonfucosylated seed amyloid xyloglucan or fucosylated stem xyloglucan as substrate with equal facility, XET from cotyledon, NXG1, has a significantly higher activity against nonfucosylated xyloglucan [11]; <18> 2 isoenzymes: sXET from seeds acts on xyloglucan molecules stochastically along the length of their polyglucose main chain and prefers low-MW xyloglucan-derived oligosaccharides as acceptors, eXET from epicotyls attacks the substrate predominantly near the reducing end and shows no preference for a size of xyloglucan oligosaccharide acceptors [21]; <1> tamarind seed xyloglucan as donor substrate, oligosaccharidyl-alditol as acceptor [12]; <18> acceptor: fucosylated xyloglucan nonasaccharide [14]; <18> investigation of substrate subsite recognition requirements, xylose substitution at Glc +2 and -3 of the NXET cleavage site at the backbone of the xyloglucan substrate is a requirement, Gal substitution of a Xyl at +1 prevents, and at -2 modifies, chain-cleavage [15]; <11> acceptors: XXXG-ol, a reduced xyloglucan heptasaccharide, its dimer XXXGXXXG-ol, and other xyloglucan oligosaccharides, reaction probably involves an enzyme-donor-acceptor complex, in which the en533

Xyloglucan:xyloglucosyl transferase

2.4.1.207

zyme has two binding sites separately for donor and acceptor, mechanism [16]; <27> enzyme cleaves and religates xyloglucan polymers [17]; <22, 25> pronounced preference for xyloglucan oligosaccharides with backbones of 4 over 3 over 2 Glc residues as acceptors [18]; <1, 18> XET is present in cell walls in form of a competent glycosyl-enzyme complex which decomposes by transglycosylation of its glycan moiety to added xyloglucan-oligosaccharide acceptors [22]; <22, 25> XET forms a stable donor xyloglucan-XET complex [23]; <18, 20-22, 25> enzyme chooses its donor substrate independently of size and attacks it, once only, at a randomly selected cleavage site [24]; <29> NtXET-1 has a prefence for smaller xyloglucan molecules as acceptors [25]; <20,30> acceptor and donor substrate specificities of the isoenzymes TCH4, Meri-5, EXGT and XTR9 [26]; <30> isoenzyme XTR9 has a clear preference for non-fucosylated xyloglucan polymers as donor, but not isoenzymes TCH4, Meri-5 and EXGT [26]; <31> acceptors: sulforhodamine-labeled xyloglucan-oligosaccharides [27]; <13> donor xyloglucan cleavage sites of XTHs from stems and epicotyls [28]; <5, 20, 28, 33-87> acceptors: xyloglucan oligosaccharide-SR mixture [29]) (Reversibility: ? <1-87> [1-29]) [1-29] P ? S Additional information <1, 5, 13, 14, 17-20, 28, 30, 31, 33-87> (<1> no acceptors: a-d-Xylp(1-6)d-Glc and isoprimeverose [1]; <13> no glycosidase or glycanase activity, no donors: xyloglucans smaller than 10 kDa, carboxymethylcellulose, Avena glucan, maize xylan, Rhodymenia xylan, no acceptors: pyridylamino cellohexaose, pyridylamino laminarihexaose [2]; <1, 14, 18> not: cellotetraose [3, 15]; <1, 14> not: Xyl1 Glc3 , XylGlc2 [3]; <5, 20, 28, 33-87> not: cellobiose-SR [29]; <18> no action on other poly- and oligosaccharides than xyloglucan, such as cellulose, its soluble derivatives, mixed linkage b-glucans, cello-oligosaccharides, enzyme catalyzes, additional to the powerful and specific xyloglucan endo-transglycosylase action, the hydrolysis of tamarind xyloglucan to a mixture of oligosaccharides of different sizes, hydrolysis increases at low substrate concentrations [7]; <19> in absence of reduced xyloglucan-derived heptasaccharide XXXG-ol enzyme depolymerizes xyloglucan by hydrolysis and in the presence of it by both hydrolysis and endotransglycosylation [8]; <20> not: carboxymethylcellulose, barley glucan [9]; <17> tXET-B2 has no hydrolytic activity [13]; <18> NXET has also a xyloglucan-specific hydrolase activity, cleavage sites for hydrolysis and endotransglycosylation are identical, hydrolase action is significantly only under conditions, where suitable acceptor chain-end concentrations are limiting [15]; <1, 18> oligosaccharides of cello-, chito- and/or oligoglucurono-series are much less effective than xyloglucan-derived oligosaccharides [22]; <20, 30, 31> contains strongly conserved sequence motif of XETs: DEIDFEFLG [26, 27]; <20, 30> no endoglucanase activity [26]; <31> PttXET16A has 4 conserved Cys residues [27]) [1-3, 7-9, 13, 15, 22, 26, 27, 29] P ?

534

2.4.1.207

Xyloglucan:xyloglucosyl transferase

Inhibitors (NH4 )2 SO4 <22> (<22> 1 M, about 50% inhibition [5]) [5] Ag+ <1, 19> (<1,19> 1 mM [1,8]; <1> partial inhibition [1]) [1, 8] CaCl2 <22> (<22> 300 mM, about 50% inhibition [5]) [5] H2 O2 <22> (<22> 1 M, about 50% inhibition [5]) [5] Hg2+ <1, 19> (<1> 1 mM, partial inhibition [1]; <19> at least 10 mM [8]) [1, 8] La3+ <1> (<1> 10 mM, partial inhibition [1]) [1] NaCl <22> (<22> 1 M, about 50% inhibition [5]) [5] NaN3 <22> (<22> 1 M, about 50% inhibition [5]) [5] Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2)Xyla(16))Glcb(1-4)Glc-ol <20, 30> (<20> slight substrate inhibition of isoenzymes Meri-5 and EXGT at high concentrations [26]; <30> significant substrate inhibition of isoenzyme XTR9 at high concentrations [26]) [26] Zn2+ <1> (<1> 10 mM, partial inhibition [1]) [1] ethanol <22> (<22> 15%, about 3 M, about 50% inhibition of product formation [5]) [5] phosphate <22> (<22> 300 mM, about 50% inhibition [5]) [5] pyridine <22> (<22> 300 mM, about 50% inhibition [5]) [5] Additional information <1, 22> (<1> not inhibited by 10 mM d-gluconolactone, chelating agents, cello-oligosaccharides, cellopentaose [1]; <22> not inhibited by 1 M fluoride, 1 M urea, 1 M DMSO, 300 mM borate, 100 mM EDTA, 100 mM Triton X-100 [5]) [1, 5] Activating compounds 2-mercaptoethanol <1> (<1> slightly activates [1]) [1] ascorbate <1> (<1> 10 mM, slightly activates [1]) [1] bovine serum albumin <20> (<20> enhances activity by stabilizing TCH4 protein conformation [9]) [9] spermidine <1> (<1> 1 mM, slighly activates [1]) [1] urea <20> (<20> enhances activity of TCH4 protein [9]) [9] xyloglucan-derived oligosaccharide <18> (<18> stimulates, highest stimulating effect with the nonasaccharide Glc4 Xyl3 Gal2 , lowest one with the pentasaccharide Glc3 Xyl2 [14]) [14] Additional information <1, 20, 26> (<26> LeEXT gene encoding XET is induced by hormone treatments that elicites elongation of hypocotyl segments, e.g. treatment with auxin, brassinolide or 2,4-D [6]; <20> TCH4 protein is not activated by reducing agents, spermidine, spermine, glycerol or carboxymethylcellulose [9]; <1> strong positive correlation between gibberellic acidenhanced length and extractable XET activity in internodes [12]; <20> AtXTH22 is strongly induced in response to mechanical perturbations such as wind or touch [28]) [6, 9, 12, 28] Metals, ions Ca2+ <1> (<1> 10 mM, slightly activates [1]) [1] Mg2+ <1> (<1> 10 mM, slightly activates [1]) [1] Mn2+ <1> (<1> 10 mM, slightly activates [1]) [1] Additional information <20> (<20> TCH4 protein is not activated by Ca2+ or other divalent cations [9]) [9] 535

Xyloglucan:xyloglucosyl transferase

2.4.1.207

Turnover number (min±1) 0.0108 <20> (tamarind xyloglucan, <20> recombinant TCH4 protein, with XLLG-ol as acceptor [9]) [9] 0.0186 <20> (pea xyloglucan, <20> recombinant TCH4 protein, with XLLG-ol as acceptor [9]) [9] Specific activity (U/mg) 0.216 <25> [23] Additional information <1, 13-16, 19, 21-25> (<16,22-25> a rapid, semiquantitative assay suitable for testing crude plant extracts directly on to the test paper [5]; <1,14,15,21> low cost, simple, high-speed colorimetric assay that allows to analyze multiple samples simultaneously [14]) [2, 5, 8, 14] Km-Value (mM) 0.0003 <20> (xyloglucan acceptor, <20> recombinant TCH4 protein [9]; <20> At-XTH22, high MW xyloglucan [28]) [9, 28] 0.005 <30> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <30> isoenzyme XTR9 [26]) [26] 0.016 <25> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <25> isoenzyme M55a [18]) [18] 0.017 <20> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <20> isoenzyme TCH4 [26]) [26] 0.019 <1> (Xyla(1-6)Glcb(1-4)(Galb(1-2)Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc) [28] 0.028 <25> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <25> isoenzyme M45 [18]) [18] 0.03 <20> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <20> isoenzyme EXGT [26]) [26] 0.031-0.035 <25> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4) (Galb(1-2)Xyla(1-6))Glcb(1-4)Glc-ol, <25> isoenzymes M35a and M35b [18]) [18] 0.032 <25> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <25> isoenzyme M55b [18]) [18] 0.033 <1> (Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glc) [28] 0.04 <20> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <20> isoenzyme Meri-5 [26]) [26] 0.05 <1> (Xyl(1-6)Glc(1-4)(Xyl(1-6))Glc(1-4)(Fuc-Gal-Xyl(1-6))Glc(1-4)Glc, <1> xyloglucan-derived nonasaccharide [1]) [1, 28] 0.071-0.082 <22> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4) (Galb(1-2)Xyla(1-6))Glcb(1-4)Glc-ol, <22> isoenzymes C45a and C45b [18]) [18] 0.073 <20> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <20> acceptor XLLG-ol, recombinant TCH4 protein [9]; <20> recombinant At-XTH22 [28]) [9, 28] 0.1 <19> (Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glcol, <19> reduced xyloglucan-derived heptasaccharide XXXG-ol, derived from kiwifruit xyloglucan [8]) [8] 536

2.4.1.207

Xyloglucan:xyloglucosyl transferase

0.13 <22> (Xyla(1-6)Glcb(1-4)(Galb(1-2)-Xyla(1-6))Glcb(1-4)(Galb(1-2) Xyla(1-6))Glcb(1-4)Glc-ol, <22> isoenzyme C30 [18]) [18] 0.2 <1> (Xyl(1-6)Glc(1-4)(Xyl(1-6))Glc(1-4)Glc) [28] 0.23 <11> (Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4) Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glcol, <11> XXXGXXXG-ol [16]) [16] 0.32 <11> (Xyla(1-6)Glcb(1-4)(Xyla(1-6))Glcb(1-4)(Xyla(1-6))Glcb(1-4)Glcol, <11> XXXG-ol [16]) [16] Additional information <11, 19, 20> (<19> Km : 0.6 mg/ml for kiwifruit xyloglucan [8]; <20> recombinant TCH4 protein, Km : 0.62 mg/ml for nonfucosylated tamarind xyloglucan as donor, 1.8 mg/ml for fucosylated pea xyloglucan [9,28]) [8, 9, 16, 28] pH-Optimum 5-5.5 <22, 25> (<22> isoenzyme C30 [18]; <25> isoenzymes M35a, M35b and M55b [18]) [18] 5-7 <25> (<25> isoenzymes M45 and M55a [18]) [18] 5.5 <1> [1] 5.5-5.8 <19> [8] 5.8 <13> [2] 6-6.5 <20, 30> (<20,30> isoenzymes TCH4, Meri-5, EXGT and XTR9, EXGT shows very sharp pH-optimum at pH 6 [26]) [9, 26] 6.5 <22> (<22> isoenzymes C45a and C45b [18]) [18] Additional information <18> (<18> pI: 5.0-5.14 [4]) [4] pH-Range 4-7 <1> (<1> pH 4: 25-35% of maximal activity, pH 7: 30-40% of maximal activity [1]) [1] 4.5-8 <20, 30> (<30> XTR9 retains approximately 35% of its maximal activity at pH 4.5 and 30% at pH 8 [26]; <20> TCH4 and Meri-5 retain approximately 10-15% of its maximal activity at pH 4.5 and 65% at pH 8 [26]) [26] 5-7.5 <20> (<20> pH 5: about 30% of maximal activity, pH 7.5: about 40% of maximal activity [9]) [9] 5-8 <19> (<19> active between [8]) [8] Temperature optimum ( C) 12-18 <20> (<20> recombinant TCH4 protein [9]) [9] 18 <20, 30> (<20,30> isoenzymes TCH4 and XTR9 [26]) [26] 20 <16, 18, 20-25> (<16,18,20-25> assay at [5,18,24]) [5, 18, 24] 22 <19> (<19> assay at [8]) [8] 24-34 <22, 25> (<22,25> broad optimum [18]) [18] 25 <1-10, 12-14, 17> (<1-10,12-14,17> assay at [1-3,12]) [1-3, 12] 28 <20> (<20> isoenzyme Meri-5 [26]) [26] 37 <1, 14, 15, 18, 20, 21> (<20> isoenzyme EXGT [26]; <1,14,15,18,21> assay at [14]) [14, 26] Additional information <20> (<20> assay at room temperature [26]) [26]

537

Xyloglucan:xyloglucosyl transferase

2.4.1.207

Temperature range ( C) 5 <20, 30> (<20,30> all 4 isoenzymes are markedly cold-tolerant retaining activity at 5 C [26]) [26] 18-26 <20> (<20> recombinant TCH4 protein, more active at lower temperatures, at or below 18 C, less active at higher temperatures, above 26 C [9]) [9] 45 <20, 30> (<20> isoenzymes EXGT and Meri-5 retain approximately 80% of maximum activity [26]; <20,30> isoenzymes TCH4 and XTR9 activities are reduced by 55-60% [26]) [26]

4 Enzyme Structure Subunits ? <13, 17-20, 22, 25, 29> (<18> x * 31000, SDS-PAGE and calculated from the amino acid sequence, the cDNA encodes a 33.5 kDa precursor polypeptide, which is subsequently processed to a 31 kDa mature protein [4]; <13> x * 33000, SDS-PAGE [2, 28]; <19> x * 34000, predicted MW of the mature protein encoded by AdXET5 is 31.8 kDa, SDS-PAGE [8]; <20> x * 33400, predicted size of TCH4 [10]; <17> x * 32000, His-tagged recombinant tXET-B2 with predicted molecular mass of 32 kDa [13]; <17> x * 30493, tXET-B1 [13]; <17> x * 30460, tXET-B2 [13]; <22, 25> x * 32000, each isoenzyme, SDSPAGE [18, 23]; <29> x * 33900, calculated from the amino acid sequence [25]; <29> x * 36000, probably glycosylated enzyme, Western blot [25]) [2, 4, 8, 10, 13, 18, 23, 25, 28] Posttranslational modification glycoprotein <13, 17, 19, 20, 29-31> (<13> with mannosyl residues [2]; <19> AdXET5 and AdXET6: asparagine-linked carbohydrate, N-glycosylated, glycosylation of the protein is not required for endo-transglycosylase activity [8]; <17> tXET-B1 and tXET-B2 with N-glycosylation sites [13]; <29> two putative glycosylation sites at amino acid positions 14 and 114 [25]; <20,30> all 4 isoenzymes, TCH4, Meri-5, EXGT and XTR9, are N-glycosylated, requirement of glycosylation for activity of the isoenzymes differs [26]; <31> PttXET16A contains a putative N-glycosylation site [27]) [2, 8, 13, 25-27] no glycoprotein <18> [4] proteolytic modification <17-20, 29, 31> (<18> hydrophobic core of 24 amino acids at the N-terminus, characteristic of a signal peptide, enzyme must be transported through the cell membrane in order to reach the cell wall, DNA encodes a 33.5 kDa precursor polypeptide, which is subsequently processed to a 31 kDa mature protein [4]; <19> 21 amino acids signal peptide at the N-terminus is cleaved to produce the mature protein [8]; <20> mature TCH4 polypeptide lacks the putative N-terminal signal sequence [9]; <17> tXET-B1 and tXET-B2 contain a hydrophobic signal peptide of 20 and 18 amino acids, respectively, typical of an exported protein [13]; <29> contains a hydrophobic N-terminus indicating a signal peptide [25]; <31> PttXET16A has a putative 22 amino acids signal peptide at the N-terminus [27]) [4, 8, 9, 13, 25, 27]

538

2.4.1.207

Xyloglucan:xyloglucosyl transferase

Additional information <20> (<20> co- and post-translational eucaryoticspecific modifications of TCH4 are critical for optimal XET activity [26]) [26]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture <11, 28> [16, 19] cotyledon <1, 14, 15, 18, 20, 21> (<18> source for mRNA isolation [4]; <1, 14, 15, 18, 21> of germinated seeds [7,14,15]; <18> NXG1 is exclusively expressed in cotyledons [11]) [4, 7, 10, 11, 14, 15] epicotyl <1, 13, 18> (<18> XET1 is highly expressed in young epicotyls [11]; <18> eXET, in epicotyls and other growing regions [21]) [1, 2, 11, 21, 22, 28] floret <22> [18, 23, 24] fruit <17, 19> (<19> core tissue of ripe kiwifruit [8]; <17> tXET-B1 is most abundant in pink fruit pericarp [13]) [8, 13] gametophyte <3> [1] hypocotyl <20, 26> (<26> LeEXT gene is expressed in outer cell layers of the hypocotyl, after auxin treatment overlapping spatial distribution in the epidermis and outer cortical cell layers [6]) [6, 10] internode <1> (<1> internodes I, II, III and V, numbered from the cotyledonary node [12]) [12] leaf <5-9, 14, 17, 18, 20, 29, 31> (<14> leaves from 5-day seedlings [3]; <20> bases [10]; <18> XET1 is expressed in young and mature leaves at lower levels than in young epicotyls and roots, 7fold higher expression in young leaves than in mature ones [11]; <29> source leaf, levels of NtXET-1 mRNA decreases in midribs with increasing age of leaves [25]; <31> PttXET16A is expressed transiently in developing leaves [27]) [1, 3, 10, 11, 25, 27] petiole <23> (<23> activity is very pronounced in the vicinity of the vascular bundles [5]; <23> young petioles, high activity in thick-walled collenchyma cells [28]) [5, 28] phloem <31> (<31> XET activity in xylem and phloem fibers at the stage of secondary wall formation, PttXET16A [27]) [27] root <5, 18, 20, 28, 31, 33-87> (<18> XET1 is highly expressed in young roots [11]; <31> PttXET16A expression in young roots and root tips [27]; <20,28> XET activity in the root cell elongation zone [28]; <20> XET activity in the initiating root hairs [28]; <5,20,28,33-87> high XET activity in the epidermis cell wall of the elongation zone and in trichoblasts in the differentation zone [29]) [11, 27-29] seed <1, 14, 15, 18, 21> (<1, 14, 15, 18, 21> cotyledons of germinated seeds [7,14,15]; <18> sXET, expressed during germination [21]) [4, 7, 14, 15, 20, 21, 24, 28] seedling <1, 17, 24, 25> (<1> 7-8-day etiolated seedlings [1]; <25> sprouting seedlings, more abundant in the growing tissues of the hypocotyls and leaves than in the cotyledons [18]) [1, 5, 18, 23] shoot <20, 25> (<20> 26 days old [9]; <25> etiolated shoots [24]) [9, 24] sprout <25> [5] 539

Xyloglucan:xyloglucosyl transferase

2.4.1.207

stem <1, 4-7, 10, 12, 13, 16-18, 31> (<1> etiolated stem, activity is positively correlated with growth rate in different zones of pea stem [1]; <10> first year stem [1]; <4,12> peduncle [1]; <1> etiolated 7-day stems [3]; <18> XET1 is expressed at lower levels in stems than in young epicotyls and roots [11]; <17> tXET-B1 is detected in stems [13]) [1, 3, 5, 11, 13, 27, 28] thallus <2> [1] vascular tissue <20> [10] xylem <31> (<31> XET activity in xylem and phloem fibers at the stage of secondary wall formation, PttXET16A [27]) [27] Additional information <18, 20, 29, 31> (<20> XETs accumulates in expanding cells, at the sites of intercellular airspace, formation, and at the bases of leaves, cotyledons and hypocotyls, detailed localization [10]; <18> XET1 is not expressed in cotyledons [11]; <29> tissue-specific NtXET-1 expression pattern, highest mRNA levels in organs highly enriched in vascular tissues [25]; <31> detailed localization of XET in poplar stems, PttXET16A expression pattern, expression in secondary vascular tissues [27]) [10, 11, 25, 27] Localization apoplast <13> [28] cell wall <1, 11, 13, 18, 19, 28, 29> (<18> localized exclusively in [4]; <19> most of the XET activity is strongly associated with the cell wall [8]; <11,28> bound to [16,28]; <1,18> cell wall marker enzyme, XET is present in the cell walls in form of a competent glycosyl-enzyme complex [22]; <29> the same gene encodes a protein, that may be both soluble and bound to the cell wall [25]) [2, 8, 16, 22, 25, 28] extracellular <13> (<13> extracellular or apoplastic space of epicotyls [2]) [2] soluble <29> (<29> the same gene encodes a protein, that may be both soluble and bound to the cell wall [25]) [25] Additional information <1, 20, 28> (<1> not ionically bound to the cell wall, little XET activity is covalently bound in the cell wall [1]; <28> during interphase EXGT is extensively secreted into the apoplast via the endoplasmic reticulum-Golgi apparatus network, during cytokinesis it is exclusively located in the phragmoblast and eventually transported to the cell plate [19]; <20> expression profiles of members of the XTH gene family, each XTH gene is likely to have a unique fingerprint of expression and regulation [28]) [1, 19, 28] Purification <1> [14] <13> (87fold, from apoplastic space of epicotyls [2]) [2, 28] <14> [14] <15> [14] <17> (His-tagged recombinant tXET-B2, expressed in Escherichia coli [13]) [13] <18> [4, 7, 14, 15, 20, 24] <19> (3000fold, AdXET6 [8]) [8] <21> [14, 24] 540

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Xyloglucan:xyloglucosyl transferase

<22> (general, mechanism-based method for purification, about 200fold, step-wise addition of (NH4 )2 SO4 reveals distinct isoforms [23]) [18, 23, 24] <25> (general, mechanism-based method for purification, about 200fold, step-wise addition of (NH4 )2 SO4 reveals distinct isoforms [23]) [18, 23, 24] <27> (recombinant I, expressed in Pichia pastoris [17]) [17] <29> (recombinant NtXET-1, expressed in Escherichia coli [25]) [25] <31> (recombinant His6-tagged PttXET16A, expressed in Escherichia coli BL21(DE3) [27]) [27] <20, 30> (recombinant TCH4 protein expressed in Escherichia coli [9]; TCH4 and EXGT [24]; partial, recombinant TCH4, Meri-5, EXGT and XTR9, expressed in Sf9 insect cells [26]) [9, 24, 26] Crystallization <27> (2 different crystal forms [17]) [17] Cloning <17> (small multi-gene family encodes XET: tXET-B1, -B2, -B3 and -B4, tXET-B2 is cloned, sequenced and overexpressed in Escherichia coli, nucleotide and amino acid sequence of tXET-B1 [13]) [13] <18> (single copy gene, cDNA encoding XET is cloned, sequenced and encodes a 33.5 kDa precursor polypeptide, which is subsequently processed to a 31 kDa mature protein, NXG1 and NXG2 may be different alleles of the XET gene [4]; genes XET1 and NXG1 encode 2 enzyme forms, XET1 cDNA is cloned and sequenced, amino acid sequence of NGX1 [11]) [4, 11] <19> (6 isoforms, AdXET1-6 gene family is cloned, a full-length cDNA, AdXET5, is cloned, sequenced and overexpressed as active XET in Escherichia coli, purified XET is encoded by AdXET6 [8]) [8] <20> (cloning and expression of TCH4 in Escherichia coli [9]; TCH4 is a member of the XET-related gene family [9,10]; 3 insect-cell-produced XETs: EXGT, TCH4 and MERI-5 [24]; cloning and expression of TCH4, Meri-5, EXGT and XTR9 using the baculovirus/Sf9 insect cell system, XTR9 is sequenced [26]; gene structure, genomic localization and phylogenetic relationship of the XTH gene family [28]) [9, 10, 24, 26, 28] <26> (LeEXT gene encoding XET is cloned [6]) [6] <27> (recombinant I expression in Pichia pastoris [17]) [17] <29> (NtXET-1 is cloned and expressed in Escherichia coli, ORF encodes a 295 amino acids protein, cDNA is cloned in sense and antisense orientation, creation of transgenic tobacco plants with reduced NtXET-1 expression: 2 independent lines with reduced total XET activity by 56% and 37%, respectively, in midribs of tobacco plants transformed with an antisense construct, which exhibit an at least 20% increase in the average MW of xyloglucan [25]) [25] <30> (cloning and expression of XTR9 using the baculovirus/Sf9 insect cell system, XTR9 is sequenced [26]) [26] <31> (PttXET16A, a XET isoform in secondary vascular tissues and a member of XET subfamily I, is cloned, sequenced and expressed in Escherichia coli BL21(DE3), structure of the coding sequence [27]) [27]

541

Xyloglucan:xyloglucosyl transferase

2.4.1.207

Engineering E97Q <20> (<20> At-XTH22: a Glu to Gln substitution, converting the active site sequence DEIDFEFL to DQIDFEFL, abolishes XET activity [28]) [28]

6 Stability Temperature stability 5 <20, 30> (<20,30> all 4 isoenzymes are markedly cold-tolerant retaining activity at 5 C [26]) [26] 44 <20> (<20> TCH4 protein: undetectable activity, more heat-sensitive than the total XET activity [9]) [9] Additional information <1, 22, 25> (<1> boiling inactivates [1]; <22,25> isoenzymes are heat-labile, none of the isoenzymes is particularly cold-tolerant [18]) [1, 18] Organic solvent stability ethanol <22> (<22> 15%, about 3 M, about 50% inhibition of product formation [5]) [5] General stability information <18>, 0.1 mM final concentration spermine stabilizes [14] <19>, loss of activity if frozen and stored at -80 C [8] <22, 25>, progressive purification destabilizes XET activity, 10% glycerol stabilizes [23] Storage stability <19>, -80 C, partial purified XET, ConA-eluate stage, 4 weeks, stable [8] <19>, -80 C, purified XET, loss of activity [8] <19>, 4 C, purified XET, 24 h, stable [8]

References [1] Fry, S.C.; Smith, R.C.; Renwick, K.F.; Martin, D.J.; Hodge, S.K.; Matthews, K.J.: Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem. J., 282, 821-828 (1992) [2] Nishitani, K.; Tominaga, R.: Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule. J. Biol. Chem., 267, 21058-21064 (1992) [3] Lorences, E.P.; Fry, S.C.: Xyloglucan oligosaccharides with at least two a-dxylose residues act as acceptor substrates for xyloglucan endotransglycosylase and promote the depolymerisation of xyloglucan. Physiol. Plant., 88, 105-112 (1993) [4] de Silva, J.; Jarman, C.D.; Arrowsmith, D.A.; Stronach, M.S.; Chengappa, S.; Sidebottom, C.; Reid, J.S.G.: Molecular characterization of a xyloglucan-

542

2.4.1.207

[5] [6] [7]

[8] [9] [10]

[11] [12] [13] [14] [15]

[16] [17]

