Siderophores from Microorganisms and Plants (Structure and Bonding)

8 Structure

and Bonding

Editors: M. J. Clarke, Chestnut Hill • J. B. Goodenough, Oxford J. A. Ibers, Evanston • C. K. J¢rgensen, Gen6ve D. M. P. Mingos, Oxford • J. B. Neilands, Berkeley G. A. Palmer, Houston • D. Reinen, Marburg P. J. Sadler, London • R. Weiss, Strasbourg R. J. P. Williams, Oxford

Siderophores from Microorganisms and Plants

With Contributions by A. Chimiak R.C. Hider A. Liu J.B. Neilands K. Nomoto Y. Sugiura

With 35 Figures and 16 Tables

Springer-Verlag Berlin Heidelberg New York Tokyo 1984

Editorial Board

Professor Michael J. Clarke, Boston College, Department of Chemistry, Chestnut Hill, Massachusetts 02167, U.S.A. Professor John B. Goodenough, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, Great Britain Professor James A. Ibers, Department of Chemistry, Northwestern University, Evanston, Illinois 60201, U.S.A. Professor Christian K. Jcrgensen, D6pt. de Chimie Min6rale de l'Universit6, 30 quai Ernest Ansermet, CH-1211 Gen6ve 4 Professor David Michael P. Mingos, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, Great Britain Professor Joe B. Neilands, Biochemistry Department, University of California, Berkeley, California 94720, U.S.A. Professor Graham A. Palmer, Rice University, Department of Biochemistry, Wiess School of Natural Sciences, P. O. Box 1892, Houston, Texas 77251, U.S.A. Professor Dirk Reinen, Fachbereich Chemie der Philipps-Universit/it Marburg, Hans-Meerwein-Stral3e, D-3550 Marburg Professor Peter J. Sadler, Birkbeck College, Department of Chemistry, University of London, London WC1E 7HX, Great Britain Professor Raymond Weiss, Institut Le Bel, Laboratoire de Cristallochimie et de Chimie Structurale, 4, rue Blaise Pascal, F-67070 Strasbourg Cedex Professor Robert Joseph P. Williams, Wadham College, Inorganic Chemistry Laboratory, Oxford OX1 3QR, Great Britain

ISBN 3-540-13649-5 Springer-Verlag Berlin Heidelberg New York Tokyo ISBN 0-387-13649-5 Springer Verlag New York Heidelberg Berlin Tokyo

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Foreword

This is the first volume of Structure and Bonding to be devoted entirely to siderophores. These compounds have become a popular subject for study by scientists whose interests range from synthetic organic chemistry to molecular genetics. Annually a substantial number of papers on siderophore systems are presented before such bodies as the American Society for Microbiology. Siderophores were not always so much in fashion even though three decades have passed since their general presence and low-iron induction in microbes was demonstrated. Volume 1 of Structure and Bonding carried a review on the compounds now classed as "siderophores" (Gr. "iron bearers") under the somewhat cumbersome title: "Naturally Occurring Non-Porphyrin Iron compounds") ) By the mid-1950's ferrichrome had been postulated on the basis of its structure, properties and, especially, its induction at low iron growth, to function biologically as an iron carrier. 2) Mycobactin and terregens factor, which initially had no connection with iron, could be related to ferrichrome on the basis of binding of Fe(III) and/or growth tests with Pilobolus kleinii or Arthrobacter terregens.3) At the end of the decade of the 1950's Swiss workers produced a large number of ferrioxamines from Actinomyces sp. and coined the word siderochromes for all "red-brown" iron-containing compounds of this t y p e : ) This was subsequently extended to the second major chemical class of microbial products with equivalent function, the phenolates and catechols. 5) As all of these ligands yield with Fe(III) charge transfer bands in the visible, the designation siderochrome seemed appropriate. However, Lankford 6) pointed out that some of the ligands may not afford colored complexes and as a consequence he suggested the now generally accepted term of siderophore. His suggestion was timely since compounds with siderophore activity are now turning up with amino, imino and carboxylate functions only. These ligands do not generate with ferric ion much if any color. 7) By definition, therefore, a siderophore is a low molecular weight, virtually ferric ion specific ligand induced by low iron growth of a microorganism. Siderophores appear to be so uniformly present in aerobic and facultarive anaerobic microbial life that failure to detect them prompts a research paper on the fact. Clearly, tests for hydroxamate or catechol functions will miss the amino/imino carboxylates. Demonstration that the low iron grown supernatant has the power to reverse iron starvation imposed by a nonutilized chelator is a more inclusive test. Still, failure to detect a siderophore may be a consequence of the specific cultural conditions. Furthermore, it is

VI

Foreword

evident that enteric, and probably other species, possess uptake systems for exogenous siderophores. Finally, siderophores may dwell in the lipid phase of the microbial membrane, s) Notwithstanding these qualifications, failure of a microorganism to extract iron from a polymer when the macromolecule and cells are separated by a membrane has been taken as evidence for absence of siderophore. 9) Here one would have to be certain that the carrier iron does not bind to the membrane. Do siderophores occur in plants and animals? The paper by Sugiura and Nomoto in this volume suggests that higher plants do indeed synthesize a line of compounds which they designate as phytosiderophores. In plants, in contrast to microbes, genetic confirmation of function is less easily obtained. Animal tissue has yet to yield a bona fide siderophore but mutants of SV40 transfomred BALB/3 T 3 cell line adapted to growth in picolinic acid produce "siderophore-like" growth factors.I°) What are the prospects for practical applications of siderophores? The use of deferdferrioxamine B mesylate (Desferal) for deferration of siderotic patients is well known. Unfortunately, the drug is inactive via the oral route and must be injected; otherwise, it is relatively effective and non-toxic. The observation that siderophores may complex loosely bound, errant iron atoms and so eliminate or diminish the toxicity of partially reduced oxygen species, such as superoxide anion and peroxide, suggests that compounds of this type may have possibilities for treatment of rheumatoid arthritis and other degenerative diseasesJ 1) The chemotherapeutic potential of siderophores remains unexploited. Antibioses related to iron have been relegated to three classes.12) In Type I there is a simple deprivation of iron, as provoked by transferdn or deferdferdchrome A. In Type II, as exemplified by albomycin and ferrimycin, the lethal moiety is smuggled into the cell on a siderophore transport system. In Type III a compound such as iron bleomycin binds to the DNA and effects strand scission through generation of oxygen radicals. The seemingly disparate fields of infection and neoplasia, and the role therein of iron and siderophores, is discussed in a recent review by Weinberg. 13) The identifications of the tumor antigen of transformed cells as the transferrin receptor 14) and the very recent report 15) that ras proteins in human carcinoma cells complex with the receptor underline the significance of iron assimilation in the neoplastic state. Although phytopathogenic microorganisms in general synthesize siderophores, ~6)no correlation between iron assimilation and virulence has yet been made in plants as has been possible in human and animal subjectsJ 7) It is hoped that the present volume of Structure and Bonding will stimulate the interest of colleagues in both pure and applied branches of science to investigate further the properties and behaviour of the unique family of compounds grouped generically under the term siderophores. J. B. NEILANDS Berkeley, California, May 1984

Foreword

VII

References

1. Neilands, J. B.: Structure and Bonding I, 59 (1966) 2. Neilands, J. B.: Bactedol. Rev. 21, 101 (1957) 3. Burton, M. O., Sowden, F. J., Lochhead, A. G.: Can. J. Biochem. and Physiol. 32, 400 (1954) 4. Prelog, V.: Pure Appl. Chem. 6, 327 (1963) 5. Neilands, J. B.: In Inorganic Biochemistry (G. L. Eichhom Ed.), Elsevier, p. 167 (1973) 6. Lankford, C. E.: Crit. Rev. Microbiol. 2, 273 (1973) 7. Smith, M. J., Neilands, J. B.: J. Plant Nutr., in press 8. Ratledge, C., Marshall, B. V.: Biochim. Biophys. Acta 279, 58 (1972) 9. Simonson, C., Trivett, T., DeVoe, I. W.: Infect. Immun. 31, 547 (1981) 10. Fernandez-Pol, J. A.: In Microbiology 1983. (D. Schlessinger Ed.), Am. Soc. Microbiol., Washington, D. C., p. 313 11. Hoe, S., Rowley, D. A., Halliwell, B.: Chem.-Biol. Interactions 41, 75 (1982) 12. Neilands, J. B., Valenta, J. R.: In Metal Ions in Biological Systems, Vol. 19, (H. Sigel Ed.), Marcel-Dekker, New York, in press 13. Weinberg, E. D.: Physiol. Rev. 64, 65 (1984) 14. Trowbridge, I. S., Omary, M. B.: Proc. Natl. Acad. Sci. USA 78, 3039 (1981) 15. Finkel, T., Cooper, G. M.: Cell 136, 1115 (1984) 16. Leong, S. A., Neilands, J. B.: Arch. Biochem. Biophys. 218, 351 (1983) 17. Weinberg, E. D.: Microbiol. Rev. 42, 45 (1978)

Table of Contents

Methodology of Siderophores J. B. Neilands . . . . . . . . . . . . . . . . . . . . . . . . Siderophore Mediated Absorption of Iron R. C. Hider . . . . . . . . . . . . . . . . . . . . . . . . .

25

Lysine Analogues of Siderophores A. Chimiak, J. B. Neilands . . . . . . . . . . . . . . . . .

89

Mutational Analysis of Rhodotorulic Acid Synthesis in

Rhodotorula pilimanae A. Liu, J. B. Neilands . . . . . . . . . . . . . . . . . . . .

97

Phytosiderophores - Structures and Properties of Mugineic Acids and Their Metal Complexes Y. Sugiura, K. Nomoto . . . . . . . . . . . . . . . . . . .

107

Author Index Volumes 1-58

137

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

Methodology of Siderophores J.B. Neilands Department of Biochemistry, University of California, Berkeley, CA 94720, USA

Siderophores, defined as iron(Ill) specific transport compounds, are widely distributed in aerobic and facultative anaerobic microbial species. The list of microbes known to form siderophores includes various enteric bacteria; human, animal and plant pathogenic bacteria and fungi; soil microorganisms; Gram positive and negative species, blue green alga (Cyanobacteria) and higher algae; nitrogen fixing bacteria; and many types of fungi including all species of Penicillia, Neurospora, basidiomycetes and certain types of yeast. As natural products, siderophores are classed conveniently as hydroxamates, catechols and "miscellaneous", the latter possibly structurally related to the phytosiderophores of plants. A variety of standard methods has evolved for detection, enhanced production, isolation, characterization and synthesis of the siderophores. The cloning of the enterobactin and aerobactin systems of Escherichia coli has been reported. A very large number of siderophores remain to he characterized as either known compounds or new products. The opportunities for technical exploitation of the substantial reservoir of basic research knowledge of siderophores abound in fields as diverse as clinical medicine and plant pathology.

I.

Introduction

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

2

II.

Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Detection and Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Chemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Biological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Siderophore Auxotrophes . . . . . . . . . . . . . . . . . . . . . . . . . . b. Wild-Type CeUs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Minimal Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Carbon Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Deferration Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Structure and Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Chemical Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Molecular Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 5 5 7 7 11 12 12 13 13 14 14 16 18 18

III. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20

IV. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23

Structure and Bonding 58

© SpringeroVerlagBerlin Heidelberg 1984

2

J. B. Neilands

I. Introduction Siderophores, defined as low molecular weight, virtually Fe(III) specific ligands, are formed generally by aerobic and facultative anaerobic bacteria and by fungi. According to present knowledge they do not appear to be present in strictly anaerobic bacteria, such as the Clostridia, probably because the low potential of the growth media of these species maintains the iron, which they require for synthesis of their iron-sulfur and other proteins, in a more soluble Fe(II) state. Siderophores may not occur in strict lactic acid bacteria since these lack cytochromes of all types, hydroperoxidases, and substitute the cobalt-containing ribonucleotide reductase for the more usual iron-containing form of this enzyme. Indeed, Lactobacillus plantarum, when grown in low iron media, has been shown 1) to contain only 1.7 atoms of iron per bacterial cell and hence it is possible that this species has zero requirement for this usually biologically precious metal. A species of Legionella2) does not appear to form siderophores of any kind when cultured in laboratory media and it is not known how the pathogen acquires iron in the host. No siderophore has thus far been reported from Saccharomyces spp., but other fungi commonly make siderophores of the ferrichrome type. So, apart from the few exceptions just noted, siderophores seem to be almost universally present in the microbial world. It is assumed that iron was firmly established as a bio-essential element during the anaerobic phase of life on planet Earth 3). In view of the substantial solubility of Fe(II) at biological pH, it is further speculated that specific ligands were not required and that iron was assimi!atedby pathways analogous to those followed by other divalent metal ions. However, once a prokaryotic species, probably Cyanobacteria (blue-green algae), achieved the capacity to generate O2 gas, the surface iron oxidized and precipitated as the oxyhydroxide polymer, the analytical composition of which is FeOOH. The relevant reactions leading to a trimer are Fe3+ + 3 (OH-) = Fe(OH)3 3Fe(OH)3 = HO-Fe--[O-Fe-]O-Fe-OH + 2H20

I

OH

f

OH

I

OH

Taking 10-38 M as an approximation of the solubility product constant (Ksp) for Fe 3+, the maximum concentration of soluble ferric ion at pH 7 is given by K,p

= (Fe3+)(OH-)3 = 10-3SM

(Fe 3+) ---- 10-38/10-21

= 10-17M

This very small number would be diminished even further by taking 7.4 as a more reasonable value for biological pH. Besides hydroxyl ion, specific iron (Ill) binding proteins such as transferrin, ovotransferrin, lactotransferrin and ferritin will serve to decrease even further the level of free ferric ions. It is clear from this semi-quantitative analysis that the appearance of siderophores concomitantly with the switch from a reducing to an oxidizing atmosphere makes good evolutionary sense. The versatile role of iron as an electron transfer catalyst - the redox potentials from the ferredoxins to cytochrome c oxidase span more or less 1000 m y - and the requirement

MethodologyofSiderophores

3

of the metal ion in such crucial biological reactions as fixation of dinitrogen, photosynthesis, deoxyribotide synthesis4) and oxygen metabolism underscore the need for an effective assimilation system. But precious though iron may be in its physiological roles, it can also be extremely toxic5). Its capacity to generate OH" greatly exacerbates the toxicity of H202 and O~. Thus in the Fenton reaction H 2 0 2 + F e 2+ =

HO" + HO- + Fe3+

iron acts as a catalyst to produce the highly reactive OH" radical. For this reason the uptake of iron is carefully regulated at the membrane level. This appears to be true in bacteria, fungi, plants and animals. In the last named species it is well established that iron is recycled internally and this may be the case for lower organisms as well. Although living species may be expected - in view of the physiological importance of the element - to have diverse pathways for the uptake of iron, it is the high affinity siderophore-mediated process in microbial cells which is the most amenable to study. This is because the system is comprised of two parts, namely, the biosynthetic pathway leading to the siderophore and the transport system for the chelated iron. It is thus possible to insert independent genetic blocks in both parts of the siderophore system. The microbe for which we have the greatest depth of knowledge vis-a-vis its genetics is Escherichia coli, and hence considerable progress has been made in unraveling the various iron assimilation systems in this microorganism. No less than four such systems have thus far been described in E. coli, namely, (i) enterobactin, (ii) ferrichrome and other hydoxamate siderophores, (iii) citrate, and (iv) plasmid encoded systems such as the aerobactin system found on pColV. The molecular mechanism whereby iron regulates at least one of these systems will soon be known in detail and it is an article of faith among the microbial iron mongers that these data may suggest how the corresponding controls operate in fungi, plants and animals. Simple iron deficiency anemia is the single most important nutritional deficiency in much of the world and basic research with microorganisms may indicate how this condition can be corrected in the human, where it seems to be at root an inability to use efficiently the dietary iron. Genetic analysis of E. coli shows beyond doubt that the role of siderophores is in iron metabolism. Thus genetic blocks in either biosynthesis or transport of enterobactin can be overcome by administration of massive doses of iron salts or by trace levels of a metalfree siderophore that is utilized. This demonstrates that the role of the ligand is to make iron available to the cell. Iron assimilation can be correlated with public health in many other ways. The tumor antigen has now been established with confidence to be the transferrin receptor6). Is is assumed that the enhanced iron requirement associated with the neoplastic state can be assigned to the higher energy requirement associated with unbridled protein synthesis, higher levels of ribotide reductase, and other demands for iron. The transfusion induced siderosis consequent to the particular therapy for thalasemmia (Cooley's anemia), aplastic anemia and, to some extent, sickle cell anemia, is now treated with deferriferrioxamine B, a siderophore from Streptomyces pilosus. Although a number of other siderophores and a wide range of synthetic compounds have been screened, the S. pilosus siderophore, which is marketed as the mesylate salt by CibaGeigy as Desferal, remains the drug of choice7). There is still a need for an orally effective deferration drug. The theory of using the siderophores as the reservoir in which

4

J.B. Neilands

to search for such a nostrum is based primarily on the biological origin of these compounds and their specificity for Fe(III). It has been shown8) recently that a substantial fraction of clinical isolates of E. coli synthesizes an hydroxamate type siderophore, aerobactin, as well as the usual catechol type siderophore, enterobactin, the latter commonly encountered among Gram negative bacteria. In E. coli the aerobactin system is encoded on the ColV ("virulence") plasmid, but in other enterics the evidence suggests that the genetic determinants for the siderophore system may, like enterobactin, be on the chromosome9). Bacteria with the capacity to transport siderophores are frequently susceptible to attack by a variety of naturally occurring lethal agents. These outer membrane receptor systems 1°) might be exploited in chemotherapy as they provide specific sites of attachment for siderophore analogues. The high affinity of the receptor for the drug imparts specificity and sensitivity. While resistance to antibiotics, such as albomycin, has been cited as a serious defect to the "illicit transport" approach to drug design, this may not be a serious drawback in the siderophore series. An organism which underwent a genetic variation to no longer use the high affinity iron assimilation pathway would grow well in laboratory media but would be at a serious competitive disadvantage inside living cells where the iron is much less readily available. There is convincing evidence that one of the biological values of fever is interference with the iron assimilation system in the invading pathogen TM12). Exactly how elevated temperatures selectively block the iron gathering pathways of microorganisms remains to be determined but apparently the siderophore system is involved. It has been known for many years that there is a close correlation between availability of iron and pathogenicity in both bacteria and fungi attacking man t3). The list of microorganisms includes, but is not limited to, Yersinia pestis, Pasteurella multocida, Clostridium perfringens, Pseudomonas aeruginosa, E. coli, Listeria monocytogenes, Vibrio cholerae, Neisseria meningitidis, Neisseria gonorrhoeae, Salmonella typhimurium, Klebsiella pneumoniae, Clostridium septicum, and Erysipelothrix rhusiopathiae. In addition, the toxin of Corynebacterium diphtheriae and other organisms is only produced at low iron. It has been shown that the complete high affinity transport system of E. coli is expressed in infections in experimental animals. Moving out of the arena of public health, we see that the struggle for iron among all living species has an impact on biological selection. The fact that siderophore outer membrane receptors in enteric bacteria are "parasitized" by lethal agents constitutes the debit side of the high affinity iron transport system. Siderophore mediated antibiosis based on iron starvation appears to be one mechanism underlying selection and blooming in mixed populations of alga 14). A marine dinoflagellate, Prorocentrum minimum, produces a trihydroxamate type siderophore 15), but the subject of high affinity iron assimilation in marine and estuarine environments is in urgent need of further development. Iron is a limiting nutrient for growth in the blue water of the deep ocean. Similarly, plant growth promoting rhizobacteria are believed to excrete siderophores which render iron unavailable to noxious soil organisms 16). Mycorrhizal fungi, which have long been implicated in phosphorus nutrition in the host plant, may also produce siderophores which assist the plant to acquire iron tT). Soil Pseudomonads produce siderophores which display antifungal activity based on iron complexation, thus holding out the hope for development of novel pesticides ~s).

Methodologyof Siderophores

5

Magnetotactic bacteria have been shown to possess strings of cuboid particles rich in magnetite, Fe304, and it is believed that the microorganisms use this device for orientation to food sources19). The magnetic bacteria contain orders of magnitude more iron than non-magnetic species, but it has not been ascertained if they use the high affinity iron assimilation which is the subject of this review. The discovery of magnetic material in microbes, algae and animals makes the subject of general interest. In sum, the profound biological importance of iron coupled with its quantitative insolubility in aerobic environments at biological pH are the twin factors first eliciting interest in the siderophores. Secondarily, interest has centered on the practical applications of siderophores, as represented by the use of Desferal for treatment of siderosis. This review has been prepared in the hope that it will make available to future workers the expertise which has been accumulated gradually over three decades of siderophore research. It is safe to say that a very large number of siderophores remain to be isolated and characterized. Some of these compounds, such as the factor produced by plasmid bearing strains of Vibrio anguillarum2°), will have both theoretical and practical importance.

H. Methodology A. Detection and Determination 1. Chemical Methods We now recognize three general classes or types of siderophores, namely, hydroxamates, catechols and "miscellaneous"2t). In some instances, such as in the mycobactins, hydroxamate and phenolate ligands are present in the same molecule. The alpha hydroxycarboxylate function occurs rather frequently in the bacterial siderophores, but it is doubtful that this bidentate ligand would stand alone without assistance from the more powerful ligands, hydroxamate and catechol, present in the same molecule. The hydroxamates are, in some respect, the classical variety of siderophore since ferrichrome is the first member of the series to be recognized as an iron transporting natural product from microbial sourcesn). As hydroxamic acids are acylated organic hydroxylamines, digestion of the sample in sulfuric acid and oxidation with iodine to nitrous acid, a modification of the Blom test introduced by Csaky for detection of bound hydroxylamine, is generally employed. The test is sensitive since the nitrous acid is diazotized and coupled to 1-naphthylamine to yield a highly conjugated, intensely colored dye. The reaction depends on the release of hydroxylamine and since all hydroxamate siderophores are of the secondary variety, i.e., the hydroxylamino N is bound to a carbon chain, it is a little mysterious as to how this occurs. The presence of an alpha amino group and a chain length favorable for its cyclization to a potential aldehyde function enhances the yieldsn~. Nonetheless, the Csaky test can only be regarded as semi-

6

J.B. Neilands

quantitative in nature and it further suffers from the fact that it is quite ceremonial and requires several steps using noxious reagents. As an alternative to the Csaky test, reaction with ferric perchlorate in dilute acid, as introduced by Atkin et al. z4), is frequently employed. Hydroxamic acids form di, tetra and sexadentate derivatives with Fe(III) according to the following reactionsZS): F e II1

/

xl-3 O O-

I1 I

Fe3+ + 1-3 R-CO==-N(OH) - R' : R-C--N-R' + 1-3 H + The 1 : I complex is produced in acid solution and is purplish in color with a maximum of 500-520 rim. The 1 : 3 complex is generated in neutral pH and is orange in color with a maximum at ca. 420-450 rim. The intensity of absorption is about 1000 per ring so that the a,,M of ferrichrome, for example, is 2.9 at 425 rim. The perchlorate is used as counterion since its large size precludes coordination with Fe 3÷ and generation of the familiar yellow color of ferric chloride. The ferric ion reaction of the hydroxamic acids can be made to reveal certain intimate details of the structure of siderophores. A trihydroxamate such as ferrichrome remains orange colored even at pH 2. At still lower pH, in mineral acid, the color fades through a fleeting purple and the solution becomes colorless. In contrast, a ferric monohydroxamic acid, such as aceto- or benzo-hydroxamic acid, is purplish already at pH 5; at neutral pH these simple hydroxamic acids have absorption spectra as their ferric derivatives which are quantitatively indistinguishable from that of ferrichrome. A dihydroxamic such as rhodotorulic acid displays intermediate behavior. It is thus apparent that a careful analysis of the effect of the pH on the intensity and position of the absorption peak of the ferric hydroxamate can reveal much about the number of R-CO-N(OH)R' groups per mole and their disposition vis-a.vis the iron atom. In one study of eight different opportunistic and systemic fungal pathogens, the presence of hydroxamate siderophore in the cell-free supernatant was measured by the generation of the sharp peak at 264 nm following periodate oxidation (see below)26). All catechol containing sideroph0res display fluorescence in the ultraviolet that is reminiscent of that observed with 2,3-dihydroxybenzoic acid. The 2,3-dihydroxybenzoyl moiety has three absorption bands in the ultraviolet, one at about 320 rim, one at around 250 nm and the third and most intense in the stepwise sequence, at about 210 nm. The intensity of the band in the near ultraviolet is such that the extinction is 3.2 per millimole. All catechol type siderophores yield wine colored Fe(III) complexes at neutral pH. The very high pKa's of the ring hydroxyls mean that these are not good chelating agents for Fe(III) at slightly acid pH and, in addition, there is a tendency for the metal ion to oxidize the ligand. This has been suggested as one mode of biological unloading of the ironr0; it is also the basis of one test for catechol where the product measured is the ferrous ion28). It is possible to determine hydroxamic acid type siderophores in the presence of the catechol type with the perchlorate reagent since the pH in this medium is low enough to block binding to the aromatic ligand. Oxidation products of the catechol may, however, interfere with quantitation of the reaction. Thus a safer procedure may be first to lower the pH of the cell free supernatant, extract the catechols with ethyl acetate and then apply the ferric perchlorate reagent to the aqueous phase. This avoids dealing

Methodology of Siderophores

7

with any insoluble neutral iron complexes that may be generated in the course of decreasing the pH of the assay solution. The third category of siderophores, designated above as "miscellaneous", probably are amino or imino carboxylic acids. The Fe(III) complexes of these compounds lack the charge transfer bands of ferric hydroxamates and ferric catechols in the visible and are hence not highly colored. In this respect they resemble the ferric derivatives of EDTA and nitrilotriacetate. Although these chelates display considerable absorbancy in the ultraviolet, this is more difficult to quantitate. Thus we are here left with some type of bioassay as the only practical method of detection. There is a need for a universal method for detection of siderophore ligands in cell-free culture supernatants. One possibility is to add excess iron, check the pH to 7, spin out the insoluble excess iron as Fe(OH)3 and measure the dissolved iron by ferrozine (after reduction) or directly by atomic absorption. Attempts in the author's laboratory to perfect this test have been frustrated by refusal of the excess iron to precipitate quantitatively. In a few rare cases the siderophore ligand bears a chromophore. Thus certain of the siderophores produced by the Pseudomonads and variously referred to as pseudobactins 10 and pyoverdines29) are yellow-green fluorescent ligands and may be detected directly by their light absorption in the visible, and by their fluorescence. Fekete et al. a°) described a novel method for detection of siderophore iigands excreted into agar surfaces. The colony is grown up on purified agar prepared from low iron media and after growth a segment adjacent to the colony is cut out and placed on filter paper. After electrophoretic separation, the paper is viewed in ultraviolet light for detection of catechols and sprayed with iron solution for detection of hydroxamate siderophores. Using a number of different species known to form defined siderophores, the expected compounds were detected together with some spots of unknown substances.