Xyloglucan:xyloglucosyl transferase

specific endo-(1!4)-b-d-glucanase (xyloglucan endo-transglycosylase) from nasturtium seeds. Plant J., 3, 701-711 (1993) Fry, S.C.: Novel ªdot-blotª assays for glycosyltransferases and glycosylhydrolases: Optimization for xyloglucan endotransglycosylase (XET) activity. Plant J., 11, 1141-1150 (1997) Catala, C.; Rose, J.K.C.; Bennett, A.B.: Auxin regulation and spatial localization of an endo-1,4-b-d-glucanase and a xyloglucan endotransglycosylase in expanding tomato hypocotyls. Plant J., 12, 417-426 (1997) Fanutti, C.; Gidley, M.J.; Reid, J.S.G.: Action of a pure xyloglucan endotransglycosylase (formerly called xyloglucan-specific endo-(1!4)-b-d-glucanase) from the cotyledons of germinated nasturtium seeds. Plant J., 3, 691-700 (1993) Schröder, R.; Atkinson, R.G.; Langenkämper, G.; Redgwell, R.J.: Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit. Planta, 204, 242-251 (1998) Purugganan, M.M.; Braam, J.; Fry, S.C.: The Arabidopsis TCH4 xyloglucan endotransglycosylase. Substrate specificity, pH optimum, and cold tolerance. Plant Physiol., 115, 181-190 (1997) Antosiesicz, D.M.; Purugganan, M.M.; Polisensky, D.H.; Braam, J.: Cellular localization of Arabidopsis xyloglucan endotransglycosylase-related proteins during development and after wind stimulation. Plant Physiol., 115, 1319-1328 (1997) Rose, J.K.C.; Brummell, D.A.; Bennett, A.B.: Two divergent xyloglucan endotransglycosylases exhibit mutually exclusive patterns of expression in nasturtium. Plant Physiol., 110, 493-499 (1996) Potter, I.; Fry, S.C.: Xyloglucan endotransglycosylase activity in pea internodes. Effects of applied gibberellic acid. Plant Physiol., 103, 235-241 (1993) Arrowsmith, D.A.; de Silva, J.: Characterisation of two tomato fruit-expressed cDNAs encoding xyloglucan endo-transglycosylase. Plant Mol. Biol., 28, 391-403 (1995) Sulova, Z.; Lednicka, M.; Farkas, V.: A colorimetric assay for xyloglucanendotransglycosylase from germinating seeds. Anal. Biochem., 229, 80-85 (1995) Fanutti, C.; Gidley, M.J.; Reid, J.S.G: Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1!4)-b-dglucanase from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds. Planta, 200, 221-228 (1996) Takeda, T.; Mitsuishi, Y.; Sakai, F.; Hayashi, T.: Xyloglucan endotransglycosylation in suspension-cultured poplar cells. Biosci. Biotechnol. Biochem., 60, 1950-1955 (1996) Johansson, P.; Denman, S.; Brumer, H.; Kallas, A.M.; Henriksson, H.; Bergfors, T.; Teeri, T.T.; Jones, T.A.: Crystallization and preliminary x-ray analysis of a xyloglucan endotransglycosylase from Populus tremula * tremuloides. Acta Crystallogr. Sect. D, D59, 535-537 (2003)

543

Xyloglucan:xyloglucosyl transferase

2.4.1.207

[18] Steele, N.M.; Fry, S.C.: Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET). Phytochemistry, 54, 667-680 (2000) [19] Yokoyama, R.; Nishitani, K.: Endoxyloglucan transferase is localized both in the cell plate and in the secretory pathway destined for the apoplast in tobacco cells. Plant Cell Physiol., 42, 292-300 (2001) [20] Baran, R.; Sulova, Z.; Stratilova, E.; Farkas, V.: Ping-pong character of nasturtium-seed xyloglucan endotransglycosylase (XET) reaction. Gen. Physiol. Biophys., 19, 427-440 (2000) [21] Sulova, Z.; Baran, R.; Farkas, V.: Xyloglucan endotransglycosylase (XET) isoenzymes with different modes of action on xyloglucan. 12th European Carbohydrate Symposium, Grenoble, France, July 6-11 (2003) [22] Sulova, Z.; Baran, R.; Farkas, V.: Release of complexed xyloglucan endotransglycosylase (XET) from plant cell walls by a transglycosylation reaction with xyloglucan-derived oligosaccharides. Plant Physiol. Biochem., 39, 927-932 (2001) [23] Steele, N.M.; Fry, S.C.: Purification of xyloglucan endotransglycosylases (XETs): A generally applicable and simple method based on reversible formation of an enzyme-substrate complex. Biochem. J., 340, 207-211 (1999) [24] Steele, N.M.; Sulova, Z.; Campbell, P.; Braam, J.; Farkas, V.; Fry, S.C.: Ten isoenzymes of xyloglucan endotransglycosylase from plant cell walls select and cleave the donor substrate stochastically. Biochem. J., 355, 671-679 (2001) [25] Herbers, K.; Lorences, E.P.; Barrachina, C.; Sonnewald, U.: Functional characterisation of Nicotiana tabacum xyloglucan endotransglycosylase (NtXET-1): Generation of transgenic tobacco plants and changes in cell wall xyloglucan. Planta, 212, 279-287 (2001) [26] Campbell, P.; Braam, J.: In vitro activities of four xyloglucan endotransglycosylases from Arabidopsis. Plant J., 18, 371-382 (1999) [27] Bourquin, V.; Nishikubo, N.; Abe, H.; Brumer, H.; Denman, S.; Eklund, M.; Christiernin, M.; Teeri, T.T.; Sundberg, B.; Mellerowicz, E.J.: Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues. Plant Cell, 14, 3073-3088 (2002) [28] Rose, J.K.C.; Braam, J.; Fry, S.C.; Nishitani, K.: The XTH family of enzymes involved in xyloglucan endotransglucosylation and endohydrolysis: Current perspectives and a new unifying nomenclature. Plant Cell Physiol., 43, 1421-1435 (2002) [29] Vissenberg, K.; Van Sandt, V.; Fry, S.C.; Verbelen, J.-P.: Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays. J. Exp. Bot., 54, 335-344 (2003)

544

Diglucosyl diacylglycerol synthase

2.4.1.208

1 Nomenclature EC number 2.4.1.208 Systematic name UDP-glucose:1,2-diacyl-3-O-(a-d-glucopyranosyl)-sn-glycerol (1!2) glucosyltransferase Recommended name diglucosyl diacylglycerol synthase Synonyms DGlcDAG synthase MgGlcDAG (1!2) glucosyltransferase diglucosyldiacylglycerol synthase glucosyltransferase, uridine diphosphoglucose-monoglucosyldiacylglycerol monoglucosyl diacylglycerol (1!2) glucosyltransferase monoglucosyldiacylglycerol glucosyltransferase CAS registry number 168680-19-1

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

Acholeplasma laidlawii [1-5, 7] Staphylococcus aureus (strain RN4220 [6]) [6] Bacillus subtilis [6] Streptococcus pneumoniae [7]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + 1,2-diacyl-3-O-(a-d-glucopyranosyl)-sn-glycerol = 1,2-diacyl-3-O-[a-d-glucopyranosyl(1!2)-O-a-d-glucopyranosyl]-sn-glycerol + UDP Reaction type hexosyl group transfer

545

Diglucosyl diacylglycerol synthase

2.4.1.208

Natural substrates and products S UDP-glucose + 1,2-diacyl-3-O-(a-d-glucopyranosyl)-sn-glycerol <1, 2> (<1> at physiological concentrations of phosphatidylglycerol different nonlamellar-promoting pertubants can strongly enhance the synthesis of diglycosyldiacylglycerol in relation to their nonbilayer propensities [2]; <1> modulation of the activity by non-bilayer-forming lipids [3]; <2> enzyme provides glycolipid for lipoteichoic acid assembly [6]) (Reversibility: ? <1, 2> [3, 6]) [2, 3, 6] P UDP + 1,2-diacyl-3-O-(a-d-glucopyranosyl(1!2)-O-a-d-glucopyranosyl)-sn-glycerol <1, 2> [2, 3, 6] Substrates and products S UDP-glucose + 1,2-diacyl-3-O-(a-d-glucopyranosyl)-sn-glycerol <1, 2, 3, 4> (<1> no activity if the sugar is in b-position [4]) (Reversibility: ? <1, 2, 3, 4> [1-3, 6, 7]) [1-7] P UDP + 1,2-diacyl-3-O-(a-d-glucopyranosyl(1!2)-O-a-d-glucopyranosyl)-sn-glycerol <1, 2, 3, 4> [1-7] S UDP-glucose + 1,2-diheptadecanoyl-3-O-(a-d-glucopyranosyl)-sn-glycerol <1> (<1> best substrate [4]) (Reversibility: ? <1> [4]) [4] P UDP + 1,2-diheptadecanoyl-3-O-(a-d-glucopyranosyl(1!2)-O-a-d-glucopyranosyl)-sn-glycerol <1> (<1> 73% of activity with monoglucosyldioleoylglycerol [4]) [4] S UDP-glucose + 1,2-dioleoyl-3-O-(a-d-glucopyranosyl)-sn-glycerol <1> (Reversibility: ? <1> [4]) [4] P UDP + 1,2-dioleoyl-3-O-(a-d-glucopyranosyl(1!2)-O-a-d-glucopyranosyl)-sn-glycerol <1> [4] Inhibitors 1,2-diacyl-3-O-(b-d-galactopyranosyl)-sn-glycerol <1> [4] 1,2-diacyl-3-O-[6-O-acyl(a-d-glucopyranosyl)]-sn-glycerol <1> [4] 1,2-diacyl-3-O-[a-d-glucopyranosyl-1,2-O-(6-O-acyl-a-d-glucopyranosyl)]sn-glycerol <1> [4] ATP <1> (<1> above 1.5 mM, stimulation below [5]) [5] phosphenolpyruvate <1> (<1> above 1.5 mM [5]) [5] sn-glycero-3-phosphate <1> (<1> 20 mM, complete inhibition [5]) [5] Activating compounds 1,2-dioleoylphosphatidylethanolamine <1> (<1> maximal activity at 4.5% [4]) [4] 1,2-dioleoylphosphatidylglycerol <1, 4> (<4> maximal activation at 30 mol% [7]; <1> maximal activation at 25 mol% [7]) [4, 7] 1,2-dioleoylphosphatidylserine <1> (<1> 33% of activation with 1,2-dioleoylphosphatidylglycerol [4]) [4] 1,3-diacylglycerol <1> [3] 1,3-dioleoylglycerol <1> (<1> 11.5 mol%, 5fold activation [4]) [4] ATP <1> (<1> approx. 12fold stimulation at 0.75 mM, inhibition above 1.5 mM [5]) [5]

546

2.4.1.208

Diglucosyl diacylglycerol synthase

cardiolipin <1> (<1> very strong stimulation in the presence of 1,3-dioleoylglycerol [7]) [7] diphosphatidylglycerol <1> (<1> from bovine, 50% of activation with 1,2dioleoylphosphatidylglycerol [4]) [4] dsDNA <1> (<1> 0.0007 mg/ml, 7fold activation [5]) [5] fructose-1,6-bisphosphate <1> (<1> 1 mM, approx. 18fold stimulation [5]) [5] lipid <1> (<1> activates [2]; <1> activity is influenced in a dose-dependent manner by the nonbilayer propensities of several amphiphilic and hydrophilic lipids in two different bilayer matrices [2]; <1> activation by non-lamellar forming lipids [3]) [2, 3] phosphate <1> (<1> 40 mM, approx. 8fold activation [4]; <1> 20 mM, 5fold stimulation in the presence of 5 mM Mg2+ [5]) [4, 5, 7] phosphatidylglycerol <1> (<1> liquid-crystalline phosphatidylglycerol [2]; <1> enzyme needs a polar activator lipid, preferably phosphatidylglycerol at concentrations of 25-30 mol% in zwitterionic mixed CHAPS bilayer-like aggregates [4]; <1> in vivo amounts are not enough for efficient diglucosyldiacylglycerol synthesis [5]) [2, 4, 5] phosphatidylinositol <1> (<1> strong activation in the presence of 1,2-dioleoylphosphatidylglycerol [5]) [5] phosphatidylinositol-4,5-bisphosphate <1> (<1> strong activation in the presence of 1,2-dioleoylphosphatidylglycerol [5]) [5] phosphoenolpyruvate <1> (<1> 0.5 mM, 4fold stimulation, inhibition above 1.5 mM [5]) [5] sn-glycero-1-phosphate <1> (<1> stimulates above 15 mM [5]) [5] Additional information <4> (<4> not activated by cardiolipin [7]) [7] Metals, ions Mg2+ <1> (<1> essential for activity [1]; <1> maximal activity at 10 mM, inhibition above may be due to interaction with UDP-glucose [5]) [1, 5] Specific activity (U/mg) 8.05 <1> [4] Km-Value (mM) 0.14 <1> (UDP-glucose) [4] 0.7 <2> (UDP-glucose) [6] pH-Optimum 7.5 <2> [6]

5 Isolation/Preparation/Mutation/Application Localization membrane <1> [1] Purification <1> (solubilization, SP-Sepharose, hydroxyapatite, Green A agarose [4]) [4]

547

Diglucosyl diacylglycerol synthase

2.4.1.208

Renaturation <1> (enzyme can be reconstituted in liposome bilayers by the dialyse techique [4]) [4] Cloning <1> (expression <2> (expression <3> (expression <4> (expression

in Escherichia in Escherichia in Escherichia in Escherichia

coli coli coli coli

[7]) [6]) [6]) [7])

[7] [6] [6] [7]

6 Stability General stability information <2>, activity of the recombinant enzyme does not change during 3 months of storage and upon multiple cycles of freezing and thawing [6]

References [1] Karlsson, O.P.; Rytömaa, M.; Dahlqvist, A.; Kinnunen, P.K.L.; Wieslander, A.: Correlation between bilayer lipid dynamics and activity of the diglucosyldiacylglycerol synthase from Acholeplasma laidlawii membranes. Biochemistry, 35, 10094-10102 (1996) [2] Dahlqvist, A.; Nordström, S.; Karlsson, O.P.; Mannock, D.A.; McElhaney, R.N.; Wieslander, A.: Efficient modulation of glucolipid enzyme activities in membranes of Acholeplasma laidlawii by the type of lipids in the bilayer matrix. Biochemistry, 34, 13381-13389 (1995) [3] Cornell, R.B.; Arnold, R.S.: Modulation of the activities of enzymes of membrane lipid metabolism by non-bilayer-forming lipids. Chem. Phys. Lipids, 81, 215-227 (1996) [4] Vikstrom, S.; Li, L.; Karlsson, O.P.; Wieslander, A.: Key role of the diglucosyldiacylglycerol synthase for the nonbilayer-bilayer lipid balance of Acholeplasma laidlawii membranes. Biochemistry, 38, 5511-5520 (1999) [5] Vikstrom, S.; Li, L.; Wieslander, A.: The nonbilayer/bilayer lipid balance in membranes: Regulatory enzyme in Acholeplasma laidlawii is stimulated by metabolic phosphates, activator phospholipids, and double-stranded DNA. J. Biol. Chem., 275, 9296-9302 (2000) [6] Kiriukhin, M.Y.; Debabov, D.V.; Shinabarger, D.L.; Neuhaus, F.C.: Biosynthesis of the glycolipid anchor in lipoteichoic acid of Staphylococcus aureus RN4220: role of YpfP, the diglucosyldiacylglycerol synthase. J. Bacteriol., 183, 3506-3514 (2001) [7] Edman, M.; Berg, S.; Storm, P.; Wikstrom, M.; Vikstrom, S.; Ohman, A.; Wieslander, A.: Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae. J. Biol. Chem., 278, 8420-8428 (2003)

548

cis-p-Coumarate glucosyltransferase

2.4.1.209

1 Nomenclature EC number 2.4.1.209 Systematic name UDP-glucose:cis-p-coumarate b-d-glucosyltransferase Recommended name cis-p-coumarate glucosyltransferase CAS registry number 196887-88-4

2 Source Organism <1> Sphagnum fallax (peat moss [1]) [1] <2> Fagus grandifolia (american beech [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + cis-p-coumarate = 4'-O-b-d-glucosyl-cis-p-coumarate + UDP (cis-caffeic acid also serves as a glucosyl acceptor with the enzyme from Sphagnum fallax kinggr. The corresponding trans-isomers are not substrates [1]) Reaction type hexosyl group transfer Natural substrates and products S UDP-glucose + cis-coniferyl alcohol + H2 O <2> (Reversibility: ? <2> [1]) [1] P ? S UDP-glucose + cis-p-coumarate + H2 O <1> (Reversibility: ? <1> [1]) [1] P 4'-O-b-d-glucosyl-cis-p-coumarate + UDP Substrates and products S cis-caffeic acid + UDP-glucose + H2 O <1> (<1> enzyme activity of 10% compared with that using cis-p-coumaric acid as substrate [1]) (Reversibility: ? <1> [1]) [1]

549

cis-p-Coumarate glucosyltransferase

2.4.1.209

P 4'-O-b-d-glucosyl-cis-caffeate + UDP S cis-p-coumarate + UDP-glucose + H2 O <1> (Reversibility: ? <1> [1]) [1] P 4'-O-b-d-glucosyl-cis-p-coumarate + UDP Inhibitors 2-mercaptoethanol <1> (<1> 14 mM, 30% inhibition [1]) [1] NaCl <1> (<1> 0.4 M, 40% inhibition) [1] cis-p-coumaric acid <1> (<1> inhibits the rection above 1 mM concentration [1]) [1] Specific activity (U/mg) 1.054 <1> [1] Km-Value (mM) 0.044 <1> (cis-p-coumaric acid) [1] 0.11 <1> (UDP-glucose, <1> cosubstrate [1]) [1] pH-Optimum 9.3 <1> (<1> 100 mM glycine KOH [1]) [1] pH-Range 8.5-9.3 <1> [1]

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

5 Isolation/Preparation/Mutation/Application Purification <1> (partially, 600fold [1]) [1]

6 Stability pH-Stability 7.8-9.5 <1> (<1> below pH 7.8 and above pH 9.5 rapid drop in enzyme activity) [1] Storage stability <1>, -18 C, freezing results in a loss of activity of more than 50% [1] <1>, 4 C, loses more than 25% of its activity within 24 h, 5% glycerol causes total loss of activity after 48 h [1]

550

2.4.1.209

cis-p-Coumarate glucosyltransferase

References [1] Rasmussen, S.; Rudolph, H.: Isolation, purification and characterization of UDP-glucose:cis-p-coumaric acid-b-d-glucosyltransferase from Sphagnum fallax. Phytochemistry, 46, 449-453 (1997)

551

Limonoid glucosyltransferase

2.4.1.210

1 Nomenclature EC number 2.4.1.210 Systematic name uridine diphosphoglucose-limonoid glucosyltransferase Recommended name limonoid glucosyltransferase Synonyms LGTase limonoid GTase limonoid glucosyltransferase UDP-d-glucose:limonoid glucosyltransferase CAS registry number 195836-82-9

2 Source Organism <1> Citrus sinensis (Frost navel orange, Newhall navel orange [1]; sweet navel orange Osb. cv. Washington [2]) [1, 2] <2> Fragaria vesca (strawberry [1]) [1] <3> Citrus unshiu (mandarin orange [1]; satsuma mandarin Marc. cv. Miyagawa-wase [2]) [1, 2] <4> Citrus grandis (pommelo [1]) [1] <5> Citrus limonia (citron [1]) [1] <6> Citrus maxima (grapefruit, pommelo hybrid Oroblanco [1]) [1] <7> Citrus aurantium (pommelo hybrid [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + limonin = glucosyl-limonin + UDP (<1> the enzyme purified from navel orange albedo tissue also acts on the related tetranortriterpenoid nomilin [1,2]) Reaction type hexosyl group transfer

552

2.4.1.210

Limonoid glucosyltransferase

Natural substrates and products S limonin + UDP-glucose + H2 O <1-7> (Reversibility: ? <1-7> [1, 2]) [1, 2] P glucosyl-limonin + UDP S nomilin + UDP-glucose + H2 O <1, 3> (Reversibility: ? <1, 3> [1, 2]) [1, 2] P ? Substrates and products S limonin + UDP-glucose + H2 O <1> (Reversibility: ? <1> [1]) [1] P glucosyl-limonin + UDP S nomilin + UDP-glucose + H2 O <1> (Reversibility: ? <1> [1]) [1] P glucosyl-nomilin + UDP Inhibitors PO34- <2> [1] Metals, ions Mn2+ <1> (<1> 0.05 mM stimulates enzyme activity by 66% over basal activity [1]) [1] Specific activity (U/mg) 0.018 <1> [1] pH-Optimum 8 <1> [1] pH-Range 6.5-9 <1> [1]

4 Enzyme Structure Molecular weight 56000 <1> (<1> Newhall navel orange, SDS-PAGE [1]) [1] 57500 <3> (<3> predicted from amino acid sequence [2]) [2] 58000 <1> (<1> Frost navel orange, SDS-PAGE [1]) [1] Subunits monomer <1> (<1> 56000-58000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue albedo <1> (<1> inner spongy part of rind [2]) [1, 2] Localization membrane <1> [2] Purification <1> [1, 2]

553

Limonoid glucosyltransferase

2.4.1.210

Cloning <3> (cDNA clone encoding limonoid UDP-glucosyltransferase isolated, single copy gene in the citrus genome [2]) [2] Application medicine <1> (<1> pharmacological application of limonoid GTase gene [2]) [2] nutrition <1> (<1> limonoid glucosides are important compounds not only for the processing industry but also for the consumer, bitterness due to limonoids is an important economic problem in commercial citrus juice production, limonoid aglycones are converted to nonbitter glucosides by the GTase, enhancement of the limonoid GTase activity through genetic engineering could reduce aglycone concentration, insertion of a gene encoding for GTase into commercial cultivars could create transgenic citrus varieties producing fruits potentially free of limonoid bitterness [1]) [1, 2]

6 Stability pH-Stability 6.5 <1> (<1> activity below pH 6.5 is inhibited by a closed D-ring [1]) [1] Storage stability <1>, -80 C, 10 mM Mes-KOH, pH 7 allows frozen storage with full recovery of activity [2] <1>, -80 C, 50 mM Tris-HCl, pH 8.0, all acivity is lost [1]

References [1] Sim, M.K.; Lim, B.C.: Purification of limonoid glucosyltransferase from navel orange albedo tissue. Phytochemistry, 46, 33-37 (1997) [2] Kita, M.; Hirata, Y.; Moriguchi, T.; Endo-Inagaki, T.; Matsumoto, R.; Hasegawa, S.; Suhayda, C.G.; Omura, M.: Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus. FEBS Lett., 469, 173-178 (2000)

554

1,3-b-Galactosyl-N-acetylhexosamine phosphorylase

2.4.1.211

1 Nomenclature EC number 2.4.1.211 Systematic name b-d-galactopyranosyl-(1!3)-N-acetyl-d-hexosamine:phosphate galactosyltransferase Recommended name 1,3-b-galactosyl-N-acetylhexosamine phosphorylase Synonyms b-d-galactopyranosyl-(1-3)-N-acetyl-d-hexosamine:phosphate transferase

galactosyl-

CAS registry number 224427-06-9

2 Source Organism <1> Bifidobacterium bifidum (DSM 20082 [1]) [1]

3 Reaction and Specificity Catalyzed reaction b-d-galactopyranosyl-(1!3)-N-acetyl-d-glucosamine + phosphate = a-d-galactopyranose 1-phosphate + N-acetyl-d-glucosamine (<1> reaction also occurs with b-d-galactopyranosyl-(1!3)-N-acetyl-d-galactosamine as the substrate, giving N-acetyl-d-galactosamine as the product [1]) Reaction type hexosyl group transfer Natural substrates and products S b-1,3-galactooligosaccharides + H2 O <1> (Reversibility: r <1> [1]) [1] P a-d-galactose-1-phosphate + N-acetylhexosamine Substrates and products S b d-galactosido-(1,3)-N-acetylgalactosamine + H2 O <1> (Reversibility: r <1> [1]) [1] P a-d-galactose-1-phosphate + GalNAc

555

1,3-b-Galactosyl-N-acetylhexosamine phosphorylase

2.4.1.211

S b-d-galactosido-(1,3)-N-acetylglucosamine + H2 O <1> (<1> hydrolysis involves a Walden inversion [1]) (Reversibility: r <1> [1]) [1] P a-d-galactose-1-phosphate + GlcNAc S galactose-1-phosphate + GalNAc + H2 O <1> (Reversibility: r <1> [1]) [1] P b-d-galactosido-(1,3)-N-acetylglucosamine S galactose-1-phosphate + GlcNAc + H2 O <1> (Reversibility: r <1> [1]) [1] P b-d-galactosido-(1,3)-N-acetylgalactosamine Km-Value (mM) 1.6 <1> (galactose-1-phosphate) [1] 2.7 <1> (phosphate) [1] pH-Optimum 5.5-6 <1> (<1> reverse reaction [1]) [1] 6-6.5 <1> (<1> galactophosphorolytic activity [1]) [1] Temperature optimum ( C) 50-55 <1> (<1> phosphorolysis and reverse reaction [1]) [1]

4 Enzyme Structure Molecular weight 140000 <1> (<1> gel filtration [1]) [1]

5 Isolation/Preparation/Mutation/Application Localization intracellular <1> [1] Purification <1> (partly) [1] Application medicine <1> (<1> galactosyl-N-acetylhexosamine phosphorylase has a predominant role in the digestive tract of human and in the metabolism of galactose [1]) [1] nutrition <1> (<1> dairy industry production of different fermented bifidobacteria milks [1]) [1] synthesis <1> (<1> production of dissaccharidic structures instead of chemical synthesis [1]) [1]

6 Stability Temperature stability 20-60 <1> [1]

556

2.4.1.211

1,3-b-Galactosyl-N-acetylhexosamine phosphorylase

General stability information <1>, unstable after cell lysis [1]

References [1] Derensy-Dron, D.; Krzewinski, F.; Brassart, C.; Bouquelet, S.: b-1,3-Galactosyl-N-acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterization, partial purification and relation to mucin degradation. Biotechnol. Appl. Biochem., 29, 3-10. (1999)

557

Hyaluronan synthase

1 Nomenclature EC number 2.4.1.212 Recommended name hyaluronan synthase Synonyms CHAS2 CHAS3 DG42 protein HA synthase HuHAS1 XHAS1 XHAS2 XHAS3 hyaluronan synthethase hyaluronate synthase hyaluronate synthetase hyaluronic acid synthase hyaluronic acid synthetase CAS registry number 39346-43-5

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

558

Streptococcus pyogenes [1, 2, 8, 10, 12] Mus musculus [3, 6, 14] Streptococcus equisimilis [4, 10] Paramecium bursaria Chlorella virus 1 [5] Xenopus sp. DG42 [7] Pasteurella multocida [9, 11] Homo sapiens [13, 15]