2. Biological Methods a. Siderophore A u x o t r o p h e s Bioassays for siderophores are many orders of magnitude more sensitive than the best chemical methods. In essence, we have a choice of use of either a "natural" or artificial (mutant) auxotrophe on the one hand, vs. a wild-type cell grown under iron stress on the other hand. The organism most commonly used for assay of hydroxamate siderophores is the soil isolate Arthrobacter flavescens JG-9 ATCC 29 091. Lochhead 31) and co-workers showed that certain strains of Arthrobacter produced a factor required by other strains of Arthrobacter. Subsequently, a soil enrichment culture growing on the antibiotic puromycin as sole source of carbon yielded an Arthrobacter species named JG-9. It was shown to require hydroxamate type siderophores. Although it does not respond to catechol type siderophores, it is quite omnivorous as regards the hydroxamate type and even uses, at the proper level, synthetic chelating agents. Some caution must hence be used in the interpretation of bioassays based solely on the use of this tester strain.

8

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Rhodotorulic Acid (picomol) Fig. 1. Growth response of Arthrobacterflavescens JG-9 ATCC 29091 to rhodotorulic acid. Small volumes (1 to 20 p.l) of an aqueous solution of rhodotorulic acid were pipetted onto 6 mm paper discs. American Type Culture Collection (ATCC) Medium No. 424 consisting of 1 g Bacto Peptone, 1 g Difco Yeast Extract, 0.2 g K2HPO4 and 1.5 g Bacto Agar were dissolved in 100 ml of distilled water, cooled to 40 °C and seeded with two drops of an overnight culture of A. flavescens growing in Medium 424 supplemented with 2 Ixg of rhodotorulic acid per 100 ml. After impregnation with the siderophore solution, the paper discs were placed on the surface of the seeded agar plates and the latter incubated at 30 *C. The diameter of the halo of exhibition of growth was measured. Other hydroxamate siderophores, such as ferrichrome and deferriferrioxamine B are comparably potent in their capacity to stimulate the growth of A. flavescens which, however, does not respond to eatechol type siderophores

The assay can be performed on agar surfaces containing ATCC medium or in solution 32) using a formula of defined medium. Somewhat more precise assays can be achieved via the latter route. However, in this instance the sample should be filter sterilized since hydroxamates, although reasonably stable, will suffer some degradation in the autoclave. Figure I shows the growth response of Arthrobacterflavescens JG 9 to rhodotorulic acid. As an alternative to the use of Arthrobacter flavescens JG 9, a mutant enteric bacterium can be employed for the assay of hydroxamate siderophores. Although it does not synthesize any detectable siderophore other than the catechol, enterobactin, Salmonella typhimurium LT-2 strains use a wide variety of siderophores of this type 33). Mutants

Methodology of Siderophores

9

blocked in the biosynthesis of enterobactin have been placed in two major Classes, I and I134). The latter are blocked between chorismate and 2,3-dihydroxybenzoate while Class I mutants are defective in the conversion of this catechol to enterobactin. Mutants enb-1 and enb-7 are typical of Class I and II, respectively. The inability of these strains to synthesize enterobactin means that, under certain conditions, a siderophore will be required for growth. These conditions are easily achieved in S. typhimurium since, although it uses citrate as a carbon source, it cannot transport ferric citrate. [In E. coli the converse is true - it does not use citrate as an energy source but has an inducible outer membrane receptor for ferric citrate.] Thus the use of high levels of citrate in the assay medium ties up the adventitious iron in a form unavailable to the enb mutants and, at the same time, provides a metabolizeable substrate for the microorganism. It must be stressed that the enb mutants will respond to enterobactin as well as to the range of hydroxamates tested by Luckey et al. 33). In addition, Class II mutants such as enb-7 will respond to enterobactin precursors at the level of 2,3-dihydroxybenzoic acid since these can be converted to the finished siderophore. In the test of Luckey et al. 33) the siderophores are pipetted onto 6 mm paper discs which are then placed on the surface of the agar medium. While the point does not seem to have been researched systematically, it is likely that the enteric bacteria are somewhat more fastidious than Arthrobacter spp. in their requirements for chelated iron. For some siderophores a more specialized bioassay must be adopted. This is the case for the aerobactin formed by ColV plasmids of E. coli. Fortunately, mutant plasmids are available 35), designated iuc, which are blocked in the biosynthesis of aerobactin but which retain full capacity to use the siderophore. The test organism bearing the iuc plasmid can be seeded into warm agar and the sample applied on the 6 mm paper disc or in wells scooped out of the agar. In addition, a quantity of cells can be patched onto the surface of the seeded agar and the plate then examined for a halo of exhibition of growth. No general assay organism has been described for either the catechols or the "miscellaneous" group of siderophores. In these cases resort must be made to the source organism. As mutants are readily acquired in the enteric bacteria as a class, this is the solution of choice in, for example, the enterobactin produced by E. coli. Here we have two useful mutants 36), RW 193 and RWB 18, both derivatives of E. coli K 12. These have now been desposited with the ATCC and bear the numbers ATCC 33 475 and 33 476, respectively. Both are entA and are hence blocked between chorismate and 2,3-dihydroxybenzoate; neither can achieve the synthesis of enterobactin. The simultaneous use of both tester strains in an assay is recommended in order to confirm that the activity measured is, in fact, enterobactin. Strain ATCC 33 475 forms the 81,000 molecular weight outer membrane receptor for ferric enterobactin and responds to low levels of this siderophore. Strain ATCC 33 476, on the other hand, lacks this outer membrane protein and is not stimulated by low levels of enterobactin, although very high levels may give some activity. Both of these tester strains use ferrichrome and a range of other hydroxamate siderophores comparable to that seen with the enb mutants of S. typhimurium. Figure 2 shows the results of a bioassay for enterobactin as performed in this laboratory. The test depends on the ~use of a chelating agent incapable of supplying iron to E. coli. Our favorite compound of this type is deferriferrichrome A; it is available in this laboratory and its antibiosis against E. coli appears to be based entirely on iron complexation. Ferrichrome A 37) is isolated from low iron grown Ustilago sphaerogena and the

10

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E n t e r o b a c t i n ( p i c o tool) Fig. 2. Growth response of Escherichia coli RW 193 ATCC 33475 to enterobactin. The assay was performed essentially as described in the legend to Fig. 1 except that the medium consisted of 0.8 g Bacto Nutrient Broth, 1.5 g of Bacto Agar and 1 ml of 10 mM deferriferrichrome A in 100 ml of distilled water. Inoculum was two drops of an overnight culture of E. coli RW 193 growing in nutrient broth. The incubation was at 30 *C. In practice it is useful to set up a companion plate seeded with E. coli RWB 18 ATCC 33476 which, owing to the absence of the outer membrane receptor for ferric enterobactin, is not stimulated by low levels of this siderophore. As an additional control, 10 ~1 of 10 IxM ferrichrome can be applied to discs set on all test plates, where the response should be ca. 15 mm for both strains

traces of ferrichrome removed by extraction of a neutral solution with benzyl alcohol. The pure ferrichrome A is treated with cyanide and sodium hydrosulfite in the usual way to remove the iron atom 3a). A 0.1 mM solution of deferriferrichrome A, which retains some yellow color despite the apparent absence of iron, is stored in the refrigerator. In a 100 ml erlenmeyer flask are placed 50 ml of water and 0.4 g of Difco nutrient broth powder and 0.75 g agar. The flask is heated to dissolve the agar; completely aseptic conditions are not required. To the still hot solution is added 0.5 ml of the stock 0.1 mM deferriferrichrome A and the resulting solution divided into two 25 ml portions and placed in the 40 °C water bath. After temperature equilibration, one drop of an overnight culture of A T C C 33 475 and A T C C 33 476 are added to individual flasks, the flasks are swirled and poured into separate petri plates. When the agar has solidified, 6 mm paper discs impregnated with siderophore solution are deftly placed on the surface. Five or six discs can be placed on each plate. The sterile paper discs can imbibe up to about 20 Ixl of solution in a single application. If the siderophore is applied in an organic fluid, such as one of the lower alcohols, the solvent should be allowed to evaporate before the discs are placed on the agar surface. The initial working range, which can subsequently be raised or lowered, is around 10 ~tl of 10 ~tM

Methodology of Siderophores

11

siderophore solution. After the discs have been placed, the plates are inverted and incubated at 37 °C overnight. The readout, which may be visible after a half day of incubation, is a halo of exhibition of growth. Although the diameter of the halo varies monotonically with siderophore concentration, the test is not very precise in quantitative terms. In the absence of deferriferrichrome A, bipyridyl or ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA or EDDHA) 39) may be substituted as the deferration agent applied in situ. As the last named agent is certain to show an appreciable affinity for divalent metal ions in general, it can be expected and, indeed, does have substantial toxicity. One has simply to experiment with a range of concentrations from 0.1 mg up to a few mg/ml. The EDDA should be purified by the methods of Rogers39a). The substantial level of iron in nutrient broth absolutely requires that some such deferration agent be added to suppress low affinity iron uptake and overgrowth in these E. coli strains. This service is performed by citrate in the test of Luckey et al. 33) based on S. typhimurium. The E. coli tester strains require several amino acids, which can be supplied at 40 ~tg/ml for growth in minimal medium. These additions are not required in nutrient broth agar. Yet another deferration agent often employed is one of the transferrins. Some four decades ago4°) it was discovered that the bacteriostatic effect of serum could be reversed with iron - the effect was therefore attributed to binding of iron by transferrin, which is normally only 30-35% saturated. The particular transferrin to use is the ovotransferrin or conalbumin of egg white since this is commercially available at modest cost. It is usually prepared in 5 mM sodium bicarbonate, filter sterilized and diluted into the medium. For short periods of growth, such as overnight growth with relatively heavily seeded plates, it may not be necessary to work under totally asceptic conditions.

b. Wild-Type Cells In the absence of a suitable siderophore auxotrophe, either natural or laboratory acquired, the sole option for the bioassay is the wild-type cell. Negative activity for A. flavescens and E. coli and negative chemical tests for hydroxamate and catechol will require such examination of the wild-type strain before it can be concluded that siderophores are absent. By all accounts the most commonly used deferration agent is EDDA, again used at a concentration that can only be determined by trial and error experimentation. In working with the wild-type cell it must be borne in mind that such cells probably synthesize some level of siderophore regardless of the level of iron in the culture medium. In order to avoid excessive cross feeding and overgrowth of the test plates, it is hence essential to use a light seedling, ca. 103 colony forming units/ml. The zone or halo of exhibition of growth around the paper disc will not be confluent growth, as seen above for the 1 drop of nutrient broth culture of E. coli seeded into 25 ml agar, but a constellation of small, individual colonies. A good illustration of the use of the EDDA method is given by Vandenberg et al. 18~, who employed it for detection of siderophores from Pseudomonas spp. believed to exert anti-fungal activity based on iron starvation. Some of the compounds they detected appear to be superior to known siderophores in their ability to complex iron. Mutants blocked in production of siderophore were, as expected, the most sentitive to EDDA. In all of these biological tests it seems immaterial whether the siderophore is applied as ligand or as iron complex. There will be enough environmental iron available for

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J.B. Neilands

reconstitution of the complex in any growth medium. It is little wonder that most workers have opted for the paper disc assay introduced by Luckey et al. 33) over the more elaborate solution assay. Some of the siderophores, such as the catechols, only dissolve readily in organic solvents. This presents no problem with the paper discs since the solvent can simply be allowed to evaporate before the discs are placed on the agar.

B. Enhancement 1. Minimal Media It has been known for almost three decades that iron starvation elicits overproduction of both hydroxamate and catechol type siderophores. The focus of this discussion will hence be on the various techniques used for derepression of the siderophore synthesizing gene complexes of microorganisms. While a completely generalized relationship between iron concentration and siderophore production cannot be made, it is apparent that many systems are optimally derepressed at ca. 0.1 IxM iron. A certain variation can be expected depending on the

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Time (days) Fig. 3. Generalized relationship between microbial growth and siderophore production. Siderophore appearance awaits depletion of the iron concentration of the medium and generally reaches a maximum in 2-7 days. Vigorous aeration enhances the yield. Most hydroxamates remain stable in the medium (an exception may be prorocentrin - see reference No. 15), but catechols are rapidly oxidized and polymerized. Iron is the most important nutrient affecting production, its regulatory effect generally being evidenced at a concentration of about 1.0 I~M

Methodologyof Siderophores

13

particular species under investigation. Obviously, some cell growth will be required in order to obtain any metabolic products from the microorganism. An iron concentration of 10 ~M is considered to he "high" in iron and generally results in excellent cell mass with only modest yields of siderophore. The relationship between cell growth and siderophore yield is further illustrated in Fig. 3. Obviously the culture must become iron starved before the gene products (enzymes) responsible for siderophore production are optimally expressed. It is apparent that the first task in dealing with a new siderophore system is to devise a purely synthetic or defined (minimal) medium for growth of the cells. This is the most convenient means, not always easy with pathogenic microorganisms or other species with complex nutritional requirements, of achieving a medium low in iron. Regardless of what type of medium is adopted, it is necessary initially to demonstrate enhanced growth in the presence of graded levels of iron salts. This is the only way to be certain that the medium has been made poor in iron.

2. Carbon Source It is often possible to offer the aerobic and facultative anaerobic microorganisms that synthesize siderophores a choice of energy substrate. Glucose, while generally the preferred substrate, may not be optimum for siderophore production. This is well illustrated for aerobactin formation in enteric bacteria such as Aerobacter aerogenes8). Growth of this organism on glucose affords very low and erratic yields of aerobactin. Growth on succinate, on the other hand, gives, quite reproducibly, higher yields. Apparently this is because the succinate can only be metabolized via an aerobic metabolic pathway requiring iron-containing enzymes. As a consequence, the cells rapidly become depleted in iron. A secondary advantage in using a substrate such as succinate or citrate is that these can be added to the culture medium prior to sterilization. The potential aldehyde group of glucose can attach a nitrogen atom in Schiff base linkage and in this way initiate the well known "chemical browning" reactions. Thus glucose should not be added to any culture medium containing nitrogen prior to sterilization; sucrose, or other metabolizeable saccharides without the potential aldehyde function, present no problems of this type.

3. Deferration Methods Should the organism under investigation have complex nutritional requirements, it is then only possible to deferrate the medium in some way. Lankford4t) has published a number of ways in which this can be achieved, dating from the classic work of Waring and Werkman42), who extracted contaminating iron with 8-hydroxyquinoline. A 1% solution of the synthetic chelator is prepared in chloroform and the medium extracted until the gray-green color of the ferric :complex is no longer visible in the solvent layer. The medium is then extracted with pure chloroform in order to remove any residue of the chelator. Since 8-hydroxyquinoline binds a large number of metal ions, it may be necessary to add back some of those essential for growth.

14

J.B. Neilands

Filtration through Chelex, a polyaminocarboxylate resin related in structure of EDTA, has proven to be an effective means of iron removal. Complexation to ovotransferrin followed by ultrafiltration through a protein-proof filter has proven satisfactory for small scale deferration 43).

4. Additives The addition of metal ions such as AI3+ or Cr 3+ to culture media sometimes results in overproduction of siderophores. Both of these ions would be exptected to compete for iron sites although Cr 3+, being subsitution inert, would only slowly enter into complexation in the absence of an externally supplied energy source. It is believed to co-polymerize with iron and in this way to tie up the nutritious metal ion in a form unavailable to the cell. One of the common ways to render enteric and other Gram negative bacteria iron deficient is to employ media containing up to 0.1 M trihydroxymethylaminomethane (Tris). The pKa of this organic base, about 8.1, maintains the pH in the neutral range even with glucose added as energy source. The neutral pH helps to keep iron in an insoluble state and, in addition, the "Iris may have a direct effect on the cell which enhances its iron deficiency.

C. Isolation Siderophores may be recovered from the spent culture fluid either as the free ligand or as the iron complex. In general, it is found that charged species do not extract and crystallize well as their iron complexes and hence such compounds, e.g., aerobactin, are best recovered as the uncomplexed ligand. In any case, it will be necessary to remove the iron before any NMR measurements can be made since the paramagnetism of Fe 3+ will eliminate all signals via line broadening. If desired, AI3÷ or Ga 3+, both diamagnetic ions, can be substituted for the paramagnetic Fe 3+. Gallium is particularly useful since both its charge and radius are closely parallel to those of Fe z+ 44). Some success has attended the direct adsorption of neutral ferric hydroxamates to XAD resins 15), from which they may be eluted with an organic solvent such as methanol. This provides a convenient means of rapid concentration of the siderophore from a large volume of culture fluid. Detailed descriptions have been given elsewhere 4s) for the current methods used in this laboratory for the isolation of ferrichrome and enterobactin, prototypical representatives of the hydroxamate and catechol type siderophores, respectively. A few additional examples will be given here. Rhodotorulic acid. Rhodotorula pilimanae or one of the related basidiomycetous yeasts producing the siderophore is grown in the same low iron medium used for production of the ferrichromes, except with double the sugar and ammonium ion concentration 46). The medium contains,~per liter: K2SO4, 1 g; K2HPO4, 3 g; ammonium acetate, 6 g; sucrose, 40 g; citric acid, 1 g; thiamine, 2 mg; Cu 2+, 5 ~tg; Mn 2+, 35 ttg; Zn z+, 2 mg und Mg2+, 80 rag. The metal ions were supplied as their sulfates. The pH was adjusted to 6.8 by addition of about 0.7 ml of concentrated ammonium hydroxide and the medium

Methodology of Siderophores

15

dispensed in 200 ml volumes contained in I flasks. Growth was carried out with vigorous aeration for about one week. The cells were removed and the supernatant flash evaporated to ca. 0.1 volume of the original culture. After standing overnight, crude rhodotornlic acid precipitated. Recrystallization was achieved from hot water. The yield was about 1-2 g/1. As an alternative means of isolation, the rhodotorulic acid can be extracted into 1 : 1 :: CHCI3 : phenol and then returned to water as described for fen'ichrome. The maximum solubility in water is about 3 mM; most hydroxamic acids are substantially more water soluble.

Aerobactin. A. aerogenes 62-I or another enteric strain producing aerobactin is cultured in "Iris47) medium containing 1% sodium succinate as carbon source, pH 7.4. If A. aerogenes 62-I is used, the aromatic amino acids phenylalanine, tyrosine and tryptophane must be added at a level of 40 mg each per liter. A trace of added iron, 0.5 to 1.0 0M may improve cell yield and hence aerobactin synthesis; iron levels of 10 ~tM or higher will result in repression and should be avoided. After about two days at 37 °C with vigorous aeration the supernatant solution is freed from cells and passed through a column of anion exchanger, such as AG 1-X2, of sufficient capacity to retain all of the siderophore. The aerobactin is eluted with 1 N NH4CI, the effluent fractions giving a ferric chloride positive reaction on the spot plate are pooled and passed through a column of cation exchanger, A G 50 W-X 2. The already acidic solution was adjusted to pH 2, the solution saturated with (NH4)2SO 4 and extracted with benzyl alcohol to remove all hydroxamate from the aqueous phase. Three extractions are generally sufficient to remove all of the aerobactin. The pooled benzyl alcohol extracts are centrifuged to clarity and then filtered through a double layer of filter paper. The clear filtrate is diluted with ten volumes of diethyl ether and the aerobactin extracted into a small volume of water. The aqueous extract containing the aerobactin is swirled with ether to remove traces of benzyl alcohol and then lyophilized. The yield of aerobactin varies from 20-60 mg/J and consists of a slightly tan colored, hygroscopic and deliquescent solid. Aerobactin moves as a trivalent anion on paper electrophoresis at neutral pH. On field desorption mass spectrometry the major line for M + 1 occurs at 565 mass units. Some cultures producing aerobactin have been found to form the side chain, N ~hydroxy-N~-acetyl-L-lysine9). As this product is neutral it comes directly through the ion exchange resins. It is extracted into chloroform: phenol from saturated ammonium sulfate solution at pH 6.5 as the ferric complex. It was deferrated with 8-hydroxyquinoline and crystallized from absolute ethanol to yield prisms with mp 210-211 °C, with evolution of gas. The recovery of this compound suggests that aerobactin arises by oxidation of lysine on the N% followed by the acetylation of the resulting hydroxylamine and, finally, condensation of the a-amino groups with the distal carboxyl groups of citrate. Quite a fair number of enteric bacteria make both aerobactin and enterobactin 9), and Vibrio vulnificus has just been shown to make hydroxamate and catechol, neither yet identified43). The method given above for isolation of aerobactin differs from the original procedure of Gibson and Magrath 4s) essentially by inclusion of a solvent extraction step. In our hands this step has proven indispensable for obtaining hydroxamate type siderophores completely free from salts and other impurities. In lieu of benzyl alcohol, a 1 : 1 :: phenol : chloroform solution may be used as the organic extraction fluid. In this case the organic phase may be the lower one, even in the presence of a relatively high salt

16

J.B. Neflands

concentration. The phenol:chloroform mixture probably has greater solvation power than benzyl alcohol but the latter, which is relatively expensive, can be recovered by distillation. Vibriobactin. In addition to enterobactin, the catechol type siderophores known at this date include agrobactin, parabactin and vibriobactin49). All of these are isolated as the ligands by extraction into ethyl acetate, followed by precipitation with hexane or benzene. The preparation of vibriobactin is illustrative of the general procedures involved. Vibrio cholerae Lou 15 is maintained on 3.7% Difco Brain Heart Infusion agar. This strain is only weakly pathogenic and it is believed that an infective dose would require ingestion of about 1 ml of fully grown culture fluid. A broth culture of V. cholerae Lou 15 is transferred into 6 x 40 ml of "Iris medium containing, as carbon source, 0.4% each of the sodium salts of lactic and succinic acids. After overnight growth at 37 °C with vigorous shaking, the culture was transferred into 6 x 1 1of Tris medium, pH 6.8. During the course of growth the pH drifts upward owing to the consumption of the organic acid. The pH is not allowed to exceed 8.0 since catechols are sensitive to air oxidation, especially at alkaline pH, with consequent formation of quinones and brown polymers. The catechol concentration is monitored by the Arnow reaction which, after 1.5 to 2 days of growth, will reach a maximum value. The pH of the cell free supernatant solution is reduced to about 6.0 by addition of HC1 and then extracted with 3 x 1 1 portions of ethyl acetate. The combined ethyl acetate extract is concentrated to about 100-200 ml on the rotary evaporator, washed with 0.1 M citrate buffer pH 5.5, water and dried over anhydrous MgSO4. The dried ethyl acetate is concentrated to a few ml and the vibriobactin precipitated with excess n-hexane. The crude product is dissolved in the least volume of methanol and again precipitated with hexane. To remove traces of bound ethyl acetate, which NMR spectroscopy shows cling tenaciously to catechol type siderophores, the product is dissolved in the smallest volume of methanol and precipitated with water. After lyophilization the vibriobactin is obtained as a white, amorphous powder in a yield of ca. 20 mg per 6 1. An unusual feature of vibriobactin is the presence of norspermidine rather than spermidine, as in agrobactin and parabactin, as the polyamine backbone. A survey5°) of a large number of Vibrio spp. indicates that norspermidine is very common in these bacteria and its presence there has been suggested to have some taxonomic value.

D. Structure and Characterization The usual methods of structural analysis in the natural product series may be applied to the siderophores. Such methods include the use of standard instrumental techniques such as mass spectrometry and NMR, guided by, in the first instance, knowledge of the C, H and N composition of the sample. A simple flat bed paper electrophoresis is most convenient for study of both the intact siderophores and any hydrolysis products released by digestion in 6 N HCI. Many siderophores, for example, all of those elaborated from citrate as the skeletal backbone, will carry charges and will move on paper electrophoresis with or without the iron. The rate of migration will depend on, primarily, charge, and secondarily, molecular weight. The frictional coefficient has a cube root dependency on the molecular weight, while mobility is directly dependent on charge.

Methodology of Siderophores

17

Thus by use of known standards it is a relatively simple matter to arrive at a postulation regarding size and charge of the unknown. Hydrolysis of hydroxamic acids will yield an acyl moiety and an organic hydroxylamine, both of which will move nicely in an electric field. Similarly, amide and ester groups are present in catechol type siderophores. The bidentate metal-coordinating functional groups are held together by subunits which are themselves condensed together and hence labile to cleavage. The technique of periodate oxidation is most useful for the hydroxamic acids since it cleaves the -CO--N(OH)- bond while leaving intact the amide bonds, which are invariably present elsewhere in the molecule. Total hydrolysis should not be performed when iron is present in the siderophore since this will lead to extensive decomposition of the hydroxylamino moiety, which is sensitive to oxidoreduction. The iron can be removed by treatment with excess 8-hydroxyquinoline in methanol, evaporation of the solvent and extraction of the excess reagent with chloroform. This lability of the -N(OH)H group has prompted de novo use of a variety of methods for its reduction to the amide level. While the hydroxamic acid bond is notoriously difficult to reduce, Raney nickel at 50 lbs pressure will give satisfactory results if the target bond is not too hindered. Treatment with hot 50% HI will both cleave and reduce the -CON(OH)-function. Recently, Akers 51) has suggested the use of TiCI3 as reductant. The recently improved technology of mass spectrometry enables direct examination of a range of siderophores as their iron complexes. If the complex crystallizes, the preferred structural analysis is via X-ray diffraction. A special virtue of the X-ray diffraction method of structural analysis lies in the fact that it reveals at once the sites of attachment of the iron. In determining the nature of the complex ion, the first step is to measure the proton count and then to calculate the overall charge on the complex. The iron must be linked to O, N or S and the base "hardness" of the first of these atoms means that it will be the preferred site of transition metal binding in siderophores. The stability constants are measured by the standard methods of inorganic and coordination chemistry. For biological studies it is sometimes desirable to insert a kinetically inert ion such as Cr 3÷. In the case of ferrichrome, this was accomplished at reflux temperature in methanol 52). The pale green needles of the chromic complex are virtually indistinguishable from the natural product, apart from the change in color. A good illustration of the identification of iron binding sites in a siderophore is the work of Plowman et al. 53) on ferric schizokinen. They showed by a variety of techniques that a deprotonated hydroxyl and carboxyl group of the citrate moiety are linked directly to the iron. The siderophore ligand will enter into complex formation with the-actinides 54), and the binding to AI3÷ and Ga 3÷ have just been cited. The latter element is rare and the aluminum, while abundant, is orders of magnitude weaker than Ga 3+ or Fe 3+ in its affinity for the usual oxygen ligands in siderophores. Thus we are left with the high spin d5 octahedral Fe(III) complex as essentially the only form in which the siderophore ligand can be expected to be encountered in nature. A special feature of siderophore ligands containing optically active centers is the capacity to form A or A coordination isomers. These are in addition to the geometrical isomers, c/s and trans, which are also possible, although c/s is the most usual because of constraints imposed by length of the ligand arms. The enantio isomers of both enterobactin 55) and ferrichrome56) have been synthesized. In E. coli the synthetic A, c/s ferric enterobactin is totally inactive and the ligand, which is prepared from D-serine, com-

18

J.B. Neilands

pletely denies iron to the bacteria. The linear catechols agrobactin and parabactin apparently form isomers which are, as expected, inactive in E. coli. However, on hydrolysis of the oxazoline ring to afford the "A" analogues, which still contain L-threonine, the complex appears to be of the A variety and, interestingly, now acquires some activity for E. coli 56a). This specificity may reside in the siderophore attachment sites in the outer membrane receptor proteins. Siderophore ligands which are 6-coordinate hydroxamates or catechols are often accompanied in the culture fluid by bidentate ligands which appear to be the building stones of the complete ligand. Thus 2,3-dihydroxybenzoic acid is nearly always found in the cell free supernatant of bacteria forming the catechol type siderophores. It has been suggested that these simpler ligands may be involved in interllgand exchange as a device for unloading siderophore iron 57). Low though the redox potentials5s) of ferric siderophores may be, microbial cells do appear to have the capacity for their reduction59).

E. Chemical Synthesis Most of the siderophores, including such relatively complicated structures as mycobactin6°), have been obtained by chemical synthesis. The list includes aerobactin61) and schizokinen 62), and various members of the ferrioxamine series 63). Among the catechols, enterobactin55) has been synthesized from both L- and D-serine. Much of this work has been inspired by the search for an orally effective substitute or replacement for Desferal. The classical chemical route to the hydroxamic acid bond is over the nitro compound, which can be reduced with zinc dust in ammonium chloride solution to the hydroxylamino stage. Acylation of the latter generally results in an N,O-diacyl product, but the O-acyl bond can be cleaved in dilute ammonium hydroxide leaving intact the substantially more stable hydroxamic acid function. Unfortunately, very few nitro compounds useful as siderophore synthons are commercially available. Oxidation of the amino group is difficult to arrest at the hydroxylamino stage. However, the special oxidation method introduced by Keller-Schiedein64) and his colleagues shows considerable promise. Here the amino group is first condensed in Schiff base linkage with benzaldehyde, the Schiff base is then oxidized and hydrolyzed to yield the organic hydroxylamine. These gentle series of reactions can be applied to optically active to-amino acids suitably blocked on the alpha amino group to afford good yields of products and retention of configuration. Some details of the chemical synthesis of specific catechol type siderophores are discussed elsewhere in this volume (Chimiak and Neilands, p. 89). The first member of the series, 2,3-diliydroxy-N-benzoylglycine, was prepared by direct condensation of 2,3dihydroxybenzoic acid with glycine in the presence of dicyclohexycarbodiimide. Generally, however, some type of reversible blocking on the ring hydroxyls will be required for good yields.

F. Molecular Cloning The simple expedient of lowering the iron level of the medium will generally suffice to give reasonable yields of siderophores. While iron starvation also, and apparently coordi-

Methodology of Siderophores

19

Table 1. Siderophore outer membrane receptors in Escherichia coli K 12

Receptor

MoleculaP weight

Ferrichrome

78,000

Ferric enterobactin Ferric aerobactin

81,000 74,000

Gene locus 2.5 rain

13.0 rain plasmid ColV

Lethal agents Albomycin; bacteriophages T 1, T5, 80 and UC-167); Colicin M Colicin B Cloacin

' Relative molecular weight as measured in sodiumdodecylsulfate-polyacrylamidegel electrophoresis

natelyr5, 66), induces overproduction of the transport system, it is still desirable to further enhance this segment of the siderophore system by molecular cloning. Indeed, a complete understanding of the mechanism of action of the high affinity system will require possession of all of the DNA and gene products involved in the system. Cloning in the siderophore series is greatly facilitated by the fact, already noted, that siderophore transport systems are often "parasitized" by specific lethal agents. Table 1 lists the outer membrane receptors in E. coli for three siderophores which are known to act as common binding sites for a variety of bacteriophages, bacteriocins and antibiotics. The enterobactin gene complex of E. coli has been cloned68'69) on bacteriophage Mu and shown to be organized into several transcriptional units across some 26 kb of DNA. The tonA 7°) protein or outer membrane receptor for ferrichrome type siderophores has been cloned in a number of laboratories. The cloning of the aerobactin system from ColV bearing, hospital isolates of E. coli illustrates the general principles 71). ColV DNA is separated by density gradient sedimentation and subjected to complete digestion with restriction enzyme HindIII. This afforded 11 fragments which ranged in size from 1.2 to 30 kb and which summed to about 90 kb, the known size of pColV. The fragments were mixed in 10 fold molar excess with the expression vector pPlac, opened with HindIII. Strain 294 of E. coli K 12 was then transformed with this mixture, after ligation with T 4 ligase. Since the vector carries ampicillin resistance, a primary selection screen was based on resistance to this antibiotic. In a second, negative selection, the ampicillin resistance strains were replicated to plates containing cloacin. This served as an indicator for the presence of the ferric aerobactin receptor, which would make the colonies sensitive to cioacin. From 500 penicillin-resistant transformants, one clone was isolated and sized to I8.3 kb. This plasmid, pABN 1, contained the entire aerobactin gene complex of pColV-K 30 inserted into the single Hind III restriction site of pPlac. A sub-clone of the plasmid yielded pABN5, which contained only the regulatory and biosynthetic genes of the complex on 8.7 kb of DNA, of which 1.9 kb represents the vector programming origin of replication of the plasmid and the 30,000 molecular weight 13-1actamase protein. In minicell preparations pABN 1 was shown to form five proteins 72), in addition to ~-lactamase, with molecular weights ranging from 27,000 to 74,000, the latter the outer membrane ferric aerobactin-cioacin receptor. A sys/,ematic deletion analysis of the largest plasmid revealed the gene order and some information about the function of each

20

J.B. Neilands

of the proteins encoded in the complex, which appears to be a single operon. The promoter73) region was detected by S 1 mapping and the transcription start site identified by sequence analysis of the transcript. The nucleotide base sequence through the promoter region indicates it to be unusually "strong" and closely related to the concensus sequence for E. coli promoters. Thus virulent and invasive strains of E. coli have become equipped with a very efficient mechanism for expression of aerobactin. Since the stability constant of this siderophore is many orders of magnitude below that observed for enterobactin, the question arises as to why the former is at all necessary. At least part of the answer seems to be that enterobactin, the catechol type siderophore, does not function well in a protein aceous environment 74'75). In fact, serum albumin appears to form a relatively stable 1:1 complex with enterobactin easily detected by either equilibrium dialysis or via shift of the near ultraviolet absorption band of the siderophore to longer wavelengths. According to this view, E. coli would maintain a chromosomally encoded siderophore, enterobactin, which it uses for survival in the environment and then resorts to the plasmid encoded aerobactin, non-aromatic in character and with no detectable affinity for serum proteins, for invasion of the host. There is increasing evidence that in enteric bacteria other than E. coli, the aerobactin system may exist side-by-side with the enterobactin system on the bacterial chromosome. It is hence conceivable that the former is on a transposon or other type of mobile genetic element9).

HI. Summary and Conclusions Although at the moment of writing many dozens of siderophores have been characterized, it is apparent that many more such compounds remain to be identified from microbial sources. Hydroxamate and catechol bidentate ligands, as found in ferrichrome and enterobactin, respectively, are the most usual functional groups encountered in siderophores. The preponderance of oxygen atoms at the metal ion binding center of the siderophore provides the specificity for iron(III) and, at the same time, affords a release mechanism via reduction of the bound metal to iron(II). That oxygen is not the exclusive atom linked to iron is illustrated by mycobactin, where coordination to an oxazoline nitrogen has been proven by electron diffraction structural analysis. This type of bonding also probably occurs in agrobactin, parabactin and vibriobactin, all siderophores from Gram negative bacteria. The a-hydroxycarboxylic center occurs in a number of siderophores derived from citrate, namely, aerobactin, arthrobactin (Terregens Factor) and schizokinen. The same grouping occurs in the pseudobactins, where it is supplemented by a brace of catechol and hydroxamate functions. There remain certain compounds with siderophore activity which contain neither catechol nor hydroxamate groups; these may be more closely related to the phytosiderophores described elsewhere in this volume (Sugiura and Nomoto, p. 107) or to the opines. The only clinical application of siderophores of note is the use of deferriferrioxamine B for treatment of transfusion induced siderosis, which is sold by Ciba-Geigy as the mesylate under the trade name of Desferal. Unfortunately, it is not effective via the oral route. There is thus still a requirement for development of a cheap, non-toxic orally active deferration drug. The identification of aerobactin as the siderophore of virulent

Methodology of Siderophores

21

E. coli opens up some interesting possibilities for illicit transport of chemotherapeutic drugs, the latter yet to be synthesized. Optimum production of siderophores will depend primarily on selection of the most propitious microorganism and, secondarily, on choice of a medium readily depleted in iron. The objective is to secure a reasonably robust growth of iron-starved cells. Hydroxamate type siderophores can usually be allowed to accumulate in the growth medium but catechols are much more readily destroyed by oxidation, especially at alkaline pH. Structural determination in the siderophore series is most efficiendy accomplished by X-ray diffraction of the crystals of the iron complex, if these are forthcoming. While most siderophores are now available by chemical synthesis, the biosynthetic method generally yields sufficient material for purposes of basic research. A variety of lethal agents gain access to the cell via outer membrane receptors designed for the transport of siderophores. There would thus seem to exist a unique opportunity for the pharmaceutical industry to design and synthesize an array of drugs which could be smuggled into the cell on this uptake pathway. Cloning of the siderophore systems of E. coli is relatively easily achieved, thanks to the existence of specific lethal agents, and it remains to apply such modern methods of molecular genetics to the fungi and yeasts. The biosynthetic enzymes for the siderophores will prove to be of considerable interest as this will provide another target for crippling the iron-gathering function of the pathogenic microorganism. Finally, the molecular mechanism whereby iron represse s the siderophore system of iron assimilation will soon be known in E. coli, and we can then await the rapid extension of this line of research to fungi, plants and animals. Table 2 gives a list of siderophores which have been characterized to date. Although there is a growing tendency to now isolate known siderophores from new species - thus dimerum acid turned up in Verticillium dahliae, schizokinen in Anabaena sp. andferrioxamine E in Pseudomonas stutzeri- the list given in Table 2 is certain to be elaborated and will include new compounds of clinical interest. It is hoped that this cursory review of the field will be of some assistance in this fascinating work.

Table

2. List of siderophores and related compounds~ Sourceb

L Hydroxamic Acids Ferrichrome Ferrichrome A Ferrichrome C Ferrichrysin Ferricrocin Ferrirubin Ferrirhodin Albomycins (gdsein)76) Ferribactin Sake colorant A Verticillins Pseudobactins16)

Penicillia spp. Ustilago sphaerogena Cryptococcus raelibiosum Aspergillus melleus Aspergillus fumigatus Paecilomyces varioti Aspergillus nidulans Actinomyces subtropicus Pseudornonas fluorescens Aspergillus oryzae Verticillium dahliae Pseudomonas fluorescens

J. B. Neilands

22 Table 2 (continued)

Sourceb Pyoverdines 29) Rhodotomlic acid Dimemm acid Coprogen Triornoicin ~ Isotriornicin TM Schizokinen Terregens Factor (Arthrobactin) Aerobactins) Fnsarinine (Fusigen) Fusarinine A, B, C Triacetyl fusarinine Ferrioxamine A1, A2, B, D1, D2, E, G, H ~) Ferrimycin AI Mycobactins Nocobactins Aspergillic acids (neo-, meta, hydroxy, neohydrory) Mycelianamide Pulcherrimic acid Hadacidin Actinonin

Thioformin (Fluopsin)

Pseudomonas fluorescens Rhodotorula pilimanae Fusarium dimerum Neurospora crassa Epicoccum purpurascens Epicoccum purpurascens Bacillus megaterium Arthrobacter terregens Escherichia coli ColV Fusarium roseum Fusarium roseum Fusarium roseum Streptomyces spp. Streptomyces spp. Mycobacterial spp. Nocardia spp. AspergiUus flavus Penicillium griseofulvum Candida pulcherimma Penicillium aurantioviolaceum Streptomyces sp. Pseudomonas fluorescens

II. Catechols Enterobactin 2,3-dihydroxy-N-benzoyl lysine 2,3-dihydroxy-N-benzoyl serine 2,3-dihydroxy-N-benzoyl glycine 2,3-dihydroxy-N-benzoyl threonlne 2,3-dihydroxybenzoic acid Compound II Agrobactin s°) parabactin sl) Vibriobactin 49) Pyochelinsz)

Enteric bacteria Azotobacter vinelandii Escherichia coli Bacillus subtilis Klebsiella oxytoca Accompanies conjugates Paracoccus denitrificans Agrobacterium tumefaciens Paracoccus deni~flcans Vibrio cholerae Pseudomonas aeruginosa

Unless otherwise referenced, see Neilands, J. B., Ratledge, C. In Handbook of Microbiology, Vol. IV, 2nd ed., Laskin, A. I., Lechevalier, H. A. (eds.), CRC Press, Cleveland, p. 565 (1982), for structures and original literature citations b Not all of these compounds have been shown to meet the definition of a siderophore: low molecular weight, virtually iron(III) specific ligands induced by growth of microorganisms at suboptimal levels of iron. In addition, only a single source is listed. Thus ferrichrome is produced by Aspergillus niger, Ustilagosphaerogena, other fungi and all PeniciUia. Similarly, schizokinen is the product of various cyanobacteria as well as Bacillus megaterium

Methodology of Siderophores

23

IV. References Archibald, F.: FEMS Microbiol. Lettr. 19, 29 (1983) Reeves, M. W., Pine, L., Neilands, J. B., Balows, A.: J. Bacteriol. 154, 324 (1983) Neilands, J. B.: Structure and Bonding 11, 145 (1972) Lammers, M., FoUmann, H.: ibid. 54, 27 (1983) Flitter, W., Rowley, D. A., Halliwell, B.: FEBS Lettr. 158, 310 (1983) Trowbridge, I. S., Bishr Omary, M.: Proc. Natl. Acad. Sci. USA 78, 3039 (1981) Martell, A. E., Anderson, W. F., Badman, D. G. (eds.): Development of Iron Chelators for Clinical Use, Elsevier/North Holland, New York 1981 8. Warner, P. J., Williams, P. H., Bindereif, A., Neilands, J. B.: Infect. Immun. 33, 540 (1981) 9. Neilands, J. B.: Microbiology 1983 (Schlessinger, D., ed.), Am. Soc. Microbiol., Washington, D.C., p. 284 (1983) 10. Neilands, J. B.: Ann. Rev. Microbiol. 36, 285 (1982) 11. Garibaldi, J. A.: J. Bacteriol. 110, 262 (1972) 12. Kluger, M. J., Rothenburg, B. A.: Science 203, 374 (1978) 13. Bullen, J. J.: Rev. Infect. Dis. 3, 1127 (1981) 14. Murphy, T. P., Lean, D. R. S., Nalewajko, C.: Science 192, 900 (1976) 15. Trick, C. G., Andersen, R. J., Gillam, A., Harrison, P. J.: ibid. 219, 306 (1983) 16. Teintze, M., Hossain, M. B., Barnes, C. L., Leong, J., Vander Helm, D.: Biochemistry 20, 6446 (1982) 17. Powell, P. E., Szaniszlo, P. J., Cline, G. R., Reid, C. P. P.: J. Plant Nutr. 5, 653 (1982) 18. Vandenberg, P. A., Gonzalez, C. F., Wright, A. M., Kunka, B. S.: Appl. Environ. Microbioi. 46, 128 (1983) 19. Maugh, T. H.: Science 215, 1492 (1982) 20. Crosa, J. H.: Nature 284, 566 (1980) 21. Neilands, J. B.: Ann. Rev. Biochem. 50, 715 (1981) 22. Neilands, J. B.: Bacteriol. Rev. 21, 101 (1957) 23. Tomlinson, G., Cruickshank, W. H., Viswanatha, T.: Anal. Biochem. 44, 670 (1971) 24. Atkin, C. L., Phaff, H., Neilands, J. B.: J. Bacteriol. 103, 722 (1970) 25. Neilands, J. B.: Structure and Bonding 1, 59 (1965) 26. Holzberg, M., Artis, W. M.: Infect. Immun. 40, 1134 (1983) 27. Hider, R. C., Silver, J., Neilands, J. B., Morrison, I. E. G., Rees, L. V. C." FEBS Lettr. 102, 325 (1979) 28. Rioux, C., Jordan, D. C., Rattray, J. B. M.: Anal. Biochem., 133, 163 (1983) 29. Philson, S. B., Llinas, M.: J. Biol. Chem. 257, 808 (1982) 30. Fekete, F. A., Spence, J. T., Emery, T.: Anal. Biochem. 131, 516 (1983) 31. Lochhead, A. G., Burton, M. O., Thexton, R. H.: Nature 170, 282 (1952) 32. Morrison, N. E., Antoine, A. D., Dewbrey, E. E.: J. Bacteriol. 89, 1630 (1965) 33. Luckey, M., Pollack, J. R., Wayne, R., Ames, B. N., Neilands, J. B.: ibid. 111, 731 (1972) 34. Pollack, J. R., Ames, B. N., Neilands, J. B.: ibid. 104, 635 (1970) 35. Williams, P. H., Warner, P. J.: Infect. Immun. 29, 411 (1980) 36. Wayne, R., Frick, K., Neilands, J. B.: J. Bacteriol. 126, 7 (1976) 37. Garibaldi, J. A., Neilands, J. B.: J. Am. Chem. Soc. 77, 2429 (1955) 38. Emery, T., Neilands, J. B.: Org. Chem. 27, 1075 (1962) 39. Miles, A. A., Khimji, P. L.: J. Med. Microbiol. 8, 477 (1975) 39a. Rogers, H. J.: Infect. Immun. 7, 445 (1973) 40. Schade, A. L., Caroline, L.: Science 100, 14 (1944) 41. Lankford, C. E.: Critical Reviews of Microbiology 2, 273 (1973) 42. Waring, W. S., Werkman, C. H." Arch. Biochem. 1, 303 (1942) 43. Simpson, L. M., Oliver, J. D.: Infect. Immun. 41, 644 (1983) 44. Llinas, M., Wilson, D. M., Neilands, J. B.: Biochemistry 12, 3836 (1973) 45. Neilands, J. B.: In Development of Iron Chelators for Clinical Use (Martell, A. E., Anderson, W. F., Badman, D. G., eds.), Elsevier/North Holland, New York, p. 13 (1981) 46. Atkin, C. L., Neilands, J. B.: Biochemistry 7, 3734 (1968) 1. 2. 3. 4. 5. 6. 7.

24

J.B. Neilands

Simon, E. H., Tessman, I.: Proc. Natl. Acad. Sci. USA 50, 526 (1963) Gibson, F., Magrath, D. I.: Biochim. Biophys. Acta 192, 175 (1969) Griffiths, G., Sigel, S. P., Payne, S. M., Neilands, J. B.: J. Biol. Chem., 259, 383 (1984) Yamamoto, S., Shinoda, S., Kawaguchi, M., Wakamatsu, K., Makita, M.: Can. J. Microbiol. 29, 724 (1982) 51. Akers, H. A.: Appl. Environ. Microbiol. 45, 1704 (1983) 52. Leong, J., Raymond, K. N.: J. Am. Chem. Soc. 96, 6628 (1974) 53. Plowman, J. E., Loehr, T. M., Goldman, S. J., Sanders-Loehr, J.: J. Inorg. Biochem., 20, 183 (1984) 54. Bulman, R. A.: Structure and Bonding 34, 39 (1978) 55. Rastetter, W. H., Erickson, T. J., Venuti, M. C.: J. Org. Chem. 45, 5011 (1980) 56. Winkelmann, G.: FEBS Lettr. 97, 43 (1979) 564. Neilands, J. B., Peterson, T., Leong, S. A.: In ACS Symposium Series 140 (Martell, A. E., ed.) American Chemical Society, Washington, DC p 263 (1980) 57. Monzyk, B., Crumbliss, A. L.: J. Inorganic. Biochem. 19, 19 (1983) 58. Raymond, K. N., Carrano, C. J.: Acc. Chem. Res. 12, 183 (1979) 59. Lodge, J. S., Gaines, C. G., Arceneaux, J. E. L., Byers, B. R.: J. Bacteriol. 149, 771 (1982) 60. Manrer, P. J., Miller, M. J.: J. Am. Chem. Soc. 105, 240 (1983) 61. Manrer, P. J., Miller, M. J.: ibid. 104, 3096 (1982) 62. Lee, B. H., Miller, M. J.: J. Org. Chem. 48, 24 (1983) 63. Keller-Schierlein, W., Prelog, V., Zahner, H.: Prog. Chem. Org. Nat. Products 22, 279 (1964) 64. Naegeli, H.-U., Keller-Schierlein, W.: Helv. Chim. Acta 61, 2089 (1978) 65. Uemura, J., Mizushima, S.: Biochim. Biophys. Acta 413, 163 (1975) 66. Mclntosh, M. A., Earhart, C. F.: Biochem. Biophys. Res. Commun. 70, 315 (1976) 67. Lundrigan, M. D., Lancaster, J. H., Earhart, C. F.: J. Virol. 45, 700 (1983) 68. Laird, A. J., Ribbons, D. W., Woodrow, G. C., Young, I. G.: Gene 11, 347 (1980) 69. Laird, A. J., Young, I. G.: ibid. 11, 359 (1980) 70. Menichi, B., Buu, A.: J. Bacteriol. 154, 130 (1983) 71. Bindereif, A., Neilands, J. B.: ibid. 153, 1111 (1983) 72. Bindereif, A., Thorsness, P. E., Neilands. J. B.: Inorganica Chimica Acta 79, 78 (1983) 73. Bindereif, A., Neilands, J. B.: Unpubfished 74. Konopka, K., Bindereif, A., Neilands, J. B.: Biochemistry 21, 6503 (1982) 75. Konopka, K., Neilands, J. B.: ibid., 23, 2122 (1984) 76. Benz, G., Schr6der, T., Kurz, J., Wiinsche, C., Karl, W., Steffens, G., Pfitzner, J., Schmidt, D.: Angew. Chem. Suppl., 1322 (1982) 77. Frederick, C. B., Szaniszlo, P. J., Vickrey, P. E., Bentley, M. D., Shive, W.: Biochemistry 20, 2432 (1981) 78. Frederick, C. B., Bentley, M. D., Shive, W.: Biochem. Biophys. Res. Commun. 105, 133 (1982) 79. Adapa, S., Huber, P., KeHer-Schiedein, W.: Helv. Chim. Acta 65, 1818 (1982) 80. Ong, S. A., Peterson, T., Neilands, J. B.: J. Biol. Chem. 254, 1860 (1979) 81. Peterson, T., Neilands, J. B.: Tetrahedron Lettr. 50, 4805 (1979) 82. Cox, C. D., Rinehart, K. L., Moore, M. L., Cook, J. C.: Proc. Natl. Acad. Sci. USA 78, 4256 (1981) 47. 48. 49. 50.

Siderophore Mediated Absorption of Iron Robert C. Hider Department of Chemistry, Essex University, Wivenhoe Park, Colchester, Essex, UK

A brief presentation of iron chemistry is made with emphasis on those aspects relevant to siderophore biochemistry. Siderophore structure and biosynthesis is described. The underlying chemistry associated with, 1, the movement of iron(III) complexes across membranes and 2, the removal of iron from such complexes is discussed in detail. The ability of siderophores to interact with other metals is considered. Finally, the role of siderophores in infection and their clinical potential as iron scavenging molecules are reviewed.

Symbols and Abbreviations 1 Introduction

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

26

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

27

2 Siderophore Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Bidentate Ligands Possessing High Affinity for Iron(III) . . . . . . . . . . . . . . 2.2 Hexadentate Siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Kinetic Lability of Iron(III) Complexes . . . . . . . . . . . . . . . . . . . . . . . 2.4 Redox Activity of Catechol Ligands . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Stereochemistry of Octahedral Hydroxamate and Catecholate Complexes . . . . .

34 34 38 40 42 46

3 Siderophore Structure and Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Siderophore Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Siderophore Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48 48 50

4 Iron Transport in Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Iron(III) Siderophore Uptake by Fungi . . . . . . . . . . . . . . . . . . . . . . . 4.1.1 Iron(III) Siderophore Uptake by Ustilago . . . . . . . . . . . . . . . . . . 4.1.2 Iron(III) Siderophore U p t a k e by Neurospora . . . . . . . . . . . . . . . . 4.1.3 Iron(III) Siderophore Uptake by Rhodotorula . . . . . . . . . . . . . . . . 4.1.4 Iron(III) Siderophore Uptake by Fusaria . . . . . . . . . . . . . . . . . . . 4.1.5 General Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Iron(III) Siderophore Uptake by Enteric Bacteria . . . . . . . . . . . . . . . . . 4.2.1 Iron(III) Siderophore Translocation of the Outer Membrane . . . . . . . . 4.2.1.1 Iron(III) enterobactin Receptor . . . . . . . . . . . . . . . . . . 4.2.1.2 Ferrichrome Receptor . . . . . . . . . . . . . . . . . . . . . . . 4.2.1.3 Iron(III) citrate Receptor . . . . . ................. 4.2.1.4 Iron(III) aerobactin Receptor . . . . . . . . . . . . . . . . . . . 4.2.1.5 Regulationof the Outer Membrane Transport Systems . . . . . . . 4.2.1.6 Outer Membrane Proteins of Enteric Bacteria General Comments . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1.7 Outer Membrane Proteins of Non Enteric Bacteria . . . . . . . . . 4.2.2 Iron(III) Siderophore Translocation of the Cytoplasmic Membrane . . . . .

53 53 53 57 57 57 58 59 59 60 61 61 61 62 63 63 63

St~cture and Bonding58 © Springer-VerlagBerlinHeidelberg1984

26

R.C. Hider 4.2.2.1 Iron(III) enterobactin Permease . . . . . . . . . . . . . . . . . . 4.2.2.2 Ferrichrome Permease . . . . . . . . . . . . . . . . . . . . . . . 4.2.3 Iron(III) Uptake by M y c o b a c t e r i a c e a e . . . . . . . . . . . . . . . . . . . . 4.3 Antibiotics Based on Siderophore Structure . . . . . . . . . . . . . . . . . . . . . 4.3.1 The Ferrimycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 The Albomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

64 67 68 68 68 69

5 Removal of Iron From Siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Removal of Iron From Hydroxamate Siderophores . . . . . . . . . . . . . . . . . 5.2 Removal of Iron From Phenolate Siderophores . . . . . . . . . . . . . . . . . . . 5.3 Utilisation of Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 70 71 74

6 The Interaction of Siderophores and Siderophore-like Molecules with Metals Other Than Iron(III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Copper(II) Siderophore Complexes . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Molybdenium(VI) Siderophore Complexes . . . . . . . . . . . . . . . . . . . . .