2.4.1.212

2.4.1.212

Hyaluronan synthase

3 Reaction and Specificity Catalyzed reaction n UDP-N-acetyl-d-glucosamine + n UDP-d-glucuronate = [b-N-acetyl-dglucosaminyl(1!4)b-d-glucuronosyl(1!3)]n + 2n UDP (The enzyme from Streptococcus Group A and Group C requires Mg2+ . It is highly specific for UDP-GlcNAc and UDP-GlcA; no copolymerization is observed if either is replaced by UDP-Glc, UDP-Gal, UDP-GalNAc or UDP-GalA. Similar enzymes have been found in a variety of organisms) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetyl-d-glucosamine + UDP-d-glucuronate <1-7> (Reversibility: ? <1-7> [1-15]) [1-15] P UDP-d-glucuronate Substrates and products S UDP-N-acetyl-d-glucosamine + UDP-d-glucuronate <1-7> (Reversibility: ? <1-7> [1-15]) [1-15] P UDP-d-glucuronate Inhibitors N-ethylmaleimide <1> [12] Metals, ions Co2+ <4> (<4> 2% as effective as Mn2+ at similar concentrations [5]) [5] Mg2+ <4, 5> (<4> 20% as effective as Mn2+ at similar concentrations [5]) [5, 7] Mn2+ <4, 6> (<4> essential for activity [5]) [5, 9] Km-Value (mM) 0.032 <2> (UDP-d-glucuronate, <2> HAS2 [6]) [6] 0.034 <2> (UDP-d-glucuronate, <2> HAS3 [6]) [6] 0.04 <1> (UDP-d-glucuronate) [10] 0.051 <3> (UDP-d-glucuronate) [10] 0.06 <5> (UDP-d-glucuronate) [7] 0.06 <3> (UDP-N-acetyl-d-glucosamine) [10] 0.073 <2> (UDP-d-glucuronate, <2> HAS1 [6]) [6] 0.08 <2> (UDP-N-acetyl-d-glucosamine, <2> HAS3 [6]) [6] 0.11 <2> (UDP-N-acetyl-d-glucosamine, <2> HAS2 [6]) [6] 0.14 <6> (UDP-d-glucuronate) [11] 0.149 <1> (UDP-N-acetyl-d-glucosamine) [10] 0.16 <6> (UDP-N-acetyl-d-glucosamine) [11] 0.23 <5> (UDP-N-acetyl-d-glucosamine) [7] 0.79 <2> (UDP-N-acetyl-d-glucosamine, <2> HAS1 [6]) [6] Additional information <2> (<2> values for other substrate concentrations [6]) [6]

559

Hyaluronan synthase

2.4.1.212

pH-Optimum 7.2 <4> [5] 7.6-8.1 <5> [7] pH-Range 7-8.4 <5> [7]

4 Enzyme Structure Molecular weight 42000 <1, 3> (<1> SDS-PAGE [1,2,4]) [1, 2, 4] 47780 <1, 3> (<1,3> calculation from sequence [4,8]) [4, 8] 48000 <2> (<2> northern blot [3]) [3] 52000 <2> (<2> SDS-PAGE [14]) [14] 66000 <7> (<7> gel filtration [13]) [13]

5 Isolation/Preparation/Mutation/Application Source/tissue breast adenocarcinoma cell <2> (<2> B6-cell line [14]) [14] glioma cell <7> (<7> cell line [13]) [13] keratinocyte <7> [15] Localization membrane <1, 2> (<1> enzyme is predicted to be an integral membrane protein [1]) [1, 2, 3, 6, 8, 12] Purification <7> (partial [13]) [13] Cloning <2> (expression in COS-1 cells and rat 3Y1 fibroblasts [6]) [6] <5> (expression in yeast [7]) [7] <1, 3, 4, 6> (expression in Escherichia coli [1,2,4,5,8-12]) [1, 2, 4, 5, 8-12] Engineering D196N <6> (<6> mutants possess UDP-d-glucuronate-transferase activity [11]) [11] D477K <6> (<6> mutants possess UDP-N-acetyl-d-glucosamine-transferase activity [11]) [11] Additional information <1> (<1> variety of cystein mutatants [12]) [12]

6 Stability Storage stability <7>, 4 C, Na-phosphate buffer, 10% glycerol, 96 h, 18% [13]

560

2.4.1.212

Hyaluronan synthase

References [1] DeAngelis, P.L.; Papaconstantinou, J.; Weigel, P.H.: Molecular cloning, identification and sequence of the hyaluronan synthase gene from Group A Streptococcus pyogenes. J. Biol. Chem., 268, 19181-19184 (1993) [2] DeAngelis, P.L.; Weigel, P.H.: Immunochemical confirmation of the primary structure of streptococcal hyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme. Biochemistry, 33, 90339039 (1994) [3] Spicer, A.P.; Augustine, M.L.; McDonald, J.A.: Molecular cloning and characterization of a putative mouse hyaluronan synthase. J. Biol. Chem., 271, 23400-23406 (1996) [4] Kumari, K.; Weigel, P.H.: Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis. J. Biol. Chem., 272, 32539-32546 (1997) [5] DeAngelis, P.L.; Jing, W.; Graves, M.V.; Burbank, D.E.; Van Etten, J.L.: Hyaluronan synthase of chlorella virus PBCV-1. Science, 278, 1800-1803. (1997) [6] Itano, N.; Sawai, T.; Yoshida, M.; Lenas, P.; Yamada, Y.; Imagawa, M.; Shinomura, T.; Hamaguchi, M.; Yoshida, Y.; Ohnuki, Y.; Miyauchi, S.; Spicer, A.P.; McDonald, J.A.; Kimata, K.: Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties. J. Biol. Chem., 274, 2508525092 (1999) [7] Pummill, P.E.; Achyuthan, A.M.; DeAngelis, P.L.: Enzymological characterization of recombinant Xenopus DG42, a vertebrate hyaluronan synthase. J. Biol. Chem., 273, 4976-4981 (1998) [8] Tlapak-Simmons, V.L.; Baggenstoss, B.A.; Clyne, T.; Weigel, P.H.: Purification and lipid dependence of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis. J. Biol. Chem., 274, 4239-4245 (1999) [9] DeAngelis, P.L.: Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase. J. Biol. Chem., 274, 26557-26562 (1999) [10] Tlapak-Simmons, V.L.; Baggenstoss, B.A.; Clyne, T.; Weigel, P.H.: Purification and lipid dependence of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis. J. Biol. Chem., 274, 4239-4245 (1999) [11] Jing, W.; DeAngelis, P.L.: Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: two active sites exist in one polypeptide. Glycobiology, 10, 883-889 (2000) [12] Heldermon, C.D.; Tlapak-Simmons, V.L.; Baggenstoss, B.A.; Weigel, P.H.: Site-directed mutation of conserved cysteine residues does not inactivate the Streptococcus pyogenes hyaluronan synthase. Glycobiology, 11, 10171024 (2001) [13] Asplund, T.; Brinck, J.; Suzuki, M.; Briskin, M.J.; Heldin, P.: Characterization of hyaluronan synthase from a human glioma cell line. Biochim. Biophys. Acta, 1380, 377-388 (1998)

561

Hyaluronan synthase

2.4.1.212

[14] Klewes, L.; Prehm, P.: Intracellular signal transduction for serum activation of the hyaluronan synthase in eukaryotic cell lines. J. Cell.Physiol., 160, 539-544 (1994) [15] Sayo, T.; Sugiyama, Y.; Takahashi, Y.; Ozawa, N.; Sakai, S.; Ishikawa, O.; Tamura, M.; Inoue, S.: Hyaluronan synthase 3 regulates hyaluronan synthesis in cultured human keratinocytes. J. Invest. Dermatol., 118, 43-48 (2002)

562

Glucosylglycerol-phosphate synthase

2.4.1.213

1 Nomenclature EC number 2.4.1.213 Systematic name ADP-glucose:sn-glycerol-3-phosphate 2-b-d-glucosyltransferase Recommended name glucosylglycerol-phosphate synthase Synonyms GG-phosphate synthase GGPS Glucosyl-glycerol-phosphate synthase CAS registry number 161515-13-5

2 Source Organism <1> Synechocystis sp. (PCC 6803 [1-5]) [1-5]

3 Reaction and Specificity Catalyzed reaction ADP-glucose + sn-glycerol 3-phosphate = 2-(b-d-glucosyl)-sn-glycerol 3-phosphate + ADP (acts with EC 3.1.3.69: glucosylglycerol phosphatase to form glucosylglycerol, an osmolyte that endows cyanobacteria with resistance to salt) Reaction type hexosyl group transfer Natural substrates and products S ADPglucose + sn-glycerol 3-phosphate <1> (Reversibility: ? <1> [1-5]) [1-5] P 2-(b-d-glucosyl)-sn-glycerol 3-phosphate + ADP Substrates and products S ADPglucose + sn-glycerol 3-phosphate <1> (Reversibility: ? <1> [1-5]) [1-5] P 2-(b-d-glucosyl)-sn-glycerol 3-phosphate + ADP

563

Glucosylglycerol-phosphate synthase

2.4.1.213

Activating compounds NaCl <1> [3, 4]

5 Isolation/Preparation/Mutation/Application Purification <1> (partial [4]) [3, 4] Cloning <1> (expression in Escherichia coli [2,4]) [2, 4]

6 Stability Storage stability <1>, 4-6 C, 10 days, 40% [4]

References [1] Hagemann, M.; Erdmann, N.: Activation and pathway of glucosylglycerol biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology, 140, 1427-1431 (1994) [2] Marin, K.; Zuther, E.; Kerstan, T.; Kunert, A.; Hagemann, M.: The ggpS gene from Synechocystis sp. strain PCC 6803 encoding glucosylglycerol-phosphate synthase is involved in osmolyte synthesis. J. Bacteriol., 180, 48434849 (1994) [3] Schoor, A.; Hagemann, M.; Erdmann, N.: Glucosylglycerol-phosphate synthase: target for ion-mediated regulation of osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803. Arch. Microbiol., 171, 101106 (1999) [4] Hagemann, M.; Effmert, U.; Kerstan, T.; Schoor, A.; Erdmann, N.: Biochemical characterization of glucosylglycerol-phosphate synthase of Synechocystis sp. strain PCC 6803: Comparison of crude, purified, and recombinant enzymes. Curr. Microbiol., 43, 278-283 (2001) [5] Marin, K.; Huckauf, J.; Fulda, S.; Hagemann, M.: Salt-dependent expression of glucosylglycerol-phosphate synthase, involved in osmolyte synthesis in the cyanobacterium synechocystis sp. strain PCC 6803. J. Bacteriol., 184, 2870-2877 (2002)

564

Glycoprotein 3-a-L-fucosyltransferase

2.4.1.214

1 Nomenclature EC number 2.4.1.214 Systematic name GDP-l-fucose:glycoprotein (L-fucose to asparagine-linked N-acetylglucosamine of N4 -{N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl-(1!3)-[Nacetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl-(1!6)]-b-d-mannosyl(1!4)-N-acetyl-b-d-glucosaminyl-(1!4)-N-acetyl-b-d-glucosaminyl}asparagine) 3-a-l-fucosyl-transferase Recommended name glycoprotein 3-a-l-fucosyltransferase Synonyms AtFUT11 Core a-(1,3)-fucosyltransferase Fuc-T C3 FucT1 FucTA GDP-l-fuc:Asn-linked GlcNAc a1,3-fucosyltransferase GDP-l-fuc:N-acetyl-b-d-glucosaminide a1,3-fucosyltransferase GDP-fucose:b-N-acetylglucosamine (Fuc to (Fuca1-6GlcNAc)-Asn-peptide) a1-3-fucosyltransferase

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

Arabidopsis thaliana [1] Drosophila melanogaster [2] Vigna radiata [3, 10] Lymnaea stagnalis [4] Apis mellifera [5] Homo sapiens [6, 8, 9, 11] Cricetulus griseus [7] Schistosoma mansoni [12] Caenorhabditis elegans [13]

565

Glycoprotein 3-a-L-fucosyltransferase

2.4.1.214

3 Reaction and Specificity Catalyzed reaction GDP-l-fucose + N4 -{N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl(1!3)-[N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl-(1!6)]-b-d-mannosyl-(1!4)-N-acetyl-b-d-glucosaminyl-(1!4)-N-acetyl-b-d-glucosaminyl} asparagine = GDP + N4 -{N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl(1!3)-[N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl-(1!6)]-b-d-mannosyl-(1!4)-N-acetyl-b-d-glucosaminyl-(1!4)-[a-l-fucosyl-(1!3)]-N-acetyl-b-d-glucosaminyl}asparagine Reaction type hexosyl group transfer Natural substrates and products S GDP-l-fucose + N4 -[N-acetyl-b-d-glucosaminyl-(1-2)-a-d-mannosyl-(13)-[N-acetyl-b-d-glucosaminyl-(1-2)-a-d-mannosyl-(1-6)]-b-d-mannosyl-(1-4)-N-acetyl-b-d-glucosaminyl-(1-4)-N-acetyl-b-d-glucosaminyl]asparagine <1-7> (Reversibility: ? <1-7> [1-7]) [1-7] P GDP + N4 -[N-acetyl-b-d-glucosaminyl-(1-2)-a-d-mannosyl-(1-3)-[Nacetyl-b-d-glucosaminyl-(1-2)-a-d-mannosyl-(1-6)]-b-d-mannosyl-(1-4)N-acetyl-b-d-glucosaminyl-(1-4)-[a-l-fucosyl-(1-3)]-N-acetyl-b-d-glucosaminyl]asparagine <1-7> [1-7] S Additional information <1, 4> (<1>, the core a1-3-fucosyltransferases are involved in the synthesis of glycans specific to plants and invertebrates which are known to be immunogenic and allergenic [1]; <4>, the enzyme functions in the synthesis of core a1-3-fucosylated complex-type glycans [4]) [1, 4] P ? Substrates and products S GDP-l-fucose + N4 -[N-acetyl-b-d-glucosaminyl-(1-2)-a-d-mannosyl-(13)-[N-acetyl-b-d-glucosaminyl-(1-2)-a-d-mannosyl-(1-6)]-b-d-mannosyl-(1-4)-N-acetyl-b-d-glucosaminyl-(1-4)-N-acetyl-b-d-glucosaminyl]asparagine <1-7> (Reversibility: ? <1-7> [1-10]) [1-10] P GDP + N4 -[N-acetyl-b-d-glucosaminyl-(1-2)-a-d-mannosyl-(1-3)-[Nacetyl-b-d-glucosaminyl-(1-2)-a-d-mannosyl-(1-6)]-b-d-mannosyl-(1-4)N-acetyl-b-d-glucosaminyl-(1-4)-[a-l-fucosyl-(1-3)]-N-acetyl-b-d-glucosaminyl]asparagine <1-7> [1-10] S GDP-fucose + Fuca1-2Galb1-4GlcNAc <6> (Reversibility: ? <6> [8]) [8] P GDP + Fuca1-2Galb1-4(Fuca1-3)GlcNAc <6> [8] S GDP-fucose + Fuca1-2Galb1-4GlcNAc <6> (Reversibility: ? <6> [9]) [9] P GDP + Fuca1-2Galb1-4(Fuca1-3)GlcNAc <6> [9] S GDP-fucose + GalGal-N-glycan <2> (<2>, 4.6% relative conversion, FucTA gene [2]) (Reversibility: ? <2> [2]) [2] P GDP + a1-3-fucosylated GalGal-N-glycan <2> [2] S GDP-fucose + GalGalF6-N-glycan <2> (<2>, 19.8% relative conversion, FucTA gene [2]) (Reversibility: ? <2> [2]) [2]

566

2.4.1.214

Glycoprotein 3-a-L-fucosyltransferase

P GDP + a1-3-fucosylated N-glycan <2> [2] S GDP-fucose + GalNAcb1-4GlcNAcb1-3Galb1-4Glc <9> (<9>, incubation with extracts from transfected COS7 cells [13]) (Reversibility: ? <9> [13]) [13] P GDP + GalNAcb1-4[Fuca1-3]GlcNAcb1-3Galb1-4Glc <9> (<9>, product obtained at a level of about 50% of that obtained with the acceptor GalNAcb1-4GlcNAcb1-R [13]) [13] S GDP-fucose + GalNAcb1-4GlcNAcb1-R <9> (Reversibility: ? <9> [13]) [13] P GDP + GalNAcb1-4[Fuca1-3]GlcNAcb1-R <9> [13] S GDP-fucose + Galb1-4Glc <6> (Reversibility: ? <6> [8]) [8] P GDP + Galb1-4 (Fuca1-3)Glc <6> [8] S GDP-fucose + Galb1-4GlcNAc <6> (Reversibility: ? <6> [8]) [8] P GDP + Galb1-4 (Fuca1-3)GlcNAc <6> [8] S GDP-fucose + Galb1-4GlcNAc-R <8, 9> (Reversibility: ? <8, 9> [12, 13]) [12, 13] P GDP + Galb1-4(Fuca1-3)GlcNAc-R <8, 9> (<8,9> Lewis x determinant [12,13]) [12, 13] S GDP-fucose + GlcNAcb1-2-Mana1-3(GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb1-Asn <3> (Reversibility: ? <3> [3]) [3] P GDP + GlcNAcb1-2-Mana1-3(GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4 (Fuca1-6)(Fuca1-3)GlcNAcb1-Asn <3> [3] S GDP-fucose + GlcNAcb1-2-Mana1-3(GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb1-Asn <3> (Reversibility: ? <3> [3]) [3] P GDP + GlcNAcb1-2-Mana1-3(GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4 (Fuca1-3)GlcNAcb1-Asn <3> [3] S GDP-fucose + GlcNAcb1-2-Mana1-3[Mana1-3(Mana1-6)Mana1-6]Manb1-4-GlcNAcb1-4GlcNAcb1-Asn <3> (Reversibility: ? <3> [3]) [3] P GDP + GlcNAcb1-2-Mana1-3[Mana1-3(Mana1-6)Mana1-6]Manb1-4GlcNAcb1-4 (Fuca1-3)GlcNAcb1-Asn <3> [3] S GDP-fucose + GlcNAcb1-2-Mana1-6(GlcNAcb1-2Mana1-3)Manb1-4 GlcNAcb1-4(Fuca1-6)GlcNAcb1-N-Asn-peptide(Nac) <5> (Reversibility: ? <5> [5]) [5] P GDP + GlcNAcb1-2-Mana1-6(GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb14(Fuca1-6)(Fuca1-3)GlcNAcb1-N-Asn-peptide(Nac) <5> (<5>, the product shows a difucosylated structure with two Fuc residues at the Asnbound GlcNAc residue, 40% yield [5]) [5] S GDP-fucose + GnGn + F(Ac) <5> (Reversibility: ? <5> [5]) [5] P GDP + a1-3-fucosylated peptide <5> [5] S GDP-fucose + GnGn-oligosaccharide <3> (<3>, 93% relative activity of pure enzyme [10]) (Reversibility: ? <3> [10]) [10] P GDP + a1-3 fucosylated GnGn-oligosaccharide <3> [10] S GDP-fucose + GnGn-peptide <3> (<3>, 100% relative activity of pure enzyme and of crude extract [10]) (Reversibility: ? <3> [10]) [10] P GDP + a1-3 fucosylated GnGn-peptide <3> [10]

567

Glycoprotein 3-a-L-fucosyltransferase

2.4.1.214

S GDP-fucose + GnGnF6-oligosaccharide <3> (<3>, 95% relative activity of pure enzyme [10]) (Reversibility: ? <3> [10]) [10] P GDP + a1-3 fucosylated GnGnF6-oligosaccharide <3> [10] S GDP-fucose + GnGnF6-peptide <3> (<3>, 87% relative activity with recombinant enzyme [3]) (Reversibility: ? <3> [3]) [3] P GDP + a1-3-fucosylated GnGnF6-peptide <3> [3] S GDP-fucose + GnGnF6-peptide <3> (<3>, 99% relative activity of pure enzyme and 70% relative activity of crude extract [10]) (Reversibility: ? <3> [10]) [10] P GDP + a1-3 fucosylated peptide <3> [10] S GDP-fucose + LacNAc <6> (Reversibility: ? <6> [11]) [11] P GDP + ? <6> [11] S GDP-fucose + LacNAcb-O-(CH2 )5 CO2 CH3 <6> (Reversibility: ? <6> [11]) [11] P GDP + ? <6> [11] S GDP-fucose + M5Gn-Asn <3> (<3>, 71% relative activity with recombinant enzyme [3]) (Reversibility: ? <3> [3]) [3] P GDP + a1-3-fucosylated M5Gn-Asn <3> [3] S GDP-fucose + NeuAca2 -3Galb1 -4GlcNAc <6> (Reversibility: ? <6> [9]) [9] P GDP + NeuAca2-3Galb1-4(Fuca1-3)GlcNAc <6> [9] S GDP-fucose + NeuAca2-3Galb1-4GlcNAc <8, 9> (<8>, rate of reaction below 5% of the rate obtained toward nonsialylated acceptor [12]) (Reversibility: ? <8, 9> [12, 13]) [12, 13] P GDP + NeuAca2-3Galb1-4(Fuca1-3)GlcNAc <8, 9> (<8,9> sialyl Lewis x [12,13]) [12, 13] S GDP-fucose + a1 -acid glycoprotein <7> (<7>, the activity of the LEC11 cells enzyme is considerably higher than the activity of the LEC12 cells enzyme [7]) (Reversibility: ? <7> [7]) [7] P GDP + a1-3 fucosylated glycoprotein <7> [7] S GDP-fucose + asialo-a1 -acid glycoprotein <7> (Reversibility: ? <7> [7]) [7] P GDP + a1-3 fucosylated glycoprotein <7> [7] S GDP-fucose + asialo/agalacto-glycopeptide-F2 <4> (<4>, asialo/agalactoglycopeptide-F2: from human fibrinogen [4]) (Reversibility: ? <4> [4]) [4] P GDP + a1-3-fucosylated glycopeptide <4> [4] S GDP-fucose + asialo/agalacto-glycopeptide-IgGF6 <4> (<4>, asialo/agalacto-glycopeptide-IgGF6: from core a1-6-fucosylated human IgG [4]) (Reversibility: ? <4> [4]) [4] P GDP + a1-3-fucosylated glycopeptide <4> [4] S GDP-fucose + asialofetuin <7> (Reversibility: ? <7> [7]) [7] P GDP + a1-3 fucosylated glycoprotein <7> [7] S GDP-fucose + asialotransferrin <7> (Reversibility: ? <7> [7]) [7] P GDP + a1-3 fucosylated glycoprotein <7> [7] S GDP-fucose + bisected agalacto-glycopeptide-IgGF6 <4> (Reversibility: ? <4> [4]) [4]

568

2.4.1.214

Glycoprotein 3-a-L-fucosyltransferase

P GDP + a1-3-fucosylated glycopeptide <4> [4] S GDP-fucose + dabsyl-GnGn <1, 2> (<1>, FucTA gene, dabsylated glycopeptide derived from fibrin carrying a GnGn oligosaccharide [1]; <2>, GnGn ist the most suitable substrate for FucTA [2]) (Reversibility: ? <1, 2> [1, 2]) [1, 2] P GDP + a1-3-fucosylated dabsyl-GnGn <1, 2> [1, 2] S GDP-fucose + dabsylated-GalGal-peptide <3> (<3>, 0.7% relative activity with recombinant enzyme [3]) (Reversibility: ? <3> [3]) [3] P GDP + a1-3-fucosylated dabsyl-GalGal-peptide <3> [3] S GDP-fucose + dabsylated-GalGn-peptide <3> (<3>, 50% relative activity with recombinant enzyme [3]) (Reversibility: ? <3> [3]) [3] P GDP + a1-3-fucosylated dabsyl-GalGn-peptide <3> [3] S GDP-fucose + dansyl-GnGn <1, 2> (<1>, FucTA gene, dansylated glycopeptide derived from IgG, 40% relative conversion [1]) (Reversibility: ? <1, 2> [1, 2]) [1, 2] P GDP + a1-3-fucosylated dansyl-GnGn <1, 2> [1, 2] S GDP-fucose + dansyl-GnGnF6 <2> (<2>, prefucosylation of GnGn with chicken heart extract results in a superior conversion of GnGnF6 by FucTA than with GnGn, 100% relative conversion [2]) (Reversibility: ? <2> [2]) [2] P GDP + a1-3-fucosylated dansyl-GnGnF6 <2> [2] S GDP-fucose + dansylated mono-b1,3-galactosylated glycopeptide <1> (<1>, FucTA gene [1]) (Reversibility: ? <1> [1]) [1] P GDP + a1-3-fucosylated glycopeptide <1> [1] S GDP-fucose + fetuin <7> (<7>, the activity of the LEC11 cells enzyme is considerably higher than the activity of the LEC12 cells enzyme [7]) (Reversibility: ? <7> [7]) [7] P GDP + a1-3 fucosylated fetuin <7> [7] S GDP-fucose + lacto-N-neotetraose <8, 9> (Reversibility: ? <8, 9> [12, 13]) [12, 13] P GDP + lacto-N-fucopentaose III <8, 9> [12, 13] S GDP-fucose + lacto-N-neotetraose <6> (Reversibility: ? <6> [9]) [9] P GDP ? <6> [9] S GDP-fucose + transferrin <7> (Reversibility: ? <7> [7]) [7] P GDP + a1-3 fucosylated transferrin <7> [7] S Additional information <1-4, 6, 8, 9> (<1>, FucTA gene, amongst the dansyl glycopeptides tested, only those with one free non-reducing terminal N-acetyl-glucosamine residue act as substrates: specifically GnGn lacking one GlcNAc, GnGn substituted with one galactose residue or GnGn substituted with one Lewis-a epitope [1]; <2>, the enzyme is able to fucosylate N-glycan structures of human transferrin in vitro [2]; <2>, a1-6-fucosylated glycans are better substrates than b-1-4-galactosylated glycans, FucTA gene [2]; <4>, compounds lacking the peptide-linked GlNAc or outer arm GlcNAc do not serve as acceptors [4]; <6>, lactose and its fucose-substituted derivative are very poor acceptors for the enzyme [9]; <3>, the enzyme acts upon N-glycopeptides and related oligosaccharides with the glycan structure GlcNAc2 Man3 GlcNAc2 , no transfer 569

Glycoprotein 3-a-L-fucosyltransferase

2.4.1.214

to N-glycans is observed when the terminal GlcNAc residues are either absent or substituted with galactose, N-acetyllactosamine, lacto-N-biose and N-acetylchito-oligosaccharides do not function as acceptors for the enzyme [10]; <6>, catalyzes the transfer of the l-fucose moiety from guanosine diphosphate-b-l-fucose to acceptor sugars to form biologically important fucoglycoconjugates, including sialyl Lewis x, the enzyme processes acceptor sugars with either oxygen or sulfur in the GlcNAc ring, but a similar sugar structure with nitrogen in the GlcNAc ring, is an inhibitor [11]; <8>, the enzyme does not transfer efficiently to the isomeric oligosaccharide lacto-N-tetraose [12]; <8,9>, the enzyme has acceptor specifity properties similar to that of the human myeloid enzyme FTIV [12,13]; <9>, the recombinant enzyme has a clear preference for nonsialylated type-2 acceptors over either neutral type-1 acceptors or sialylated type-2 acceptors [13]) [1, 2, 4, 8, 10-13] P ? Inhibitors GDP <6, 7> (<7>, inhibits both LEC11 and LEC12 cells enzyme activity to approximately the same extent [7]; <6>, causes 22% inhibition [11]) [7, 11] GDP-a-d-glucose <6> [11] GDP-a-d-mannose <6> [11] GDP-fucose <6> [11] GMP <6, 7> (<7>, inhibits both LEC11 and LEC12 cells enzyme activity to approximately the same extent [7]; <6>, 80% inhibition at 0.4 mM [8]; <6>, causes 23% inhibition [11]) [7, 8, 11] GTP <6> (<6>, potent inhibitor [11]) [11] l-fucal <6> (<6>, modest inhibitor, causes 26% inhibition, combined with GDP causes 40% inhibition [11]) [11] Mn2+ <5> (<5>, at concentrations above 40 mM [5]) [5] N-ethylmaleimide <6-8> (<6>, at 3 mM [6]; <7>, at 1 mM completely inhibits the enzyme activity in LEC11 cells, in LEC12 cells: not inhibitory [7]; <6>, not inhibitory at 5 mM, 30 min. [8]; <3>, not inhibitory [10]; <8>, 14 mM causes 50% inhibition [12]) [6-8, 12] NaCl <6> (<6>, 40% inhibition at 300 mM [8]) [8] Triton X-100 <5> (<5>, at concentrations above 0.5% [5]) [5] deoxyfuconojirimycin <6> (<6>, modest inhibitor, causes 21% inhibition, synergism: combined with GDP at their Ki levels results in 80% inhibition, combined with GMP causes 50.4% inhibition [11]) [11] inosine diphosphate <6> (<6>, potent inhibitor [11]) [11] p-chloromercuribenzoate <6> (<6>, at 0.05 mM: almost 30% of the original enzyme activity remains [6]) [6] Additional information <6> [11] Activating compounds Triton X-100 <3> (<3>, 0.1-0.5% slightly enhances activity [10]) [10] b-mercaptoethanol <6> [9]