75 75 76

7 The Role of Siderophores in Infection

77

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

8 Clinical Applications of Siderophores and Their Analogues

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

9 Conclusions and Suggestions for Further Work . . . . . . . . . . . . . . . . . . . . . .

79 80

10 Notes Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

80

11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81

Symbols and Abbreviations DNP CCCP NEM I~ I I~

2,4-dinitrophenol (Proton unconpier) Carbonylcyanide phenylhydrazone (Proton uncoupler) N-ethylmaleimide Solubility product59~ Formation constant59)

I(1

First equilibrium constant of a multistep association of more than one ligand molecule with the same cation59)

1~

Cumulative equilibrium constant of a multistep association of more than one ligand molecule with the same cation 59)

Siderophore Mediated Absorption of Iron

27

1 Introduction Siderophores are compounds produced by microorganisms for scavanging iron from the environment 1). They have been defined as low molecular weight compounds (500-100 daltons) possessing a high affinity for iron(III) (Ke > 103°), the biosynthesis of which is regulated by iron levels and the function of which is to supply iron to the cell2). During the period 1949-52, four different siderophores were isolated and identified as growth factors. Mycobactin and arthrobactin (Terregens factor) were isolated as free ligands 3, 4), while ferrichrome5) and coprogen6) were isolated as iron complexes. Snow, in two classical papers, largely characterised the structure of mycobactin, correctly identifying it's coordination groups as two hydroxamates and 2-hydroxyphenyloxazoline7, 8). He also demonstrated that mycobactin possesses a high affinity for AI3+, Cu2+ and Fe 3+. This seminal work was followed in 1960 by the discovery of the ferrioxamines9), which like the ferrichromes1°) possess hydroxamate ligands. A key observation concerning the mode of action of these growth factors was made by Garibaldi and Neilands in 19561~),when it was demonstrated that the production of ferrichrome A was enhanced by growing the organism Ustilago sphaerogena in medium deficient in iron. This finding was subsequently confirmed for mycobactin12) and many other siderophores1). The complete structures of the ferrioxaminet3) and ferrichrome14) classes were finally elucidated in 1962 and 1963 respectively. Thus by the early 1960's, three types of structurally diverse hydroxamate containing siderophores had been characterised as iron binding growth factors. Characterisation of the catecholato siderophores was initiated by the discovery, in 1958, that an iron(III) binding agent, the glycine conjugate of 2,3-dihydroxybenzoic acid, was secreted by Bacillus subtilis when grown under low iron conditions ~5). Analogous serine 16) and threonine 17)conjugates were subsequently isolated but it was not until 1970 that the first tricatechol siderophore was identified. O'Brien, Cox and Gibson reported evidence for condensed units of 2,3-dihydroxybenzoylserine in culture fluids of both Escherichia coli and Aerobacter aerogenes is) and in the same year enterobactin was independently isolated by Pollock and Neilands from Salmonella typhimurium 19) and O'Brien, and Gibson from Escherichia coli2°). The latter workers term this siderophore enterochelin. Since 1970 the number of well characterised siderophores has risen to over forty (Table 1). The majority of these molecules fall into either the hydroxamate or the phenolate classes and despite considerable structural variation, chelate iron in a hexadentate fashion. Siderophores, coordinated to iron, are accumulated by microorganisms by specific translocation mechanisms and the tightly bound iron is removed for utilisation by the cell1, 51, 52). Siderophore uptake varies between the different classes of molecule, some enter the cytoplasm, while others apparently donate iron at the cytoplasmic membrane surface. Indeed some siderophores may be secreted in order to deprive competing organisms of iron2°' 53) and as such will influence the ecology of the environment occupied by the secreting colony of microorganisms. An analysis of the experimental data concerning membrane permeation by siderophores is presented in this review and methods by which iron may be actively accumulated by organisms against considerable concentration gradients is discussed. Because of the high affinity of siderophores for iron(III) the mechanism of iron removal is of considerable chemical interest. Indeed, the manner in which iron is

6

7

8 (a) (b) (c) (d) (e) (f) (g) (h)

Pseudomonas fluorescensputida

Pseudomonas Bi0

Pseudomonas aeruginosa

Mycobacteria Mycobacterium phlei M. aurum M. fortuitum M. thermoresistible M. marinum M. terrae M. smegmatis M. tuberculosis

Pseudobactin u)

Pseudobactin A zs)

Pyoverdine ~)

Mycobactins~) Mycobactin P Mycobactin A Mycobactin F Mycobactin H Mycobactin M Mycobactin R Mycobactin S Mycobactin T

5

Pseudomonas aeruginosa

Pyochelin~)

6 (a)

4

Vibrio Cholerae

Vibriobactin ")

3

2(a) 2(c) 2(b) 2(d)

1

Azotobacter vinelandii

Paracoccus denitrificans

Agrobacterium tumefacie~

Aerobacter aerogenes, Escherichia coil Salmonella typhimurim

Ri F.A? F.A. F.A. F.A. CH3 F.A. F.A. F.A.

Structure

a, e-bis-2,3-dihydroxybenzoyllysine21)

-

Agrohactin49) Agrohactin A Parabactin49, 5o) - Parahactin A

Catechol type Enterobactin (enterochelin)tT. 1~)

Bacterium

1 a. Bacterial siderophores

Siderophore

Table

R2 CH3 CH3 H CH3 H H H H

R3 H H CH3 CH3 CH3 H H H

IL C2H5 CH3 CH3 CH3 F.A. C.zH5 CH3 CHa

R5 CH3 H H H CH3 CH3 H H

2-Hydroxyphenyloxazoline and hydroxamate

Catechol, hydroxamate and a-hydroxy acid Catechol and hydroxamate

Catechol, hydroxamate and a-hydroxy acid

Phenol and thiazoline

Catechol and 2-hydroxyphenyloxazoline

Catechol

Catechol and 2-hydroxyphenyloxazoline

Catechol and 2-hydroxyphenyloxazoline

Catechol

Ligands

to oo

B 33)

Nocardia, Micromonospom, Streptomyces, Acfinomyces

Bacillus megaterium, Anabaena sp. E. coli, Aerobacter aerogenes (now Klebsiella pneumoniae) Arthrobacter sp. R=H,n=2

CH = CH; n = 12, 14, 16

n = 5 R1 = H, R2 = CH3, n = 5 RI=CH3CO, R2=CH3, n=5 RI = H, R2 = CH2CH2CO2H, n = 5 R1=H, R2=CH3, n=4

n = 5

R=H,n=4

-= C H 3 • ( C H 2 ) n •

10(a) 10(b) ll(a) ll(b) 11(c) 11(d)

9(c)

9(b) R = C O 2 H , n = 4

9(a)

a F.A. = mixture of long chain fatty acids, for example Mycobactin P; F.A.

Desferrioxamine D134) Desferrioxamine G 35) Desferrioxamine A: 32)

Desferrioxamine

Desferrioxanune E 31) Desferrioxamine D232)

Ferrioxamines

Arthrobactin 3°) (Terregens factor)

Citrate-Hydroxamates Schizokinen2s) Aerobactin 29)

Hydroxamate

Hydroxamate and a-hydroxycarboxylate

o

O

O

>.

r~

~t 2o.

t~

fD

R. C. Hider

30 Table 1 b, Fungal siderophores (All hydroxamates) Siderophore

Fungi

Structure

Ferriehrome (name refers to iron complex)

Aspergillus, Neurospora Penicillium, Ustilago, Actinomyces, Streptomyces

12

R1 R2 Ferrichromet3, ~) Ferrichrome A ~s)

(a) H (b) H

R3 R H H CH3 H CH2OH CH2OH "C /

(c) H (d) H

CH3~'C02 H CH2OH CH2OH CH3 H CH2OH CH3

(e) H

CH2OH CH2OH

(f) H

CH2OH CH2OH

(g) H (h) H

CH3

Ustilago sphaerogena

Ferrichrysin37. 3s) Ferricrocin37, 3s) Ferrirubin3S. 39)

Aspergillus sp.

Ferrirhodin ~, 39) Ferrichrome C~) Malonichrome 41) Fusarinines (Fusigens) Triacetyi fusarine 42) (Triacetyl fusigen) Fusarinine A 43) } Fusarinine B43) Fusafinine C~) Rhodotorulic acid derivatives Rhodotorulic acid45)

Dimerumic acid~

Cryptococcus melibiosum Fusarium roseum

oc~H ]I CH~ "CH~OH H~C. jl. CH~ -CH2OH

H H

CH 3

CH3

•CH2CO2H

Fusaria, Aspergillus Penicillium, Gibberella Penicillium sp.

13

R = CH3CO

Fusarium roseum F. cubense

14(a) R = H , n = 2

(b) R = H , n = 3 13(b) R = H

Rhodotorula, Sporobolomyces Leucosporidium

15(a) R = CH3

Fusarium dimerum

15(b) R : C---~

H

?H2OH

CHa = cH

Coprogen47)

16(a) R=CHaCO; RI

--IO H CHa

PeniciUium sp. Neurospora sp.

(b) R=H, RI =~==~__.OH

Epicoccum purpurascens

(c) R = CHACO, R1 = CH3

CH3

Coprogen B 47)

(name refers to iron complex) Tfiornicin ~)

31

Siderophore Mediated Absorptionof Iron

HO

NH OH OH

O~

~..o.r..O o~o

l l / ~ y "OH

r~"...~/OH

~OH o~>--~,~o. 0 ~'H~

N"~ N H ~ O a.

,o,

X = OH

b, X = H

o

@ HO

0

Me

OH

~NH~0 C,

X = OH

d,

x

=

H

OH

OH OH

OH

N~<.o ®

~

~.OH

GOH .~"o ,~--_,~OH

o O~NH~~

~NH,"~O ([igands not established)

3)

o

®

~

""

O~Z

X

w

I

w

g

Z

z

~A.z

o

I

I

O~Z

-r-

-I-

~

°

@,

:~

\~

~--

._~

i

~,/~

r"-

o

-1-

\

z

Z

/

0

7

P

%-d

O.

=-r"°

o

,~-'~

~o

~\

~

z

®=

o

z

/o

0

7.

o,,

0

,-

N "" ~

0

/

-~

z

z

el.

til

33

Siderophor¢ Mediated Absorption of Iron R3

~ICH~N OyNH /

¢CH2~

OH

o

O

R

y

,~

NH

R

NH

OH

. o ~o

,/

| II / ~'~'~ / vN~c R ~)'- NH /

HO--N

H2)s NH R~

o

N r ' ~ ~ " ~°

0

o~,. ( ...IN.oH

I

_oH..,. II

NH

\CH

O~==~ Rz

R1

~01

L.,,I

0

OH

® OH

CH 3

0

/~--(CH~l-°J~-/NH~ ~

~,NF "0 ICH'F \

R K,H

"y,

o---~,,o

~CHz)z

HO] I

~

HU

@

L R.H

N---G( / \\

OH

0

<~

°

OH

®

H H

O.

I

o

~

I

N

~--N~~

.o/"

OH ~

N

~

A

N,-,R

~,0. I

O- iH--~O~[H3v

H

Jn

R

I

OH

\ , I (CHz)2--O-I-H

RNH 0

H OH 0 'I' R

T I

°" (c,~.c,~

~yH

CH3

~o

\ ._~N_

OH

~'~

O

®

t

AIN--~

p

~0 v -- "R,

34

R.C. Hider

removed from phenolate siderophores is under active debate at the time of writing. In this review, the chemistry of iron coordinated to catechol, hydroxamate and related ligands is presented in such a manner as to clarify and distinguish these various points of view. Although by definition, siderophores possess extremely high affinities for iron(III), many also form relatively stable complexes with copper(II)54), aluminium5s), molybdenum 56) and some transuranic elements57). The coordination of metals other than iron could have relevance to the microbial accumulation of these metals and to clinical applications of siderophores. These possibilities are considered in the present review.

2 Siderophore Chemistry Iron(III), by virtue of it's high charge density is a powerful Lewis acid and like protons, forms most stable bonds with ligands containing weakly poladsable atoms, for instance oxygen.

2.1 Bidentate Ligands Possessing High Affinity for Iron(Ill) There is a wide range of bidentate oxygen ligands and it is important to understand why hydroxamate and catecholate have been predominantly selected for siderophore construction. Figure 1 shows the Irving-Williams series of divalent transition metals5s) and the binding constants (K1) for different oxygen containing bidentate ligands 59, 6o)

14 B OH

m (~I~

-

OH

12

OH IX

10

~,~

OH

F1 CH~OOH0

8

0

CH) CH) HN~==/H OH

6 •

/.

NHz 0~--~"OH 0/I'-'~-OH

f Mn2+

) Fe2+

l Co2+

l Ni z+

i Cu2+

i Zn2+

Fig. 1. Affinity constants (Kt) of the first row transition metals with a range of bidentate oxygen containing ligands

Siderophore MediatedAbsorptionof Iron

35

There is a typical uniform sequence of stability for the replacement of water by the ligands. The smaller metal ions polarise the ligand electrons more strongly. Thus progressively smaller cations form more stable complexes. Significantly, ortho-dihydroxybenzene (catechol) is capable of bonding to cations more strongly than the other bidentate ligands (Fig. 1). The relationship between ligand affinity and charge density of the cation and hence Lewis acid strength is illustrated in Fig. 2. It is clear that electrostatic interactions dominate the interaction between the cations and the two selected ligands. Furthermore such interaction favours iron(III) over the other biologlcially important metals due to its small ionic radius (0.65/~,) (Fig. 2). On metal chelation, both catechol and hydroxamate form five membered rings with similar 0--0 bite distances, 2.6A for catecho161) and 2.55/~ for hydroxamate62). Therefore it is not the ligand geometry, but the charge density on each of the two ligating oxygens which is largely responsible for the differential affinities (Figs. 1 and 2). Indeed, the geometry of bidentate ligands has remarkably little influence on their selectivity. This point is adequately illustrated by the finding that the ratios of log K1 for iron(III) and iron(II) fall in the range 2.2-2.5 for a range of dioxygen containing ligands (catechol, acetohydroxamic acid, salicylate, acetylacetone, oxalate and hydroxyacetate). For monobasic bidentate high affinity iron(III) ligands, the pKa must be greater than 6.5, i.e. the ligand possesses a relatively high affinity for protons (Table 2). The presence of two dissociatable protons of high pKa, for instance catechol, is indicative of two oxygen atoms possessing a high electron density when deprotonated. Thus at non acid pH values, catechol is predicted to be a more powerful ligand for iron(III) than hydroxamates.

Fe~ Zn~ Ni~ CJ

AI~

•

....li

i"

!

i

l

20

FeTM !

I /

Q/

18

16

~I~

f 12

10

8

6

Fig. 2. The dependenceof affinityconstants (K1)for catechol and acetohydroxamicacid, on cationcharge density.The value of the tetravalent radii was used for copper(II)

/, 0-1

I

I

I

I

]

0"2

0.3

04

05

0-6

e ~-2

charge density of cation

R. C. Hider

36 Table 2. pKa values of ligands possessing high affinity for iron(III)

Catechol N-Methylacetohydroxamic acid 3-Hydroxy-2-methyl-4-pyrone Tropolone Ascorbic acid 3-Hydroxy-l,2-dimethylpyrid-4-one 3-Hydroxy-l-methylpyrid-2-one Salicylic acid Acetylacetone

pKa~

pKa2

9.2 9.0 8.6 6.6 4.2 9.7 8.8 3.9 8.9

13.0

11.6 13.1

O-

~

~.0

O-

m

I

Il

0

0

®

\~ /"\Rz

7, o

~-

®

N+

\R~

RI

j!

oe

oe

N

R

-0

o) /~

/

O-

@

e

RI

O-

®

R

/ N \

O

Siderophore MediatedAbsorptionof Iron

37

Resonance stabilisation of the anion can further influence the coordinating properties of the ligand. Thus 5-nitrocatechol (17) and N,N-dimethyl-2,3-dihydroxy benzamide (18) have reduced affinities for iron(III) as indicated by their log K1 values of 17.1 and 17.8 respectively (catechol, log K1 -- 20). Similar trends are noted for hydroxamates where stabilization of the resonance form corresponding to nitrogen lone pair delocalisation into the carbonyl function (19) stabilises the iron(III) complex 63). Structure 19 is strongly influenced by the electron donor ability via either resonance or induction effects of R2. Thus the replacement of hydrogen by an alkyl group at this position enhances the ligand's affinity for iron(HI). Delocalisation of electrons from conjugated side chains (20) can also enhance the electron density on the carbonyl oxygen and thus increase the affinity for iron(III). Such modification is observed in mycobactin (8 a), some ferrichromes (12 b, 12e, 120 and rhodotorulic acid derivatives (15 b, 16 a, 16 b). O)

@

(a)

(b)

Although bidentate ligands possessing two ligating oxygen atoms predominate, 2hydroxyphenyloxazoloneis frequently incorporated into bacterial siderophores (2 a, 2 b, 4 and 8). The C-O and C-N bond lengths indicate that an appreciable contribution is made by form (21 b) 64). Due to the charged nature of this resonance form, nitrogen might be expected to be the preferred ligand atom, the oxazolium oxygen being relatively electron deficient. Such coordination is observed in iron(III) mycobactin (8) 12) and iron(III) agrobactin (2 a)65) where 2-hydroxyphenyloxazolone binds iron in a bidentate fashion.

Single bond Oxazofinering64) Double bond

C-O

C-N

1.47 1.33 1.23

1.46 1.29 1.27

Clearly, dioxygen ligands, possessing a high electron density on both ligating oxygen atoms favour the selective binding of iron(III) in the biosphere by virtue of both the strong electrostatic interaction and resonance stabilisation of the resulting complex. Catechol would appear to be the ligand of choice, but has the disadvantage of being susceptible towards oxidation. Hydroxamates, although possessing a lower affinity for iron(III) than catecholates, are capable of forming uncharged complexes (Eq. (ii)). This difference from iron catecholato complexes (Eq. (i)) has implications for membrane translocation, (Sect. 4). The selection of 2-hydroxyphenyloxazolonemay well be associated with its potential susceptibility towards hydrolysis 49). Under controlled conditions such hydrolysis could facilitate the release of iron.

38

3

[~

OH +

...."~,

Fe]+

[ ~

0}3-

R.C. Hidcr

4"

em + 6H

(i)

N

OH

o

R1

0

3

+

Fe

3H+

--.

(~)

Rz/N~o'~3

Rz~N~oH

2.2 Hexadentate Siderophores For iron(HI), donor-acceptor bond energies largely determine the enthalpy contribution to the stability of the complex, but entropy can also make a considerable contribution with multidentate ligands. A favourable entropic contribution from displacing coordinated water molecules on going from a tris (bidentate) to a hexadentate complex can account for an increase in the formation constant, Kf, of up to 6 log unitsss). Precisely this increment is observed when the formation constants of tr/s (N,N-dimethyl-2,3-dihydroxybenzamide)iron(III) and the synthetic tricatechol ligand MECAM (22) are compared. The log values are 40.267) and 45.9s) respectively. However, an even greater differential is observed with entcrobactin (1), the formation constant of which is the highest ever measured for an iron complex, log Kf = 5267). This additional increment is not only due to entropy effects but must also involve an enthalpy change associated with the central triester ring.

°@0N NH

OH

I

CH2

OH

~

NH ~ CHz

OH

CH~

A similar transition is also observed for hydroxamate ligands, although the difference is not so marked, for instance the log formation constants of tr/s-(acetohydroxamic acid) and ferdoxamine E (10a) are 28.3 and 32.5 respectively69). With deferriferdchrome

Siderophore Mediated Absorption of Iron

39

(12a) (log Kf = 29.1) and ferrioxamine B (11 a) (log Kf = 30.6) the differential is even less 67), indicating that although chelation provides a favourable entropic contribution, hexadentate formation can involve molecular strain, which in turn gives rise to unfavourable enthalpic contributions. The major reason for microorganisms commonly utilising hexadentate siderophores is probably not for the increased affinity as measured by formation constants, but rather the increased holding power at low iron concentrations (< 1 ~tM). This point can be demonstrated by comparing the position of equilibrium of Eqs. (iii) and (iv), the equilibrium constants of which are very similar. If these two ligands are scavenging iron at a ligand concentration of 10-SM in an aqueous solution containing colloidal iron at a concentraFe 3+ + Trihydroxamate. 3 H -~ for deferriferrichrome log

~1 =

F e In °

Trihydroxamate + 3 H +

(iii)

29.1

Fe 3+ + 3 CH3CONOH ~ FelII(CH3CONO)3 + 3 H +

(iv)

log [53 = 28.3 tion of 10-1°M, then the equilibrium non-coordinated iron(III) concentrations will be quite different. For the hexadentate ligand the free iron concentration will approach 10-3°M but in the presence of the bidentate hydroxamate the colloidal iron concentrations will remain the same. The equilibrium concentration of non-coordinated iron(III) at pH 7.0 is about 10-is M, due to the extreme insolubility of iron(III) hydroxide (K~l = 10-38). At this concentration of iron(III) the monohydroxamate is unable to compete with hydroxide anions. Although the formation constants for the binding of iron(III) to tricatechols are enormous, they have little direct relevance to physiological conditions. By definition I-I6(ent) + Fe 3+ ~- [FenI(ent)]3- + 6 H + where ent = enterobactin

(v)

formation constants exclude the hydrogen ion concentration, an important term in Eq. (v). The effective equilibrium constant K~ of the reaction depicted by Eq. (v) is given by:

[Fe(ent)3-][H+] 6 K~' =

[Fe3+][H6ent]

and has been determined to be 10-1° over the pH range 4--667). Thus at pH 6.0 the value of the ratio [Fe(ent)3-]/[Fe3+][H6ent] = 1026. This value, although large, is considerably smaller than the formation constant (1052).

40

R.C. Hider

2.3 Kinetic Lability of lron(III) Complexes In addition to the range of thermodynamic stabilities of metal chelates there is considerable variation in kinetic stability. Some rate constants for the water exchange reaction (Eq. (vi)) are presented in Fig. 3. Although this reaction has zero thermodynamic driving M(H20)~+ + H20* ~ M(H20)m-] (H20*) "+ + H20

(vi)

force, the rate constants vary over some 17 orders of magnitudev°). Since electrostatic binding forces between the metal ion and ligands are larger for a high cation charge density, there is a general trend for tripositive cation complexes to be less kinetically labile than those of divalent complexes (Fig. 3). However, geometrical distortion can speed up the exchange of ligands because the rapid interconversion of axial and equatorial ligands, through molecular vibration, stretches the metal ligand bonds. Thus copper(II) complexes, which generally possess a distorted coordination sphere due to the Jahn-Teller effect, possess a high kinetic lability71). Whereas the extreme slowness of chromium(III) and rhodium(III) octahedral complexes can be attributed to crystal field effects. The d-electron configurations are such that d 3 and d 6 octahedral complexes are considerably destabilised by any reaction that temporarily changes their geometry. Highspin iron(III) possesses a d 5 configuration which lacks any crystal field stabilisation energy thus rendering the complexes kinetically labile despite the high charge density of the trivalent cation (Fig. 3). Such lability can he further enhanced if the ligand forms a distorted complex. Thus iron(III) chelates, possessing small differences in thermody-

I lO0 lO6

~

I

I

~n2-*;',, Zn2+ _

lO~. I

I

. Cu z÷

Ni2%

• In3*~

~o2 At3+,

% -r-

C03+e 10-2

10-.~ _

10-6 _

• Cr 3÷ Rh 3+

lO-e o.1

I

I

I

I

0.2

0"3

O-t~

0"5

Cation charge densify e / ~ 2

0"6

Fig. 3. The dependence of the rate of substitution of inner coordination sphere water molecules on cation charge density. The value of the hexadentate radii was used for copper(II)

41

Siderophore Mediated Absorption of Iron Table 3. Kinetic stability of iron(HI) chelates 7z)

Iron(III) ligand

Thermodynamic stability constant

Half time of iron exchange with apotransferrin

Nitrilotriacetic acid Citric acid EDTA

10z3 102z 1025

3s 8h 4 days

namic stability, can have very different kinetic stabilities (Table 3). By virtue of this kinetic lability, siderophores are able to rapidly abstract iron from labile complexes and colloidal iron hydroxide. For instance ferrichrome removes iron(III) from citrate with a half life of 10 min 73) and from iron(III) hydroxide with a half life of 32 min 74). When in competition with the mammalian plasma iron binding protein transferrin, catechol containing siderophores are kinetically more efficient than the hexadentate hydroxamates. The mechanism of iron removal is believed to involve a ternary complex as shown in Eq. (vii) where Tr is apotransferrin and L is the hexadentate siderophore. (vii)

FezTr + 2 L ~ 2 LFeTr ~ 2 FeL + Tr

The rate determining step is probably the displacement of the HCO~ anion from the transferrin iron inner coordination sphere by the incoming ligand 75). Hydroxamates are kinetically hindered in this reaction as indicated by the data in Table 4.3,4-LICAMS (23) a synthetic siderophore is much more effective at removing iron from transferrin than desferrioxamine B and yet it possesses a similar affinity for iron(III). The introduction of a catechol function to desferrioxamine B (24) removes this kinetic barrier (Table 4). Presumably, the 3-hydroxylcatechol function, being more nucleophilic than the hydroxamate hydroxyl function, facilitates attack. By virtue of their multidentate nature, siderophores once given a hold on iron, will rapidly envelope it forming a high affinity complex. The kinetic exchange between two

Table 4. Relative kinetic ability to remove iron(III) from transferrin 76' 77)

Ligand

log Kf

P[Fe3+]"

K2 (ligand) b I(2 (desferrioxamine B)

Enterobactin (1) 3,4-LICAMS (22) Desferrioxamine B (11 a) N(2,3-dihydroxybenzoyldesferrioxamine B) (23)

51 42 3I -

35.6 27.6 26.6 -

120 120 1 180

' Hexaaquo Fe concentrations m equilibrium with iron complex where total ligand concentration is 10-5M, total iron(III) concentration is 10-6M and pH is 7.4 b K2 is second-order rate constant for the removal of iron from transferrin 3+

•

•

42

R.C. Hider

,~ e03S

%,s,.

~

..OH

..o.

y

e.

"OH O~-------(

II'L~ "

@ OH

0~/

OH (CHT)s "NH~ "N

/ OH

(.C (

o

N ~ (C~2)S N

/ OH

/(CH2)z

( '

(CHz)s

\ N"

o

/ OH

(

CH3

0

@ siderophores is extremely slow. The half time for exchange of 59iron between desferrioxamine B and ferrichrome A (4 mM, pH 7.4) is over 200 h76) although the rate can be greatly increased by acidification. Thus iron(III) complexes of hexadentate siderophores are both kinetically and thermodynamically stable, which is ideally suited for their iron scavenging role. However, in principle, this presents a problem to the microorganism during the iron assimulation stage. As siderophore-mediated iron transport and release is much more rapid than exchange kinetics of iron(III) siderophores, redox reactions have been implicated7s). The resulting iron(II) is kinetically (Fig. 3) and thermodynamically (Fig. 2) much less stable than iron(III).

2.4 Redox Activity of Catechol Ligands In contrast to hydroxamates, catechols are susceptible towards oxidation (Eq. (viii)). The oxidation products, semiquinone and quinone are also able to coordinate cations, but generally with reduced affinity79-s2). Assignment of a formal oxidation state to the coordinated metal is not always unambiguous as the orbitals involved are delocalised over the metal and at least one ring. The electronic structure of the metal quinone chelate ring can be viewed in terms of three isoelectronic forms (25). These various complex types can be induced electrochemically by the addition or removal of electrons via an electrode surface or a chemical agent. Tris(catecholato)chromium(III) for instance, displays a 7 membered redox series corresponding to the stepwise oxidation to the tri(obenzoquinone)chromium(III) (Eq. (ix)) 83). The cyclic voltammogram for tris(9-10

43

Siderophore Mediated Absorption of Iron e-

.