570

2.4.1.214

Glycoprotein 3-a-L-fucosyltransferase

Metals, ions Ca2+ <6> (<6>, alternative divalent metal cofactor [11]) [11] Co2+ <6> (<6>, alternative divalent metal cofactor [11]) [11] Mg2+ <6> (<6>, alternative divalent metal cofactor [11]) [11] Mn2+ <3, 5, 6, 8, 9> (<5>, required, optimal activity at 20 mM [5]; <6>, optimal activity at 5 mM [8]; <3>, optimal activity at 10-15 mM [10]; <6> acts as electrophilic catalyst in the nonenzymatic hydrolysis of GDP-fucose [11]) [5, 8, 10-13] Zn2+ <3, 6> (<6>, at 10 mM stimulates the activity more than Mn2+ [8]; <6>, no activity [11]) [8, 10] Specific activity (U/mg) 0.000001 <7> (<7>, LEC12 mutant, substrate: a1 -acid glycoprotein [7]) [7] 0.000002 <7> (<7>, LEC11 mutant with transferrin as substrate and LEC12 mutant with fetuin as substrate [7]) [7] 0.000003 <7> (<7>, LEC12 mutant, substrate: transferrin [7]) [7] 0.000007 <7> (<7>, LEC11 mutant, substrate: asialotransferrin [7]) [7] 0.000013 <7> (<7>, LEC11 mutant, substrate: asialofetuin [7]) [7] 0.000014 <7> (<7>, LEC11 mutant with fetuin as substrate and LEC12 mutant with asialofetuin as substrate [7]) [7] 0.000015 <7> (<7>, LEC11 mutant, substrate: a1 -acid glycoprotein [7]) [7] 0.000016 <7> (<7>, LEC12 mutant, substrate: asialo-a1 -acid glycoprotein or asialotransferrin [7]) [7] 0.000019 <7> (<7>, LEC11 mutant, substrate: asialo-a1 -acid glycoprotein [7]) [7] 0.071 <3> [10] 43.35 <3> [3] Additional information <4, 6, 9> [4, 9, 13] Km-Value (mM) 0.011 <2> (dansyl-GnGnF6, <2>, glycopeptide derived from IgG, FucTA gene [2]) [2] 0.025 <6> (GDP-fucose, <6>, LacNAcb-O-(CH2 )5 CO2 CH3 as substrate [11]) [11] 0.043 <6> (GDP-fucose) [8, 9] 0.046 <2> (dansyl-GnGn, <2>, glycopeptide derived from IgG, FucTA gene [2]) [2] 0.06 <6> (GDP-fucose, <6>, LacNAc as substrate [11]) [11] 0.06 <3> (GnGnF6-peptide) [10] 0.065 <4> (GDP-fucose) [4] 0.067 <1> (dansyl-GnGn, <1>, FucTA gene [1]) [1] 0.11 <3> (GDP-fucose) [3] 0.13 <3> (GnGnF6-peptide) [3] 0.17 <6> (LacNAcb-O-(CH2 )5 CO2 CH3 ) [11] 0.19 <3> (GnGn-peptide) [3] 0.23 <3> (M5Gn-Asn) [3] 0.26 <3> (GnGn-peptide) [10] 0.3 <8> (lacto-N-neotetraose) [12] 571

Glycoprotein 3-a-L-fucosyltransferase

2.4.1.214

0.4 <6> (Galb1-4GlcNAc) [8] 0.49 <4> (bisected agalacto-glycopeptide-IgGF6) [4] 0.53 <6> (N-acetyl-b-lactosamine) [9] 0.76 <6> (lacto-N-neotetraose) [9] 0.96 <6> (NeuAca2-3Galb1-4GlcNAc) [9] 1 <6> (Fuca1-2Galb1-4Glc) [8] 1.2 <6> (Co2+ ) [11] 6.1 <6> (Mn2+ ) [11] 6.7 <6> (Ca2+ ) [11] 6.7 <5> (GnGn-peptide, <5>, GnGn + F(Ac) [5]) [5] 8.1 <6> (Galb1-4Glc) [8] 8.6 <6> (Mg2+ ) [11] 8.8 <6> (LacNAc) [11] Ki-Value (mM) 0.0062 <6> (GDP-fucose, <6>, LacNAc as substrate [11]) [11] 0.029 <6> (GDP) [11] 0.031 <6> (GDP-fucose, <6>, LacNAcb-O-(CH2 )5 CO2 CH3 as substrate [11]) [11] 0.069 <6> (inosine diphosphate) [11] 0.29 <6> (GDP-a-d-mannose) [11] 0.38 <6> (GDP-a-d-glucose) [11] 0.7 <6> (GMP) [11] 45 <6> (deoxyfuconojirimycin) [11] 71 <6> (l-fucal) [11] Additional information <6> [11] pH-Optimum 5-7 <6> (<6>, in both phosphate and cacodylate buffers [11]) [11] 6.25 <6> [8] 6.5-7 <5> [5] 7 <3> [10] 7.2 <6> [6] Temperature optimum ( C) 37 <6> [6]

4 Enzyme Structure Molecular weight 45000 <6> (<6>, gel filtration [9]) [9] 55000 <1> (<1>, FucTB gene, MALDI peptide mapping and Western blotting [1]) [1] 56210 <1> (<1>, FucTA gene, amino acid sequence analysis [1]) [1] 64000 <3> (<3>, gel filtration [10]) [10]

572

2.4.1.214

Glycoprotein 3-a-L-fucosyltransferase

Subunits ? <3> (<3>, x * 54000, major isoform, SDS-PAGE [3]) [3] ? <3> (<3>, x * 56000, represents the same enzyme as the band with 54 kDA, SDS-PAGE [3]) [3] ? <6> (<6>, x * 40000-45000, SDS-PAGE [8]) [8] monomer <3, 6> (<6>, 1 * 45000, SDS-PAGE [9]; <3>, 1 * 65000, SDS-PAGE [10]) [9, 10]

5 Isolation/Preparation/Mutation/Application Source/tissue cervical epithelium <6> [6] neuroblastoma cell <6> (<6>, CHP 134 [8]) [8] ovary <7> (<7>, glycosylation mutants LEC11 and LEC12 [7]) [7] prostate gland <4> [4] seed <3> [10] serum <6> [8] venom gland <5> [5] Localization microsome <3> [3] Purification <3> (of Fuc-T C3, using Triton X-100 extraction and column chromatography on DE52 cellulose, Affi-Gel blue, S-Sepharose, GnGn-Sepharose and GDP-hexanolamine-Sepharose [3]; by chromatography on DE52 cellulose and Affigel Blue, chromatofocusing using PBE 94 and polybuffer 96, gel filtration on a Sephacryl S200 column and affinity chromatography on a GnGncolumn [10]) [3, 10] <6> (using method A: column chromatography on DEAE-Sephadex, SP-Sephadex, ASTD-Sepharose and GTP-Agarose, method B: column chromatography on DEAE-Sephacel, SP-Sephadex and Synsorb-Galb1-4GlcNAc [8]; using ammonium sulfate precipitation, hydrophobic chromatography in phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-heanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration [9]) [8, 9] Cloning <3> (cDNA cloning of Fuc-T C3 from mRNA, recombinant enzyme expressed in Sf21 insect cells using baculovirus vector [3]) [3] <9> (putative cDNA amplified by PCR from a cDNAlZAP library, cloned into the mammalian expression vector pCR3.1 and sequenced, transfection of COS7 kidney cells from the African green monkey with cDNA encoding the enzyme results in about 20fold level of activity over control cells, using lactoN-neotetraose as acceptor [13]) [13] <1, 2> (three genes FucTA, FucTB and FucTC encode proteins similar to a13-fucosyltransferases, expression in Pichia pastoris [1,2]) [1, 2]

573

Glycoprotein 3-a-L-fucosyltransferase

2.4.1.214

References [1] Wilson, I.B.H.; Rendic, D.; Freilinger, A.; Dumic, J.; Altmann, F.; Mucha, J.; Müller, S.; Hauser, M.T.: Cloning and expression of a1,3-fucosyltransferase homologues from Arabidopsis thaliana. Biochim. Biophys. Acta, 1527, 8896 (2001) [2] Fabini, G.; Freilinger, A.; Altmann, F.; Wilson, I.B.H.: Identification of core a1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural antihorseradish peroxidase epitope. J. Biol. Chem., 276, 28058-28067 (2001) [3] Leiter, H.; Mucha, J.; Staudacher, E.; Grimm, R.; Glossl, J.; Altmann, F.: Purification, cDNA cloning, and expression of GDP-l-Fuc:Asn-linked GlcNAc a1,3-fucosyltransferase from mung beans. J. Biol. Chem., 274, 21830-21839 (1999) [4] Van Tetering, A.; Schiphorst, W.E.C.M.; van den Eijnden, D.H.; van Die, I.: Characterization of core a1-3-fucosyltransferase from the snail Lymnaea stagnalis that is involved in the synthesis of complex type N-glycans. FEBS Lett., 461, 311-314 (1999) [5] Staudacher, E.; Altmann, F.; Glössl, J.; März, L.; Schachter, H.; Kamerling, J.P.; Hård, K.; Vliegenthart, J.F.G.: GDP-fucose:b-N-acetylglucosamine (Fuc to (Fuca1-6GlcNAc)-Asn-peptide) a1-3-fucosyltransferase activity in honeybee (Apis mellifica) venom glands. The difucosylation of asparaginebound N-acetylglucosamine. Eur. J. Biochem., 199, 745-751 (1991) [6] Scudder, P.R.; Chantler, E.N.: Glycosyltransferases of the human cervical epithelium. I. Characterization of a b-galactoside a-2-l-fucosyltransferase and the identification of a b-N-acetylglucosaminide a-3-l-fucosyltransferase. Biochim. Biophys. Acta, 660, 128-135 (1981) [7] Campbell, C.; Stanley, P.: The Chinese hamster ovary glycosylation mutants LEC11 and LEC12 express two novel GDP-fucose:N-acetylglucosaminide 3a-l-fucosyltransferase enzymes. J. Biol. Chem., 259, 11208-11214 (1984) [8] Foster, C.S.; Gillies, D.R.B.; Glick, M.C.: Purification and characterization of GDP-l-Fuc-N-acetyl-b-d-glucosaminide a1!3 fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme. J. Biol. Chem., 266, 3526-3531 (1991) [9] Sarnesto, A.; Kohlin, T.; Hindsgaul, O.; Vogele, K.; Blaszczyk-Thurin, M.; Thurin, J.: Purification of the b-N-acetylglucosaminide a1,3-fucosyltransferase from human serum. J. Biol. Chem., 267, 2745-2752 (1992) [10] Staudacher, E.; Dalik, T.; Wawra, P.; Altmann, F.; Maerz, L.: Functional purification and characterization of a GDP-fucose:b-N-acetylglucosamine (Fuc to Asn linked GlcNAc) a1,3-fucosyltransferase from mung beans. Glycoconjugate J., 12, 780-786 (1995) [11] Murray, B.W.; Takayama, S.; Schultz, J.; Wong, C.H.: Mechanism and specificity of human a-1,3-fucosyltransferase V. Biochemistry, 35, 11183-11195 (1996)

574

2.4.1.214

Glycoprotein 3-a-L-fucosyltransferase

[12] DeBose-Boyd, R.A.; Nyame, A.K.; Cummings, R.D.: Schistosoma mansoni: characterization of an a1-3 fucosyltransferase in adult parasites. Exp. Parasitol., 82, 1-10 (1996) [13] DeBose-Boyd, R.A.; Nyame, A.K.; Cummings, R.D.: Molecular cloning and characterization of an a1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans. Glycobiology, 8, 905-917 (1998)

575

cis-Zeatin O-b-D-glucosyltransferase

2.4.1.215

1 Nomenclature EC number 2.4.1.215 Systematic name UDP-glucose:cis-zeatin O-b-d-glucosyltransferase Recommended name cis-zeatin O-b-d-glucosyltransferase CAS registry number 123644-76-8

2 Source Organism <1> Zea mays [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + cis-zeatin = UDP + O-b-d-glucosyl-cis-zeatin (The enzyme from maize can use cis-zeatin and UDPglucose as substrates, but not cis-ribosylzeatin, trans-zeatin or trans-ribosylzeatin. Unlike EC 2.4.1.203, transzeatin O-b-d-glucosyltransferase, UDPxylose cannot act as a donor) Reaction type transfer of hexosyl group Substrates and products S UDPglucose + cis-zeatin <1> (<1> recombinant protein [1]) (Reversibility: ? <1> [1]) [1] P UDP + O-b-d-glucosyl-cis-zeatin S Additional information <1> (<1> no substrates are sugar donors transzeatin, dihydrozeatin, respective nucleosides and sugar donors UDP-xylose, UDP-glucuronic acid, UDP-galactose, ADP-glucose, UDP-mannose [1]) [1] P ?

576

2.4.1.215

cis-Zeatin O-b-D-glucosyltransferase

4 Enzyme Structure Molecular weight 51100 <1> (<1> estimated from gene sequence [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue cob <1> (<1> lower level of mRNA [1]) [1] kernel <1> (<1> lower level of mRNA [1]) [1] leaf <1> (<1> very low levels of mRNA [1]) [1] root <1> (<1> highest level of mRNA [1]) [1] Purification <1> (recombinant protein [1]) [1] Cloning <1> (expression in Escherichia coli [1]) [1]

References [1] Martin, R.C.; Mok, M.C.; Habben, J.E.; Mok, D.W.S.: A maize cytokinin gene encoding an O-glucosyltransferase specific to cis-zeatin. Proc. Natl. Acad. Sci. USA, 98, 5922-5926 (2001)

577

Trehalose 6-phosphate phosphorylase

1 Nomenclature EC number 2.4.1.216 Systematic name trehalose 6-phosphate:phosphate b-d-glucosyltransferase Recommended name trehalose 6-phosphate phosphorylase Synonyms TrePP <26> [2] CAS registry number 403512-51-6

2 Source Organism <1> <2> <3> <4> <5> <6> <7> <8> <9> <10> <11> <12> <13> <14> <15> <16> <17> <18> <19> <20> <21> <22>

578

Carnobacterium sp. (strain 496) [1] Carnobacterium divergans [1] Carnobacterium piscola [1] Enterococcus faecalis [1] Enterococcus faecium (high activity [1]) [1] Lactobacillus casei ssp. casei [1] Lactobacillus delbrueckii ssp. delbrueckii [1] Lactobacillus pentosus [1] Lactobacillus plantarum [1] Lactobacillus reuteri [1] Lactococcus lactis [1] Lactococcus lactis ssp. cremoris [1] Lactococcus lactis ssp. lactis [1] Lactococcus plantarum [1] Lactococcus raffinolactis [1] Leuconostoc mesenteroides ssp. dextranicum [1] Leuconostoc pseudomesenteroides [1] Leuconostoc weissella (high activity [1]) [1] Oenococcus oeni [1] Pediococcus acidilactici [1] Pediococcus damnosus [1] Pediococcus pentosaceus [1]

2.4.1.216

2.4.1.216

<23> <24> <25> <26>

Trehalose 6-phosphate phosphorylase

Tetragenococcus halophilus [1] Vagococcus salmonarium [1] Weissella viridescens [1] Lactococcus lactis (CAA77100) [2]

3 Reaction and Specificity Catalyzed reaction trehalose 6-phosphate + phosphate = glucose 6-phosphate + b-d-glucose 1phosphate (The enzyme from Lactococcus lactis is specific for trehalose 6phosphate. Differs from EC 2.4.1.64, a,a-trehalose phosphorylase, in that trehalose in not a substrate) Reaction type glycosyl group transfer phospho group transfer phosphorylation Natural substrates and products S trehalose 6-phosphate + phosphate <1-26> (<26> formation of trehalose 6-phosphate is favoured [2]) (Reversibility: ? <1-25> [1]; r <26> [2]) [1, 2] P glucose 6-phosphate + b-d-glucose 1-phosphate <1-26> [1, 2] Substrates and products S trehalose 6-phosphate + phosphate <1-26> (<26> formation of trehalose 6-phosphate is favoured [2]) (Reversibility: ? <1-25> [1]; r <26> [2]) [1, 2] P glucose 6-phosphate + b-d-glucose 1-phosphate <1-26> [1, 2] Specific activity (U/mg) 0.8 <18> [1] 0.82 <5> [1] 32.4 <26> [2] Km-Value (mM) 0.9 <26> (b-d-glucose 1-phosphate) [2] 4 <26> (b-d-glucose 6-phosphate) [2] 6 <26> (trehalose 6-phosphate) [2] 32 <26> (phosphate) [2] pH-Optimum 6.3 <26> [2] Temperature optimum ( C) 35 <26> [2]

579

Trehalose 6-phosphate phosphorylase

2.4.1.216

4 Enzyme Structure Molecular weight 94000 <26> (<26> gel filtration [2]) [2] Subunits monomer <26> (<26> 1 * 94000, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <1-26> [1, 2] culture condition:trehalose-grown cell <26> [2] Purification <26> (to homogeneity [2]) [2] Cloning <26> (overexpression in Escherichia coli and Lactococcus lactis [2]) [2]

References [1] Andersson U.; Radström, P.: b-Glucose 1-phosphate-interconverting enzymes in maltose- and trehalose-fermenting lactic acid bacteria. Environ. Microbiol., 4, 81-88 (2002) [2] Andersson, U.; Levander, F.; Radström, P.: Trehalose-6-phosphate phosphorylase is part of a novel metabolic pathway for trehalose utilization in Lactococcus lactis. J. Biol. Chem., 276, 42707-42713 (2001)

580

Mannosyl-3-phosphoglycerate synthase

2.4.1.217

1 Nomenclature EC number 2.4.1.217 Systematic name GDP-mannose:3-phosphoglycerate 3-a-d-mannosyltransferase Recommended name mannosyl-3-phosphoglycerate synthase Synonyms MPG synthase MPGS mannosyl-3-P-glycerate synthase synthase, mannosyl-3-phosphoglycerate CAS registry number 393512-63-5

2 Source Organism <1> Rhodothermus marinus [1] <2> Pyrococcus horikoshii [2]

3 Reaction and Specificity Catalyzed reaction GDP-mannose + 3-phospho-d-glycerate = GDP + 2-(a-d-mannosyl)-3-phosphoglycerate Reaction type hexosyl group transfer Natural substrates and products S GDPmannose + 3-phospho-d-glycerate <1, 2> (<1,2>, involved in a pathway for synthesis of mannosylglycerate [1,2]) [1, 2] P GDP + 2-(1-d-mannosyl)-3-phosphoglycerate <1, 2> (<2>, mannosyl-3phosphoglycerate is subsequently dephosphorylated by a specific phosphatase, EC 3.1.3.70, producing mannosylglycerate [2]) [1, 2]

581

Mannosyl-3-phosphoglycerate synthase

2.4.1.217

Substrates and products S GDPmannose + 3-phospho-d-glycerate <1, 2> (<2>, transfer of the mannosyl group with retention of configuration [2]) [1, 2] P GDP + 2-(a-d-mannosyl)-3-phosphoglycerate <1, 2> [1, 2] Inhibitors KCl <2> (<2>, 150 mM, 18% inhibition [2]) [2] NaCl <2> (<2>, 150 mM, 35% inhibition [2]) [2] Metals, ions KCl <1> (<1>, or NaCl required for optimal activity [1]) [1] Mg2+ <2> (<2>, activity in absence is 46% of that in presence of 15 mM Mg2+ [2]) [2] NaCl <1> (<1>, or KCl, required for optimal activity [1]) [1] Km-Value (mM) 0.14 <2> (3-phospho-d-glycerate) [2] 0.17 <2> (GDPmannose) [2] pH-Optimum 6.4-7.4 <2> (<2>, recombinant enzyme [2]) [2] pH-Range 6-8 <2> (<2>, pH 6.0: about 60% of maximal activity, pH 8.0: about 75% of maximal activity [2]) [2] Temperature optimum ( C) 90-100 <2> (<2>, recombinant enzyme [2]) [2] Temperature range ( C) 80-105 <2> (<2>, 80 C: about 60% of maximal activity, 105 C: about 70% of maximal activity [2]) [2]

5 Isolation/Preparation/Mutation/Application Purification <2> [2] Cloning <2> (overexpression in Escherichia coli [2]) [2]

6 Stability Temperature stability 98 <2> (<2>, half-life: 16 min [2]) [2]

582

2.4.1.217

Mannosyl-3-phosphoglycerate synthase

References [1] Martins, L.O.; Empadinhas, N.; Marugg, J.D.; Miguel, C.; Ferreira, C.; Da Costa, M.S.; Santos, H.: Biosynthesis of mannosylglycerate in the thermophilic bacterium Rhodothermus marinus. Biochemical and genetic characterization of a mannosylglycerate synthase. J. Biol. Chem., 274, 35407-35414 (1999) [2] Empadinhas, N.; Marugg, J.D.; Borges, N.; Santos, H.; Da Costa, M.S.: Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii. Biochemical and genetic characterization of key enzymes. J. Biol. Chem., 276, 43580-43588 (2001)

583

Hydroquinone glucosyltransferase

2.4.1.218

1 Nomenclature EC number 2.4.1.218 Systematic name UDP-glucose:hydroquinone-O-b-d-glucosyltransferase Recommended name hydroquinone glucosyltransferase Synonyms arbutin synthase arbutin synthetase glucosyltransferase, uridine diphosphoglucose-hydroquinone hydroquinone O-glucosyltransferase hydroquinone:O-glucosyltransferase uridine diphosphate-glucose:hydroquinone glucosyltransferase CAS registry number 37341-98-3

2 Source Organism <1> Rauvolfia serpentina [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + hydroquinone = UDP + hydroquinone-O-b-d-glucopyranoside Reaction type phospho group transfer Natural substrates and products S Additional information <1> (<1>, because hydroquinone and arbutin seem not to be naturally occuring compounds in Rauvolfia plants or cell culture, the enzyme must have another substrate specificity [1]) [1] P ?

584

2.4.1.218

Hydroquinone glucosyltransferase

Substrates and products S CDPglucose + hydroquinone <1> (<1>, 2% of the activity with UDPglucose [2]) (Reversibility: ? <1> [2]) [2] P CDP + hydroquinone-O-b-d-glucopyranoside <1> [1] S TDPglucose + hydroquinone <1> (<1>, 68.8% of the activity with UDPglucose [2]) (Reversibility: ? <1> [2]) [2] P TDP + hydroquinone-O-b-d-glucopyranoside <1> [1] S UDPglucose + 2-methoxy-hydroquinone <1> (<1>, 16.7% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + 1-O-(4-hydroxy-2-methoxyphenyl)-b-d-glucopyranoside S UDPglucose + 3-hydroxyflavone <1> (<1>, 5.4% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + 3-b-d-glucopyranosyloxy-2-phenylchromen-4-one S UDPglucose + 3-methoxyphenol <1> (<1>, 10.9% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + 1-O-(3-methoxyphenyl)-b-d-glucopyranoside S UDPglucose + 4-benzyloxyphenol <1> (<1>, 1.4% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + (4-benzyloxyphenyl)-b-d-glucopyranoside S UDPglucose + 4-chloro-2-methylphenol <1> (<1>, 19.0% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + 1-O-(4-chloro-2-methylphenyl)-b-d-glucopyranoside S UDPglucose + 4-chlorophenol <1> (<1>, 7.5% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + (4-chlorophenyl)-b-d-glucopyranoside S UDPglucose + 4-hydroxyacetophenone <1> (<1>,1.8% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + (4-acetophenyl)-b-d-glucopyranoside S UDPglucose + 4-hydroxybenzaldehyde <1> (<1>, 8.6% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + 4-b-d-glucopyranosyloxy-benzaldehyde S UDPglucose + 4-hydroxythiophenol <1> (<1>, 1.2% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + (4-thiophenyl)-b-d-glucopyranoside S UDPglucose + 4-methylphenol <1> (<1>, 2.8% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + (4-methylphenyl)-b-d-glucopyranoside S UDPglucose + 4-nitrophenol <1> (<1>, 6.2% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + (4-nitrophenyl)-b-d-glucopyranoside S UDPglucose + 7-hydroxyflavone <1> (<1>, 3.0% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + 7-b-d-glucopyranosyloxy-2-phenylchromen-4-one S UDPglucose + apigenin <1> (<1>, 5.8% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + ?

585

Hydroquinone glucosyltransferase

2.4.1.218

S UDPglucose + biphenol-2-ol <1> (<1>, 1.1% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + ? S UDPglucose + carvacrol <1> (<1>, 1.0% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + (2-methyl-5-isopropylphenyl)-b-d-glucopyranoside S UDPglucose + coniferyl alcohol <1> (<1>, 1.2% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + ? S UDPglucose + eugenol <1> (<1>, 6.2% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + (4-allyl-2-methoxyphenyl)-b-d-glucopyranoside S UDPglucose + hydroquinone <1> (Reversibility: ? <1> [1, 2, 3]) [1, 2, 3] P UDP + hydroquinone-O-b-d-glucopyranoside <1> [1] S UDPglucose + kaempferol <1> (<1>, 1.0% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + ? S UDPglucose + methylvanillate <1> (<1>, 2.2% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + ? S UDPglucose + phenol <1> (<1>, 5.1% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + phenyl-b-d-glucopyranoside S UDPglucose + quercetin <1> (<1>, 2.6% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + ? S UDPglucose + resorcinol <1> (<1>, 8.7% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + (3-hydroxyphenyl)-b-d-glucopyranoside S UDPglucose + saligenin <1> (<1>, 9.4% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + saligenin-b-d-glucopyranoside S UDPglucose + scopoletin <1> (<1>, 3.0% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + 7-b-d-glucopyranosyloxy-6-methoxychromen-2-one S UDPglucose + thymol <1> (<1>, 7.6% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + (2-isopropyl-5-methyl-phenyl)-b-d-glucopyranoside S UDPglucose + umbelliferone <1> (<1>, 3.6% of the activity with hydroquinone [2]) (Reversibility: ? <1> [2]) [2] P UDP + 7-b-d-glucopyranosyloxy-chromen-2-one S UDPglucose + vanillic acid <1> (<1>, 1.0% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2, 3]) [2, 3] P UDP + vanillic acid b-d-glucoside S UDPglucose + vanillin <1> (<1>, 9.4% of the activity with hydroquinone [2,3]) (Reversibility: ? <1> [2]) [2, 3] P UDP + 4-b-d-glucopyranosyloxy-3-methoxy-benzaldehyde 586

2.4.1.218

Hydroquinone glucosyltransferase

S Additional information <1> (<1>, no activity with UDPmannose, UDPgalactose or UDPglucuronic acid [2]) [2] P ? Inhibitors Ca2+ <1> (<1>, 10 mM, 24.4% inhibition [2]) [2] Co2+ <1> (<1>, 10 mM, 54.2% inhibition [2]) [2] Hg2+ <1> (<1>, 10 mM, complete inhibition, reversed by addition of 20 mM 2-mercaptoethanol [2]) [2] Mn2+ <1> (<1>, 10 mM, 24.7% inhibition [2]) [2] Zn2+ <1> (<1>, 10 mM, 87.4% inhibition [2]) [2] dichlorophen <1> [2] Metals, ions Mg2+ <1> (<1>, 10 mM, increases the conversion rate to 168.3% [2]) [2] Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 0.077 <1> (UDPglucose) [2] 0.44 <1> (vanillin) [2] 0.55 <1> (umbelliferone) [2] 1.1 <1> (TDPglucose) [2] Additional information <1> (<1>, Km value for hydroquinone is below 0.001 mM [2,3]) [2, 3] Ki-Value (mM) 0.00009 <1> (dichlorophen) [1] pH-Optimum 4.5 <1> (<1>, and a second optimum at pH 6.8 in phosphate buffer, and a second optimum at pH 7.5 in citric acid buffer [2]) [2] 6.8 <1> (<1>, and a second optimum at pH 4.5, phosphate buffer [2]) [2] 7.5 <1> (<1>, and a second optimum at pH 4.5, citric acid buffer [2]) [2] Temperature optimum ( C) 50 <1> (<1>, recombinant enzyme [2]) [2, 3]

4 Enzyme Structure Subunits ? <1> (<1>, x * 51800, calculation from nucleotide sequence [3]; <1>, x * 52000, SDS-PAGE [1]) [1, 3]

587

Hydroquinone glucosyltransferase

2.4.1.218

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture <1> [1, 2] Purification <1> [1, 2, 3] Cloning <1> (expression in Escherichia coli [2,3]; cDNA with 6xHis tag [2]) [2, 3] Application synthesis <1> (<1>, formation of arbutin, which is a potent inhibitor of human melanin biosynthesis with commercial value [3]) [3]

6 Stability Temperature stability 50 <1> (<1>, 1 h, stable [1]) [1]

References [1] Arend, J.; Warzecha, H.; Stöckigt, J.: Hydroquinone:O-glucosyltransferase from cultivated Rauvolfia cells: enrichment and partial amino acid sequences. Phytochemistry, 53, 187-193 (2000) [2] Hefner, T.; Arend, J.; Warzecha, H.; Siems, K.; Stöckigt, J.: Arbutin synthase, a novel member of the NRD1b glycosyltransferase family, is a unique multifunctional enzyme converting various natural products and xenobiotics. Bioorg. Med. Chem., 10, 1731-1741 (2002) [3] Arend, J.; Warzecha, H.; Hefner, T.; Stöckigt, J.: Utilizing genetically engineered bacteria to produce plant-specific glucosides. Biotechnol. Bioeng., 76, 126-131 (2001)

588

Vomilenine glucosyltransferase

2.4.1.219

1 Nomenclature EC number 2.4.1.219 Systematic name UDP-glucose:vomilenine 21-O-b-d-glucosyltransferase Recommended name vomilenine glucosyltransferase Synonyms d-glycosyltransferase UDPG:vomilenine 21-b-d-glycosyltransferase b-glycosyltransferase exo-b-glycosyltransferase glycose transferase raucaffricine O-b-d-glucosidase raucaffricine b-d-glucosidase raucaffricine b-glucosidase transglucosidase transglucosylase vomilenine glycosyltransferase CAS registry number 102925-37-1 9031-48-5

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

Rauwolfia serpentina [1-5] Rauwolfia caffra [1] Rauwolfia mannii [1] Rauwolfia verticillata [1]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + vomilenine = UDP + raucaffricine

589

Vomilenine glucosyltransferase

2.4.1.219

Reaction type glycosyl group transfer Natural substrates and products S UDP-glucose + vomilenine <1> (Reversibility: r <1> [1, 4, 5]) [1-5] P UDP + raucaffricine Substrates and products S UDP-glucose + vomilenine <1> (Reversibility: r <1> [1, 4, 5]) [1-5] P UDP-glucose + raucaffricine S strictosidine + UDP <1> (<1> recombinant enzyme [5]) (Reversibility: ? <1> [5]) [5] P ? Inhibitors (phenylmethyl)sulfonyl fluoride <1> (<1> strong inhibition at 10 mM [2]) [2] 1,10-phenanthroline <1> (<1> strong inhibition at 5 mM [2]) [2] 5,5'-dithiobis(2-nitrobenzoic acid) <1> (<1> strong inhibition at 1.25 mM [2]) [2] Cu2+ <1> (<1> complete inhibition at 10 mM [2]) [2] d-fructose <1> [1] d-glucose <1> [1] EDTA <1> (<1> strong inhibition at 5 mM [2]) [2] Fe3+ <1> (<1> complete inhibition at 10 mM [2]) [2] Hg2+ <1> (<1> complete inhibition at 10 mM [2]) [2] Zn2+ <1> (<1> complete inhibition at 10 mM [2]) [2] p-hydroxybenzaldehyde <1> (<1> strong inhibition at 5 mM [2]) [2] Specific activity (U/mg) Additional information <1-4> (<1> 538 pkat/mg [1]; <1> 469 nkat/mg [4]; <1> 2.6 nkat/mg [5]; <1> 0.5 *kat/mg [5]; <2> 253 pkat/mg [1]; <3> 52 pkat/ mg [1]; <4> 136 pkat/mg [1]) [1, 4, 5] Km-Value (mM) 0.04 <1> (vomilenine) [2] 0.8 <1> (UDP-glucose) [2] 1.3 <1> (raucaffricine) [5] 1.8 <1> (strictosidine) [5] pH-Optimum 5.1 <1> [1] 6.3 <1> [1] pH-Range 4-7 <1> [1] Temperature optimum ( C) 38 <1> [1] 50 <1> [2]

590

2.4.1.219

Vomilenine glucosyltransferase

4 Enzyme Structure Molecular weight 61000 <1> (<1> gel filtration [4]) [4] 66600 <1> (<1> gel filtration [1]) [1] Posttranslational modification Additional information <1> (<1> no glycosylation [4]) [4]

5 Isolation/Preparation/Mutation/Application Source/tissue root <1> [1] Localization membrane <1> [3] microsome <1> [1] Purification <1> [2, 4] Cloning <1> (expression in Escherichia coli [5]) [5]

6 Stability Temperature stability 55 <1> (<1> 30 min, 50% of activity left [1]) [1] General stability information <1>, 2-mercaptoethanol stabilizes transferase activity [2] <1>, ascorbic acid stabilizes transferase activity [2] <1>, dithioerythritol stabilizes transferase activity [2] Storage stability <1>, -20 C, 3 weeks, no loss in activity [2] <1>, 4 C, 6 month, 30% loss in activity [1]

References [1] Schuebel, H.; Stoeckigt, J.; Feicht, R.; Simon, H.: Partial purification and characterization of raucaffricine b-d-glucosidase from plant cell-suspension cultures of Rauwolfia serpentina Benth. Helv. Chim. Acta, 69, 538-547 (1986) [2] Ruyter, C.M.; Stoeckigt, J.H.H.: Enzymatic formation of Raucaffricine, the major indole alkaloid of Rauwolfia serpentina cell-suspension cultures. Helv. Chim. Acta, 74, 1707-1712 (1991)

591

Vomilenine glucosyltransferase

2.4.1.219

[3] Lutterbach, R.; Ruyter, C.M.; Stoeckigt, J.: Isolation and characterization of an UDPG-dependent glucosyltransferase activity from Rauwolfia serpentina Benth. cell suspension cultures. Can. J. Chem., 72, 51-55 (1994) [4] Warzecha, H.; Obitz, P.; Stockigt, J.: Purification, partial amino acid sequence and structure of the product of raucaffricine-O-b-d-glucosidase from plant cell cultures of Rauwolfia serpentina. Phytochemistry, 50, 1099-1109 (1999) [5] Warzecha, H.; Gerasimenko, I.; Kutchan, T.M.; Stockigt, J.: Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis. Phytochemistry, 54, 657-666 (2000)

592

Indoxyl-UDPG glucosyltransferase

2.4.1.220

1 Nomenclature EC number 2.4.1.220 Systematic name UDP-glucose:indoxyl 3-O-b-d-glucosyltransferase Recommended name indoxyl-UDPG glucosyltransferase Synonyms UDP-glucose:indoxyl glucosyltransferase hydroxyindole glucosyltransferase indican synthase indoxyl glucosyltransferase indoxyl-UDPG-glucolsyltransferase CAS registry number 258339-72-9

2 Source Organism <1> Baphicacanthus cusia [1] <2> Polygonum tinctorium (indigo plant [2]) [2]

3 Reaction and Specificity Catalyzed reaction UDP-glucose + indoxyl = UDP + indican Reaction type glycosyl group transfer Natural substrates and products S UDP-glucose + indoxyl <1, 1> (<1> indole is the biosynthetic precursor to the indoxyl derivates indican isatan B [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1H-indole-3-yl-b-d-glucopyranoside <1> (<1> i.e. indican [1]) [1]

593

Indoxyl-UDPG glucosyltransferase

2.4.1.220

Substrates and products S ADP-glucose + indoxyl <2> (Reversibility: ? <2> [2]) [2] P ADP + indican <2> [2] S GDP-glucose + indoxyl <2> (Reversibility: ? <2> [2]) [2] P GDP + indican <2> [2] S UDP-glucose + 4-hydroxyindole <1> (<1> minimal activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1H-indol-4-yl-b-d-glucopyranoside S UDP-glucose + 5-hydroxyindole <1> (<1> activity as 3-OH-indole [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1H-indol-5-yl-b-d-glucopyranoside S UDP-glucose + 6-hydroxyindole <1> (<1> minimal activity [1]) (Reversibility: ? <1> [1]) [1] P UDP + 1H-indol-6-yl-b-d-glucopyranoside S UDP-glucose + 7-hydroxyindole <1> (Reversibility: ? <1> [1]) [1] P UDP + 1H-indol-7-yl-b-d-glucopyranoside S UDP-glucose + indoxyl <1, 2> (<1> i.e. indole-3-ol or 3-OH-indole [1]; <1,2> best substrate [1,2]) (Reversibility: ? <1, 2> [1, 2]) [1, 2] P UDP + 1H-indole-3-yl-b-d-glucopyranoside <1, 2> (<1,2> i.e. indican [1,2]) [1, 2] S Additional information <1> (<1> not: 2-OH-indole [1]) [1] P ? Turnover number (min±1) Additional information <1> [1] Specific activity (U/mg) Additional information <1> [1] Km-Value (mM) 0.13 <2> (UDP-glucose) [2] 0.5 <1> (5-hydroxyindole) [1] 1.2 <1> (indoxyl) [1] 1.4 <1> (7-hydroxyindole) [1] 1.7 <1> (UDP-glucose) [1] 1.9 <1> (4-hydroxyindole) [1] 3.3 <1> (6-hydroxyindole) [1] pH-Optimum 8.5 <1> (<1> pI: 6.5 [1]) [1] 10 <2> [2] pH-Range 6.8-10.2 <1> (<1> 6.8 and 10.2 half-maximal-activity [1]) [1] Temperature optimum ( C) 30 <1> [1] 45-48 <1> [1]

594

2.4.1.220

Indoxyl-UDPG glucosyltransferase

Temperature range ( C) 30-50 <2> (<2> 30 and 50 half-maximal-activity [2]) [2]

4 Enzyme Structure Molecular weight 48000 <2> (<2> gel-filtration [2]) [2] 53000 <2> (<2> SDS-PAGE [2]) [2] 60000 <1> (<1> SDS-PAGE [1]) [1]

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

6 Stability Oxidation stability <1>, anaerobic conditions, since under aerobic conditions indoxyl dimerizes spontaneously to indigo [1]

References [1] Marcinek, H.; Weyler, W.; Deus-Neumann, B.; Zenk, M.H.: Indoxyl-UDPGglucosyltransferase from Baphicacanthus cusia. Phytochemistry, 53, 201-207 (2000) [2] Minami, Y.; Nishimura, O.; Hara-Nishimura, I.; Nishimura, M.; Matsubara, H.: Tissue and intracellular localization of indican and the purification and characterization of indican synthase from indigo plants. Plant Cell Physiol., 41, 218-225 (2000)

595

Peptide-O-fucosyltransferase

1 Nomenclature EC number 2.4.1.221 Systematic name GDP-b-l-fucose:polypeptide O-a-l-fucosyltransferase Recommended name peptide-O-fucosyltransferase Synonyms EC 2.4.1.68 (formerly) GDP-l-fucose-glycoprotein fucosyltransferase GDP-l-fucose:polypeptide fucosyltransferase GDP-fucose glycoprotein fucosyltransferase GDP-fucose protein O-fucosyltransferase GDP-fucose:polypeptide fucosyltransferase N-acetyl-b-d-glucosaminide a1 !6-fucosyltransferase N-glycan a-6-fucosyltransferase O-fucT-1 a-6-fucosyltransferase core a6FucT fucosyltransferase, guanosine diphosphofucose-glycoprotein glycoprotein fucosyltransferase guanosine diphosphofucose-glycoprotein fucosyltransferase CAS registry number 9033-08-3

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

596

Homo sapiens [1, 3] Rattus norvegicus (rat [2]) [2] Cricetulus griseus (Chinese hamster, CHO cells [2,4]) [2, 4] Candida elegans [3] Mus musculus [3] Drosophila melanogaster [3]

2.4.1.221

2.4.1.221

Peptide-O-fucosyltransferase

3 Reaction and Specificity Catalyzed reaction transfers an a-l-fucosyl residue from GDP-b-l-fucose to the serine hydroxy group of a protein acceptor Reaction type glycosyl group transfer transglycosylation Substrates and products S Additional information <1, 2, 3> (<1> o-fucosylation of thrombospondin-1 at Ser 377, Thr 432 and Thr 489 [1]; <2,3> links fucose through an O-glycosidic linkage to a conserved serine or threonine residue in of the EGF-1 domain of human factor VII, various synthetic peptides serve as substrates [2]; <1> adds o-fucose to epidermal growth factor-like repeats [3]; <3> fucosylates various synthetic peptides of EGF-1 domain of human factor VII, GDP-mannose, UDP-glucose, UDP-N-acetylglucosamine, UDP-galactose, UDP-H-acetylgalactosamine and UDP-xylose can replace GDP-fucose [4]) [1, 2, 3, 4] P ? Inhibitors factor VII EGF-1 <3> (<3> inhibition above 0.015 mM [4]) [4] Metals, ions Ca2+ <2, 3> (<2,3> 2fold increase at 10 mM [2]) [2] Mn2+ <2, 3> (<2,3> 10fold increase at 50 mM, 4fold increase at 10 mM [2]; <3> 17fold increase at 50 mM [4]) [2, 4] Specific activity (U/mg) 0.784 <3> [4] Km-Value (mM) 0.004 <1> (GDP-fucose) [3] 0.006 <3> (GDP-fucose) [4] 0.006 <1> (factor VII EGF) [3] 0.015 <3> (factor VII EGF) [4] pH-Optimum 7 <3> (<3> assay at [4]) [4] pH-Range 5.5-8 <3> [4] Temperature optimum ( C) 37 <3> (<3> assay at [4]) [4]

597

Peptide-O-fucosyltransferase

2.4.1.221

4 Enzyme Structure Molecular weight 44000 <3> (<3> SDS-PAGE [4]) [4] Posttranslational modification glycoprotein <3> (<3> glycoprotein with more than one high mannose type oligosaccharide chain [4]) [4]

5 Isolation/Preparation/Mutation/Application Source/tissue CHO cell <3> [2, 4] heart <1> [3] liver <2, 3> [2] Additional information <1> (<1> gene is widely expressed in human tissues [3]) [3] Localization membrane <2, 3> [2] Purification <1> [3] <3> (partial [2]) [2, 4] Cloning <1, 4, 5, 6> [3]

References [1] Hofsteenge, J.; Huwiler, K.G.; Bacik B.; Hess, D.; Lawler, J.; Mosher, D.F.; Peter-Katalinic, J.: C-mannosylation and o-fuxosylation of the thrombospondin type 1 module. J. Biol. Chem., 276, 6485-6498 (2001) [2] Wang, Y.; Lee, G.F.; Kelley, R.F.; Spellman, M.W.: Identification of a GDP-lfucose:polypeptide fucosyltransferase and enzymic addition of O-linked fucose to EGF domains. Glycobiology, 6, 837-842 (1996) [3] Wang, Y.; Shao, L.; Shi, S.; Harris, R.J.; Spellman, M.W.; Stanley, P.; Haltiwanger, R.S.: Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase. J. Biol. Chem., 276, 40338-40345 (2001) [4] Wang, Y.; Spellman, M.W.: Purification and characterization of a GDP-fucose:polypeptide fucosyltransferase from Chinese hamster ovary cells. J. Biol. Chem., 273, 8112-8118 (1998)

598

O-Fucosylpeptide 3-b-Nacetylglucosaminyltransferase

2.4.1.222

1 Nomenclature EC number 2.4.1.222 Systematic name UDP-d-GlcNAc:O-l-fucosylpeptide 3-b-N-acetyl-d-glucosaminyltransferase Recommended name O-fucosylpeptide 3-b-N-acetylglucosaminyltransferase Synonyms Fringe O-fucose-b1,3-N-acetylglucosaminyltransferase O-fucosylpeptide b-1,3-N-acetylglucosaminyltransferase UDP-GlcNAc:fucose b1,3 N-acetylglucosaminyltransferase UDP-acetylglucosamine:fucosylglycoprotein UDP-glucose:O-linked fucose b1,3-glucosyltransferase acetylglucosaminylferase, uridine diphosphoacetylglucosamine:fucosyglycoprotein b1-3b1,3-acetylglucosaminyltransferase fringe b1,3 N-acetylglucosaminyltransferase fringe glycosyltransferase lunatic fringe glycosyltransferase manic fringe glycosyltransferase radical fringe glycosyltransferase CAS registry number 299203-70-6

2 Source Organism <1> Drosophila melanogaster [1, 2, 3] <2> mammalia [1, 3]

3 Reaction and Specificity Catalyzed reaction transfers a b-d-GlcNAc residue from UDP-d-GlcNAc to the fucose residue of a fucosylated protein acceptor (<1> GlcNAc-transfer activity of Fringe is es-

599

O-Fucosylpeptide 3-b-N-acetylglucosaminyltransferase

2.4.1.222

sential for it to modulate Notch signalling [1]; <1> co-expressing of Notch and Fringe in Drosophila SL2 cells results in an increase of binding of ligand delta to Notch [2]) Reaction type hexosyl group transfer Substrates and products S l-fucose + GlcNAc <1> (Reversibility: ? <1> [2]) [2] P GlcNAc-b-1,3-fucitol S O-fucose residues on d Notch ligand + UDP-GlcNAc <1, 2> (<2> rat delta1 ligand, exogenous expressed in Chinese hamster ovary Lec1 cells, experiment in vivo [3]; <1> in vitro [3]) (Reversibility: ? <1, 2> [3]) [3] P GlcNAc-b-1,3-fucitol-d Notch <2> [3] S O-fucose residues on Jagged1 Notch ligand + UDP-GlcNAc <2> (<2> human Jagged1 ligand, exogenous expressed in Chinese hamster ovary Lec1 cells, experiment in vivo [3]) (Reversibility: ? <2> [3]) [3] P GlcNAc-b1,3-fucitol-Jagged1 Notch <2> [3] S O-fucose residues on Serrate Notch ligand + UDP-GlcNAc <1> (<1> in vitro [3]) (Reversibility: ? <1> [3]) [3] P ? S O-linked fucose on the epidermal growth factor-like sequence repeats of Notch + GlcNAc <1, 2> (<2> endogenous Notch1, Manic Fringe [1]) (Reversibility: ? <1, 2> [1]) [1] P GlcNAc-b-1,3-fucitol-Notch <1, 2> [1] S UDP-b-d-GlcNAc + fucosyl-protein <1, 2> (Reversibility: ? <1, 2> [1-3]) [1-3] P UDP + O-b-d-GlcNAc-fucosyl-protein S epidermal growth factor-like sequence repeats-O-fucose + UDP-GlcNAc <1, 2> (<1,2> Drosophila Fringe, Lunic Fringe, Manic Fringe [1]) (Reversibility: ? <1, 2> [1]) [1] P ? S p-nitrophenyl-a-l-fucose + UDP-GlcNAc <1, 2> (<1,2> p-nitrophenyl-al-fucose structurally mimics O-linked fucose, Lunic Fringe, Manic Fringe, Drosophila Fringe [1]) (Reversibility: ? <1, 2> [1]) [1] P GlcNAc-b-1,3-fucitol + p-nitrophenol + UDP <1, 2> [1] S Additional information <1, 2> (<1,2> no substrates: p-nitrophenyl-galactose, p-nitrophenyl-glucose [1]; <1,2> further substrate: recombinant epidermal growth factor repeat from the serum protein factor VII modified with O-linked fucose [1]) [1] P ? Metals, ions manganese <1, 2> [1] Specific activity (U/mg) Additional information <1, 2> (<1,2> Lunic Fringe has a higher specific activity than either Drosophila Fringe and Manic Fringe [1]) [1]

600

2.4.1.222

O-Fucosylpeptide 3-b-N-acetylglucosaminyltransferase

Temperature optimum ( C) 29 <1> (<1> enzyme assay [3]) [3] 37 <1, 2> (<1,2> enzyme assay [1,2]) [1, 2]

5 Isolation/Preparation/Mutation/Application Localization microsome <1> (<1> microsomal fraction enriched for Golgi membrane [2]) [2] Cloning <1> (expressed in Drosophila S2 cells [1,3]; Notch alone or Fringe alone expressed in Drosophila SL2 cells or Notch and Fringe co-expressed in Drosophila SL2 cells [2]) [1, 2, 3] <2> (Lunatic Fringe and Manic Fringe expressed in Chinese hamster ovary Lec1 cells [1]; Manic Fringe stable expressed in Chinese hamster ovary Lec1 cells [3]) [1, 3]

References [1] Moloney, D.J.; Panin, V.M.; Johnston, S.H.; Chen, J.; Shao, U.; Wilson, R.; Wang, Y.; Stanley, P.; Irvine, K.D.; Haltwanger, R.S.; Vogt, T.F.: Fringe is a glycosyltransferase that modifies Notch. Nature, 406, 369-375 (2000) [2] Bruckner, K.; Perez, L.; Clausen, H.; Cohen, S.: Glycosyltransferase activity of Fringe modulates Notch-delta interactions. Nature, 406, 411-415 (2000) [3] Panin, V.M.; Shao, L.; Lei, L.; Moloney, D.J.; Irvine, K.D.; Haltiwanger, R.S.: Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe. J. Biol. Chem., 277, 29945-29952 (2002)

601

Glucuronyl-galactosyl-proteoglycan 4-a-N-acetylglucosaminyltransferase

2.4.1.223

1 Nomenclature EC number 2.4.1.223 Systematic name UDP-N-acetyl-d-glucosamine:b-d-glucuronosyl-(1!3)-b-d-galactosyl4IV-a-N-acetyl-d(1!3)-b-d-galactosyl-(1!4)-b-d-xylosyl-proteoglycan glucosaminyltransferase Recommended name glucuronyl-galactosyl-proteoglycan 4-a-N-acetylglucosaminyltransferase Synonyms a-N-acetylglucosaminyltransferase I a1,4-N-acetylglucosaminyltransferase glucuronosylgalactosylproteoglycan 4-a-N-acetylglucosaminyltransferase CAS registry number 145539-84-0 336193-98-7

2 Source Organism <1> Drosophila melanogaster (gene DEXT3 [1]) [1] <2> Homo sapiens (genes EXTL1 and EXTL3 [2]) [2] <3> Caenorhabditis elegans (gene rib-2 [3]) [3]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + b-d-glucuronosyl-(1!3)-b-d-galactosyl(1!3)-b-d-galactosyl-(1!4)-b-d-xylosyl-proteoglycan = UDP + a-N-acetyld-glucosaminyl-(1!4)-b-d-glucuronosyl-(1!3)-b-d-galactosyl-(1!3)-b-dgalactosyl-(1!4)-b-d-xylosyl-proteoglycan Reaction type hexosyl group transferase

602

2.4.1.223

Glucuronyl-galactosyl-proteoglycan 4-a-N-acetylglucosaminyltransferase

Natural substrates and products S UDP-N-acetyl-d-glucosamine + b-d-glucuronosyl-(1-3)-b-d-galactosyl(1-3)-b-d-galactosyl-(1-4)-b-d-xylosyl-proteoglycan <1, 2, 3> (<1, 2> essential in the heparan sulfate biosynthesis [1, 2]; <3> initiation and elongation of heparan sulfate [3]) [1, 2, 3] P UDP + a-N-acetyl-d-glucosaminyl-(1-4)-b-d-glucuronosyl-(1-3)-b-d-galactosyl-(1-3)-b-d-galactosyl-(1-4)-b-d-xylosyl-proteoglycan Substrates and products S UDP-N-acetyl-d-glucosamine + b-d-glucuronosyl-(1-3)-b-d-galactosyl(1-3)-b-d-galactosyl-(1-4)-b-d-xylosyl-proteoglycan <1, 2> (Reversibility: ? <1,2> [1,2]) [1, 2] P UDP + a-N-acetyl-d-glucosaminyl-(1-4)-b-d-glucuronosyl-(1-3)-b-d-galactosyl-(1-3)-b-d-galactosyl-(1-4)-b-d-xylosyl-proteoglycan S UDP-N-acetyl-d-glucosamine + b-d-glucuronosyl-(1-3)-b-d-galactosyl-1O-benzyloxycarbonyl <1, 2, 3> (Reversibility: ? <1,2,3> [1,2,3]) [1, 2, 3] P UDP + a-N-acetyl-d-glucosaminyl-(1-4)-b-d-glucuronosyl-(1-3)-b-d-galactosyl-1-O-benzyloxycarbonyl

5 Isolation/Preparation/Mutation/Application Cloning <1> (expression in COS-1 cells [1]) [1] <2> (expression in COS-1 cells, lacking the putative NH2 -terminal transmembrane and cytoplasmic domains [2]) [2] <3> (expression in COS-1 cells, truncated form , lacking the first 58 N-terminal amino acids [3]) [3]

References [1] Kim, B.-T.; Kitagawa, H.; Tamura, J.-I.; Kusche-Gullberg, M.; Lindahl, U.; Sugahara, K.: Demonstration of a novel gene DEXT3 of Drosophila melanogaster as the essential N-acetylglucosamine transferase in the heparan sulfate biosynthesis: Chain initiation and elongation. J. Biol. Chem., 277, 1365913665 (2002) [2] Kim, B.-T.; Kitagawa, H.; Tamura, J.-I.; Saito, T.; Kusche-Gullberg, M.; Lindahl, U.; Sugahara, K.: Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode a1,4-N-acetylglucosaminyltransferases that likely are involved in heparan sulfate/heparin biosynthesis. Proc. Natl. Acad. Sci. USA, 98, 7176-7181 (2001) [3] Kitagawa, H.; Egusa, N.; Tamura, J.-I.; Kusche-Gullberg, M.; Lindahl, U.; Sugahara, K.: rib-2, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes encodes a novel a1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. J. Biol. Chem., 276, 4834-4838 (2001)

603

Glucuronosyl-N-acetylglucosaminylproteoglycan 4-a-N-acetylglucosaminyltransferase

2.4.1.224

1 Nomenclature EC number 2.4.1.224 Systematic name UDP-N-acetyl-d-glucosamine:b-d-glucuronosyl-(1!4)-N-acetyl-a-d-glucosaminyl-proteoglycan 4-a-N-acetylglucosaminyltransferase Recommended name glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-a-N-acetylglucosaminyltransferase Synonyms N-acetyl-d-glucosaminyl-(N-acetyl-d-glucosamine) transferase UDP-GlcNAc:oligosaccharide b-N-acetylglucosaminyltransferase a-N-acetylglucosaminyltransferase II glucuronyl-N-acetylglucosaminoproteoglycan 4-a-N-acetylglucosaminyltransferase heparan sulfate polymerase Additional information (<1> rib-2 protein, no b1,4-glucuronyltransferase activity (EC 2.4.1.225), second activity is a-N-acetylglucosaminyltransferase I activity that is required for initiation of heparan sulfate precursor [1]; <2-4, 8> EXT1 and EXT2 proteins, second activity is b1,4-glucuronyltransferase activity (EC 2.4.1.225) [1, 2, 3, 8]; <2> , EXTL1 shows transferase II activity, EXTL3 shows transferase I and II activities [10]; <6> DEXT3 is the ortholog of human EXTL3, shows transferase I and II activities [11]) [1, 2, 3, 8, 10, 11] CAS registry number 145539-84-0