°"

O-

(viii) o

O-

Car

B(/

SQ

[ Cr=(B(2)3 ]3+ ~ -

[c=(Ba)2(sa)]~+ _e- [C=(SQ)(SQ)2]~+ (a)

.e% [crm{so)3]0

e-

[£r=(SQ)2 (Cat)]'-

e-~

[Cr=(Sa)(Cat)2]z-

e\ [Cr~(Cat)~]3_

dihydroxyphenanthrene) chromium(III) (26) is shown in Fig. 4. The 3 negative reduction potentials correspond to reduction to the species with the net charges -1, -2 and -3. The position of the reduction potential varys with ligand and therefore in principle can be tuned to requirement82). Confirmation of the formal oxidation state of the metal can be established by the use of e.s.r, measurements (copper and chromium)s2) and M6ssbauer spectroscopy (iron)s*' ss). The distorted octahedral stereochemistries of tris(catecholato)chromium(III) and tris(3,5-ditert-butyl-o-benzosemiquinone)chromium(III) are very similar possessing chromium-oxygen distances of 1.9961) and 1.93 A s6) respectively. Proportionately slightly larger differences are found for the C-O bond lengths, mean values being 1.35 A for the catecholato complexes and 1.29 A for the semiquinone complex. Essentially the same bond lengths have been determined for analogous chromium(III), iron(III) and cobalt (III) semiquinone complexess7-9°).

I

I

l

I

1

I

+ 1"2

I

I

I

I

+0.8

I

I

I

I

+04

I

I

I

I

0

l

I

I

I

-0.6,

I

I

I

I

-0.8

,

I

-I,2

Voffs vsSCE

Fig. 4. Cyclic voltammogram for Chromium(o-phenanthrosemiquinone)3(26). The voltage slope was 50 mV s-1 and the solvent was CH2C12containing(n-But4N)CIO4(0.1 M). The reaction was run under nitrogen~)

44

R.C. Hider

0/

~

0~0 0/

@

/~-0,

@ Chelate bonding by the semiquinone structure is formally analogous to acetylacetohate bonding and as such is quite strong. Indeed chelation can protect semiquinones from further oxidation to the corresponding quinones. Zinc(II) has been demonstrated to stabilise o-benzosemiquinones in the presence of air for several hours, conditions where the uncomplexed radical decomposes rapidly79). Indeed zinc(II) also catalyses the reverse disproportionation of catechol and benzoquinone (Eq. (x))91). Metal catechol complexes are able to undergo intramolecular electron transfer reactions. The position of equilibrium of such reactions are influenced by the solution pH, the dielectric strength of the medium and the presence of other ligands. Copper complexes of

~o

~oH 0

+

OH

r'~'-,#o2zg+ ~ o , , ~

*-~"++~-kMA~ ~.k.~k~ o/°

(x)

of

SiderophoreMediatedAbsorption Iron

45

3,5-ditert-butylcatechol show such a ligand dependence. With nitrogen donors a copper(II) catecholato complex (27 a) is favoured92), whereas with phosphorus donors, a copper(I) o-benzosemiquinone (27 b) predominates 93).The formal transfer of an electron between a ligand and metal ion was first observed with a cobalt complex9°). The bipyridine adduct of [Cobalt(3,5-ditertbutyl-o-benzosemiquinone)2]4is a cobalt(III) catecholate semiquinone complex, in the solid state at room temperature, but in toluene and diethylether solutions (- 60"C to + 60 °C) an equilibrium exists (Eq. (xi)) with the cobalt (II) species forming at the higher temperatures. This process is completely reversible82, 90)

)_d/°%

( bi Py ~

h'

\ O A , , ~

(b)

(a)

t2"t-

(H~O)~ H (a)

I

12+ H

~

(b)

A similar phenomenon has been observed for iron(III) catechol complexes, where an internal redox reaction is triggered by a change in pHe~). Over the pH range 2.0 to 4.5, the green iron(II) semiquinone complex (28 b) is the dominant species as determined by M6ssbauer spectroscopya4) and magnetic moment measurements94). Negatively charged ligands tend to stabilise iron(III), as electrostatic repulsion will favour removal of an

~j

~f

~

j

46

R.C. Hider

electron from iron(II). However, neutral unsaturated ligands, like the monoprotonated semiquinone (28 b) will stabilise the divalent state. This inversion is largely due to the greater crystal field stabilisation of a d6 system (iron(II)) compared to that of a d 5 system (iron(III)) in strong fields95). In this pH range the coordinated iron(II) prevents the semiquinone undergoing disproportionation to catechol and benzoquinone. However, at pH values below 2.0, disproportionation does occur9~). Thus the redox state of iron coordinated to catechol is dependent on the pH of the solution and can be repeatedly cycled between iron(II) and iron(III) by manipulation of pH. As a result of the ability to undergo internal redox reactions, the electrochemical activity of iron(III) catechol complexes is unlike those of chromium(III), only two or three reversible couples being identified by cyclic voltammetrys2' 97). The reduction potentials of those cations which have been demonstrated to be capable of undergoing an internal redox reaction with catechol are all less than -0.1 volt with respect to the hydrogen electrode. They are copper (II/I), -0.17 V; iron (IIUII), -0.77 V and cobalt (III/II), -1.81 V. In contrast the chromium III/II redox potential is +0.41. Thus chromium(HI) is a much weaker oxidising agent than the other cations and is unable to oxidise catechol to the semiquinone.

2.5 Stereochemistryof OctahedralHydroxamate and Catecholate Complexes The tr/s chelate of a symmetrical bidentate ligand such as catechol can exist as an enantiomeric pair (Fig. 5). With an assymetric ligand such as hydroxamate both geometric and optical isomers are possible: A-cis, A-trans, A-cis and A-trans. However, many hexadentate hydroxamates are stereochemically restricted to the cis isomers. Since iron(III) complexes are quite labile many stereochemical assignments have been made using the kinetically inert chromium(III) and rhodium(III) analogues (Fig. 3) 98). Both these cations possess similar radii to that of iron(III). Raymond and coworkers have separated, isolated and characterised diastereoisomeric pairs of bidentate hydroxamate complexes of chromium(III)99). Having eharacterised each enatiomer, application of circular dichroism spectroscopy facilitates the determination of the stereochemistry the related coordination centres of hydroxamate siderophores in solutiont°°-1°2). The stereochemistry of some siderophores had previously been determined by X-ray diffraction methods, but in view of the kinetic lability of iron(III) complexes it is conceivable that both isomers could exist in solution. For hexadentate hydrox-

0 A "left hand propeller . . . .

A right hand propeller"

Fig. 5. Enantiomersofhexacoordinated metal complexes

47

Siderophore Mediated Absorption of Iron Table 5. Stereochemistry of siderophore metallate(III) complexes

Siderophore

Stereochemistry

Ferrichrome Ferrichrysin Ferrichrome A Malonichrome Rhodotorulic acid Aerobactin Iron(III) myobactin P N,N,N-triacetylfusarinine

(12a) (12c) (12b) (12h) (15a) (9b) (8a) (13)

Ferrioxamine E Ferrioxamine B Ferrioxamine D Enterobactin Agrobactin Parabactin Enantioenterobactin

(10) (lla) (llb) (1) (2a) (2b)

Ref.

A-cis 100 A-cis 100 A-cis 99 A-cis 41 A-cis 101 A 102 A-cis 103 A-cis (solid state); Predominantly 104 A-cis (solution) Racemate-cis 105 Racemate-cis and -trans 106 Racemate-cis and -trans 106 A-cis 98 A-cis 65 A-cis 65 A-cis 109

m a t e s , this appears not to be the case, although bidentate hydroxamates undergo rapid racemisation in methanol and aqueous solutions 98). The known stereochemistries of hexadentate siderophores are presented in Table 5. Unlike most known siderophores, the ferrioxamines are not optically active. It is not surprising therefore, that there is no clear preference for a particular coordination propeller type for this class of molecule (Table 5). The optical isomers of both tr/s(catecholato)chromate(III) and -rhodate(III) have been resolved and characterised by circular dichroism 1°7' 108). Using this data, the stereochemistry the Chromium(III), Rhodium(III) and Iron(III) complexes of enterobactin have been determined as A-cis (29) 98).

0

H

48

R.C. Hider

3 Siderophore Structure and Biosynthesis The known fungal siderophore structures (Table lb) lack both 2,3-dihydroxybenzoic acid and 2-hydroxyphenyloxazoline as ligands and are largely restricted to hydroxamates. In contrast bacteria are more versatile, utilising a range of high affinity iron(III) ligands (Table 1 a).

3.1 Siderophore Structure The general principles underlying the detection and isolation of siderophores has been outlined by Lankford 1) and Neilands 2). In addition, a new test for catechol detection has been reported u°). This assay depends on the iron(III) catechol internal redox reaction ~). Characterisation is greatly facilitated by high resolution NMR techniques zs' 49) and mass spectrometry 26). Fast atom bombardment mass spectrometry (FAB) is particularly useful for the determination of siderophore molecular weights m). Until recently, siderophores had proved intractable to mass spectrometric analysis partly because of their polar nature and thermal instability and partly because many are most conveniently isolated as a metal complex. Many siderophore structures have been confirmed by chemical synthesis (1)1°9), (2 a) 49), (8) n2), (10)32), (11)3s), (12) 37-39), (15)46) and (16)46' 47) The basic design of siderophores is such that the bidentate functional units are placed at suitable distances in order to create an intramolecular hexadentate complex. This spacing is achieved via cyclic (1, 13) and acyclic esters (14, 16); by cyclic (12) and acyclic ~t-aminoacid amide bonds (6, 7), by cyclic (10, 13) and acyclic o~-aminoacid amide bonds (11) and by mixtures of ester and amide bonds (8). The structural framework can also be based on spermidine and related polyamines (2, 4) and citrate (9). The advantage of a cyclic structure appears to be two-fold. They are more resistant to attack by proteolytic enzymes and secondly, due to a decreased entropy difference between the free ligand and the complex as compared with the acyclic analogue, they possess a higher affinity constant for iron(HI). This latter point is exemplified by the finding that enterobactin possesses a higher affinity for iron(III) than both its natural acyclic analogue m) and the synthetic acyclic siderophore LICAMS (Table 4) 76). With the dihydroxamate, rhodotorulic acid (15 a), intermolecular complexes of the type (Iron(III))z (Rhodotorulate)3 are favoured 1°1). Seven siderophore iron(III) complexes have been subjected to X-ray diffraction methods, (12c) 114), (8a) 1°3), (12b) n5), (10a, 30) 1°5), (13) 1°4), (12a) lx6) and (6) 24). All possess a distorted octahedral coordination, the distortion being greater in complexes possessing non-equivalent ligands, for instance mycobactin P (8a, 31) 1°3). The X-ray derived structures of uncomplexed agrobactin (2 d) has also been determined 64). Although most hydroxamate siderophores possess 3 identical ligands (structures 10--16), enterobactin is the only phenolate siderophore isolated to date, containing a coordination sphere which possesses a 3-fold symmetry. The phenolate siderophore types 2, 4, 6, 7 and 8 all contain either 2 or 3 different bidentate ligands covalently linked together. The iron IIUII reduction potentials of these non-regular octahedrally coordinated complexes are likely to be less negative than that of iron enterobactin as they contain coordination groups with lower electron density than that of catecholate 95).

Siderophore MediatedAbsorption of Iron

49

® Furthermore these complexes will be more kinetically labile (Sec. 2.3). Both properties are critical factors for the removal of iron and could account for the high proportion of ,non-regular phenolate siderophores. It is likely that some microbial siderophores contain neither hydroxamate or phenolate ligands. Low molecular iron(III) binding compounds have been isolated from Corynebacterium diphtheriae nT) and Aeromonassalmonicida ns) which give negative Arnow and C'saky tests. Not all microorganisms produce detectable levels of siderophoresrig}.

50

R.C. Hider

3.2 Siderophore Biosynthesis The influence of iron on siderophore production is dramatic as demonstrated by hydroxamate (ferrichrome and ferrichrome A) synthesis by Ustilago spherogena (Fig. 6)78). Although, iron has been shown to inhibit the synthesis of the majority of siderophores presented in Table 1, the method by which this control is achieved is unknown at present. Cell free extracts capable of enterobactin (1) 20' 120)and parabactin (2 b)~) biosynthesis require only ATP as a cofactor. The presence of Coenzyme A is not necessary. At least four proteins are required for enterobactin synthesis from dihydroxybenzoic acid and Lserine (Scheme 1) 121). A similar stepwise synthesis has been observed for parabactin (2b) (S-cheme 2) 5°' 65). D-Threonine and L-serine are unable to replace L-theonine, although N-(3-aminopropyl) 1,3-diaminopropane can replace spermidine as a substrate. An indication of the location of the control step in this synthesis is given by the observation that formation of the dicatechol intermediate is inhibited in the presence of 35 I~M iron(II) 5°). In contrast copper(H), cobalt(II) and chromium(III) are not inhibitory at 200 l~M5°). Despite the structural diversity of hydroxamate siderophores, their biosynthesis almost certainly follow a similar pattern. The biosynthesis of ferrichrome and fen'ichrome A has been extensively studied in U. sphaerogena 7a' 122) and is presented in Scheme 3. The acetogenin precursor of deferriferrichrome A, methylgiutaconic acid is derived from acetyl CoA and in principle similar acetogenins could give rise to a wide range of cyclic and oligomeric hydroxamate siderophores (Scheme 3) 78). An enzyme which catalyses the acetylation of N-hydroxyornithine has been isolated from cell free extracts of U. sphaerogena 7a). Significantly although it is unable to acetylate ornithine it readily derivatives e-N-hydroxylysine. Thus analogous biosynthetic routes to those shown in Scheme 3 appear likely for the ferrioxamines (10 and 11) and have been demonstrated for aerobactin (9 b) 12a). The mechanism by which iron represses hydroxamate siderophore biosynthesis is unknown. It is most probable that iron is removed from

1-0 /o_

0.8

/

o

"

~_.o_~_7 ~ ~

0-6

10- H Fe

o-

"

,,/

o.~ 2

~

o~

5 x 10-7 H Fe

o1__~0"6M ~e

0 0

2

6

6

days

Fig. 6. Effect of different iron concentrations on hydroxamate synthesis by Ustilago sphaerogena TM

51

Siderophore Mediated Absorption of Iron

OH

(

OH

Serine [ OzH

~C02H

~

Enterobnctin

(1)

OH

OH

OH

O~'~L"~O O ~ C02H OH

Scheme 1. Biosynthesis of enterobactin

OH

OH

oH/

>

COzH

Spermidine

~o. f C

OH

0

NH HN (CH23) ~(CHz)4

°H

OzH

Threonine

Scheme 2. Biosynthesis of parabactin

Me~'~[ 0:,H OH

Parebactin (2b)

52

R. C. Hider

CoA

~H2N.~ NHOH

NHz I

(c.,)= HzN' ' ~ COzH

Ace~ytCoA

I I I

~deferrlferrtchrome (12a)

213 'CO.zH

~ " ' - ~ R h o d o t o r u l i c acid (15a)

C02H

c~

I I I I

c

deferriferrAchrome (12b)

HO--N

HzN

il I

co~ Glyclne + SerAne

Ii II Ii

SCoA

Glyc£ne

C02H

O=

HOo'~CH3

CH~ ~==0 NOH

l

A

CO~

O C ~ OH

I I C~ 0 ~

Ho--N

\ \

HO

~

0H j

~

HZN ' ~ C 02H

0~'~ CH3 HO--N

_.....~deferriferr£rhod£n (12f} "~'~Fusarlnes (13 and 14)

H2N"~" C02H

ROUTESNOTESTABLISHED Scheme 3. Biosynthesis of hydroxamate sidcrophores

deferr£ferrirub£n (12e) > d£merumlcacid (ISb)

Coprogen(16a)

Siderophore Mediated Absorption of Iron

53

the siderophore complex before acting as a repressor or inhibitor124'12s).It is conceivable that in some organisms, siderophore biosynthesis is regulated indirectly by cellular oxidative metabolism7a, nO

4 Iron Transport in Microorganisms In view of the variety of siderophores that have been identified it is not surprising that a corresponding diversity of iron(III) transport mechanisms exists. Indeed there are several examples of more than one system operating in a single species, and in the presence of relatively high levels of iron (> 10-5 M) a low affinity iron translocation mechanism operates. Thus the kinetic interpretation of iron uptake can be complicated. This difficulty is compounded by the different levels and types of trace iron present in the incubation media. Lankford 1), Neilands 124), and Winkelmann 126) present clear discussion of these problems. Uptake sudies have been carded out with fungi (for instance Ustilago sphaerogena and Fusarium roseum) and bacteria (mainly enteric bacteria for instance Escherichia coli and Salmonella typhimurium). As the structures of these two classes of organism are quite different they will be considered separately.

4.1 Iron(Ill) Siderophore Uptake by Fungi Fungi are eukaryotic organisms and possess a single cytoplasmic membrane usually surrounded by a hyphal wall. This hyphal wall does not contain an integral lipid membrane and thus unlike enteric bacteria, the iron only has to permeate a single bilayer structure. Siderophores have been isolated from three different fungal classes: Basidiomycetes (Ustilago); Ascomycetes (Neurospora and Rhodotorula) and Deuteromycetes (Penicillium, Aspergillus, and Fusarium). A more complete list is provided by Winkelmann 126). Iron(III) siderophore accumulation is an active transport process, being severely inhibited in the presence of metabolic poisonsTM.

4.1.1 Iron(Ill) Siderophore Uptake by Ustilago Emery and his coworkers have made a major contribution to our understanding of ferrichrome uptake by Ustilago sphaerogenaTM. Washed cells of this organism rapidly accumulate ferrichrome from the medium which can be monitored by the loss of extracellular colour and the formation of highly pigmented cells127). When [14C]- and [59Fe]ferrichrome are utilised in such experiments both isotopes are initially accumulated at the same rate but as the experiment progresses [14C]-deferriferdchrome is released from the cell and thus is capable of scavenging more iron (Fig. 7) 127). That ferrichrome is accumulated independently of the removal of iron from the siderophore is confirmed by the finding that gallium(III) ferrichrome is also actively accumulated by U. sphaerogena128).This property is most readily explained by the exist-

54

R.C. Hider I

100

I

I

I

[s9 Fe ] Ferrichrome 80 \

@1

\

60

/

_

e-s

20

[ 16C ]

.......

0 0

Desferriferrichrorne 4-. . . . .

"t"-. . . . .

I

1

2

3 houFs

I

Fig. 7. Uptake of ferrichrome by Us. tilago sphaerogena IzO. Percentage radioactivity (59Feor 14C)taken up by a cell suspension upon addition of [i:C]ferrichrome, [SgFe]ferrichromeor [ C]deferriferrichrome. Siderophore concentration, 36 ~M; incubation carried out at 30 °C and pH 7.0. After 2 h the medium iron(HI) level was increased to 6.8 mM in the samples containing [l~C]ferrichrome. Additional uptake of 14C was observed ( . . . )

ance of a shuttle mechanism for iron as originally suggested by Neilands 129). Ferrichrome is accumulated against a concentration gradient 127)and therefore it is to be expected that anaerobic conditions and the presence of low concentrations of azide and cyanide will inhibit the transport process. Ferrichrome uptake shows saturation kinetics and thus is likely to be mediated by a protein carrier. The specificity of the process confirms this view (Table 6). Both molecular features of the ferrichrome molecule, namely the metal centre and the cyclic hexapeptide appear to be involved in receptor interaction. When the metal centre is blocked by branched side chains poor uptake rates result. Furthermore, when the complex adopts a net charge of 1-, as for instance with the hexadentate Cobalt(II)deferriferrichrome, no uptake is observed. Slight modification of the hexapep-

Table 6. Relative Rates of Uptake of Iron Chelates by Ustilago 1~' 1~o,131) Compound

Relative rate of uptake (%)

Ferrichrome deferriferrichrome Ferrichrome A Ferrichrome A trimethylester Ferrichrysin Ferrirhodin Ferrirubin Tripropionylferdchrome Tributylferrichrome Ferrioxamine B Fusarine Rhodotorulic acid Ahminium(IH)deferriferrichrome Gallium(III)deferriferrichrome Chromium(II)deferriferrichrome Cobalt (III)deferriferrichrome

100 0 8 2 55 very low very low approx. 50 220 very low very low very low 74 70 90 0

Siderophore Mediated Absorption of Iron

55

tide ring decreases the uptake rate as demonstrated by ferrichrysin and siderophores lacking the cyclic structure have little or no affinity for the uptake mechanism (Table 6). The hexapeptide ring of ferrichrome is capable of coordinating the alkaline earth metals Mg 2+ and Ca 2÷ 132)and it has been suggested that by doing so, ferrichrome uptake would become sensitive to membrane potential. The cell membrane of fungal cells supports an electrical potential which in yeast is generated by a proton pump 133' 134). In yeasts, nutrients are accumulated by symport mechanisms, for instance aminoacids135, 136), phosphateZ37) and sugars 137). In principle a similar mechanism could operate for iron(III) siderophore uptake. However, Emery has demonstrated that 2,4dinitrophenol, (DNP) at the relatively high concentration of 10-3M, only decreased ferrichrome uptake to 75% that of normal. Clearly then, proton symport cannot be involved. However, with magnesium symport (Fig. 8) DNP would not be predicted to completely inhibit ferrichrome uptake. Although its presence would discharge the membrane potential, it would not directly influence the trans-membrane distribution of Mg 2+ ions. If the intracellular uncomplexed magnesium levels are lower than those on the outside, a reasonable supposition as indicated in Fig. 8, ferrichrome accumulation could still occur. Such a symport process is in agreement with the finding that the uptake system does not exhibit exchange diffusion properties. Furthermore, the observation that it is possible to stimulate the release of accumulated ferrichrome by the addition of alkaline phosphate solutions 127), can be explained in terms of this mechanism. The extracellular magnesium would be decreased to low levels (10 -6 M) by alkaline-induced precipitation of magnesium phosphate x3s), thus stimulating the symport mechanism to run in reverse. A likely structure for the magnesium ferrichrome complex involves coordination by 3 amide oxygen atoms (32). Such a structure offers an explanation for the conserved nature of glycine-1 in the entire series of ferrichromes (Table 1 b). Glycine in this position of the

in

out

+

AT p " ~ ' ~ _ Mg2+(H20)n AOP+P l~Keq=106 M g 2+(H2p

Mg2+(HzO)n

-

~

I~ Keq= 102"s ~

Mg 2+

Hg2+(H2PO~)(H20)n-1

Ferrichrome CeLL

membrane Fig. 8. Possible symport mechanism for ferrichrome uptake by Ustilago. By analogy with yeast cells the cytoplasmic membrane potential is probably generated by an electrogenic proton pump. An asymmetric distribution of non complexed magnesium ions will be maintained by the differential affinities of magnesium for orthophosphate and pyrophosphate (e.g. ADP). Under the incubation conditions used by Emery 127) the extracellular [Mg2+]totalis approximately 3 mM. When a magnesium ion enters the cell via the symport carrier it will move down an electrochemical gradient and therefore can "pull" the ferrichrome molecule with it. The advantage of utilising a divalent anion in a symport process is that a small membrane potential is able to support a large distribution ratio. In the above system a membrane potential of 100 mV would support a ferrichrome concentration gradient of approximately l&

56

R.C. Hider

@ cyclohexapeptide favours the ~-turn to adopt a Type II conformation which is ideal for cation coordination 132). The binding of alkaline earth cations to synthetic cyclohexapeptides has been reported previously 139' 140). U. sphaerogena synthesises deferriferrichrome A as well as deferriferdchrome, indeed under low iron conditions the secretion of ferrichrome A predominates over ferrichrome. When grown in culture, their synthetic rates are initially similar, however, as the culture progresses and becomes more iron deficient, deferriferrichrome A is preferentially synthesised 141). In contrast at higher iron levels, whereas deferriferrichrome A synthesis is completely repressed at 10-SM iron, deferriferrichrome can be isolated from cultures grown in 5 x 10-SM iron 141). Ferrichrome A, by virtue of the conjugated nature of its hydroxamate functions, possesses a higher affinity for iron than ferrichrome (1030 vs 1029)141). This, together with its acidic nature renders deferriferrichrome A more efficient than deferriferrichrome at solubilising ferric oxides74). Thus the highly efficient scavenging properties of defferriferrichrome A are only called upon when the organism senses severe stress due to low iron levels. The uptake of ferrichrome A is much slower than that of ferrichrome (Table 6) and yet iron associated with ferrichrome A enters cells rapidly and as demonstrated by ESR spectroscopy, is released as iron(II) 142). Originally it was thought that ferrichrome A donated iron(III) to deferriferrichrome and indeed this does occur to some extent, but at an insufficient rate to account for the accumulation of iron from ferrichrome A 143). From a number of lines of evidence it has become apparent that ferrichrome A supplies iron to the cell by directly interacting with a specific protein in the cytoplasmic membrane. This protein reduces the siderophore bound iron, which dissociates into the cytoplasm and the deferrifenichrome A is returned to the extracellular fluid. If the metal cannot be released by reduction, the complex dissociates from the receptor. This additional uptake system for iron presents U. sphaerogena with a strong competitive advantage over other organisms. In more favourable environments ([Fe] > 1 ~tM) the cells are able to utilise deferriferrichrome as an iron scavenger. Ferrichrome has an advantage over ferdchrome A in that it may also be stored intracellularly 142). Thus whereas there are conditions where deferriferrichrome A is not synthesised by the cell, the presence of deferriferrichrome is always required. This pattern of iron uptake may well exist for other fungal species.

Siderophore Mediated Absorption of Iron

57

4.1.2 lron(III) Siderophore Uptake by Neurospora The first uptake studies with Neurospora crassa were reported by Padmanaban and Sarma in 1965144)when they demonstrated that 59Fe coordinated to the organism's major siderophore, coprogen, is taken up much more quickly than [59Fe] citrate and that the iron is efficiently utilised for haeme synthesis. Ferrichrysin (12 c) and ferricrocin (12 d) are also efficiently transported by the cells, but rather surprisingly ferrichrome is not 145). With the aid of Mtssbauer spectroscopy it has been shown that iron(III) coprogen enters the cell where it is reduced. The resulting iron(II) dissociates and the coprogen becomes available for recycling146). A large proportion of iron(III) coprogen is not reduced and remains unchanged in the cytoplasm, possibly as a storage form of iron 146). Thus the uptake mechanism would appear to be similar to that operating in Ustilago for ferrichrome. Presumeably ferricrocin, a minor siderophore of N. crassa147)and ferrichrysin are transported in a similar manner. As with the ferrichrome receptor in Ustilago, ferrichrome A (12b) is not effectively transported by Neurospora and likewise ferrirubin (12e). Rather surprisingly, ferrirubin is a powerful competitive inhibitor of iron(III) coprogen (Ki = 5 × 10-7M). That there is such a difference between the uptake rates of ferrichrome and ferricrotin indicates that the cytoplasmic membrane receptor interacts with the peptide backbone. However, the metal centre must also be involved in the binding process since the hexapeptide sections of ferrirubin and ferrichrysin are identical. Although enantioferrichrome possesses a markedly lower rate of uptake than ferrichrome in N. crassa148),it is not clear whether this difference results from the different stereochemistry of the iron, the peptide or both.