2 Source Organism <-1> no activity in yeast [2] <1> Caenorhabditis elegans [1] <2> Homo sapiens [1, 9, 10] <3> Bos taurus (EXT2 protein [2]) [2, 3, 5, 6, 8] <4> Mus musculus (EXT1 protein [2]; wild type mouse fibroblasts and deficiency mutant cell line gro2C [3]; wild type mouse fibroblasts and deficiency mutant cell line Sog9, EXT1 protein [12]) [2, 3, 5, 7, 9, 12] <5> Dictyostelium discoideum [4]

604

2.4.1.224

Glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-a-N-acetylglucosaminyltransferase

<6> Drosophila melanogaster [11] <7> Escherichia coli (K 5 [7]) [7] <8> hamster (deficiency mutant CHO-K1 used for experiments [8,9]; mutation in pgsD gene locus [9]) [8, 9, 12]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetyl-d-glucosamine + b-d-glucuronosyl-(1!4)-N-acetyl-a-d-glucoseaminyl-proteoglycan = UDP + N-acetyl-a-d-glucosaminyl-(1!4)-b-dglucuronosyl-(1!4)-N-acetyl-a-d-glucosaminyl-proteoglycan Reaction type transfer of a-N-acetylglucosamine Natural substrates and products S UDP-N-acetyl-d-glucosamine + b-d-glucuronosyl-(1!4)-N-acetyl-a-dglucosaminyl-proteoglycan <1-4, 6-8> (<1-4,6-8> elongation of growing chains of heparin and heparan sulfate, tumor suppressor [1-3,5-12]) (Reversibility: ? <1-4, 6-8> [1-3, 5-12]) [1-3, 5-12] P UDP + N-acetyl-a-d-glucosaminyl-(1!4)-b-d-glucuronosyl-(1!4)-Nacetyl-a-d-glucosaminyl-proteoglycan Substrates and products S UDP-N-acetyl-d-glucosamine + N-acetylheparosan oligosaccharide <6> (<6> with terminal nonreducing glucuronic acid [11]) (Reversibility: <6> ? [11]) [11] P ? S UDP-N-acetyl-d-glucosamine + [d-glucuronic acid-N-acetyl-d-glucosamin]n -d-glucuronic acid-2,5-anhydro-d-mannose <3, 4, 7> (<3,4> substrate is produced by E. coli K5, acceptor ability increases with increasing chain length [5]; <3> octasaccharide but not nonasaccharide serves as acceptor [6,7]; <4,7> different activities observed for acetylated and sulfated acceptors [7]; <7> heptasaccharide and nonasaccharide serve as acceptors [7]) (Reversibility: ? <3, 4, 7> [5-7]) [5-7] P UDP + N-acetyl-a-d-glucosaminyl-(1!4)-N-acetyl-d-glucoseamine-[dglucuronic acid-N-acetyl-d-glucosamine]n -d-glucuronic acid-2,5-anhydro-d-mannose <3> [6] S UDP-N-acetyl-d-glucosamine + [mannose]5 -N-acetyl-d-glucosamine <5> (Reversibility: ? <5> [4]) [4] P ? (<5> N-acetyl-d-glucosamine is transfered to an intersecting position [4]) [4] S UDP-N-acetyl-d-glucosamine + [mannose]9 -N-acetyl-d-glucosamine <5> (Reversibility: ? <5> [4]) [4] P ? (<5> N-acetyl-d-glucosamine is transfered to an intersecting position [4]) [4]

605

Glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-a-N-acetylglucosaminyltransferase

2.4.1.224

S UDP-N-acetyl-d-glucosamine + b-d-glucuronosyl-(1!4)-N-acetyl-a-dglucosaminyl-proteoglycan <1-4, 6-8> (Reversibility: ? >1-4, 6-8 [1-3, 512]) [1-3, 5-12] P UDP + N-acetyl-a-d-glucosaminyl-(1!4)-b-d-glucuronosyl-(1!4)-Nacetyl-a-d-glucosaminyl-proteoglycan S Additional information <1> (<1,2> different acceptors were tested for activity with UDP-N-acetyl-d-glucosamine [1]; <2-4> EXT1 and EXT2 proteins also acts as b(1,4)-glucuronyltransferase (EC 2.4.1.225) [1-3,5,6]) [13, 5, 6] P ? Activating compounds detergent <4> (<4> required, range from 0.03-1% [9]) [9] Metals, ions Mn2+ <4> (<4> required, can not be substituted by other divalent cations [5,9]) [5, 9] Specific activity (U/mg) Additional information <4> (<4> specific activities of different pgsD mutants tested [9]) [9] Km-Value (mM) 0.006 <4> ([d-glucuronic acid-N-acetyl-glucosamine]14 -d-glucuronic acid2,5-anhydro-d-mannose) [5] 0.06 <4> ([d-glucuronic acid-N-acetyl-glucosamine]4 -d-glucuronic acid-2,5anhydro-d-mannose) [5] 0.3 <4> (UDP-N-acetyl-d-glucosamine) [9] 0.4 <4> (UDP-N-acetyl-d-glucosamine) [5] 0.65 <5> ((mannose)9 -N-acetyl-d-glucosamine) [4] 1.2 <5> (UDP-N-acetyl-d-glucosamine) [4] pH-Optimum 6-7.2 <4> (<4> broad optimum [5]) [5] pH-Range 5-8 <4> [9] 5.2-8.4 <4> [5]

4 Enzyme Structure Molecular weight 69000-71000 <3> (<3> SDS-PAGE [8]) [8] 70000 <3> (<3> SDS-PAGE, soluble form [3]; <3> SDS-PAGE, purified enzyme [3]) [3] 80000 <3, 4> (<3,4> SDS-PAGE of expressed EXT1 and EXT2 proteins, weak band at 90000 D [2]) [2] 88000 <8> (<8> SDS-PAGE, species two with second species with MW 91000 [12]) [12] 606

2.4.1.224

Glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-a-N-acetylglucosaminyltransferase

90000 <3, 4> (<3,4> SDS-PAGE of coexpressed EXT1 and EXT2, no second band detected [2]) [2] 91000 <8> (<8> SDS-PAGE, species one with second species with MW 88000 [12]) [12] 110000 <1> (<1> expressed fusion protein from COS-1 after N-glycosidase treatment, SDS-PAGE [1]) [1] 130000 <1> (<1> expressed fusion protein from COS-1, SDS-PAGE [1]) [1] Posttranslational modification glycoprotein <1, 3, 4> (<1> N-linked glycosylation, molecular weight of expressed fusion protein is reduced from 1300000 to 110000 by N-glycosidase treatment [1]; <3,4> N-linked glycosylation, Endo H treatment does not effect 80000 Da species but reduces size of 90000 Da species that is found mainly when both proteins are coexpressed [2]) [1, 2, 12]

5 Isolation/Preparation/Mutation/Application Source/tissue fibroblast <4> [3, 5, 7, 12] mastocytoma cell <4> [5, 7] serum <3> [3, 5] Localization endoplasmic reticulum <4, 8> [12] membrane <4, 7, 8> (<4,8> transmembrane protein [12]) [7, 12] microsome <4> [5, 7] Purification <2> (EXTL1 and EXTL3 proteins from COS-1 cells [10]) [10] <3> [3, 5, 8] Cloning <1> (DV1, expression of soluble form of rib-2 protein in COS-1 cells [1]) [1] <2> (expression of soluble form of EXT1 and EXT2 proteins in COS-1 cells [1]; expression of EXTL1, EXTL2 and EXTL3 in COS-1 cells, EXTL2 was found to show only N-acetyl-d-glucose transferase I activity, EXTL1 shows transferase II activity, EXTL3 shows transferase I and II activities [10]) [1, 10] <3> (soluble form expressed in COS-7 cells [3]) [3] <4> (EXT1 used to complement deficiency mutant gro2C [3]; EXT1 and EXT2 from wild type CHO used to complement deficiency mutant CHO-K1, EXT2 failed to complement functional deficiency while it was fully corrected by EXT1 [9];) [3, 9] <4> (completion of mutant sog9 with HeLa cDNA, restores susceptibility to HSV-1 infection [12]) [12] <6> (expression of soluble form in COS-1 cells [11]) [11]

607

Glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-a-N-acetylglucosaminyltransferase

2.4.1.224

<3, 4> (expression of full length and truncated form in Pichia pastoris and COS-1 cells, dramatic increase of activity when full length EXT1 and EXT2 are coexpressed, no increase of activity when mixing extract containing EXT1 and EXT2 protein [2]) [2] Engineering C339D <8> (<8> resistant in HSV-1 infection assay [12]) [12] R340C <8> (<8> resistant in HSV-1 infection assay [12]) [12] Additional information <4, 8> (<4,8> d620EXT1myc deletion mutant without 620 C-terminal amino acids, no enzymatic activity, ER localization [12]) [12]

References [1] Kitagawa, H.; Egusa, N.; Tamura, J.I.; Kusche-Gullberg, M.; Lindahl, U.; Sugahara, K.: rib-2, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes encodes a novel a-1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. J. Biol. Chem., 276, 4834-4838 (2001) [2] Senay, C.; Lind, T.; Muguruma, K.; Tone, Y.; Kitagawa, H.; Sugahara, K.; Lidholt, K.; Lindahl, U.; Kusche-Gullberg, M.: The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis. EMBO Rep., 1, 282-286 (2000) [3] Lind, T.; Tufaro, F.; McCormick, C.; Lindahl, U.; Lidholt, K.: The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate. J. Biol. Chem., 273, 26265-26268 (1998) [4] Sharkey, D.J.; Kornfeld, R.: Identification of an N-acetylglucosaminyltransferase in Dictyostelium discoideum that transfers an intersecting N-acetylglucosamine residue to high mannose oligosaccharides. J. Biol. Chem., 264, 10411-10419 (1989) [5] Lidholt, K.; Lindahl, U.: Biosynthesis of heparin. The d-glucuronosyl- and N-acetyl-d-glucosaminyltransferase reactions and their relation to polymer modification. Biochem. J., 287, 21-29 (1992) [6] Lind, T.; Lindahl, U.; Lidholt, K.: Biosynthesis of heparin/heparan sulfate. Identification of a 70-kDa protein catalyzing both the d-glucuronosyl- and the N-acetyl-d-glucosaminyltransferase reactions. J. Biol. Chem., 268, 20705-20708 (1993) [7] Lidholt, K.; Fjelstad, M.; Jann, K.; Lindahl, U.: Biosynthesis of heparin. XXV. Substrate specificities of glucosyltransferases involved in formation of heparin precursor and E. coli K5 capsular polysaccharides. Carbohydr. Res., 255, 87-101 (1994) [8] Lind, T.: Enzymes involved in heparan sulfate chain elongation. Trends Glycosci. Glycotechnol., 11, 221-225 (1999) [9] Wei, G.; Bai, X.; Gabb, M.M.G.; Bame, K.J.; Koshy, T.I.; Spear, P.G.; Esko, J.D.: Location of the glucuronosyltransferase domain in the heparan sulfate

608

2.4.1.224

Glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-a-N-acetylglucosaminyltransferase

copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants. J. Biol. Chem., 275, 27733-27740 (2000) [10] Kim, B.T.; Kitagawa, H.; Tamura, J.I.; Saito, T.; Kusche-Gullberg, M.; Lindahl, U.; Sugahara, K.: Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode a-1,4-N-acetylglucosaminyltransferases that likely are involved in heparan sulfate/heparin biosynthesis. Proc. Natl. Acad. Sci. USA, 98, 7176-7181 (2001) [11] Kim, B.T.; Kitagawa, H.; Tamura, J.I.; Kusche-Gullberg, M.; Lindahl, U.; Sugahara, K.: Demonstration of a novel gene DEXT3 of Drosophila melanogaster as the essential N-acetylglucosamine transferase in the heparan sulfate biosynthesis: Chain initiation and elongation. J. Biol. Chem., 277, 1365913665 (2002) [12] McCormic, C.; Leduc, Y.; Martindale, D.; Mattison, K.; Esford, L.E.; Dyer, A.P.; Tufaro, F.: The putative tumor suppressor EXT 1 alters the expression of cell surface heparan sulfate. Nat. Genet., 19, 158-161 (1998)

609

N-Acetylglucosaminyl-proteoglycan 4-b-glucuronosyltransferase

2.4.1.225

1 Nomenclature EC number 2.4.1.225 Systematic name UDP-a-d-glucoronate:N-acetyl-a-d-glucosaminyl-(1!4)-b-d-glucuronosylproteoglycan 4-b-glucuronosyltransferase Recommended name N-acetylglucosaminyl-proteoglycan 4-b-glucuronosyltransferase Synonyms N-acetylglucosaminylproteoglycan b-1,4-glucuronosyltransferase UDP-glucuronate:oligosaccharide diphosphoglucuronate:oligosaccharide uridine glucuronosyltransferase exostosin-1 exostosin-2 exotose-2 gene EXTL1 glycosyltransferase gene EXTL2 glycosyltransferase glucuronosyltransferase heparan glucuronosyltransferase II heparan sulfate co-polymerase heparan sulfate glucuronosyltransferase CAS registry number 145539-84-0

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

610

Bos taurus [1, 2] Escherichia coli (K5 [3]) [3] Mus musculus [3] Oncorhynchus mykiss [4]

2.4.1.225

N-Acetylglucosaminyl-proteoglycan 4-b-glucuronosyltransferase

3 Reaction and Specificity Catalyzed reaction UDP-a-d-glucuronate + N-acetyl-a-d-glucosaminyl-(1!4)-b-d-glucuronosyl-proteoglycan = UDP + b-d-glucuronosyl-(1!4)-N-acetyl-a-d-glucosaminyl-(1!4)-b-d-glucuronosyl-proteoglycan Natural substrates and products S UDP-a-d-glucuronate + N-acetyl-a-d-glucosaminyl-(1-4)-b-d-glucuronosyl-proteoglycan <1, 3, 4> (Reversibility: ? <1, 3, 4> [1-4]) [1-4] P UDP + b-glucuronosyl-(1-4)-N-acetyl-a-d-glucosaminyl-(1-4)-b-d-glucuronosyl-proteoglycan Substrates and products S K5 heptasaccharide + UDP-a-d-glucuronate <2> (Reversibility: ? <2> [3]) [3] P ? S UDP-a-d-glucuronate + N-acetyl-a-d-glucosaminyl-(1-4)-b-d-glucuronosyl-proteoglycan <1, 3, 4> (Reversibility: ? <1, 3, 4> [1-4]) [1-4] P UDP + b-glucuronosyl-(1-4)-N-acetyl-a-d-glucosaminyl-(1-4)-b-d-glucuronosyl-proteoglycan Specific activity (U/mg) 0.000012 <4> [4] Additional information <1> (<1> 12000000 dpm 3 H/mg [2]; <1> 6700000 dpm 14 C/mg [2]) [2]

4 Enzyme Structure Molecular weight 81900 <1> (<1> calculation from amino acid sequence [1]) [1] Subunits monomer <1> (<1> 1 * 70000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue blood serum <1> [1] mastocytoma cell <3> [3] ovary <4> [4] Localization Golgi apparatus <1> [2] membrane <2> [3] microsome <3> [3]

611

N-Acetylglucosaminyl-proteoglycan 4-b-glucuronosyltransferase

2.4.1.225

Purification <1> [1, 2] Cloning <1> (expression in COS-7 cells as His/FLAG fusion protein [1]) [1] <4> (expression in CHO cells [4]) [4]

References [1] Lind, T.; Tufaro, F.; McCormick, C.; Lindahl, U.; Lidholt, K.: The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate. J. Biol. Chem., 273, 26265-26268 (1998) [2] Lind, T.; Lindahl, U.; Lidholt, K.: Biosynthesis of heparin/heparan sulfate. Identification of a 70-kDa protein catalyzing both the d-glucuronosyl- and the N-acetyl-d-glucosaminyltransferase reactions. J. Biol. Chem., 268, 20705-20708 (1993) [3] Lidholt, K.; Fjelstad, M.; Jann, K.; Lindahl, U.: Biosynthesis of heparin. XXV. Substrate specificities of glucosyltransferases involved in formation of heparin precursor and E. coli K5 capsular polysaccharides. Carbohydr. Res., 255, 87-101 (1994) [4] Wei, G.; Bai, X.; Gabb, M.M.G.; Bame, K.J.; Koshy, T.I.; Spear, P.G.; Esko, J.D.: Location of the glucuronosyltransferase domain in the heparan sulfate copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants. J. Biol. Chem., 275, 27733-27740 (2000)

612

N-Acetylgalactosaminyl-proteoglycan 3-b-glucuronosyltransferase

2.4.1.226

1 Nomenclature EC number 2.4.1.226 Systematic name a-d-glucuronate:N-acetyl-b-d-galactosaminyl-(1!4)-b-d-glucuronosyl-proteoglycan 3-b-glucuronosyltransferase Recommended name N-acetylgalactosaminyl-proteoglycan 3-b-glucuronosyltransferase Synonyms UDP-glucuronate:chondroitin glucuronyltransferase chondroitin b-glucuronyltransferase chondroitin glucuronyltransaferase II chondroitin glucuronyltransferase chondroitin sulfate glucuronyltransferase glucuronyltransferase, uridine diphosphoglucuronate-chondroitin CAS registry number 176023-59-9

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

Bos taurus (fetal, newborn calf, calf, adult animals [1]) [1, 2] Rattus norvegicus (Wistar rats [1]) [1] Gallus gallus gallus (White Leghorn [1]) [1] Homo sapiens [1] Homo sapiens (further cDNA: Kazusa DNA Research Institute number KIAA1402, SwissProt-ID: Q9P2E5 [3]) [3]

3 Reaction and Specificity Catalyzed reaction UDP-a-d-glucuronate + N-acetyl-b-d-galactosaminyl-(1!4)-b-d-glucuronosyl-proteoglycan = UDP + b-d-glucuronosyl-(1!3)-N-acetyl-b-d-galactosaminyl-(1!4)-b-d-glucuronosyl-proteoglycan (<1,2,3,4> transfer to polymer most likely through a b(1-3) linkage [1]; <1> transfer to the acceptor through a b-linkage [2])

613

N-Acetylgalactosaminyl-proteoglycan 3-b-glucuronosyltransferase

2.4.1.226

Reaction type glucuronyl group transfer glycosyl group transfer Natural substrates and products S UDP-a-d-glucuronate + N-acetyl-b-d-galactosaminyl-(1-4)-b-d-glucuronosyl-proteoglycan <1, 2, 3, 4> (Reversibility: ? <1, 2, 3, 4> [1, 2]) [1, 2] P UDP + b-d-glucuronosyl-(1-3)-N-acetyl-b-d-galactosaminyl-(1-4)-b-dglucuronosyl-proteoglycan <1, 2, 3, 4> [1, 2] Substrates and products S UDP-glucuronic acid + chondroitin <1, 2, 3, 4> (Reversibility: ? <1, 2, 3, 4> [1, 2]) [1, 2] P UDP + N-acetylchondrosine glucuronic acid b(1-3)N-acetylgalactosamine <1, 2, 3, 4> [1] S UDP-glucuronic acid + chondroitin heptasaccharide <5> (<5> heptasaccharide having a N-acetylgalactosamine residue at its non-reducing terminus [3]) (Reversibility: ? <5> [3]) [3] P ? S UDP-glucuronic acid + chondroitin polysaccharide <5> (<5> polysaccharide substrate used after b-glucuronidase treatment [3]) (Reversibility: ? <5> [3]) [3] P ? S UDP-glucuronic acid + chondroitin sulfate heptasaccharide <5> (<5> heptasaccharide having a N-acetylgalactosamine residue at its non-reducing terminus [3]) (Reversibility: ? <5> [3]) [3] P ? S UDP-glucuronic acid + chondroitin sulfate undecasaccharide <5> (Reversibility: ? <5> [3]) [3] P ? S Additional information <1, 5> (<1> further substrates: chondroitin heptaserine, pentaserine, non-sulfated odd-numbered oligosaccharides with a N-acetylgalactosamine residue at the non-reducing terminus except for a trisaccharide [2]; <1,5> the glucuronic acid transfer rate roughly increases with increasing chain length [2,3]; <1> no substrates: a trisaccharide-serine, an a-N-acetylgalactosamine-capped pentasaccharide-serine [2]; <1> 6-O-sulfation of non-reducing terminal N-acetylgalactosamine markedly enhances glucuronic acid transfer [2]; <5> no substrates: chondroitin, chondroitin sulfate, dermatan sulfate, N-acetylheparosan, hyaluronan, heparan sulfate, heparin, hyaluronan heptasaccharides [3]) [2, 3] P ? Metals, ions Mn2+ <1, 2, 3, 4, 5> [1, 2, 3]

614

2.4.1.226

N-Acetylgalactosaminyl-proteoglycan 3-b-glucuronosyltransferase

Specific activity (U/mg) Additional information <1, 2, 3, 4, 5> (<1,2,3,4> highest at the middle prenatal stage in the bovine and chicken sera, highest at the late prenatal stage in the rat serum [1]) [1, 2, 3] Km-Value (mM) 0.051 <1> (UDP-glucuronic acid) [2] 0.0653 <5> (chondroitin sulfate undecasaccharide) [3] 0.0824 <5> (UDP-glucuronic acid) [3] pH-Optimum 5.5-6 <1> (<1> sodium acetate buffer [2]) [2] Temperature optimum ( C) 37 <1, 2, 3, 4, 5> (<1,2,3,4,5> enzyme assay [1,3]) [1, 3]

5 Isolation/Preparation/Mutation/Application Source/tissue blood serum <1, 2, 3, 4> (<1> fetal bovine serum [2]) [1, 2] Purification <1> (partially [2]) [2] <5> [3] Cloning <5> (enzyme domain containing b-3-glycosyltransferase motifs, expressed in COS-7 cells ATCCCRL 1651 [3]) [3]

References [1] Kitagawa, H.; Ujikawa, M.; Sugahara, K.: Developmental changes in serum UDP-GlcA:chondroitin glucuronyltransferase activity. J. Biol. Chem., 271, 6583-6585 (1996) [2] Kitagawa, H.; Ujikawa, M.; Tsutsumi, K.; Tamura, J.I.; Neumann, K.W.; Ogawa, T.; Sugahara, K.: Characterization of serum b-glucuronyltransferase involved in chondroitin sulfate biosynthesis. Glycobiology, 7, 905-911 (1997) [3] Gotoh, M.; Yada, T.; Sato, T.; Akashima, T.; Iwasaki, H.; Mochizuki, H.; Inaba, N.; Togayachi, A.; Kudo, T.; Watanabe, H.; Kimata, K.; Narimatsu, H.: Molecular cloning and characterization of a novel chondroitin sulfate glucuronyltransferase that transfers glucuronic acid to N-acetylgalactosamine. J. Biol. Chem., 277, 38179-38188 (2002)

615

Undecaprenyldiphosphomuramoylpentapeptide b-N-acetylglucosaminyltransferase

2.4.1.227

1 Nomenclature EC number 2.4.1.227 Systematic name UDP-N-acetyl-d-glucosamine:N-acetyl-a-d-muramyl(oyl-l-Ala-g-d-Glu-lLys-d-Ala-d-Ala)-diphosphoundecaprenol b-1,4-N-acetylglucosaminyltransferase Recommended name undecaprenyldiphospho-muramoylpentapeptide b-N-acetylglucosaminyltransferase Synonyms Lipid I acetylglucosaminyltransferase MurG MurG glycosyltransferase MurG transferase UDP-acetylglucosamine-acetylmuramoylpentapeptide pyrophospholipid acetylglucosaminyltransferase acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-acetylmuramoylpentapeptide pyrophospholipid gene murG enzyme gene murG proteins proteins, gene murG CAS registry number 60976-26-3

2 Source Organism <1> Escherichia coli (strains JM83, JM109, OV2, GS58, RM4102 lysA, OV2 lysA and GS58 lysA [1]; wild type [2]; strain OV58-pUG18 [3]; BL21-DE3-pLysS strain overexpressing MurG [5]) [1, 2, 3, 4, 5, 6, 7, 8, 9]

616

2.4.1.227

Undecaprenyldiphospho-muramoylpentapeptide b-N-acetylglucosaminyltransferase

3 Reaction and Specificity Catalyzed reaction UDP-N-acetylglucosamine + Mur2 Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-d-Ala)diphosphoundecaprenol = UDP + N-acetylglucosamine-(1!4)-Mur2 Ac(oyll-Ala-g-d-Glu-l-Lys-d-Ala-d-Ala)-diphosphoundecaprenol (<1> MurG utilizes an ordered Bi Bi mechanism in which the donor binds first [7]) Reaction type transfer of a-N-acetylglucosamine Natural substrates and products S UDP-N-acetylglucosamine + Mur2 Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-dAla)-diphosphoundecaprenol <1> (Reversibility: ? <1> [1, 3]) [1, 3] P UDP + N-acetylglucosamine-(1-4)-Mur2 Ac(oyl-l-Ala-g-d-Glu-l-Lys-dAla-d-Ala)-diphosphoundecaprenol <1> [1, 3] Substrates and products S 2'-deoxy ribosyl derivate of UDP-N-acetylglucosamine + Mur2 Ac(oyl-lAla-g-d-Glu-l-Lys-d-Ala-d-Ala)-diphosphoundecaprenol <1> (Reversibility: ? <1> [8]) [8] P ? S UDP-N-acetylglucosamine + Mur2 Ac(oyl-l-Ala-g-d-Glu-l-Lys-d-Ala-dAla)-diphosphoundecaprenol <1> (Reversibility: ? <1> [1, 3]) [1, 3] P UDP + N-acetylglucosamine-(1-4)-Mur2 Ac(oyl-l-Ala-g-d-Glu-l-Lys-dAla-d-Ala)-diphosphoundecaprenol <1> [1, 3] S UDP-N-acetylglucosamine + MurNAc(Ne -dansylpentapeptide)-pyrophosphoryl (R,S)-a-dihydrodecaprenol <1> (<1> very poor substrate [9]) (Reversibility: ? <1> [9]) [9] P N-acetylglucosamine-MurNAc(Ne -dansylpentapeptide)-pyrophosphoryl (R,S)-a-dihydrodecaprenol + UDP <1> [9] S UDP-N-acetylglucosamine + MurNAc(Ne -dansylpentapeptide)-pyrophosphoryl (R,S)-a-dihydroheptaprenol <1> (Reversibility: r <1> [9]) [9] P UDP + N-acetylglucosamine-MurNAc(Ne -dansylpentapeptide)-pyrophosphoryl (R,S)-a-dihydroheptaprenol <1> [9] S UDP-N-acetylglucosamine + citronellyl-lipid I <1> (<1> synthetic substrate, 55-carbon undecaprenol chain replaced by the 10-carbon chain of citronellol, biotin labeled substrate [2,5]; <1> undecaprenol replaced with a citronellol group [4]) (Reversibility: ? <1> [2, 4, 5]) [2, 4, 5] P UDP + N-acetylglucosamine-citronellyl-lipid I <1> [2, 4, 5] S UDP-N-acetylglucosamine + synthetic substrate analogue 2 <1> (<1> synthetic substrate analogue 2: a 10 carbon citronellyl derivative) (Reversibility: ? <1> [7]) [7] P ? S UDP-N-acetylglucosamine + synthetic substrate analogue 7 <1> (<1> synthetic substrate analogue 7: a derivative containing a 20 carbon chain with a cis-allylic double bond [7]) (Reversibility: ? <1> [7]) [7] P ?

617

Undecaprenyldiphospho-muramoylpentapeptide b-N-acetylglucosaminyltransferase

2.4.1.227

S Additional information <1> (<1> no substrates: P1-citronellyl-P2-a-dglucosyl pyrophosphate, P1-citronellyl-P2-a-d-N-acetylglucoseaminyl pyrophosphate, P1-citronellyl-P2-a-d-N-acetylmuramyl pyrophosphate [4]; <1> MurG requires substrates containing an intact amide between the muramyl lactyl ether side-chain and the pentapeptide [4]; <1> MurG binds significantly better to UDP than to CDP, ADP or GDP, E269 is an important residue in binding the nucleotide-sugar donor [5]; <1> MurG prefers substrates that contain lipid chains that are considerably shorter than that of the natural substrate, compounds that contain a cis-allylic double bond react faster than compounds that do not [7]; <1> no substrates: TDP-GlcNAc, C4-hydroxyl of UDP-GalNAc cannot form hydrogen bonds to A264 or N292 [8]) [4, 5, 7, 8, 9] P ? Inhibitors EDTA <1> [2] Ni2+ <1> [2] UDP <1> (<1> competitive inhibitor of UDP-GlcNAc donor, noncompetitive inhibitor of lipid I acceptor analogue [7]) [7] Zn2+ <1> [2] methanol <1> [9] ramoplanin <1> [3] trifluoroethanol <1> [9] tunicamycin <1> [3] Activating compounds Ca2+ <1> [2] Mg2+ <1> [2, 4, 5, 7, 9] dimethyl sulfoxide <1> [9] Metals, ions Mn2+ <1> (<1> activating compound [2]) [2] Turnover number (min±1) 7.4 <1> (biotinylated citronellyl-lipid I, <1> in the absence of additional metal ions [2]) [2] 16 <1> (biotinylated citronellyl-lipid I, <1> in the presence of Mg2+ [2]) [2] 19 <1> (biotinylated citronellyl-lipid I, <1> in the presence of Mn2+ [2]) [2] 56 <1> (MurNAc(Ne -dansylpentapeptide)-pyrophosphoryl (R,S)-a-dihydroheptaprenol) [1] 134 <1> (synthetic substrate analogue 2, <1> 10 carbon citronellyl derivative [7]) [7] 837 <1> (synthetic substrate analogue 7, <1> derivative containing a 20 carbon chain with a cis-allylic double bond [7]) [7, 8] Specific activity (U/mg) 0.000000096 <1> (<1> thermosensitive murG mutant strain GS58, 40 C [1]) [1] 0.000000336 <1> (<1> thermosensitive murG mutant strain GS58, 30 C [1]) [1] 0.0000016 <1> (<1> parental strain, 30 C [1]) [1]

618

2.4.1.227

Undecaprenyldiphospho-muramoylpentapeptide b-N-acetylglucosaminyltransferase

0.000002 <1> (<1> parental strain, 40 C [1]) [1] 0.0000064 <1> (<1> murG gene under the control of the lac promotor [1]) [1] 0.0000141 <1> [3] Km-Value (mM) 0.0028 <1> (MurNAc(Ne -dansylpentapeptide)-pyrophosphoryl (R,S)-a-dihydroheptaprenol) [9] 0.036 <1> (biotinylated citronellyl-lipid I, <1> in the presence of Mn2+ [2]) [2] 0.037 <1> (biotinylated citronellyl-lipid I, <1> in the presence of Mg2+ [2]) [2] 0.044 <1> (biotinylated citronellyl-lipid I, <1> in the absence of additional metal ions [2]) [2] 0.045 <1> (UDP-N-acetylglucosamine, <1> in the absence of additional metal ions [2]) [2] 0.046 <1> (UDP-N-acetylglucosamine, <1> in the presence of Mn2+ [2]) [2] 0.053 <1> (UDP-GlcNAc) [8] 0.053 <1> (synthetic substrate analogue 7, <1> derivative containing a 20 carbon chain with a cis-allylic double bond [7]) [7, 8] 0.058 <1> (UDP-N-acetylglucosamine, <1> in the presence of Mg2+ [2]) [2] 0.15 <1> (UDP-N-acetylglucosamine) [9] 0.553 <1> (synthetic substrate analogue 2, <1> 10 carbon citronellyl derivative [7]) [7] 1.5 <1> (2'-deoxy ribosyl analogue of UDP-GlcNAc) [8] pH-Optimum 7.5 <1> [9] 8.3 <1> (<1> HEPES buffer [2]) [2] Temperature optimum ( C) 25 <1> (<1> at room temperature [3]; <1> enzyme assay [3,4]) [3, 4] 35 <1> (<1> enzyme assay [1]) [1] 37 <1> (<1> enzyme assay [9]) [9]

4 Enzyme Structure Molecular weight 37640 <1> (<1> calculated from the nucleotide sequence, gel filtration [1]) [1] 76000 <1> (<1> gel filtration [2]) [2] Subunits dimer <1> (<1> 2 * 38000, SDS-PAGE [2]) [2]

619

Undecaprenyldiphospho-muramoylpentapeptide b-N-acetylglucosaminyltransferase

2.4.1.227

5 Isolation/Preparation/Mutation/Application Localization membrane <1> [1, 3] soluble <1> (<1> cytoplasmic fraction [2]) [2, 4] Purification <1> [2] Crystallization <1> (hanging-drop vapor-diffusion method, X-ray structure [8]) [8] Cloning <1> (expressed in Escherichia coli cells BL21-DE3-pLysS [2,4,5,7,8]) [2, 4, 5, 7, 8]

6 Stability Storage stability <1>, -70 C [8] <1>, -80 C [3] <1>, 4 C, stable for at least one month [2]

References [1] Mengin-Lecreulx, D.; Textier, L.; Rousseau, M.; van Heijenoort, J.: The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine:N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis. J. Bacteriol., 173, 4625-4636 (1991) [2] Ha, S.; Chang, E.; Lo, M.C.; Men, H.; Park, P.; Ge, M.; Walker, S.: The kinetic characterization of Escherichia coli MurG using synthetic substrate analogues. J. Am. Chem. Soc., 121, 84158426 (1999) [3] Branstrom, A.A.; Midha, S.; Longley, C.B.; Han, K.; Baizman, E.R.; Axelrod, H.R.: Assay for identification of inhibitors for bacterial MraY translocase or MurG transferase. Anal. Biochem., 280, 315-319 (2000) [4] Cudic, P.; Behenna, D.C.; Yu, M.K.; Kruger, R.G.; Szewczuk, L.M.; McCafferty, D.G.: Synthesis of P1-citronellyl-P2-a-d-pyranosyl pyrophosphates as potential substrates for the E. coli undecaprenyl-pyrophosphoryl-N-acetylglucoseaminyl transferase MurG. Bioorg. Med. Chem. Lett., 11, 3107-3110 (2001) [5] Men, H.; Park, P.; Walker, S.: Substrate synthesis and activity assay for MurG. J. Am. Chem. Soc., 120, 2484-2485 (1998) [6] Ha, S.; Gross. B.; Walker, S.: E. coli MurG: A paradigm for a superfamily of glycosyltransferases. Curr. Drug Targets Infect. Disord., 1, 201-213 (2001)

620

2.4.1.227

Undecaprenyldiphospho-muramoylpentapeptide b-N-acetylglucosaminyltransferase

[7] Chen, L.; Men, H.; Ha, S.; Ye, X.Y.; Brunner, L.; Hu, Y.; Walker, S.: Intrinsic lipid preferences and kinetic mechanism of Escherichia coli MuG. Biochemistry, 41, 6824-6833 (2002) [8] Hu, Y.; Chen, L.; Ha, S.; Gross, B.; Falcone, B.; Walker, D.; Mokhtarzadeh, M.; Walker, S.: Crystal structure of the MurG: UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases. Proc. Natl. Acad. Sci. USA, 100, 845-849 (2003) [9] Auger, G.; van Heijenoort, J.; Mengin-Lecreulx, D.; Blanot, D.: A MurG assay which utilises a synthetic analogue of lipid I. FEMS Microbiol. Lett., 219, 115-119 (2003)

621

Lactosylceramide 4-a-galactosyltransferase

2.4.1.228

1 Nomenclature EC number 2.4.1.228 Systematic name UDP-galactose:lactosylceramide 4II-a-d-galactosyltransferase Recommended name lactosylceramide 4-a-galactosyltransferase Synonyms GB3 synthase Gal-b1-4Glcb1-Cer a1,4-galactosyltransferase Gb3 galactosyltransferase Gb3 synthase UDP-galactose-lactosylceramide galactosyltransferase UDP-galactose:b-d-galactosyl-b-1-R 4-a-d-galactosyltransferase UDP-galactose:lactosylceramide a-galactosyltransferase UDP-galactose:lactosylceramide a1-4-galactosyltransferase a1-4 galactosyltransferase galactosyltransferase, uridine diphosphogalactose-lactosylceramide galactosyltransferase, uridine diphosphogalactose-lactosylceramide a1-4globotriaosylceramide synthase globotriaosylceramide/CD77 synthase histo-blood group Pk UDP-galactose histo-blood group Pk antigen synthase lactosylceramide a1,4galactosyltransferase CAS registry number 52725-57-2

2 Source Organism <1> Rattus norvegicus (male Wistar rats [1]) [1] <2> Homo sapiens (NTERA-2 cl.D1 cells, a cloned EC subline of the teratocarcinoma cell line TERA-2, during retinoic acid induced differentiation, Burkitt lymphoma Ramos cells [4]; leukaemia-myeloid cell lines: K562, KG-1, HL-60, and THP-1, lymphoma-lymphoid cell lines: Reh, Daudi, Raji, RPMI 8226, CCRF-CEM, and MOLT-4 [5]; melanoma cell line SK-MEL-37 [7]) [2, 3, 4, 5, 7]

622

2.4.1.228

Lactosylceramide 4-a-galactosyltransferase

<3> Oryctolagus cuniculus (New Zealand White rabbits, 18- to 90-days-old [6]) [6] <4> Homo sapiens [8]

3 Reaction and Specificity Catalyzed reaction UDP-galactose + b-d-galactosyl-(1!4)-d-glucosylceramide = UDP + a-dgalactosyl-(1!4)-b-d-galactosyl-(1!4)-d-glucosylceramide Reaction type galactosyl group transfer Natural substrates and products S UDP-galactose + b-d-galactosyl-(1-4)-d-glucosylceramide <1, 2, 3> (Reversibility: ? <1, 2, 3> [1, 2, 3, 4, 5, 6, 7]) [1, 2, 3, 4, 5, 6, 7] P UDP + a-d-galactosyl-(1-4)-b-d-galactosyl-(1-4)-d-glucosylceramide <1, 2, 3> [1, 2, 3, 4, 5, 6, 7] Substrates and products S UDP-galactose + b-d-galactosyl-(1-4)-d-glucosylceramide <1, 2, 3> (Reversibility: ? <1, 2, 3> [1, 2, 3, 4, 5, 6, 7]) [1, 2, 3, 4, 5, 6, 7] P UDP + a-d-galactosyl-(1-4)-b-d-galactosyl-(1-4)-d-glucosylceramide <1, 2, 3> [1, 2, 3, 4, 5, 6, 7] S UDP-galactose + galactosylceramide <2> (Reversibility: ? <2> [7]) [7] P ? S UDP-galactose + lactose <1, 2> (Reversibility: ? <1, 2> [1, 2]) [1, 2] P ? S UDP-galactose + lactosylceramide <1, 2, 3> (<1,2> UDP-galactose sole sugar donor [1,2]; <2> in the small p cells in vitro only [3]; <2> reduction of enzyme activity following retinoic acid induced differentiation [4]) (Reversibility: ? <1, 2, 3> [1, 2, 3, 4, 5, 6, 7]) [1, 2, 3, 4, 5, 6, 7] P UDP + globotriaosylceramide <1, 2, 3> [1, 2, 3, 4, 5, 6, 7] S Additional information <1> (<1> no substrates: galactosylceramide, glucosylceramide, neolactotetraose, neolactotetraosylceramide [1]) [1] P ? Metals, ions Cu2+ <1> (<1> some activity [1]) [1] Mn2+ <1, 2, 3> (<1> a second divalent cation in the enzyme assay can inhibit the enzyme activity [1]; <2> THP-1 cells [5]) [1, 2, 3, 4, 5, 6, 7] Ni2+ <1> (<1> some activity [1]) [1] Specific activity (U/mg) 0.0000001 <2> (<2> lactosylceramide as acceptor [7]) [7] 0.0000247 <3> (<3> 1.5-days-old animals [6]) [6] 0.000067 <3> (<3> 14-days-old animals [6]) [6] 0.000174 <3> (<3> 18-days-old animals [6]) [6]

623

Lactosylceramide 4-a-galactosyltransferase

2.4.1.228

0.000256 <3> (<3> 6-months-old animals [6]) [6] 0.00458 <1> [1] Additional information <2, 4> (<4> cloned enzyme encodes exclusive Pk a4-galactosyltransferase activity [8]) [3, 8] Km-Value (mM) 0.0013 <2> (lactosylceramide) [5] 0.006 <1> (lactosylceramide) [1] 0.03 <1> (UDP-galactose) [1] 0.0545 <2> (lactosylceramide) [7] 0.132 <2> (UDP-galactose) [7] 0.445 <3> (lactosylceramide) [6] 0.52 <2> (UDP-galactose) [5] pH-Optimum 6.5 <3> (<3> MES buffer, 0.25-0.5% sodium cholate [6]) [6] 7 <2> (<2> THP-1 cells [5]) [5] 7.2 <1> [1] pH-Range 6.7-7.4 <1> [1] Temperature optimum ( C) 37 <1, 2, 3> (<1,2,3> enzyme assay [1,2,3,4,6]) [1, 2, 3, 4, 6]

4 Enzyme Structure Molecular weight 90000 <1> (<1> corresponding to the Stokes radius of 40 A, gel filtration [1]) [1] Subunits dimer <1> (<1> a 1 * 65000 + b 1 * 22000, linked through a disulfide bond, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue DAUDI cell <2> [5] RAJI cell <2> [5] RPMI-8226 cell <2> [5] THP-1 cell <2> [5] intestine <3> (<3> small intestine [6]) [6] kidney <2> (<2> frozen [2]) [2] liver <1> (<1> stored at -80 C until use [1]) [1] lymphoblastoid <2> (<2> blood of a small p subject and a P1 control subject [3]) [3]

624

2.4.1.228

Lactosylceramide 4-a-galactosyltransferase

Additional information <2> (<2> THP-1, RPMI 8226, Daudi, and Raji cell lines with detectable enzyme activity, no detectable enzyme activity: K562, KG-1, HL-60, Reh, MOLT-4, CCRF-CEM cell lines [5]) [5] Localization microsome <2, 3> [2, 6] microvillus <3> (<3> membrane [6]) [6] Purification <1> [1] <2> (partially [1]) [2] Cloning <2> (expressed in mouse fibroblast L cells [7]) [7] <4> (expressed in insect Namalwa cells [8]) [8]

6 Stability Storage stability <1>, -20 C, in 20% glycerol, stored at a concentration greater than 0.1 mg/ml without detectable loss of activity upon repeated freezing and thawing for 3 months [1] <2>, -20 C [3] <2>, -70 C, however enzyme activity in RPMI 8226 cells destroyed by storage at -70 C [5] <2>, 4 C, stable for several weeks in the presence of 20% glycerol [2]

References [1] Taniguchi, N.; Yanagisawa, K.; Makita, A.; Naiki, M.: Purification and properties of rat liver globotriaosylceramide synthase, UDP-galactose:lactosylceramide a1-4-galactosyltransferase. J. Biol. Chem., 260, 4908-4913 (1985) [2] Bailly, P.; Piller, F.; Cartron, J.P.; Leroy, Y.; Fournet, B.: Identification of UDPgalactose:lactose (lactosylceramide) a-4 and b-3 galactosyltransferases in human kidney. Biochem. Biophys. Res. Commun., 141, 84-91 (1986) [3] Iizuka, S.; Chen, S.H.; Yoshida, A.: Studies on the human blood group P system: an existence of UDP-Gal: lactosylceramide a1!4 galactosyltransferase in the small p type cells. Biochem. Biophys. Res. Commun., 137, 1187-1195 (1986) [4] Chen, C.; Fenderson, B.A.; Andrews, P.W.; Hakomori, S.: Glycolipid glycosyltransferases in human embryonal carcinoma cells during retinoic acid induced differentiation. Biochemistry, 28, 2229-2238 (1989) [5] Stults, C.L.M.; Larsen, R.D.; Macher, B.A.: a-1,4Galactosyltransferase activity and Gb3Cer expression in human leukemia/lymphoma cell lines. Glycoconjugate J., 12, 680-689 (1995)

625

Lactosylceramide 4-a-galactosyltransferase

2.4.1.228

[6] Mobassaleh, M.; Koul, O.; Mishra, K.; McCluer, R.H.; Keusch, G.T.: Developmentally regulated Gb3 galactosyltransferase and a-galactosidase determine Shiga toxin receptors in intestine. Am. J. Physiol., 267, G618-G624 (1994) [7] Kojima, Y.; Fukumoto, S.; Furukawa, K.; Okajima, T.; Wiels, J.; Yokoyama, K.; Suzuki, Y.; Urano, T.; Ohta, M.; Furukawa, K.: Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferase that initiates the synthesis of Globo series glycosphingolipids. J. Biol. Chem., 275, 15152-15156 (2000) [8] Steffensen, R.; Carlier, K.; Wiels, J.; Levery, S.B.; Stroud, M.; Cedergren, B.; Sojka, B.N.; Bennett, E.P.; Jersild, C.; Clausen, H.: Cloning and expression of the histo-blood group Pk UDP-galactose: Galb1-4Glcb1-Cer a1,4-galactosyltransferase. Molecular genetic basis of the p phenotype. J. Biol. Chem., 275, 16723-16729 (2000)

626

[Skp1-Protein]-hydroxyproline N-acetylglucosaminyltransferase

2.4.1.229

1 Nomenclature EC number 2.4.1.229 Systematic name UDP-N-acetyl-d-glucosamine:[Skp1-protein]-hydroxyproline N-acetyl-d-glucosaminyl-transferase Recommended name [Skp1-protein]-hydroxyproline N-acetylglucosaminyltransferase Synonyms GnT51 <1, 2> [1, 3] Skp1-HyPro GlcNAc-transferase UDP-GlcNAc:Skp1-hydroxyproline GlcNAc-transferase UDP-GlcNAc:hydroxyproline polypeptide GlcNAc-transferase UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNActransferase acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-glycoprotein serine/threonine acetylglucosaminyltransferase, uridine diphosphoacetylglucose-protein Skp1 hydroxyproline CAS registry number 256531-81-4 37277-59-1

2 Source Organism <1> Dictyostelium discoideum (gene GnT51 [1,3]) [1, 3] <2> Dictyostelium sp. [2, 3]

3 Reaction and Specificity Catalyzed reaction UDP-N-acetylglucosamine + [Skp1-protein]-hydroxyproline = UDP + [Skp1protein]-O-(N-acetyl-d-glucosaminyl)hydroxyproline (Requires dithiothreitol and a divalent cation for activity. This enzyme commences the building up of a pentasaccharide (Gala1-6Gala1-l-Fuca1-2Galb1-3GlcNAc) on Hyp-143 of

627

[Skp1-Protein]-hydroxyproline N-acetylglucosaminyltransferase

2.4.1.229

the Dictyostelium protein Skp1, which is required for the ubiquitination of cell-cycle regulatory proteins and transcription factors. The fucose residue is probably in the a configuration [3]. The specificity of the enzyme for Skp1Hyp-143 and its high affinity for this substrate suggests that it is the GlcNActransferase that modifies Skp1 in vivo) Reaction type hexosyl group transfer Natural substrates and products S UDP-N-acetylglucosamine + [Skp1-protein]-hydroxyproline <1, 2> (<2> part of complex O-linked glycosylation, which is necessary for targeting of Skp-1-protein to the nucleus [3]) (Reversibility: ? <1, 2> [1-3]) [1-3] P UDP + [Skp1-protein]-O-(N-acetyl-d-glucosaminyl)hydroxyproline <1, 2> [1-3] Substrates and products S UDP-N-acetylglucosamine + [Skp1-protein]-hydroxyproline <1, 2> (<2> recombinant protein substrate, deficient in GlcNAc [2,3]; <1,2> hydroxyproline143 [1-3]; <2> high affinity for UDP-GlcNAc [2]; <1> enzyme attaches the first sugar of pentasaccharide chain [1-3]) (Reversibility: ? <1,2> [1-3]) [1-3] P UDP + [Skp1-protein]-O-(N-acetyl-d-glucosaminyl)hydroxyproline <1, 2> [1-3] S UDP-N-acetylglucosamine + hydroxyproline-peptide <2> (<2> synthetic peptide substrate [2,3]) (Reversibility: ? <2> [2,3]) [2, 3] P UDP + (N-acetyl-d-glucosaminyl)hydroxyproline peptide <2> [2, 3] S Additional information <2> (<2> no activity with nonhydroxylated Skp1-protein or -peptide [3]) [3] P ? Inhibitors ATP <2> (<2> weak inhibition [2]) [2] CTP <2> (<2> weak inhibition [2]) [2] Ca2+ <2> [2] EDTA <2> (<2> complete inhibition at 5 mM [2]) [2] K+ <2> (<2> weak [2]) [2] Mn2+ <2> [2] Na+ <2> (<2> inhibitory at concentrations above 0.05 M [2]) [2] UDP <2> [2] UMP <2> (<2> best inhibitor [2]) [2] UTP <2> [2] Additional information <2> (<2> Skp-1-protein isoform containing unhydroxylated proline143 is probably inhibitory [2]) [2] Activating compounds Brij-35 <2> (<2> slightly stimulating [2]) [2] NP-40 <2> (<2> slightly stimulating [2]) [2] Triton X-100 <2> (<2> slightly stimulating [2]) [2]

628

2.4.1.229

[Skp1-Protein]-hydroxyproline N-acetylglucosaminyltransferase

Tween 20 <2> (<2> stimulates, less efficient than Tween 80 [2]) [2] Tween 80 <2> (<2> stimulates [2]) [2] dithiothreitol <2> (<2> activates, best at 5 mM [2]) [2] uracil <2> (<2> stimulates the purified enzyme at 0.1 mM [2]) [2] Additional information <2> (<2> requires reducing condition [2,3]) [2, 3] Metals, ions Mg2+ <2> (<2> activates, best at 10 mM [2]) [2] Additional information <2> (<2> divalent cations required [2,3]) [2, 3] Specific activity (U/mg) 0.00023 <2> (<2> purified enzyme [2]) [2] Km-Value (mM) 0.00016 <2> (UDP-GlcNAc, <2> pH 7.8, 30 C [2]) [2] 0.00056 <2> (Skp-1-protein, <2> pH 7.8, 30 C [2]) [2] 1.6 <2> (hydroxyproline-peptide, <2> pH 7.8, 30 C [2]) [2] pH-Optimum 7.5-8 <2> [2] 7.8 <1> (<1> assay at [1]) [1] Temperature optimum ( C) 30 <2> [2]

4 Enzyme Structure Molecular weight 45000 <2> (<2> gel filtration [2]) [2] Subunits ? <1> (<1> x * 47500, wild-type enzyme and mutants K23R, D102A, H104D, and L276S, SDS-PAGE [1]; <1> x * 29600, mutant GnT51(1-282), SDS-PAGE [1]; <1> x * 44000, mutant GnT51(1-369+), SDS-PAGE [1]) [1] monomer <2> (<2> 1 * 51000, SDS-PAGE [2,3]) [2, 3]

5 Isolation/Preparation/Mutation/Application Localization cytosol <1, 2> [1-3] Additional information <2> (<2> away from membranes [3]; <2> no activity in the particulate fraction [2]) [2, 3] Purification <1> (recombinant wild-type enzyme and mutants from Escherichia coli as CBD-intein tagged proteins, to near homogeneity [1]) [1] <2> (130000fold [2]) [2, 3]

629

[Skp1-Protein]-hydroxyproline N-acetylglucosaminyltransferase

2.4.1.229

Cloning <1> (gene GnT51, DNA sequence determination and analysis, functional expression of wild-type and mutants as fusion proteins with CBD-intein, in Escherichia coli strain ER2566, autocleavage of tag moiety [1]) [1, 3] Engineering D102A <1> (<1> site-directed mutagenesis, no remaining activity [1]) [1] H104D <1> (<1> site-directed mutagenesis, no remaining activity [1]) [1] K23R <1> (<1> site-directed mutagenesis, unaltered enzyme activity [1]) [1] L276S <1> (<1> site-directed mutagenesis, 7% remaining activity compared to the wild-type enzyme [1]) [1] Additional information <1> (<1> C-terminal deletion mutants GnT51(1-282) and GnT51(1-369+) show no remaining activity [1]) [1]

6 Stability Temperature stability 4 <2> (<2> partially purified enzyme, unstable [2]) [2] General stability information <2>, Tween 20 and Tween 80 stabilize [2]

References [1] van der Wel, H.; Morris, H.R.; Panico, M.; Paxton, T.; Dell, A.; Kaplan, L.; West, C.M.: Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of Dictyostelium. J. Biol. Chem., 277, 46328-46337 (2002) [2] Teng-umnuay, P.; van der Wel, H.; West, C.M.: Identification of a UDPGlcNAc:Skp1-hydroxyproline GlcNAc-transferase in the cytoplasm of Dictyostelium. J. Biol. Chem., 274, 36392-36402 (1999) [3] West, C.M.; van der Wel, H.; Gaucher, E.A.: Complex glycosylation of Skp1 in Dictyostelium: implications for the modification of other eukaryotic cytoplasmic and nuclear proteins. Glycobiology, 12, 17R-27R (2002)

630

Kojibiose phosphorylase

2.4.1.230

1 Nomenclature EC number 2.4.1.230 Systematic name 2-a-d-glucosyl-d-glucose:phosphate b-d-glucosyltransferase Recommended name kojibiose phosphorylase Synonyms KPase CAS registry number 206566-36-1

2 Source Organism <1> Thermoanaerobium brockii (ATCC 35047 [1]) [1, 2, 3]

3 Reaction and Specificity Catalyzed reaction 2-a-d-glucosyl-d-glucose + phosphate = d-glucose + b-d-glucose 1-phosphate (the enzyme from Thermoanaerobacter brockii can act with a-1,2-oligoglucans, such as selaginose, as substrate, but more slowly. The enzyme is inactive when dissaccharides with linkages other than a-1,2 linkages, such as sophorose, trehalose, neotrehalose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, gentiobiose, sucrose and lactose, are used as substrates) Reaction type hexosyl group transfer Substrates and products S d-glucose + O-b-d-fructofuranosyl-(2-1)-O-b-d-fructofuranosyl-(2-1)-Ob-d-fructofuranosyl-(2-1)-glucopyranoside <1> (Reversibility: ? <1> [3]) [3] P [O-a-d-glucopyranosyl-1(1-2)]n -O-[b-d-fructofuranosyl-(2-1)]3 -a-d-glucopyranoside + phosphate <1> (<1> n = 1 or 2 [3]) [3]

631

Kojibiose phosphorylase

2.4.1.230

S d-glucose + b-d-glucose 1-phosphate <1> (Reversibility: r <1> [1]; ? <1> [2,3]) [1, 2, 3] P 2-a-d-glucosyl-d-glucose + phosphate <1> (<1> [O-a-d-glucopyranosyl(1-2)]m -O-[b-d-fructofuranosyl-(2-1)]2 -a-d-glucopyranoside with m = 1, 2 or 3 [3]; <1> kojioligosaccharides having only the a-1,2-linkage are synthesized and the average degree of polymerization of oligosaccharides increases with decreasing proportions of glucose [2]) [1, 2, 3] Inhibitors Hg2+ <1> [1] Pb2+ <1> [1] Km-Value (mM) 0.77 <1> (b-d-glucose 1-phosphate) [1] 0.77 <1> (kojibiose) [1] 0.85 <1> (phosphate) [1] 3.52 <1> (glucose) [1] pH-Optimum 5.5 <1> [1] Temperature optimum ( C) 65 <1> [1]