4.1.3 Iron(III) Siderophore Uptake by Rhodotorula Rhodotorula pilimanae is capable of producing enormous quantities of rhodotorulic acid when grown under low iron conditions. The cell membrane of this yeast is able to transport iron, presented by rhodotorulic acid, the process being inhibited by respiratory poisons 149).The uptake of the ligand itself is very slow and thus the iron(III) rhodotulate receptor appears to be similar in mechanism to that of the ferrichrome A receptor of Ustilago143).Ferrichrome A and ferrioxamine B are unable to donate iron to this receptor, in contrast to iron(III) citrate which is equally efficient as iron(III) rhodotulate 149). Significantly neither of the active ligands are hexadentate and therefore will be kinetically more labile than hexadentate siderophores.

4.1.4 Iron(Ill) Siderophore Uptake by Fusaria As with Ustilago, Fusariurn roseum secretes two major siderophore types, the family of fusarinines (13 and 14) and malonichrome (12 h) 41). The relative production of these two classes varies with the age of the culture (Fig. 9). It is tempting to suggest that these two siderophore types have different functions, namely the fusarinines act as true siderophores and possibly form intracellular iron stores while malonichrome acts as a powerful iron scavenger. Certainly the highly negatively charged nature of deferrimalonichrome,

58

R.C. Hider

/rnnes l

E

I

I

6

o c

, , / / /

0 5

10

days

15

Fig. 9. The relative production of fusarinines and malonichrome by Fusarium roseum as a function of the culture age41)

endows the molecule with potent iron solubilising properties. Iron(III) fusarinine C by virtue of its net positive charge would be accumulated by the cell as a result of sensing the membrane potential; symport would not be necessary (Fig. 10). At present experiments which test this possibility have not been reported. Malonichrome is able to donate iron to cells of F. roseum but not in an efficient manner 40. In contrast ferrichrome, a siderophore not produced by this organism, is very much more efficient 4x). In related Aspergillus sp. two siderophores are also synthesised, namely triacetylfusarinine C and ferricrocin 151). 4.1.5 G e n e r a l C o m m e n t s

The overall picture emerging from this limited study of fungal iron transport is that fungi secrete a single potent iron scavanger into the environment. This scavanger is able to donate iron to the external face of the cytoplasmic membrane. However, in addition to in

m

our

+

2

H

ATP ~ ~ _ _ AOP+P

~

+ - ++

[iro~fusarinine]3+ Celt

membrane

Fig. 10. Possible uptake mechanism for fusarinine in Fusaria. A facilitated transport system for the positively charged iron(Ill) fusarinine will lead to accumulation of the complex. A symport mechanism is not required. Similar uptake mechanisms are known for basic aminoacids 15°)

Siderophore Mediated Absorption of Iron

59

this iron assimulation mechanism, some fungi possess additional independent uptake systems which are centred on true siderophore activity. Such molecules are capable of coordinating extracellular iron, permeating the cell membrane and of either donating the coordinated iron for biosynthetic requirements or retaining it as an intracellular store.

4.2 Iron(Ill) Siderophore Uptake by Enteric Bacteria A large proportion of bacterial siderophore uptake studies has been centred on enteric bacteria as typified by E. coli and S. typhimurium. The relative ease of mutant production and study has greatly facilitated progress in this area. These bacteria possess a cell wall consisting of both an outer membrane layer and a peptidoglycan layer152)(Fig. i1). The former acts as a molecular sieve-type barrier and the latter confers mechanical stability. Thus the metabolically active cytoplasmic membrane is protected against bile salts and hydrolytic enzymes found in mammalian gastrointestinal tract. The outer membrane basically consists of a lipid bilayer, rich in lipopolysaccharide which contains approximately 50 proteins of which only 3-5 are major species. Three of these form pore-fike structures called porins which render the outer membrane freely permeable towards hydrophilic molecules (<650 Mr) for instance aminoacids, sugars and peptides ls3). The region between the outer and cytoplasmic membranes is termed the periplasmic space 154)and contains approximately 20% of the total cell water 155). A Donnan equilibrium exists across the outer membrane as a result of fixed anions associated with the membrane. This influences the Na ÷ and C1- distributions and thus gives rise to an associated membrane potential of approximately 30 mV 155). Both membranes make contact at adhesion zones 156) and some phage receptors are located at such regions 157). Presumeably the presence of these zones facilitates the injection of viral nucleic acid.

4.2.1 Iron(Ill) Siderophore Translocation of the Outer Membrane As iron(Ill) siderophores possess molecular weights in excess of 600, their ability to permeate porin structures is low and specific carrier proteins are necessary. Studies with mutants 1) have provided indirect evidence for independent carriers being associated with adhesion zones oufer membrane

(/~÷÷÷ Em= 30 mV (Donnan membrane pofentiat) Em = 100 mV

Fig. I1. Schematic figure of a Gram-negative bacterium

60

R.C. Hider

the accumulation of iron(III)enterobactin19), ferrichromexSa) and iron(TIT)citrate 159). A clue to the identification of siderophore receptors was provided by Guterman who in 1971 demonstrated that iron(III)enterobactin could block colicin B action in sensitive E. coli cells 16°). As a result of further studies, Guterman suggested that the inhibition of colicin B adsorption by enterobactin reflected an interaction of enterobactin with either a cell surface protein (a receptor) or with colicin B molecule itself161). Subsequently a common receptor site for vitamin B12 and colicin E was located on the outer membrane of E. coli 162). However, it was not until 1975 that the tight association of a classic genetic lesion, ton A (now fhu A), with siderophore transport was made x63'164)and subsequently confirmed165). The Ton A (Fhu A) protein is located in the outer membrane and in addition to being the receptor for phages T5, T 1 and ~ 80, for colicin M and albomycin 166),it functions as a receptor for ferrichrome164't65).Thus the presence of ferrichrome protects E. coli from attack by phages 164'167).By using a range of mutants it was subsequently demonstrated that Guterman's earlier proposal of colicin B and enterobactin sharing the same receptor161)was correcd 6s' 169) In 1975, Uemura and Mizushima 17°)reported that the presence of iron, repressed the levels of some outer membrane proteins in E. coli. This finding was confirmed in several laboratories throughout the world. The presence of the 81,000 Mr protein was correlated with colicin B binding and iron(III)enterobactin transport 171)and the 78,000 Mr protein was identified as Ton A (Fhu A) protein 172).Typical profiles of E. coli outer membrane proteins, grown under high and low iron conditions are indicated in Fig. 12. Four different iron transport systems have now been characterised. 4.2.1.1 Iron(III)enterobactin Receptor The 81,000 Mr protein (Fep A 166)) is easily monitored by SDS gel electrophoresis and this property has facilitated its isolation in Triton X-100 solutions where iron(III)en...-..- 83 K 81 K

Iron I fransporf ~ profeins [ - -

- -

\\

\ "78K-/ (TonA

Porins

I

m

=

m

[Fe] < 10-6H

(Cir) / 3 4 K ~ ,33K-~ 3 0 K

,,

[Fe] > Io'SH

Fig. 12. Pattern of major outer membrane proteins of E. coli K 12 after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulphate. Molecular weights are based on band running time

Siderophore Mediated Absorption of Iron

61

terobactin binding activity is retained 1731. Further purification using ion exchange and affinity chromatography has led to the isolation of a homogeneous protein possessing a Kd for iron(III)enterobactin of 10 nM 174~.The pI is 5.5 and the protein lacks carbohydrate. A carbocyclic analogue of enterobactin 173~ and the aromatic analogue MECAM (22) 1751are both capable of providing iron to ent mutants of E. coli, indicating that the exposed side of the octahedral complex binds to the receptor rather than the triester ring of enterobactin. The stereochemistry of enterobactin is A-cis981and significantly enantioenterobactin (A-cis) is incapable of either supplying iron to ent mutants or competing with enterobactin for the Fep A protein 176~.Furthermore agrobactin (2 a) and parabactin (2 b), which are both A-cis isomers, fail to replace enterobactin, whereas the analogues possessing the opened oxazoline ring and a A-cis stereochemistry (2 c, 2 d) can do so65~. Thus it would appear that the chirality of the metal centre determines the affinity of phenolate siderophores for the outer membrane receptors. 4.2.1.2 Ferrichrome Receptor The outer membrane receptor for ferrichrome is associated with the Ton A (Fhu A) protein (78,000 Mr), which as yet has not been isolated. The closely related hydroxamates, ferrichrysin (12c) and ferrichrocin (12d), also use the Fhu A protein but ferrichrome A (12 b) does not. This is not due to ferrichrome A possessing a net negative charge as the dianionic succinate ester of ferricrocin (12 d) is transported at the same rate as ferrichromele~t. Like the enterobactin receptor therefore, it would appear that Fhu A protein recognises the metal centre of the ferrichromes. The iron of synthetic A-cis enantioferrichrome was found to be accumulated at approximately half the rate observed with natural A-cis ferrichrome177~. Iron(III) rhodotorulic acid and iron(III) aerobactin show no affinity for Fhu A proteins2~. It is strange that these enteric bacteria possess a transport system for ferrichrome, a siderophore not synthesised by them. It is not obvious why a gut organism should possess the ability to utilise a siderophore produced by a soil fungus.

4.2.1.3 Iron(III)citrate Receptor The tricarboxylate citrate can solubilise iron, but unless it is in a large excess, it forms a polymeric complex. When in high concentrations (10-3M) it will supply iron to E. coli 178~, and under these conditions, the synthesis of an outer membrane protein (80,500 Mr) termed Fec A is induced 166~. A citrate system is also present in mycobacteria, however, unlike the receptor in E. coli the uptake system is apparently not induced by growth on citrate 1791. 4.2.1.4 Iron(III)aerobactin Receptor The aerobactin receptor is present in many virulent strains of E. coli and has been demonstrated to be coded for by a plasmid (Col V). The existance of this plasmidmediated iron uptake system was originally demonstrated in 1979 by Williams, who also

62

R.C. Hider

suggested that the 74,000 Mr protein could be the outer membrane receptor associated with this plasmid-mediated phenomenon 18°). A clue to the nature of the siderophore involved in this process was provided by the observation that aerobactin (9b) is present in the culture fluid of a range of enteric bacteria tsL 182).Indeed, E. coli cells containing the plasmid Col V were subsequently shown to produce aerobactin ts3' 184).As originally suggested by Williams, the iron(III)aerobactin receptor has been identified as a 74,000 Mr outer membrane protein lsS-lsT) and is also the receptor for cloacin DF 13188). The aerobactin-mediated iron assimilation system of the plasmid Col V has been clonedtSg). 4.2.1.5 Regulation of the Outer Membrane Transport Systems The three outer membrane proteins, 83,000, 81,000 and 74,000 Mr were originally reported to be under coordinate regulation by iron ~9°) and this has been confirmed by Klebba et al. using pulse labelling techniques 19~). The 78,000 Mr and newly identified iron-dependent 25,000 Mr protein are clearly under a different regulatory control (Fig. 13). Iron-poor cells induced rapidly with a half life of 10 min, whereas iron-rich cells began induction after a lag and induced with a half time of 30 min. The synthetic burst of the 25,000 Mr protein precedes the induction of the outer 4 proteins (Fig. 13) but then returns to basal levels, regardless of the subsequent iron status. The significance of this important observation is not clear at the present time. Iron deficient asynchronous E. coli cultures have been pulse labelled and fractionated by size in order to observe the induction process in various age classes 192). No differences were found indicating that, like other proteins in the outer membrane 193)the iron regulated proteins are synthesised continously during the cell cycle. Iron(III)enterobactin completely represses the synthesis of the 81,000 and 74,000 Mr proteins with a half life of 2.5 min, however, both enterobactin and haeme are excluded from directly participating in the regulatory process and Klebba has suggested that intracellular inorganic iron is a corepressor191). I 100

I

i

i

i

i

I

1

-

i 7/, K

//\ / - .

°"-/X" "~

0

"711

I

I

"

I

78 K

TM

I

I

I

I

I

20 L,O 60 80 minutes after Oeferriferrichrome A addition

Fig. 13. Effect of iron stress on iron-regulated membrane protein synthesis191).E. coli was grown in iron-rich minimal media, subjected to deferriferrichrome A and pulse-labelledwith [14C]leucineat different time intervals. The membrane proteins were subjected to acrylamidegel electrophoresis and autoradiography.The resulting autoradiograph was scanned and the protein peaks were quantitated as percentages of their maximal synthetic rates

Siderophore Mediated Absorption of Iron

63

4.2.1.6 Outer Membrane Proteins of Enteric Bacteria - General Comments Inevitably the field is dominated by studies centred on the two organisms, E. coli and S. typhimurium, although with the discovery of the plasmid-mediated aerobactin system other bacterial families have been monitored for instance, Shigella flexneri t87). Virulent strains of Vibrio anguiUarum, contain a plasmid which influences the efficiency of iron uptake by the organism :94). When grown under low iron conditions these strains induce the synthesis of two outer membrane proteins, in the 80,000 molecular weight range195, 196). Several iron-dependent outer membrane proteins are also present in Vibrio cholerae197). A range of iron-induced outer membrane proteins (83,000, 77,500 and 76,500 Mr) has also been located in Aeromonas salmonicida, a fish pathogen 119).It seems likely that this property is widely distributed amongst enteric bacteria. E. coli and S. typhimurium possess a transport system for iron(III)enterobactin and iron(III)citrate, they can transport exogenous siderphores such as ferrichrome and may harbour plasmids for the synthesis and transport of aerobactin. The considerable evolutionary pressure to obtain iron from the environment is probably responsible for this diverse range of uptake processes.

4.2.1.7 Outer Membrane Proteins of Non Enteric Bacteria Agrobacteria are phytopathogenic organisms which are capable of inducing gall formation in a variety of plants. Many species have been demonstrated to possess siderophores:98). Growth of Agrobacterium tumefaciens under low iron conditions leads to the synthesis of several 80,000 Mr proteins in the outer membrane :99). A mutant unable to utilise agrobactin lacks one of these proteins 199).

4.2.2 Iron(Ill) Siderophore Translocation of the Cytoplasmic Membrane Iron(III) siderophores, having entered the periplasmic space via facilitated transport through the outer membrane, are actively accumulated by the cell. Iron(III)enterobactin20o), ferrichrome2°1)and Vitamin B:22°2)are transported through the cytoplasmic membrane as intact complexes. Once inside the cell, iron is removed from siderophore by reduction (Sect. 5). In general it would appear that the transport step occurs independently of iron(III) reduction. For example the kinetically inert chromium(III) deferriferrichrome accumulates at the same rate as that of ferrichrome2°:) and by using an E. coli strain incapable of removing iron from enterobactin, it is possible to obtain iron(III)enterobactin distribution ratios of greater than 502°°). In principle the energy for this accumulation could result by coupling transmembrane movement to an ion gradient and membrane potential or by direct linkage to a protein phosphorylation-dephosphorylation cycle. Pugsley and Reeves2°3) compared the influence of a wide range of metabolic inhibitors on the uptake of iron(III)enterobactin, glutamine and proline. These two aminoacids were selected because, the uptake of the former depends directly on the cleavage of phosphate anhydrides whereas that of the latter depends on the assymetric distribution of ions and the cytoplasmic membrane potential2°4). The differential susceptibility of iron(III)enterobactin to these inhibitors fell between those of the two marker

64

R. C. Hider

Table 7. Ton B-Dependent Systems

System

Substrate

Receptor

Permease

Ton B-dependent factors which utilise the system

fep

Iron(III)enterobactin Fep A

Fep B

Iron(III)enterobactin Cohcin B Colicin D Ferrichrome Albomyein Colicin M Phage T1 Phage ~ 80 Vitamin Bn Iron(IlI) citrate

fhu

Ferrichrome

Fhu A Fhu B (formerly Ton A)

btu fec

Vitamin B12 Iron(III)eitrate

Btu B Fec A

Fee B

aminoacids indicating that neither mechanism is entirely appropriate for iron(III) enterobactin. Nevertheless, it is clear that the iron(III) siderophore uptake is strongly dependent on the transmembrane proton motive force. A single point mutation in the ton B gene generates insensitivity to many phages and colicins, while at the same time preventing cytoplasmic membrane iron transport (Table 7)2°5-2°~). Significantly, the binding of phages TI and ~ 80 to E. coli, like iron(III) siderophore uptake is dependent on the presence of a cytoplasmic membrane potential2°9). This has led to the suggestion that the Ton B protein is associated with membrane energy transduction21°, 211).However, two alternate hypotheses have also been proposed. One suggests that the function of Ton B protein is to facilitate the apposition between outer and cytoplasmic membranes 2~2)and the other that the ton B gene is involved in the molecular processing of a range of cytoplasmic permeases213). Thus at present, the precise function of the Ton B protein remains unknown. The E. coli ton B gene has been cloned and found to express a relatively small protein 36,000 Mr214),40,000 Mr 215)which has been located in the cytoplasmic membrane 215). The DNA sequence has been determined321). The expression of the ton B gene is not regulated by iron216). An important observation concerning Ton B is that under conditions where its synthesis is inhibited, competition is observed between the uptake of ferrichrome, iron(III)enterobactin and vitamin B12, despite each of them possessing independent permeases (Table 7)217).If Ton B was associated with energy transduction then this observation is readily explained.

4.2.2.1 Iron(III)enterobactin Permease Spheroplasts prepared from E. coli, transport iron(III)enterobactin when derived from fep B mutants 21s). As the outer membrane becomes permeable in spheroplast preparations it seems likely that fep B mutants are defective in the cytoplasmic membrane. The uptake of iron(III)enterobactin is strongly dependent on the cytoplasmic membrane potential as demonstrated by the inhibitory effects of DNP (10-3M)2°6, 219)and CCCP

Siderophore Mediated Absorption of Iron

65

(2 x 10-5M) 2°6). This presents thermodynamic problems, because if Fep B functions solely as a facilitatory system, iron(III)enterobactin would be pumped out of the cell, by virtue of its trianionic nature. The available proton motive force of a cell is governed by the pH gradient across the membrane, together with the membrane potential. For a univalent species the energies associated with these two quantities are approximately equal 22°). However, for a trianionic species the term associated with the membrane potential will dominate. Therefore in order to achieve accumulation2°°), the net charge on iron(III)enterobactin must be decreased. One possible way of achieving this is via complexation with an alkaline earth metal. Indeed iron(III)enterobactin forms quite stable 1 : 1 complexes with calcium forming a monoanionic species221).The interaction is specific for calcium and strontium, other metals, for instance magnesium, barium and alkali cations possess markedly lower affinities. However, a similar complex is not formed with the enterobactin analogue MECAM (22) due to the presence of the central aromatic ring221). As iron(III) MECAM is utilised by E. coli mutants which are unable to synthesise enterobactin17s,z22), the complexation by calcium has almost certainly no physiologicial relevance. An alternative method for the neutralisation of iron(III)enterobactin is by protonation223). Since protons are pumped into the limited volume of the periplasmic space (Fig. 11)22°), protonation of iron(III)enterobactin is quite likely to occur. A critical series of observations by Rogers et al. provides a further clue as to the likely nature of the mechanism of iron(III)enterobactin permeation224-226). Whereas Scandium(III)enterobactin competes with iron(III)enterobactin for the permease (Fep B), in a range of E. coli strains 226), aluminium(III)enterobactin and gallium(III)enterobactin do not. Furthermore In a+ and Sc3+ complexes of enterobactin inhibit the growth of Gram negative bacteria, whereas the corresponding Ga 3+ and AP + complexes do not. 46Sc(III)enterobactin shows similar uptake characteristics to that of 59Fe(III)enterobactin, whereas 67Ga(III)enterobactin is virtually an impermeable species 226).This is in complete contrast to uptake studies with gallium(III)deferriferfichrome12s). Taken together these results are surprising as the radii of Ga 3+ and AI3+ (0.76 and 0.68A respectively) are much closer to the radius of Fe 3+ (0.65A), than those of Sc3+ and In 3+ (0.89 and 0.94A respectively). Tricatecholamide complexes of gallium(III) and aluminium(III) possess affinity constants close to 1040227)and therefore should remain intact in the incubation media. Furthermore, in view of the size of their atomic radii, the overall shape, stereochemistry and net charge of these complexes will be virtually identical to those of iron(III)enterobactin. One clear difference between the In3+/Sc3+ pair and the Ga3+/A13+ pair is the kinetic lability of the complexes (Fig. 3). The former pair exchange ligands approximately 103 times faster than the latter pair. Although, iron(Ill), by virtue of its small radii, possesses a kinetic lability similar to that of gallium(m) and aluminium(III), if it is reduced to iron(II), the kinetic lability of the complex will be similar to that of scandium(III) (Fig. 3). With this data in mind, a consistent explanation for the above apparently contradictory observations emerges. From bioenergetic arguements iron(III)enterobactin is probably triprotonated while attached to the permease. Under such conditions it is able to undergo an internal redox reaction leading to the formation of a kinetically labile iron(II) complex (Sect. 2.4) 84, 97. 221).Thus the common feature of the permeant species, namely Scandium, Indium and Iron is that they form kineticaUy labile enterobactin complexes. That Al3+-enterobactin and Ga3+-enterobactin, in addition to not being

~;~

Fig. 14. Permease Mediated H + Symport of Iron Enterobactin. A Protonation of iron(III)enterobactinat the surface of the cytoplasmic membrane forming a neutral complex. B Internal redox reaction forming a semiquinone ligand and the kinetically labile iron(II)enterobactin. C Displacement of one of the catecholato ligands by monoprotonated permease iigand. D Translocation ofiron(II)enterobactincomplex. E Substitution of permease ligand by free enterobactin ligand, internal

3 H÷

Periplasm

!

- - 0

H÷

F

j 3H

~01

Cytoplasm

3-

redox reaction and subsequent dissociation of complex as protonated iron(III)enterobactin. F Dissociation of 3 protons forming the trianionic iron(III)enterobactin.The process is associated with the net movement of a single positive charge and consequently iron(III)enterobactinwould be accumulated by the cell. Step B would be omitted during the uptake of Sc3÷ and In3+ enterobactin

Cytoplasmic Membrane

~. ~.

.~

Siderophore Mediated Absorption of Iron

67

accumulated, do not even inhibit the uptake of Fe3+-enterobactin226), indicates that the siderophore-metal bonds must be kineticially labile in order to form a complex with the permease. Such criterion is met if ligand substitution occurs at the permease binding site (Fig. 14). Although the sequence of reactions presented in Fig. 14 appears complicated, the equilibrium between the iron(III) and the iron(II) species only involves the movement of a single electron and consequently can occur extremely rapidly. Thus the proposed uptake process only involves ligand substitution on the triprotonated complex at each side of the membrane. This mechanism involves the net inward movement of 1 positive charge across the membrane and thus iron(III)enterobactin would be predicted to be accumulated by the cell. Uptake of the Scandium and Indium complexes would occur in an analogous fashion, without involvement of an internal redox reaction. The affinity of enterobactin for iron(II), although much smaller than iron(III) will still be considerable (13 > 102°). Thus iron(II) would not dissociate from the permeating complex. This mechanism predicts that, both the kinetically inert chromium(III)enterobactin and rhodium(III)enterobactin will not be accumulated by E. coli. It has been reported that the ease of oxidation of chromium enterobactin prevents its use as a kineticially inert complexm8). 4.2.2.2 Ferrichrome Permease A-cis chromium(III)deferriferrichrome is rapidly accumulated by both E. coli and S. typhimurium, in contrast to chromium(III)deferrioxamine B which is not absorbed2°3). Furthermore, in E. coli [3H]-ferrichrome is taken up at virtually the same rate as that of the chromium(III) analogue. As it is unlikely that the outer membrane could support a high ferrichrome concentration gradient, ferrichrome probably enters the cytoplasm as the intact complex. Cytoplasmic membrane vesicles isolated from E. coli are able to transport ferrichrome. The uptake system possesses a similar Km value (0.2 mM) to that found for intact cells228), indicating that the penetration of the inner membrane is the rate-limiting factor for ferrichrome transport. The uncoupling agents DNP (10-3M), CCCP (5 x 10-5 M) and valinomycin (10 -6 M) all inhibit ferrichrome uptake whereas arsenate has little or no effect228). Thus ferrichrome uptake is influenced by membrane potential, and as ferrichrome is neutral, a symport mechanism is strongly implicated. Vesicles prepared from fhu B strains are also able to transport iron(III)hydroxamates, suggesting that the fhu B product is not essential for this process229). In E. coli deferriferrichrome is acetylated on one of the hydroxylamine oxygen atoms23°). It has been suggested that this is a mechanism by which cells detoxify an exogenous ligand, but it has not been established as to whether or not such derivatisation is directly associated with the uptake of ferrichrome. The synchronous uptake of double labelled iron(III) aerobactin A by Aerobacter aerogenes indicates that, like ferrichrome in E. coli, the intact complex is accumulated in the cytoplasmz31).Iron(III)schizokinen is also accumulated as an intact complex by Bacillusmegaterium z32). After iron removal, the siderophore is excreted into medium and thus becomes available for reutilisation.

68

R.C. Hider

4.2.3 Iron(II1) Uptake By Mycobacteriaceae Many types of mycobacteria occur as free living organisms in soil, water and diary products where they actively oxidise lipids and hydrocarbons. Some are animal parasites causing disease including tuberculosis (M. tuberculosis) and leprosy (M. leprae). Characteristically they possess an extremely high lipid content233),the cell walls possessing up to 65% of their dry weight as lipidz34). This membrane endows the organisms with a marked insensitivity to many chemotherapeutic agents. The mycobactins (8) are extremely insoluble in water, partitioning into cell membranes. In cultures grown in irondeficient media mycobactin P reaches levels of 6% of the cell dry weight while the medium concentration is lower than 20 pg m1-1 235). Ratledge has proposed that the mycobactins function as true siderophores, transferring iron from one side of the membrane to the otherz36'237).They are not secreted into the extracellular environment of the microorganism and consequently cannot scavenge iron. However, salicylic acid is secreted from some mycobacteria in high concentration and this finding has led to the proposal that it scavenges iron and subsequently donates it to the membrane bound mycobactinz3s). The efficiency of this process will be limited by the relatively low affinity of salicylic acid for iron(III), for instance it is unable to successfully compete with phosphate and transferrinz39). More recently, peptide exochelins have been isolated. These compounds are reported to be water soluble, and to possess molecular weights < 80024°). They contain 3 mol of N~-hydroxylysine and are possibly lysine analogues of the ferrichrome class. Originally it was thought that these peptides scavenge iron and donate it to the membrane bound mycobactins. However, more recently, an active process utilising exochelins has been identified in M. smegmatis, which is apparently independent of mycobactins241). Thus the precise function of the mycobactins remains to be established. It is conceivable that they are not only siderophores, but also that they provide the organisms with an inert storage form of iron.

4.3 Antibiotics Based on Siderophore Structure Several potent antibiotics have been discovered which mimick the iron(III) coordination centre of siderophores and hence gain access to the cytoplasm of bacteria via siderophore translocating systems242). Once inside the cell, the active moiety, is cleaved from the antibiotic. These molecules have found little clinical application due to the presence of this labile link. Interestingly, siderophores have been used as carriers for synthetic antibiotics. Sulphonamides for instance, have been attached to ferricrocin and ferrioxamine B and the resulting conjugates were shown to inhibit the growth of Staphylococcus aereus243).The inhibition could be antagonised by the non conjugated siderophores.