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

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

6 Stability pH-Stability 5.5-9.7 <1> (<1> stable [1]) [1] Temperature stability 65 <1> (<1> stable up to [1]) [1]

632

2.4.1.230

Kojibiose phosphorylase

References [1] Chaen, H.; Yamamoto, T.; Nishimoto, T.; Nakada, T.; Fukuda, S.; Sugimoto, T.; Kurimoto, M.; Tsujisaka, Y.: Purification and characterization of a novel phosphorylase, kojibiose phosphorylase, from Thermoanaerobium brockii. J. Appl. Glycosci., 46, 423-429 (1999) [2] Chaen, H.; Nishimoto, T.; Nakada, T.; Fukuda, S.; Kurimoto, M.; Tsujisaka, Y.: Enzymatic synthesis of kojioligosaccharides using kojibiose phosphorylase. J. Biosci. Bioeng., 92, 177-182 (2001) [3] Okada, H.; Fukushi, E.; Onodera, S.; Nishimoto, T.; Kawabata, J.; Kikuchi, M.; Shiomi, N.: Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from b-d-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase. Carbohydr. Res., 338, 879-885 (2003)

633

a,a-Trehalose phosphorylase (configuration-retaining)

2.4.1.231

1 Nomenclature EC number 2.4.1.231 Systematic name a,a-trehalose:phosphate a-d-glucosyltransferase Recommended name a,a-trehalose phosphorylase (configuration-retaining) Synonyms trehalose phosphorylase CAS registry number 37205-59-7

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

Schizophyllum commune [1-4, 9] Pleurotus sajor-caju [5] Flammulina velutipes [6] Pleurotus ostreatus [7] Agaricus sp. [8] Aphyllphorus sp. [8] Grifola frondosa [10] Agaricus bisporus (strain Horst U1 [11]) [11]

3 Reaction and Specificity Catalyzed reaction a,a-trehalose + phosphate = a-d-glucose + a-d-glucose 1-phosphate (<1,8> mechanism [2,3,4,11]) Reaction type glycosyl group transfer phospho group transfer Natural substrates and products S a,a-trehalose + phosphate <1-8> (<8> key role in trehalose metabolism [11]) (Reversibility: r <1-4, 7, 8> [1, 2, 4-7, 9-11]; ? <1, 5, 6> [3, 8]) [1-11] P a-d-glucose + a-d-glucose 1-phosphate <8> [11] 634

2.4.1.231

a,a-Trehalose phosphorylase (configuration-retaining)

Substrates and products S a,a-trehalose + arsenate <1> (Reversibility: r <1> [1,2]) [1, 2] P a-d-glucose 1-arsenate + a-d-glucose <1> (<1> a-d-glucose 1-arsenate is unstable, therefore the net reaction gives two mol of a-d-glucose [1]) [1] S a,a-trehalose + phosphate <1-8> (Reversibility: r <1-4, 7, 8> [1, 2, 4-7, 9, 10, 11]; ? <1,5,6> [3,8]) [1-11] P a-d-glucose + a-d-glucose 1-phosphate <1-8> (<1> reverse reaction only utilizes the a-anomers of d-glucose-1-phosphate and d-glucose and gives exclusively the a,a-product [1]) [1-11] S a-d-glucose 1-fluoride + phosphate <1> (<1> poor substrate, a-d-glucose 1-fluoride is recognized as a substrate analogue of a,a-trehalose but not of a-d-glucose 1-phosphate [1]) (Reversibility: ? <1> [1,3]) [1, 3] P a-d-glucose 1-phosphate + fluoride Inhibitors 1,5-anhydro-d-glucitol <1> [4] 1-deoxynojirimycin <1, 8> (<8> 88% inhibition at 10 mM [11]) [9, 11] 2-amino-2-deoxy-d-glucose <1> (<1> competitive with glucose [9]) [9] 2-deoxy-2-fluoro-a-d-glucose 1-fluoride <1> (<1> inhibits reaction with a,a-trehalose [3]) [3] 2-deoxy-2-fluoro-glucose 1-phosphate <1> [3] ADP <8> (<8> 34% inhibition at 10 mM [11]) [11] ATP <8> (<8> inhibits trehalose degradation, but stimulates synthetic activity [11]) [11] CoCl2 <8> (<8> 39% inhibition at 1 mM [11]) [11] Cu2+ <7> (<7> complete inactivation at 1 mM [10]) [10] CuSO4 <8> (<8> 37% inhibition at 1 mM [11]) [11] d-galactose <1> [3] d-glucal <1> [4, 9] d-glucono-d-lactone <1> [9] d-glucose <1> [4] d-xylose <1> [3] NaCl <1> (<1> competitive with respect to phosphate [2]) [2] S-trehalose <1> [4] Zn2+ <7> (<7> inactivation at 1 mM [10]) [10] a-d-galactose 1-phosphate <1> [3] a-d-glucose 1-fluoride <1> (<1> competitive with respect to a,a-trehalose in the phosphorolysis direction [3]) [3] a-d-glucose 1-phosphate <1> [3, 4] a-d-glucuronic acid 1-phosphate <1> [3] a-d-mannose 1-phosphate <1> [3] a-d-xylose 1-phosphate <1> [3] isofagomine <1> [9] orthovanadate <1> (<1> competitive against phosphate and a-d-glucopyranosyl phosphate, inhibits forward and reverse reaction [9]) [9] phosphate <8> (<8> inhibits synthetic activity [11]) [11]

635

a,a-Trehalose phosphorylase (configuration-retaining)

2.4.1.231

validamycin A <8> (<8> 99% inhibition at 10 mM [11]) [11] vanadate <1> (<1> phosphate mimic, inhibits phosphorolysis of a-d-glucose 1-fluoride by forming a ternary complex [3]) [3] Activating compounds AMP <8> (<8> stimulates phosphorolytic activity at 10 mM [11]) [11] ATP <8> (<8> stimulates synthetic activity at 10 mM [11]) [11] Metals, ions Mg2+ <1> (<1> each molecule contains one Mg2+ [4]) [4] Turnover number (min±1) 6.6 <1> (a-d-glucose 1-fluoride, <1> 30 C, pH 6.6 [3]) [3] 570 <1> (a-d-glucose 1-phosphate, <1> 30 C, pH 6.6 [4]) [4] 600 <1> (a-d-glucose, <1> 30 C, pH 6.6 [2]) [2] 600 <1> (a-d-glucose 1-phosphate, <1> 30 C, pH 6.6 [2]) [2] 618 <1> (a-d-glucose, <1> 30 C, pH 6.6 [9]) [9] 798 <1> (a,a-trehalose, <1> 30 C, pH 6.6 [4]) [4] 840 <1> (phosphate, <1> 30 C, pH 6.6 [2]) [2] 846 <1> (a,a-trehalose, <1> 30 C, pH 6.6 [9]) [9] 900 <1> (a,a-trehalose, <1> 30 C, pH 6.6 [2]) [2] Specific activity (U/mg) 4.2 <7> [10] 4.35 <8> [11] 9.8 <1> [1] 9.85 <1> [2] 13.9 <2> [5] Km-Value (mM) 0.63 <3> (a-d-glucose, <3> 30 C, pH 6.3 [6]) [6] 2.4 <1> (phosphate, <1> 30 C, pH 6.6 [2]) [2] 2.8 <1> (a-d-glucose 1-phosphate, <1> 30 C, pH 6.6 [2]) [2] 4.2 <4> (phosphate) [7] 4.7 <8> (phosphate, <8> 30 C, pH 7 [11]) [11] 5 <3> (phosphate, <3> 30 C, pH 7 [6]) [6] 5.4 <2> (phosphate, <2> pH 7 [5]) [5] 5.9 <1> (a-d-glucose 1-fluoride, <1> 30 C, pH 6.6 [3]) [3] 6.3 <8> (a-d-glucose 1-phosphate, <8> 30 C, pH 7 [11]) [11] 24 <8> (a-d-glucose, <8> 30 C, pH 7 [11]) [11] 31 <1> (a-d-glucose, <1> 30 C, pH 6.6 [9]) [9] 34.3 <2> (a-d-glucose, <2> pH 6.3 [5]) [5] 38 <4> (a-d-glucose 1-phosphate) [7] 39 <1> (a-d-glucose, <1> 30 C, pH 6.6 [2]) [2] 44.6 <2> (a-d-glucose 1-phosphate, <2> pH 6.3 [5]) [5] 47 <3> (a-d-glucose 1-phosphate, <3> 30 C, pH 6.3 [6]) [6] 61 <8> (a,a-trehalose, <8> 30 C, pH 7 [11]) [11] 74.8 <2> (a,a-trehalose, <2> pH 7 [5]) [5] 75 <4> (a,a-trehalose) [7]

636

2.4.1.231

a,a-Trehalose phosphorylase (configuration-retaining)

75 <3> (a,a-trehalose, <3> 30 C, pH 7 [6]) [6] 87 <1> (a,a-trehalose, <1> 30 C, pH 6.6 [9]) [9] 91 <1> (a,a-trehalose, <1> 30 C, pH 6.6 [2]) [2] 505 <4> (a-d-glucose) [7] Ki-Value (mM) 0.3 <1> (a-d-glucose, <1> 30 C, pH 6.6 [3]) [3] 1 <1> (orthovanadate, <1> 30 C, pH 6.6 [9]) [9] 1.6 <1> (a-d-mannose 1-phosphate, <1> 30 C, pH 6.6 [3]) [3] 2 <8> (phosphate, <8> 30 C, pH 7 [11]) [11] 2.4 <1> (a-d-glucose 1-phosphate, <1> 30 C, pH 6.6 [3]) [3] 10.6 <1> (2-deoxy-2-fluoro-glucose 1-phosphate, <1> 30 C, pH 6.6 [3]) [3] 11 <1> (a-d-xylose 1-phosphate, <1> 30 C, pH 6.6 [3]) [3] 11.5 <1> (a-d-galactose 1-phosphate, <1> 30 C, pH 6.6 [3]) [3] 13.4 <1> (a-d-glucuronic acid 1-phosphate, <1> 30 C, pH 6.6 [3]) [3] 46 <1> (2-amino-2-deoxy-d-glucose, <1> 30 C, pH 6.6 [9]) [9] 67 <1> (NaCl, <1> 30 C, pH 6.6 [2]) [2] 1000 <1> (d-galactose, <1> 30 C, pH 6.6 [3]) [3] 1000 <1> (d-xylose, <1> 30 C, pH 6.6 [3]) [3] pH-Optimum 6-7 <8> (<8> synthesis of trehalose [11]) [11] 6-7.5 <8> (<8> phosphorolytic reaction [11]) [11] 6.2 <1> (<1> reverse reaction [2]) [2] 6.5-6.8 <7> [10] 6.6 <1> (<1> forward reaction [2]) [2] 6.7 <2> (<2> phosphorolytic reaction [5]) [5] 7 <4> (<4> phosphorolytic reaction [7]) [7] 7 <4> (<4> synthesis of trehalose [7]) [7] pH-Range 6.2-6.6 <1> (<1> decrease in enzyme activity below pH 6.2 and above pH 6.6 [2]) [2] Temperature optimum ( C) 32.5-35 <7> (<7> phosphorolysis [10]) [10] 35-37.5 <7> (<7> synthesis of trehalose [10]) [10] 36 <2> [5]

4 Enzyme Structure Molecular weight 60000 <1> (<1> SDS-PAGE [1]; <1> gel filtration [2]) [1, 2] 61000 <1> (<1> SDS-PAGE [2]) [2] 88000 <2> (<2> SDS-PAGE [5]) [5] 120000 <4, 7> (<4> sedimentation equilibrium experiments [7]; <7> gel filtration [10]) [7, 10] 240000 <8> (<8> native PAGE, gel filtration [11]) [11] 637

a,a-Trehalose phosphorylase (configuration-retaining)

2.4.1.231

Subunits dimer <4> (<4> 2 * 61000, SDS-PAGE [7]; <7> 2 * 60000, SDS-PAGE [10]) [7, 10] monomer <1> (<1> 1 * 60000, SDS-PAGE, gel filtration [2]) [2] tetramer <8> (<8> 4 * 61000, SDS-PAGE [11]) [11]

5 Isolation/Preparation/Mutation/Application Source/tissue fruit body <3, 4, 5, 6, 8> [6, 7, 8, 11] mycelium <2, 3, 4, 5, 6, 8> [5, 6, 7, 8, 11] pileus <2> [5] stipe <2> [5] Purification <1> (95% purity [1]) [1, 2] <2> [5] <3> (partial [6]) [6] <4> (homogeneity [7]) [7] <7> [10] <8> [11] Cloning <2> [5]

6 Stability pH-Stability 6-7 <2> (<2> stable [5]) [5] Temperature stability 4 <1> (<1> unstable, loses activity within a few hours, glycerol, PEG 4000 or trehalose stabilize [2]) [2] 20 <1> (<1> unstable, loses activity within a few hours, glycerol, PEG 4000 or trehalose stabilize [2]) [2] 30 <2> (<2> stable below [5]) [5] 35 <7> (<7> stable up to 35 C, decrease of activity above [10]) [10] 40 <2, 8> (<2> decrease of activity [5]; <8> almost complete loss of activity after 60 min [11]) [5, 11] General stability information <8>, highly unstable during purification, glycerol, phosphate, dithiothreitol and PMSF stabilize [11] Storage stability <8>, -80 C, stable for at least 6 months [11]

638

2.4.1.231

a,a-Trehalose phosphorylase (configuration-retaining)

References [1] Eis, C.; Albert, M.; Dax, K.; Nidetzky, B.: The stereochemical course of the reaction mechanism of trehalose phosphorylase from Schizophyllum commune. FEBS Lett., 440, 440-443 (1998) [2] Eis, C.; Nidetzky, B.: Characterization of trehalose phosphorylase from Schizophyllum commune. Biochem. J., 341, 385-393 (1999) [3] Eis, C.; Nidetzky, B.: Substrate-binding recognition and specificity of trehalose phosphorylase from Schizophyllum commune examined in steadystate kinetic studies with deoxy and deoxyfluoro substrate analogues and inhibitors. Biochem. J., 363, 335-340 (2002) [4] Eis, C.; Watkins, M.; Prohaska, T.; Nidetzky, B.: Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune. Biochem. J., 356, 757-767 (2001) [5] Han, S.E.; Kwon, H.B.; Lee, S.B.; Yi, B.Y.; Murayama, I.; Kitamoto, Y.; Byun, M.O.: Cloning and characterization of a gene encoding trehalose phosphorylase (TP) from Pleurotus sajor-caju. Protein Expr. Purif., 30, 194-202 (2003) [6] Kitamoto, Y.; Akashi, H.; Tanaka, H.; Mori, N.: a-Glucose-1-phosphate formation by a novel trehalose phosphorylase from Flammulina velutipes. FEMS Microbiol. Lett., 55, 147-149 (1988) [7] Kitamoto, Y.; Osaki, N.; Tanaka, H.; Sasaki, H.; Mori, N.: Purification and properties of a-glucose 1-phosphate-forming trehalose phosphorylase from a basidiomycete, Pleurotus ostreatus. Mycoscience, 41, 607-613 (2000) [8] Kitamoto, Y.; Tanaka, H.; Osaki, N.: Survey of a-glucose 1-phosphate forming trehalose phosphorylase and trehalase in various fungi including basidiomycetous mushrooms. Mycoscience, 39, 327-331 (1998) [9] Nidetzky, B.; Eis, C.: a-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors. Biochem. J., 360, 727-736 (2001) [10] Saito, K.; Kase, T.; Takahashi, E.; Horinouchi, S.: Purification and characterization of a trehalose synthase from the basidiomycete Grifola frondosa. Appl. Environ. Microbiol., 64, 4340-4345 (1998) [11] Wannet, W.J.; Op den Camp, H.J.; Wisselink, H.W.; van der Drift, C.; Van Griensven, L.J.; Vogels, G.D.: Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus. Biochim. Biophys. Acta, 1425, 177-188 (1998)

639

Initiation-specific a-1,6-mannosyltransferase

2.4.1.232

1 Nomenclature EC number 2.4.1.232 Systematic name GDP-mannose:oligosaccharide 1,6-a-d-mannosyltransferase Recommended name initiation-specific a-1,6-mannosyltransferase Synonyms GDP-mannose:glycolipid 1,6-a-d-mannosyltransferase GDP-mannose:oligosaccharide 1,6-a-d-mannosyltransferase a-1,6-mannosyltransferase glycolipid 6-a-mannosyltransferase CAS registry number 346003-17-6 (Schizosaccharomyces pombe gene och1+)

2 Source Organism <1> Saccharomyces cerevisiae (mnn1 mutant lacking a-1,3-mannosyltransferase [1,2]; Och1p [3,5,7]; Mnn9p [9]) [1, 2, 3, 5, 7, 9] <2> Schizosaccharomyces pombe (Och1p [4,6]) [4, 6] <3> Candida albicans (NIH B-792 serotype B strain [8]) [8]

3 Reaction and Specificity Catalyzed reaction Transfers an a-d-mannosyl residue from GDP-mannose into lipid-linked oligosaccharide, forming an a-1,6-d-mannosyl-d-mannose linkage (requires Mn2+ . In Saccharomyces cerevisiae, this enzyme catalyses an essential step in the outer chain elongation of N-linked oligosaccharides. Man8 GlcNAc and Man9 GlcNAc are equally good substrates) Reaction type hexosyl group transfer

640

2.4.1.232

Initiation-specific a-1,6-mannosyltransferase

Natural substrates and products S Additional information <1, 2> (<1> involved in glycoprotein biosynthesis, enzyme catalyzes the first step specific to N-linked oligosaccharide synthesis and the biosynthesis of the outer chain of mannoproteins [1]; <2> enzyme is required for the initiation of outer chain elongation of Nlinked oligosaccharides of cell wall mannoproteins, transcriptional control of the och1+ gene, gene expression is not regulated during the cell cycle, but is induced by salt stress through the transcription factor Atf1p [4]; <1> OCH1 gene encoded enzyme initiates the polymannose outer chain elongation of N-glycans, regulation of the OCH1 transcription [5]; <2> SpOch1p is a key enzyme of outer chain elongation of N-linked oligosaccharides [6]; <1> Och1p is essential for the outer chain elongation of Nlinked oligosaccharides [7]; <1> Mnn9p is required for the addition of the long a-1,6-mannose backbone of the complex mannan and for the complex glycosylation of secreted proteins [9]; <1> Och1p is functional in the initiation of a-1,6-polymannose outer chain addition to the N-linked core oligosaccharide in yeast, Man5 GlcNAc2 and Man8 GlcNAc2 [3]; <1> enzyme initiates outer chain formation of oligosaccharides [2]) [1-7, 9] P ? Substrates and products S GDP-mannose + Man8 GlcNAc <1> (<1> Man8 GlcNAc from thyroglobulin Man9 GlcNAc by treatment with yeast specific mannosidase, specificity, enzyme catalyzes addition of mannose to the a-1,3-mannose of the substrate, initiates outer chain formation [2]) (Reversibility: ? <1> [2]) [2] P GDP + Man9 GlcNAc <1> [2] S GDP-mannose + Man8 GlcNAc2 -PA <1> (<1> best acceptor for Och1p, initiation-specific a-1,6-mannosyltransferase that requires the intact structure of Man8 GlcNAc for efficient mannose outer chain initiation [7]) (Reversibility: ? <1> [7]) [7] P GDP + Man9 GlcNAc2 -PA <1> (<1> additional Man is attached with an a1,6-linkage at the site where mannose outer chain elongation initiates [7]) [7] S GDP-mannose + Man9 GlcNAc <1> (<1> Man9 GlcNAc from thyroglobulin, specificity, enzyme catalyzes addition of mannose to the a-1,3-mannose of the substrate, initiates outer chain formation [2]) (Reversibility: ? <1> [2]) [2] P GDP + Man10 GlcNAc <1> [2] S GDP-mannose + Man9 GlcNAc2 -PA <2> (<2> enzyme incorporates a-1,6linked mannose into the substrate [6]) (Reversibility: ? <2> [6]) [6] P GDP + Man10 GlcNAc2 -PA S GDP-mannose + Mana(1-2)Mana(1-6)[Mana(1-2)Mana(1-3)]Mana(16)[Mana(1-2)Mana(1-2)Mana(1-3)]Manb(1-4)GlcNAc <1> (<1> Man9 GlcNAc from rat liver glycoproteins and thyroglobulin, enzyme initiates outer chain synthesis, the mannose residue added is a-1,6-linked to the a-1,6-mannose residue of the substrate, removal of the a-1,2-linked man-

641

Initiation-specific a-1,6-mannosyltransferase

P S

P S

P S

P S

P

2.4.1.232

nose residue from Man9 GlcNAc is not essential for enzyme activity [1]) (Reversibility: ? <1> [1]) [1] GDP + Mana(1-2)[Mana(1-6)]Mana(1-6)[Mana(1-2)Mana(1-3)] Mana(1-6)[Mana(1-2)Mana(1-2)Mana(1-3)]Manb(1-4)GlcNAc <1> (<1> Man10 GlcNAc [1]) [1] GDP-mannose + Mana(1-2)Mana(1-6)[Mana(1-3)]Mana(1-6)[Mana(12)Mana(1-2)Mana(1-3)]Manb(1-4)GlcNAc <1> (<1> Man8 GlcNAc from rat liver glycoproteins and thyroglobulin, enzyme initiates outer chain synthesis, the mannose residue added is a-1,6-linked to the a-1,6-mannose residue of the substrate [1]) (Reversibility: ? <1> [1]) [1] GDP + Mana(1-2)[Mana(1-6)]Mana(1-6)[Mana(1-3)]Mana(1-6) [Mana(1-2)Mana(1-2)Mana(1-3)]Manb(1-4)GlcNAc <1> (<1> Man9 GlcNAc [1]) [1] GDP-mannose + Mana(1-3)Mana(1-2)Mana(1-3)Mana(1-2)Man <3> (<3> mannopentaose from Candida albicans, substrate specificity study, structural requirement of enzyme is Mana(1-3)Mana(1-) [8]) (Reversibility: ? <3> [8]) [8] GDP + Mana(1-3)[Mana(1-6)]Mana(1-2)Mana(1-2)Mana(1-2)Man <3> (<3> structure, presence of an a-1,6 branching mannose unit [8]) [8] GDP-mannose + pyridylaminated oligosaccharide acceptor <1, 2> (<2> SpOch1p [6]; <1,2> substrate specificity [6,7]; <1> various high mannose-type oligosaccharides as acceptors, Man8 GlcNAc2 -PA is the best acceptor, the loss of 1 or 2 a-1,2-mannoses from Man8 GlcNAc2 reduces the activity [7]) (Reversibility: ? <1,2> [6,7]) [6, 7] ? Additional information <1, 3> (<1> no acceptors for Och1p: Man5 GlcNAc2 completely lacking a-1,2-Man, Man8 GlcNAcOH [7]; <3> enzyme also transfers the a-1,6-linked branching mannose unit to the mannan of Saccharomyces cerevisiae [8]) [7, 8] ?

Activating compounds Triton X-100 <1> (<1> requirement, maximum activity at 0.5-2% [1]) [1] Additional information <2> (<2> och1+ gene expression is induced by NaCl and KCl through the transcription factor Atf1p [4]) [4] Metals, ions Mn2+ <1> (<1> absolute requirement, optimum concentration: 10 mM, Ca2+ , Mg2+ , Co2+ or Zn2+ cannot substitute for Mn2+ [1]) [1] Km-Value (mM) 0.35 <1> (Mana(1-2)Mana(1-6)[Mana(1-2)Mana(1-3)]Mana(1-6)[Mana(12)Mana(1-2)Mana(1-3)]Manb(1-4)GlcNAc) [1] 0.39 <1> (Mana(1-2)Mana(1-6)[Mana(1-3)]Mana(1-6)[Mana(1-2)Mana(12)Mana(1-3)]Manb(1-4)GlcNAc) [1] pH-Optimum 7.1-7.6 <1> (<1> in Tris maleate buffer [1]) [1]

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Initiation-specific a-1,6-mannosyltransferase

Temperature optimum ( C) 30 <1> (<1> assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Localization membrane <1> (<1> Mnn9p is a membrane-associated protein, type II transmembrane protein [9]) [9] Cloning <1> (transcription of the OCH1 gene encoding a-1,6-mannosyltransferase is regulated by 2 transcription factors, Swi4 and Skn7, it is controlled by the ubiquitin-dependent degradation pathway [5]; OCH1 gene encodes Och1p, an a-1,6-mannosyltransferase [3,7]; MNN9 gene encoding Mnn9p is cloned and sequenced [9]) [3, 5, 7, 9] <2> (transcriptional control of the och1+ gene, gene expression is not regulated during the cell cycle, but is induced by NaCl and KCl through the transcription factor Atf1p [4]; och1+ gene encodes a-1,6-mannosyltransferase, recombinant expression and cloning, och1+ gene knockout fission yeast [6]) [4, 6] Engineering Additional information <1, 2> (<2> och1+ gene deficient strain shows loss of mannose transfer from GDP-mannose to Man9 GlcNAc2 -PA and loss of highmannose type sugar chain glycosylation, och1 mutant that does not synthesize the outer chains containing mannose on acid phosphatase [6]; <1> Mnn9 mutant strain [9]; <1> och1 mutants, och1 mutation causes a complete loss of the a-1,6-polymannose outer chain [3]) [3, 6, 9]

References [1] Romero, P.A.; Herscovics, A.: Glycoprotein biosynthesis in Saccharomyces cerevisiae. Characterization of a-1,6-mannosyltransferase which initiates outer chain formation. J. Biol. Chem., 264, 1946-1950 (1989) [2] Reason, A.J.; Dell, A.; Romero, P.A.; Herscovics, A.: Specificity of the mannosyltransferase which initiates outer chain formation in Saccharomyces cerevisiae. Glycobiology, 1, 387-391 (1991) [3] Nakanishi-Shindo, Y.; Nakayama, K.; Tanaka, A.; Toda, Y.; Jigami, Y.: Structure of the N-linked oligosaccharides that show the complete loss of a-1,6polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae. J. Biol. Chem., 268, 26338-26345 (1993) [4] Yamamoto, K.; Okamoto, M.; Yoko-o, T.; Jigami, Y.: Salt stress induces the expression of the Schizosaccharomyces pombe och1+, which encodes an initiation-specific a-1,6-mannosyltransferase for N-linked outer chain synthesis of cell wall mannoproteins. Biosci. Biotechnol. Biochem., 67, 927-929 (2003) 643

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[5] Cui, Z.; Horecka, J.; Jigami, Y.: Cdc4 is involved in the transcriptional control of OCH1, a gene encoding a-1,6-mannosyltransferase in Saccharomyces cerevisiae. Yeast, 19, 69-77 (2002) [6] Tsukahara, K.; Watanabe, T.; Yokoo, T.; Chigami, Y.: Schizosaccharomyces pombe och1+ gene encoding a-1,6-mannosyltransferase and use of och1+ gene knockout fission yeast for production of glycoprteins with reduced glycosylation. Jpn. Kokai Tokkyo Koho, 11pp (2001) [7] Nakayama, K.; Nakanishi-Shindo, Y.; Tanaka, A.; Haga-Toda, Y.; Jigami, Y.: Substrate specificity of a-1,6-mannosyltransferase that initiates N-linked mannose outer chain elongation in Saccharomyces cerevisiae. FEBS Lett., 412, 547-550 (1997) [8] Suzuki, A.; Shibata, N.; Suzuki, M.; Saitoh, F.; Takata, Y.; Oshie, A.; Oyamada, H.; Kobayashi, H.; Suzuki, S.; Okawa, Y.: Characterization of a-1,6-mannosyltransferase responsible for the synthesis of branched side chains in Candida albicans mannan. Eur. J. Biochem., 240, 37-44 (1996) [9] Yip, C.L.; Welch, S.K.; Klebl, F.; Gilbert, T.; Seidel, P.; Grant, F.; O'Hara, P.J.; MacKay, V.L.: Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins. Proc. Natl. Acad. Sci. USA, 91, 2723-2727 (1994)

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