4.3.1 The Ferrimycins The ferrimycins are isolated from Streptomyces and are active against Gram-positive organisms244). The structure of ferrimycin A, involves a substitution on the terminal amino function of ferrioxamine B (33). Invivo experiments with mice show ferrimycin to be more effective than penicillin against some microorganisms245).

Siderophore Mediated Absorption of Iron

69

Ferrioxamine-B 0 ~ HO @ @ " ~ N HO~'NH3 C!' li

"I

C.H3/

\

H

H0 . . " ' 1 ~ N ~ N . ~

NHz

/

£t ®

6cN

@ 4.3.2 The Albomycins Albomycin was first isolated from Actinomyces subtropicus by Gause 24) and consists of a mixture of closely related trihydroxamates. Grisein isolated from Streptomycesgriseus is probably identical to one of the albomycins247).For many years the structure of this series of compounds was considered to be analogous to the ferrichrome structure248). However, more recently the structures have been elucidated as given in (34) 249). Albomycin is a potent antibiotic against both Gram-positive and Gram-negative bacteria and its action is specifically antagonized by trihydroxamic acids such as ferrichrome. Presumeably the three hydroxamate functions coordinate iron(III) in analogous fashion to that established for the ferrichromes and it is this feature that is recognised by the membrane receptor. Ferrimycin A z

o

Ho

HO\

,o,

I I

(ca,]3

ICH~)~ / N , ~ " " ' ~ /

~

....K...,~NH NR3

~

O

I1 O

n

.

/

NH" " 1 I / U

O

'

II

O

k

_

~~',.,.INvN\ HO

\

~ 0

HO

/

I

I

II

0

OH3

\ H

AI,bomycin (~2, X = N'~NHz E~ ,X =NH 51 ,X =0

5 Removal of Iron From Siderophores Although in principle iron can be released from hexadentate siderophores by proteolytic dissection of the ligating structure to three bidentate ligands, the hydrolytic products, substituted catechols and hydroxamates possess considerable affinity for iron(III).

70

R.C. Hider

These, in high concentration might well interfere with the subsequent metabolism of the metal. In contrast, if the release is achieved via a reductive process, the resulting kinetically labile iron(II) is readily displaced, regenerating the siderophore and consequently rendering it available for reutilisation. Furthermore, such a mechanism renders it much less likely for aluminium to be incorporated into the proteins of the microorganism, as aluminium is not susceptible to a reductive release mechanism.

5.1 Removal of Iron From Hydroxamate Siderophores The reduction potential (measured vs the hydrogen electrode) for iron hydroxamate complexes is in the region of--450 mV76), which is close to the standard reduction potential of the NAD+/NADH couple, namely-320 mV. Thus in principle under physiological conditions, an equilibrium can be set up as indicated in Eq. (xii)

2 [Fe In. Siderophore]° + NADH ~- 2 [FeII Siderophore]- + NAD ÷ + H ÷

(xii)

Such activity has been detected in Ustilago sphaerogena z~°)mycobacteria236),Neurospora crassa, Aspergillus fumigatus 250 and Bacillus sp. 252). Ferrimycobactin reductase was found to be highly active in the presence of EDTA which acts as a sink for the newly generated iron(II) cations253). This cytoplasmic enzyme shows no preference for either NADH or NADPH, and is sensitive to the presence of sulphydryl reagents. A similar cytoplasmic enzyme, ferrisiderophore reductase, has been detected in Bacillus megaterium 2s2)and is capable of reducing the host siderophore iron(III)schizokinin. This enzyme also reduces ferrioxamine B and iron(III)aerobactin with similar efficiency, although ferrichrome A is a poor substrate. Both these enzymes are sensitive to the presence of oxygen, but many organisms possess oxygen insensitive ferrisiderophore reductase activity254). Recently a specific polyacrylamide gel stain has been introduced in order to monitor the oxygen insensitive enzyme and such activity was located in Rhodopseudomonas spheroides, R. capsulata, E. coli and Acinetobacter sp. 254). Thus ferrisiderophore reductase activity would appear to be widely distributed. There is no evidence for the simultaneous destruction of the siderophore during this reduction step, indeed some siderophores, for instance deferriferdchrome and schizokinin, are known to be recycled. Iron uptake by U. sphaerogena from ferrichrome A indicates that a specific ferrisiderophore reductase is situated in the cytoplasmic membrane and that iron is removed from the siderophore before entering the cytoplasm142). With Penicillium and Fusaria species the reduction of ferrisiderophores probably occurs sequentially with the hydrolysis of the ligand. Although the esterases isolated from both classes of organism are capable of hydrolysing the uncoordinated siderophores, fusarinine-C and triacetylfusarinine-C, their aluminium complexes possess virtually no affinity for the enzyme~5). The iron(III) complex of fusarinine-C is also not a substrate, although iron(III) triacetylfusarinine-C is cleaved very slowly255). Significantly freshly prepared crude extracts of Fusarium roseum are capable of hydrolysing iron(III) fusadnine-C, but the activity is rapidly lost during storage or attempted purification74). Similar but less complete inhibition is observed with the enzyme isolated from Penicil-

Siderophore Mediated Absorption of Iron

71

lium sp. Emery has suggested that removal of iron is necessary for siderophore hydrolysis and that a thiol function is involved in the transient reduction step 255).This would account for the extreme sensitivity of the enzymes towards sulphydryl reagents (HgC12, p-mercuribenzoate and N-bromoacetamide).

5.2 Removal of Iron From Phenolate Siderophores Byers and coworkers have clearly demonstrated that both Bacillus subtilis z55-258) and Agrobacterium tumeaciens 259)are capable of removing iron from hexadentate catecholato siderophores via ferrisiderophore reductase. In Bacillus subtilis the reaction is NADPH dependent and stimulated by the presence of FMN and Mg2+. The enzyme is found in the cytoplasm, is active in cell free extracts and is sensitive to thiol-reagents, for instance HgC12 and N-ethylmaleimide257). Ferrisiderophore reductase is capable of removing iron from iron(III) MECAM (35) in aqueous solution (pH 7.4) without destruction of the ligand, as demonstrated by the subsequent addition of ferric chloride256). The enzyme removes iron from a range of iron(III) siderophores (Table 8)256,257)

0

O.

~", 0

@ Similar results are observed with cell extracts taken from A. tumefaciens, the redox reaction utilising NADH 259). A range of synthetic hexadentate siderophores show high activity, donating the iron to ferrozine. These hexadentate ligands lack ester bonds and consequently are not substrates for an esterase-type reaction. Presumeably, a similar ferrisiderophore reductase activity is present in the cytoplasm of both E. coli and S. typhimurium as iron(III) complexes of these synthetic siderophores are capable of supporting the growth of deficient strains 173'175,222) The above findings are difficult to explain in terms of the thermodynamic stability of trianionic iron(III) enterobactin in. neutral aqueous solution. Using cyclic voltammetry, the reduction potential of iron(III) enterobactin at pH 10 has been determined as almost -1 volt26°). When this is extrapolated to pH 7.0 an estimated reduction potential of

R. C. Hider

72

Table 8. Ferrisiderophore reductase activity of the cytoplasmic fraction of Bacillus subtilis

(WB 2802)z#' 257) Substrate

Specific activity(n • mole ofiron(II) formed min-1 mg protein-1) With FMN and Mg

Iron(III)enterobactin (29) Iron(III)agrobactin

56 39

Iron(HI) MECAM (33) Iron(IH) Me3MECAMS (Each eatechol function contains a sulphonyl anion and each amide is N-methylated)

90 25

Iron(III)ferrioxamine B Iron(III)tds(dihydroxybenzoic acid)

72 115

No addition 1

2 3 0.2

2 27

-750 mV is obtained. This value is well below the range of physiological reducing agents, unlike the corresponding values for ferrichrome A and iron(III) ferrioxamine B at pH 8.0, which are --446 and --454 mV respectively26°' 261). Clearly, the active site of the ferrisiderophore reductase must be such that the environment of the siderophore iron(III) cation changes in a manner that causes a decrease in redox potential i.e. generates a coordination sphere with an enhanced affinity for iron(II). One method of achieving this, as indicated in Sect. 2.4, is by protonation of iron(III) enterobactin. Under such conditions an internal redox reaction can occur (28). Although marked changes in pK values can be achieved in the microenvironments of enzyme active sites262), in principle monoprotonation of iron(III) enterobactin could be sufficient to render intramolecular electron transfer possible (Scheme 4). Such a mechanism readily accounts for the established properties of ferrisiderophore reductase. Thus if the enzyme orientated FMN close to the semiquinone ring, an electron could readily transfer from the reduced coenzyme to the bound siderophore. On the basis of spectral studies, transient iron(II) semiquinone species have also been postulated to occur at the active site of iron(III)-containing oxygenases263). Recently Raymond and coworkers have suggested an alternative method for catecholato siderophore iron release which involves reduction in a low pH aqueous environment 264). However, the triprotonated intermediates, proposed to be of a tr/s salicylato bonding mode, are energetically unfavourable for stereochemical reasons 97). Soon after O'Brien and Gibson isolated enterobactin (enterochelin), they reported an exceedingly high reduction potential for the iron(III) complex m) and suggested that hydrolysis of iron(III) enterobactin would facilitate the removal of iron by generating a complex with a lower reduction potential (Eq. xiii). Subsequently a mutant strain of E. coli was isolated (Fes) which lacks iron(III) enterobactin esterase 265). This mutant was found to be unable to transfer iron from iron(III)enterobactin to haeme. There has been some confusion over the specificity of the enzyme266'267) although the situation is now apparently resolved26s). The enzyme (22,000 Mr) is water soluble, inhibited by N-ethylmaleimide and rapidly denatured at 37 °C. The presence of iron(III)enterobactin and

Siderophore Mediated Absorption of Iron 0

73

2m

2-

H 0/

- i-'7"0

/H

3-

F M m ~

+ I * NADP + ~ H NADPH

Fen 2+

o~HOH OH

OH

Scheme 4

dithiotreitol enhances the stability. The enzyme is capable of hydrolysing both enterobactin and iron(III) enterobactin 26s), which is surprising in view of their quite different structures and net charge. The absolute requirement for the esterase, under low iron conditions, is difficult to reconcile with the ability of non-ester containing enterobactin synthetic analogues, for

O ~ O

3~o

3~+

\J_ HO

c~

/~~

O~-~OOH

(Nefcharge 3~-~ / ( Net charge 6 O )

Eo Fem/Fe~ :

-1000 mV

Eo Fem/FJ :

--350 mV

74

R.C. Hider

instance (35), to donate iron 17a' 175,222).Nevertheless the enzyme is intimately involved in iron metabolism as its synthesis is repressed at high levels of iron268)and thefes B gene is coded at 14 min on the E. coli chromosome, together with 7 other genes involved in iron(III)enterobactin transport. A likely explanation is that, as with the NEM-sensitive esterases isolated in Fusaria and PenicilIium sp., there is a transient reduction of the coordinated iron(III) by a sulphydryl group 255).Ester bond hydrolysis could then occur in the presence of the kinetically labile iron(II) complex and the oxidised sulphur moiety would be reduced by glutathione, or under the assay conditions, by the newly released iron(II). The mechanism of the initial reduction of the iron(III) siderophore is likely to be the same as that operating for the ferrisiderophore reductases present in B. subtilis. As pointed out in Sect. 3.1 nonsymmetrical phenolate siderophores are likely to possess less negative reduction potentials than enterobactin and its synthetic tricatechol analogues. Thus, in view of the above findings it is not surprising that Paracoccus denitrificans possesses a reductase which is capable of removing iron from parabactin 5°). This enzyme can utilise either NADH or NADPH.

5.3 Utilisation of Iron An appreciable proportion of the iron assimulated by aerobic microorganisms is directed to haeme biosynthesis via ferrochetalase269). In eukaryotes, for instance Neurospora, this enzyme is largely located in the mitochondria whereas in bacteria no additional permeability barrier exists, the enzyme being located in the cytoplasmic membrane. Iron is removed from siderophores before entering the mitochondria and in N. crassa is absorbed most efficiently as complexes of either malate or citratez~t). Haeme is a potent non-competitive inhibitor of ferrisiderophore reductase isolated from Bacillus subtilis (K i = 4 x 10-5 M) 25s). As iron is only released from siderophore complexes when required, ferrisiderophore reductase is a logical allosteric control point. Thus haeme levels in aerobic organisms may partially control the rate of delivery of iron from siderophores. Haeme has also been established as a feedback inhibitor of 6aminolevulinic acid synthase27°). When the demand for iron is low, presumeably the iron remains bound to siderophores, the iron complexes of which can be accumulated in the cytoplasm (Sect. 4.1.5) or in the lipid phase associated with the cell wall (Sec. 4.2.3). Iron(III) siderophores are not the only storage form of iron, bacterioferritin having been isolated from Azotobacter species271'273) and E. coli 274). Bacterioferritin isolated from Azotobacter vinelandii possesses a very similar structure to that of mammalian ferritin. 24 Protein subunits (17,000 Mr) form a hollow protein shell of outer diameter 105 A and inner cavity diameter, 55 A 271). A similar structure has been characterised in E. coli275). This cavity can hold up to 1,600 atoms of iron in an oxide-phosphate core. There can be little doubt that this is a storage form of iron, as up to 40% of the cell iron can be associated with ferritin 273). There is however one major difference between the mammalian and bacterial ferritins, that~is the latter contains up to 12 haemes per structure 27x). These haemes yield a cytochrome b type spectrum and possess a reduction potential of -420 mV. Significantly the bulk of the non-haeme iron present in the core also titrates with haeme in the protein shell. Iron mobilisation may be effected by the oxidation state

Siderophore MediatedAbsorptionof Iron

75

of iron in the core, the kinetically labile iron(II) probably being more mobile. A stimulating hypothesis presented by Stiefel and Watt is that in Azotobacter species, ferritin is a specific iron depot for nitrogenase and its low reduction potential ensures that only when the local reduction potential in the cytoplasm approaches the values required for nitrogenase turnover ( - -430 mV) will the iron be mobilised 271). The level of a 90,000 Mr protein present in the cytoplasmic membrane of E. coli is also iron regulated191).However, unlike the other iron regulated membrane proteins, the synthesis of this protein is inhibited by iron deprivation and stimulated by iron repletion. Thus its role may be connected with the storage of iron, but at present no functional link has been identified.

6 The Interaction of Siderophores and Siderophore-like Molecules With Metals Other Than Iron(llI) Both aluminium and gallium form trivalent cations with similar radii to that of iron(III) and therefore possess high affinities for siderophores. Gallium is a scarce metal and therefore competition with iron in the biosphere is unlikely. In contrast, aluminium is extremely plentiful, and although it binds less tightly to hydroxamate ligands than does iron(III), it possesses a similar affinity for catecholato ligands. The stability constants (13) of iron(III) and aluminium(III) for acetohydroxamic acid are 1028 and 102269) and for catechol are 1044276)and 10'~ 277)respectively. Thus there is the possibility that aluminium could be absorbed by phenolate siderophores. However, it's release from the siderophore, unlike iron, would not be facilitated by a reduction step. Thus absorbed aluminium would tend to remain bound to the siderophore and not be available for protein coordination. For these reasons it is unlikely that aluminium enters mammalian food chains via microorganisms. Some actinides for instance plutonium(IV), by virtue of possessing similar charge densities to that of iron(III), also possess high affinities for hydroxamate siderophores57) and catechol siderophores27a). Thus in principle they can be accumulated by microorganisms. Two other metals namely copper and molybdenium, possess appreciable affinities for siderophores and it is likely that siderophores or siderophore-like molecules are involved in their microbial transport.

6.1 Copper(II) Siderophore Complexes Copper(II) possesses a high affinity for hydroxamate and catechol ligands as indicated by the K1 values presented in Figs. 1 and 2. Indeed this has been confirmed by Raymond and coworkers with synthetic enterobactin analogues, where copper(II) is demonstrated to bind more tightly than any other divalent cation investigated 279).Copper(II) also forms stable complexes with hydroxamate siderophores, for instance, schizokinen (9 a) and desferrioxamine B (11 a). Copper(II)schizokinen is a substrate for the oxygen insensitive ferrisiderophore reductase of Baccilus megateriurn28°). As indicated in Sect. 2.4 cop-

76

R.C. Hider

per(II) can enter an internal redox reaction with catechol ligands and thus in principle could be accumulated by some bacteria in much the same way as postulated for iron (Fig. 14). Analogous reactions have been reported to occur at the active site of the copper enzyme tyrosinase263). However, copper(II), unlike iron(III) also possesses a high affinity for aminoacids and small peptides and this property coupled with the higher solubility product of copper(II) hydroxide (K~ l Cu(OH)2 = 10-19), renders it unlikely that copper would be critically dependent on siderophore mediated transport.

6.2 Molybdenium(VI) Siderophore Complexes Molybdenium behaves rather differently to most metals essential for living processes, in that it exists predominantly in an anionic form in neutral aqueous solutions2sl). The reason for this difference is that the most stable valence state of molybdenium in aerobic aqueous solutions is six. Because the Mo(VI) cation possesses an extremely high charge density (Radius = 0.73 A, charge density = 0.9 A -2) it reacts with water forming the MoO 2+ and MoO 2+ species (Eq. (xiv)). The MoO 2+ cation does not behave as a typical divalent cation in so far as it binds anions very strongly and thus is more like a trivalent cation. Indeed the effective charge on the molybdenium atom is 3.6 + and therefore the surface of the cation not occupied by oxygen atoms is similar to that of iron(III). Not surprisingly therefore molybdenium(VI) forms complexes with catecho1283'2s4) and hydroxamates2s5), although the affinity constants are not high (Eq. (xv)). With polycatecholato complexes, for instance enterobactin, agrobactin and ct, e-b/s(2,3-dihydroxybenzoyl)-lysine, larger affinity constants are observed, falling in the range 107_101oM-1286)

/-,H+

2 HzO + ~ H+

(xiv)

+ 2

[~OH

Keq

= 10s..

°dv0 I%

1"

+ 21-120

OH

These complexes possess a characteristic yellow colour (km~ = 325 nm). Significantly, Bacillus thuringiensis produces a phenolate compound which produces an identical spectrum56, 2s7). The compound (1280 + 50 Mr) contains 2,3-dihydroxybenzoic acid together with theonine, alanine and glycine56). More recently the nitrogen-fixing cells of Azotobacter vinelandii have been shown to produce several molybdenium binding compounds when grown in a culture medium deficient in molybdenium and iron 2ss). One of the compounds has been identified as ct, e-di(2,3-dihydroxybenzoyl)-lysine2ss). When A. vinelandii is grown under molybdenium deficient conditions two membrane proteins

77

Siderophore Mediated Absorption of Iron

(44,000 and 77,000 Mr) are induced2a9). The 77,000 Mr protein is possibly common to both iron and molybdenium transport mechanisms. Molybdenium accumulation, like that of iron, is an active process and A. vinelandii cells rapidly deplete the media of molybdate and store molybdenium apparently in excess of the nitrogenase requiremerit29°). The analogous anion tungstate WO 2- is also complexed by catecho1283'284)and significantly is a competitive inhibitor of molybdate uptake 291'292). Another nitrogen fixing organism Paracoccus denitrificans also secretes a dicatechol, N 1, NS-b/s(2,3-dihyd roxybenzoyl)spermidine. 3-

-0

w~

0

°

® Dicatechols are not ideal ligands for iron(III) as they tend to form polymers. In contrast they are perfect for the MoVaO22+cation which has four vacant coordination sites (36). The net charge of Molybdenium(VI) a, e-(b/s(2,3-dihydroxybenzoyl)lysineis identical to that of iron(III)enterobactin and consequently it is not totally out of the question for it to be translocated on the iron phenolate system. Indeed this molecule has been used as an affinity ligand for the isolation of the iron(III)enterobactin receptor of E. coli174).

7 The Role of Siderophores in Infection One simple and efficient method of protecting animals from bacterial and fungal infection is to deprive the invading organism of iron293-296). Iron bound to low molecular weight ligands is present at extremely low levels in serum, saliva, tears, milk and eggs, consequently invasion is difficult. Under normal circumstances transferrin, by virtue of its high affinity for iron(III) ([3 = 1036), maintains an extremely low level of low molecular weight iron in the extracellular fluids of mammals. As long as the transferrin/apotransferrin ratio is low ( ~ 0.25) then the growth of most bacteria is severely limited296). Under

78

R.C. Hider

such iron deficient conditions a number of enterobacterial pathogens, for instance, E. coli, S. typhimurium and Shigella sonnei secrete enterobactin which can act as a virulence factor295'297).This concept is supported by the finding that enterobactin and its close analogues can remove iron from transferfin albeit rather slowly29s) and that enterobactin can abolish the bacteriostatic effect of serum on E. coli299). However, an enterobactin-specific immunoglobulin has been detected in normal human serum 3°°) which is capable of inhibiting the uptake of iron(III)enterobactin3°1). Presumeably the hydrophobic nature of enterobactin causes it to adsorb to protein surfaces where it is recognised by the host immune system. As mentioned in Sect. 4.2.1.4, a large proportion of invasive strains of E. coli harbout the Col V plasmid which codes for the synthesis of aerobactin (9b). This siderophore, unlike typical hydroxamates (Sect. 2.3), is capable of removing iron from transferrin remarkably effectively302). Presumeably this high kinetic efficiency is associated with the presence of the negative carboxylate function on the siderophore. In the presence of human serum, aerobactin is much more efficient at moving iron from transferfin to E. coli strains (possessing plasmid Col V) than enterobactin (Fig. 15)3°2). Aerobactin possesses a higher water solubility than enterobactin and therefore is less likely to behave as a hapten. Significantly, most of the clinical isolates of Shigella flexneri fail to synthesise enterobactin but do secrete aerobactin 1~, strongly implying that the phenolate siderophore is not required for the survival of Shigella in the host. The same conclusions may well apply to many other pathogens. In addition to the presence of transferrin and the immune system, fever may also facilitate the host's resistance to bacterial infection. Increasing the body temperature above 379C is reported to dramatically decrease the production of bacterial siderophores3°a). With S. typhimurium for instance, the secretion of phenolate siderophores falls off dramatically with increasing temperature (Fig. 16). It is clear that the manipulation of siderophore levels has considerable clinical relevance.

x E

12

e E

.g 10 v._

8

a,_

~6 u~ m

0

I

I

I

30

I

60 time

I 90

I 120

(rain)

Fig. 15. The uptake of SSFefrom [SSFe]transferrinby a E. coli strain possessing a Col V plasmid~). The cells were incubated in human serum enriched with [SSFe]transferrinat 37 °C

Siderophore Mediated Absorption of Iron

79

0.5

0'4 E

0.3 o r-

0-2

0.1

Fig. 16. The influence of temperature on the production of phenolate siderophores by Salmonella typhimurium 3°3). The cells were incubated for 24 h in a medium containing 0.3 ~tM iron

I 30

I

I

I

I 35

t,0

temperature *[

8 Clinical Applications of Siderophores and Their Analogues Currently there is considerable demand for a highly specific chelator of iron, which is suitable or clinical administration to iron-overloaded patients. The only successful method of treating I~-thalassaemia and sickle cell disease is blood transfusion. However, due to the relatively short life time of an appreciable fraction of transferred cells, such patients accumulate iron. This accumulation proves to be fatal if left untreated. In 1962 Sephton Smith demonstrated that desferrioxamine B, given intramuscularly to ironoverloaded patients, increased the excretion of iron in the urine 3°4). However, early experiences with this hydroxamate siderophore were disappointing and it was not until regular bolus injections were introduced that any consistant success was obtained 3°5). Following this, administration of the drug by slow continuous infusion was introduced. Although unpleasant, the treatment is quite effective3°6"3er). Desferrioxamine is also used in the treatment of acute iron poisoning and for the removal of aluminium 3°8' 309) Unfortunately desferrioxamine is not orally active, it is expensive and has a number of side effects31°' 3u). It is important therefore to develop an efficient orally active drug. Rhodotorulic acid, which can be produced in large quantities has also been considered for clinical use, however, it induces zinc excretion and local inflammatory reactions312, 313). A wide range of other hydroxamate siderophores has been demonstrated to be ineffective at iron removal from hypertransfused mice314). Amongst the catecholate siderophores, agrobactin and N',NS-b/s(2,3-dihydroxybenzoyl)spermidine appear to have potential for iron removal315'316). However, considerable effort has been applied to the design and synthesis of catechol siderophore analogues. By using 1,2,5triaminomethylbenzene and spermidine analogues a range of tricatecholato compounds capable of coordinating iron has been prepared 317). Typical examples are MECAM (22) and 3,4-LICAMS (23). In order to enhance the water solubility of the free ligands, sulphonic acid groups can be introduced in the catechol ring. This substitution also has the beneficial effect of stabilising catechol functions against air oxidation. At present there are no reports of successful clinical trials with these compounds.

80

R.C. Hider

There are a number of potential problems associated with the use of siderophores and their analogues. Those with low solubility, for instance MECAM, although likely to be absorbed by the gastrointestinal tract, will be rapidly cleared by the liver and if not metabolised are likely to be refiuxed in the bile. Furthermore, many of these neutral analogues are also able to donate iron to enteric bacteria (Sect. 5.2) and therefore could lead to septicaemia. The analogues substituted with sulphonic acid functional groups are not likely to be orally active, as the very low pKa of sulphonate will prevent absorption very effectively3°2). Siderophore analogues may find use in the removal of aluminium-, plutonium 279)and trace levels of the radionuclides gallium-67 and indium-ill which are used as tumour imaging agents 319).

9 Conclusions and Suggestions for Further Work Hopefully it is clear from the preceeding discussion presented on siderophore biochemistry, that there have been tremendous developments in the field over the past decade. The expansion of the number of structurally characterised siderophores, the use of mutants for the characterisation of the iron-regulated membrane bound proteins and the synthesis of siderophore analogues have all catalysed this progress. The association of aerobactin production with virulent pathogen strains renders the work clinically important. The characterisation of siderophore permeation of membranes and ferrisiderophore reductase activity at the molecular level is not well advanced at present. The current position is ripe for development. An improved understanding of these processes could facilitate the development of specific uptake inhibitors for aerobactin. The possibility that plutonium(IV) is accumulated in food chains via siderophores requires urgent investigation. Siderophores are beautifully designed for the chelation and transport of iron(III). In principle it should prove possible to synthesise analogues capable of removing iron from patients suffering from siderosis. In such work it will be essential to design compounds which are water soluble and yet can permeate membranes, both as the free ligand and the iron(III) complex.

Acknowledgements. The author is indebted to Joe Neilands for introducing him to the world of siderophores and to Terry Tostevin of Essex University Library for continuous help in abstracting literature.

10 Notes Added in Proof Several novel siderophores have been characterised since this manuscript was completed (April 1984). Tetraglycylferrichrome, the first heptapeptide ferrichrome to be identified has been isolated from Neovossia india3~). This complex has identical uptake characteris-

Siderophore Mediated Absorption of Iron

81

tics to ferrichrome in Neurospora crassa, indicating that the peptide ring size does not critically influence recognition by the receptor. A group of seven asperchromes have been identified in iron-deficient culture fluids of Aspergillus ochraceous323). The majority of these siderophores are hybrids of ferrichrysin (12 c) and ferrirubin (12 e), possessing a common hexapeptide ring but different ornithine 6, N-acyl groups. The presence of nonidentical acyl groups on a ferrichrome siderophore type has not been described previously. Pseudobactin 7SR1, a cyclic octapeptide containing siderophore closely related to pseudobactin (6 a), has been isolated from an agriculturally important Pseudomonas

..$p. 324).

11 References Lankford, C. E.: Crit. Rev. Microbiol. 2, 273 (1973) Neilands, J. B.: Ann. Rev. Biochem. 50, 715 (1981) Francis, J., Madinaveitia, J., Macturk, H. M., Snow, G.: Nature 163, 365 (1949) Lockhead, A. G., Burton, M. O., Thexton, R. H.: ibid. 170, 282 (1952) Neilands, J. B." J. Am. Chem. Soc. 74, 4846 (1952) Hesseltine, C. W., Pidacks, C., Whitehill, A. R., Bohonos, N., Hutchings, B. L., Williams, J. H.: ibid. 74, 1362 (1952) 7. Snow, G. A.: J. Chem. Soc. 2588 (1954) 8. Snow, G. A.: ibid. 4080 (1954) 9. Bickel, H., Bosshardt, R., Giiumann, E., Reusser, P., Vischer, E., Voser, W., Wettstein, A., Ziihner, H.: Helv. Chim. Acta 43, 2118 (1960) 10. Emery, T., Neilands, J. B.: Nature 184, 1632 (1959) 11. Garibaldi, J. A., Neilands, J. B.: ibid. 177, 526 (1956) 12. Snow, G. A.: Biochem. J. 94, 160 (1965) 13. Prelog, V., Walser, A.: Helv. Chim. Acta 45, 631 (1962) 14. Rogers, S., Warren, R. A. J., Neilands, J. B.: Nature 200, 167 (1963) 15. Ito, T., Neilands, J. B.: J. Am. Chem. Soc. 80, 4645 (1958) 16. Brot, N., Goodwin, J., Fales, H.: Biochem. Biophys. Res. Comm. 25, 454 (1966) 17. Korth, H.: Arch. Mikrobiol. 70, 297 (1970) 18. O'Brien, I. G., Cox, G. B., Gibson, F.: Biochim. Biophys. Acta 215, 393 (1970) 19. Pollack, J. R., Neilands, J. B.: Biochem. Biophys. Res. Comm. 38, 989 (1970) 20. O'Brien, I. G., Gibson, F.: Biochim. Biophys. Acta 21, 393 (1970) 21. Corbin, J. L., Bulen, W. A.: Biochemistry 8, 757 (1969) 22. Griffiths, G. L., Sigel, S. P., Payne, S. M., Neilands, J. B.: J. Biol. Chem. 259, 383 (1984) 23. Cox, C. D., Rinehart, K. L., Moore, M. L., Cook, J. C.: Proc. Natl. Acad. Sci. 78, 4256 (1981) 24. Teintze, M., Hossain, M. B., Barnes, C. L., Leong, J., Van der Helm, D.: Biochemistry 20, 6446 (1981) 25. Teintze, M., Leong, J.: ibid. 20, 6457 (1981) 26. Wendenbaum, S., Demange, P., Dell, A., Meyer, J. M., Abdallah, M. A.: Tett. Let. 24, 4877 (1983) 27. Snow, G. A.: Bacteriol. Rev. 34, 99 (1970) 28. Mullis, K. B., Pollack, J. R., Neilands, J. B.: Biochemistry 10, 4894 (1971) 29. Gibson, F., Magrath, D. I." Biochim. Biophys. Acta 192, 175 (1969) 30. Linke, W. D., Crueger, A., Dickman, H.: Arch. Mikrobiol. 85, 44 (1972) 31. Keller-Schierlein, W., Prelog, V.: HeN. Chim. Acta 44, 1981 (1961) 32. Keller-Schierlein, W., Mertens, P., Prelog, V., Walser, A.: ibid. 48, 710 (1965) 33. Prelog, V., Walser, A.: ibid. 45, 631 (1962) 34. KeUer-Schierlein, W., Prelog, V.: ibid. 44, 709 (1961) 1. 2. 3. 4. 5. 6.

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278. Durbin, P. W., Jones, E. S., Raymond, K. N., Weitl, F. L.: Radiat. Res. 81, 170 (1980) 279. Kappel, M. J., Raymond, K. N.: Inorg. Chem. 21, 3437 (1982) 280. Arceneaux, J. E. L., Boutwell, E., Byers, B. R.: Am. Soc. Microbiology Abstract K 185 (1982) 281. Pope, M. T., Still, E. R., Williams, R. J. P.: Molybdenium and Molybdenium-containing enzymes (Coughlan, Ed.), p. 3, Pergamon Press, Oxford 1980 282. Rosenheim, A., Nernst, Ch.: Z. Anorg. u. allgem. Chem. 214, 209 (1933) 283. Halmekoski, J.: Ann. Acad. Sci. Fennicae. 96, 7 (1959) 284. Kustin, K., Liu, S. T.: J. Am. Chem. Soc. 95, 2487 (1973) 285. Rowland, D., Metoan, C. E.: Anal. Chem. 36, 1997 (1964) 286. Hider, R. C., Neilands, J. B.: Unpublished work 287. Ketchum, P. A., Somerville Owens, M.: J. Bacteriol. 122, 412 (1975) 288. Fekete, F. A., Emery, T., Spence, J. T.: Am. Soc. Microbiology Abstract N45 (1982) 289. Page, W. J., yon Tigerstrom, M.: J. Bacteriol. 151, 237 (1982) 290. Pienkos, P. T., Brill, W. J.: ibid. 145, 743 (1981) 291. Keeler, R. F., Varner, J. E.: Arch. Biochem. Biophys. 70, 585 (1957) 292. Nagatani, H. H., Brill, W. J.: Biochim. Biophys. Acta 362, 160 (1974) 293. Weinberg, E. D.: Microbiol. Rev. 42, 45 (1978) 294. Weinberg, E. D.: Science 184, 952 (1974) 295. Rogers, H. J.: Infect. Immunity 7, 445 (1973) 296. Kochan, I.: Biolnorganic Chemistry 11, 55 (1976) 297. Yancey, R. I., Breeding, S. A. E. L., Lankford, C. E.: Infect. Immun. 24, 174 (1979) 298. Carrano, C. J., Raymond, K. N.: J. Am. Chem. Soc. 101, 5401 (1979) 299. Rogers, H. J., Synge, C., Kimber, B., Bayley, P. M.: Biochim. Biophys. Acta 497, 548 (1980) 300. Moore, D. G., Yancey, R. J., Lankford, C. E., Earhart, C. F.: Infect. Immun. 27, 418 (1980) 301. Moore, D. G., Earhart, C. F.: ibid. 31, 631 (1981) 302. Konopka, K. Bindereif, A., Neilands, J. B.: Biochemistry 24, 6503 (1982) 303. Garibaldi, J. A.: J. Bacteriol. 110, 262 (1972) 304. Sephton Smith, R.: Brit. Med. J. 2, 1577 (1962) 305. Barry, M., Flynn, D. M., Letsky, E. A., Risdon, R. A.: ibid. J. 2, 16 (1974) 306. Weatherall, D. J., Pippard, M. J., CaUender, S. T.: New Eng. J. Med. 308, 456 (1983) 307. Anon: Lancet, 373 (1984) 308. Ackrill, P., Ralston, A. J., Day, J. P., Hooge, K. C.: ibid. 2, 692 (1980) 309. Brown, O. J., Dawborn, J. K., Mam, K. N., Xipell, J. N.: ibid. 2, 343 (1982) 310. Davies, S. C., Hungerford, J. L., Arden, G. B., Marcus, R. E., Miller, M. H., Huehns, E. R.: ibid. 181 (1983) 311. Borgna-Pignatti, C., De Stefano, P., Broglia, A. M.: ibid. 681 (1984) 312. Grady, R. W., Peterson, C. M., Jones, R. L., Graziano, J. H., Bhargave, K. K., Berdoukas, V. A., Kokkin, G., Loukopoulos, D., Cerami, A.: J. Pharmacol. Exp. Ther. 209, 342 (1979) 313. Jacobs, A.: Brit. J. Haematol. 43, 1 (1979) 314. Pitt, C. G., Gupta, G., Estes, W. E., Rosekrantz, H., Metterville, J. J., Crumbliss, A. L., Palmer, R. A., Nordquest, K. W., Sprinkle-Hardy, K. A., Whitcomb, D. R., Byers, B. R., Arceneaux, J. E. L., Gains, G. G., Sciortino, C. V.: J. Pharmacol. Expt. Ther. 208, 12 (1979) 315. Jacobs, A., White, G. P., Tait, G. P.: Biochem. Biophys. Res. Comm. 74, 1626 (1977) 316. Bergeron, R. J., Burton, P. S., McGovern, K. A., St. Onge, E. J.: J. Med. Chem. 23, 1130 (1980) 317. Raymond, K. N., Smith, W. L.: Struct. Bonding 43, 159 (1981) 318. Forth, W.: Acta Pharmacol. Toxicol. 29, Supp. 4, 78 (1971) 319. Percoraro, V. L., Wong, G. B., Raymond, K. N.: Inorg. Chem. 21, 2209 (1982) 320. Llinas, M., Neilands, J. B.: Biophys. Struct. Mechanism 2, 105 (1976) 321. Postle, K., Good, R. F.: Proe. Natl. Acad. Sci. USA 80, 5235 (1983) 322. Deml, G., Voges, K., Jung, G., Winkelmann, G.: FEBS Lett. 173, 53 (1984) 323. Jalal, M. A. F., Mocharla, R., Barnes, C. L., Hossain, M. B., Powell, D. R., Eng-Wilmot, D. L., Grayson, S. L., Benson, I3. A., Van Der Helm, D.: J. Bacteriol. 158, 683 (1984) 324. Yang, C. C., Leong, J.: Biochemistry 23, 3524 (1984)

Lysine Analogues of Siderophores* A. Chimiak I and J. B. Neilands 2

1 Department of Organic Chemistry, Technical University of Gdafisk, 80-952 Gdafisk, Poland 2 Department of Biochemistry, University of California, Berkeley, CA94720, USA

Various chemical syntheses of the lysine containing analogues of siderophores di-Na,N'°-(2,3-dihydroxybenzoyl)lysine (I) and di-Na,N'~-(2,3-dihydroxybenzoyl)lysyl-N'~-(2,3-dihydroxybenzoyl) lysine are reported. The most effective synthon for introduction of the catechol moiety proved to be the N-hydroxysuccinimide ester of 2,3-dibenzyloxybenzoic acid.

I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Syntheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

90 91 94 95

* Presented in part at the 7th American Peptide Symposium, Madison, Wisconsin, 1981 St~cture and Bonding 58 © Springer-Verlag Berlin Heidelberg 1984

90

A. Chimiakand J. B. Neilands

I. Introduction Since 1958, when the catechol derivative, 2,3-dihydroxy-N-benzoylglycine,was first isolated from low iron stressed cultures of Bacillus megaterium 1), a number of related compounds belonging to the siderophore series have been obtained from bacterial species. In 1969, the isolation of di-Na,N°'-(2,3-dihydroxybenzoyl)lysine (I) from Azotobacter vinelandii 2) was described. This compound is especially interesting since the seven atom spacing between the two dihydroxybenzoyl moieties allows both of these bidentate ligands to coordinate to a central iron atom. No higher homologue of I has been reported in nature but compound II, di-Na,N°'-(2,3-dihydroxybenzoyl)lysyl-N°'(2,3-dihydroxybenzoyl)tysine might be a logical candidate since it would afford a third catechol function to satisfy the six-coordinate requirements of iron. In the following year, 1970, cyclo-tri-2,3-dihydroxy-N-benzoylserine (III) was isolated from Salmonella typhimurium 3) and Escherichia coh~) and named, respectively, enterobactin and enterochelin. Tait 5), in 1975, isolated a siderophore, named Compound III, from Paracoccus denitrificans and showed that it is a conjugate of spermidine with two moles of 2,3dihydroxybenzoic acid and one mole of salicylic acid. The characterization of agrobactin (IV, R = OH) from Agrobacterium tumefaciens 0 suggested the possible presence of the oxazoline ring in Tait's Compound III. This was subsequently confirmed7) and the name of Compound III changed to parabactin (IV, R = H). Recently a new member of the series, vibriobactin (V), from Vibrio cholerae8), has been reported. This member is unique in that the polyamine is the symmetrical 3,3'-imino-b/s-propylamine, and there are two oxazoline rings in the molecule. Considerable attention has focused on the chemical synthesis of the tricatechol siderophores. For enterobactin, no X-ray structure has been reported and it was hence gratifying when the Corey9) laboratory was able to achieve its chemical synthesis. Subsequently, enterobactin and its enantiomer were prepared by other methods 1°), the former, OH OH

OH OH

COOH I

OH

OH

OH

O=C

OH

OH OH

COOH

0

Lysine Analoguesof Siderophores O II

/A- c

\<~/-u/---k

0

R OH

H

w_< OH

-] ~C--~

OH OH 0 ~

91

"OT

IC~ 0

,o, OH

~ O H

N•O

"

NH 0

OH IV, R=H,OH

III

in addition by a novel procedure based on cyclization in a tin template 11). Parabactin t2) has recently been synthesized and although agrobactin has not, its structure has been confirmed by electron diffraction analysis t3). The iron (III) binding ability of the tricatechols enterobactin, agrobactin and parabactin is impressive and is no doubt related to the biofunction of these molecules14). The structure and stability of ferric vibriobactin has yet to be assessed. The tri-catechols are often accompanied in the bacterial culture fluid by monomeric species, and in addition to the 2,3-dihydroxy-N-benzoylglycine already noted, the corresponding compounds of serine and threonine have been reported 15). The function of these is not entirely clear. They may have some rudimentary siderophore activity, may be fragments "lost" in the biosynthesis of siderophores or, finally, they may play some role in release of the bound

iront6).

HO

HO

0 H

0

H3 N

~

H3 N

~

N

H

V In the present communication we describe what we believe to be effective methods for the synthesis of di-N~,N~'-(2,3-dihydroxybenzoyl)lysine (I) and di-Na,N~-(2,3-dihydroxybenzoyl)lysyl-N'<2,3-(dihydroxybenzoyl)lysine(II). The synthetic route is shown in Scheme 1.

II. Chemical Syntheses Melting points are uncorrected. NMR spectra were recorded on a 60 MHz Varian EM360 instrument. Samples for analysis were dried at 0.01 mm Hg over P205. Thin layer chromatography (TLC) was carried out on Merck silica gel (60 Fz54) plates in: S1,

92

A. Chimiak and J. B. Neilands DBBA

DHBA I I Lys + 3,4 or 5 ~ DBBA-Lys ~ DHBA-Lys (1) I DBBA

I

Lys-lys + 3 or 5 ~ DBBA-Lys

DBBA

I

DHBA

DHBA

I

I

lys ~ DHBA-Lys (2)

lys II

DBBA = 2,3 -dibenzyloxybenzoyl DHBA = 2,3-dihydroxybenzoyl (3) = DBBA-C1 (4) = DBBA-O-C6H4-NO2(p) O (5) = D B B A - O - N ~ 0

Scheme 1. Synthetic route for the synthesis of the 2,3-dihydroxybenzoylderivatives of lysine and lysyllysine chloroform-methanol (10: 1); $2, butanol-pyridine-water (1 : 1 : 1) as solvent systems. Spots were visualized with a UV lamp or with a Fe(C104)3-spray.

2,3-dibenzyloxybenzoic acid. The method of Lindgren and Nilsson 17) was used. To a stirred solution of 25 g (78 mmol) of 2,3-dibenzyloxybenzaldehyde 18) in 300 ml of acetone, a solution of 13.6 g (140 mmol) of sulphamic acid in 60 ml of water was added. The mixture was oxidized below 30 *C by the slow addition of 8.3 g (92 mmol) of sodium chlorite. The acetone was evaporated and the crude acid was filtered. The ether soluble product was purified by reextraction into diluted aqueous ammonia. The water layer was separated and acidified. Yield, 19 g (75%). M.p. after ethanol - water recryst.: 123--124 °C. RF = 0.48 (S~).

2,3-dibenzyloxybenzoyl chloride (3). The acid chloride 3 was prepared in ether solution from 3 g (9 mmol) of 2,3-dibenzyloxybenzoic acid and 2.43 g (11.7 mmol) of phosphorus pentachloride. The crude oil was evaporated five times with dry ether and used for acylation.

2,3-dibenzyloxybenzoic acid p-nitrophenyl ester (4). 15 g of acid (45 mmol), 6.3 g of p-nitrophenol (45 mmol) and 11 g of dicyclohexylcarbodiimide were dissolved with stirring in 200 ml of ethyl acetate at 5 °C. After 12 h the dicyclohexylurea was filtered off and the filtrate evaporated. Yield of ester, 12.33 g (58%) recyrstallized from ethanol, m.p. 108-109 °C. Anal.

Calc. for C27H2106N:

C, 71.20; H, 4.65; N, 3.07;

Found:

C, 71.16; H, 4.71; N, 2.99.

LysineAnaloguesof Siderophores

93

2,3-dibenzyloxybenzoic acid-N-hydroxysuccinimide ester (5). Dicyclohexylcarbodiimide, 3.1 g (15 mmol) was added at 0° to a solution of 2,3-dibenzyloxybenzoic acid, 5.0 g (15 retool), and 1.72 g (15 mmol) of N-hydroxysuccinimide in 30 ml of dioxane and left to stand overnight. Then the dicyclohexylurea was filtered and the filtrate evaporated to afford an oil, which crystallized after scratching with a few drops of isopropanol. Yield 6.5 g (98%), m.p. 112-114 °C. Anal.

Calc. for C~H2106N:

C, 69.60; H, 4.90; N, 3.24;

Found:

C, 69.54; H, 5.04; N, 3.21.

l~-N~-di-(2,3-dibenzyloxybenzoyl)lysine (1) (a) To a stirred mixture of 400 mg (2.2 mmol) of lysine hydrochloride in 10 ml of water with 10 ml of ether were added for 1 h at 0° simultaneously 4.5 ml of 1 N NaOH and 4.5 mmol of 2,3-dibenzytoxybenzoyl chloride in ether solution. Stirring was continued for 1 h. The oily sodium salt of the product was observed at the bottom of the flask. The mixture was acidified with HC1 and extracted with ethyl acetate. The organic layer was washed with aqueous 5% NaHCO3, saturated aqueous NaCI, dried over MgSO4 and evaporated to leave 1.4 g (83%) crude yellow oil of compound (1). Column chromatography on 20 g of silica gel with solvent system $2 gave: 2,3-dibenzyloxybenzoic acid ethyl ester (165 mg), 2,3-dibenzyloxybenzoic acid (440 mg) and 700 mg of lysine derivative (1) as a chromatographically pure, white oil. Rv = 0.32 ($2). NMR (CDCI3): 1.2 (m), 6H, aliph.; 3.2 (m), CH~; 4.6 (m), CH~; 5.2 (s), CH2-C6Hs; 7.2-7.5 (m), 26H, arom.; 7.9 (m), CONH-C~; 8.7 (m), CO--NH-C~; 9.1 (s), COOH. MS: 778 (1.6%) (M) theoret: 778; 760 (< 1%) (M - 18); 687 (2.5%) (M - 91); 669 (< 1%) (M - 18 - 91); 578 (8.1%) (M - 18-/2 x 91); 487 (20.3%) (M - 18-/3 x 91); 461 (7.5%) (M - acyl.); 91 (100%) CrHsCH2. (b) To the solution of 183 mg (1.0 mmol) of lysine hydrochloride and 0.6 ml (43 mmol) triethylamine in 8 ml of dioxane-water (1 : 1) mixture, 910 mg (2 mmol) ofp-nitrophenyl ester (4) was added. The solution was stirred for 24 h at room temperature and evaporated to dryness. The residue was acidified with HC1 and extracted with ethyl acetate. The acetate layer was washed with saturated NaCI solution, dried and evaporated. The crude oil (1.28 g) was dissolved in chloroform and subjected to Sephadex LH-20 column chromatography (3 x 30 cm) for nitrophenol separation using chloroform- ethyl acetate (1 : 1) as eluent. Yield, 1.1 g pure oil product identical with (1) obtained in (a) above. (c) N-hydroxysuccinimide ester method. The product (1) was prepared in the same way as product (2) (see below). Yield 3.28 g (84%) after purification on silica gel column with solvent system Sx.

N~-N~-di-(2,3-dibenzyloxybenzoyl)-lysyl-N°'-(2,3-dibenzyloxybenzoyl)-lysine (2) (a) The dipeptide derivative was prepared from 250 mg of lysyl-lysine dihydrochloride (Sigma Chem. Co.) with acid chloride (3) by a procedure analogous to that used for (1,a). After two consecutive silica gel column (30 g, 3 × 10 cm) purifications; 470 mg (54%) of a dry foam product was isolated with R f = 0.36 ($1). NMR (CDC13) : 1.1-1.4 (m), 12H, CH2; 3.3 (m), 4H, CH2-N; 4.4 (m), 2H, CH, 5.2 (s), 12H, CHz--CrHs, 6.6--6.8 (s), CO-NH, 7.2-7.8 (m), 39H, arom., 9.0-9.2 (s), CONH. MS: 676 (theoret.: M - 6 x 91 = 676).

94 Anal.

A. Chimiak and J. B. Neilands Calc. for

C75H74012N4

Found:

C, 73,63; H, 6.09; N, 4.57; C, 72.79; H, 6.14; N, 4.33 .

(b) A solution of 1.86 g (4.32 mmol) of N-hydroxysuccinimide ester (5) in 15 ml of acetone was added to a solution of lysyl-lysine dihydrochloride (500 rag, 1.44 mmol) in 1.32 ml of triethylamine (9.4 mmol) and 4 ml of water. The homogenous reaction mixture was monitored by TLC in $1. After 12 h the acetone was evaporated. The oily residue was dissolved in chloroform, washed with saturated aqueous NaCI solution, dried and evaporated. Yield, 1.70 (97%) of dry foam. Column chromatography on silica gel (13 g, 3 x 8 cm. Malinckrodt, 100 mesh) gave as a second fraction 1.13 g (65%) of pure dipeptide derivative (2) as a hard, amorphous foam and properties identical to those obtained above (2 a). R~ = 0.36 in $2.

N~-N~-di-(2,3-dihydroxybenzoyl)-lysine (I) 1 g of oily Na-N~-di-(2,3-dibenzoyloxybenzoyl)-lysine (1) was dissolved in 3 ml of benzene and the solution was diluted with 80 ml of ethanol. The sample was hydrogenated for 5 h at 40 °C with 10% Pd on charcoal (Aldrich Chem. Co.) until no O-protected compound (1) existed (TLC test, $2). The mixture was filtered and the solvents evaporated. Yield, 520 mg (89%) of product I with m.p. 82-88 °C (Ref. 2) 81-86 °C). It gave a positive Arnow test. R~ = 0.77 (Ref. 2) 0.79), $2. MS: 418 (1.35%) (M) Theoret., 418; 382 (3.0%) ( M - H 2 0 , 282 (2.1%) M + 1 - acyl; 264 (5.9%) M + 1 - acyl - H 2 0 ; 219 (10.1%), 136 (94%), 110 (68.4%) 84 (100%). Anal.

Calc. for C20H22OsN2:

N, 6.69.

Found:

N, 6.38.

1W-NW-di-(2,3-dihydroxybenzoyl)-lysyl-N~-(2,3-dihydroxybenzoyl)-lysine (II) 960 mg of compound (2) was hydrogenated as above for compound (1). The product, 530 mg of white, dry foam, had m.p. 128-151 °C. Rf = 0.69, with a positive Arnow test. Molecular mass determination (field desorption) gave M + 1 = 684; M + Na = 707. Anal.

Calc. for C33H38012N4 (682.66):

C, 58.06; H, 5.61;

Found:

C, 57.72; H, 6.03.

HI. Discussion We have described a practical synthesis of the siderophore analogue, di-Na-N'~-(2,3dihydroxybenzoyl)-lysyl-N'~-(2,3-dihydroxybenzoyl)-lysine,II. This dipeptide, equipped with the six phenolic hydroxyl groups spatially disposed for effective iron coordination, is able to form a stable, mononuelear complex. We present also a convenient synthesis of di-Na-N'~-(2,3-dihydroxybenzoyl)-lysine, I. In the synthetic process we employed direct acylation of lysine and lysyllysine in order to obviate the need to hydrolyze an ester in the

LysineAnaloguesof Siderophores

95

final step. From the several possibilities for protection of the phenolic groups of the 2,3dihydroxybenzoic acid residues, we chose O,benzyl because of the ease with which it suffers hydrogenolysis. The acid chloride (3) is not recommended for initial use because hydrogen chloride, inevitably generated in its formation, creates good conditions for acidolysis of the O-benzyl function. Application of the p-NO2-phenyl ester (4) was also unsatisfactory and resulted in a product containing p-nitrophenol, which must be removed by chromatography on Sephadex LH-20 prior to biological tests. Finally, 2,3dihydroxybenzoic acid-N-hydroxysuccinimide ester (5) appears to be a superior synthon for preparation of catechol derivatives of amino acids. Acylation with the active ester (5) proceeds quickly and efficiently, and the products (1) and (2) purify easily. During the synthesis of (1) and (2) we observed some difficulties with the extractions, at higher pH, which seemed to be attributable to the preponderance of benzyl groups in a small, hydrophobic molecule. In the ultimate step, benzyl groups were removed by catalytic hydrogenation. The structures of compounds I and II were confirmed by mass spectrometry and NMR. The ferric complex of II in methanol solution did not give CD effects in the region of 400-500 nm. Therefore, it may not have a dominant A or A configuration around the iron atom, but both forms may exist in equilibrium. A preliminary qualitative inspection of the spectra of mixtures such as: II plus ferrichrome, and enterobactin plus ferrichrome, suggests that compound II and enterobactin may have an approximately equal affinity for ferric ion. Biological tests showed that the dipeptide II inhibits growth of E. coli mutants RW 193 (with enterobactin receptor) and RWB 18 (without the receptor). The ferric complex of compound II did not compete with colicin B for the receptor of RW 193. The carboxyl-containing compounds I and II were used2°) for coupling with Affigel 102. The iron complex was generated on the modified resin and the resulting affinity column then applied successfully for isolation of the 81,000 molecular weight ferric enterobactin outer membrane receptor of E. coli. The availability of compounds I and II will facilitate further experiments of this nature, which should include a mutational analysis of A. vinelandii to determine if the synthesis of compound I is a component of the iron assimilation system of the bacterial cell.

IV. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Ito, T., Neilands, J. B.: J. Am. Chem. Soc. 80, 4645 (1958) Corbin, J. L., Bulen, W. A.: Biochemistry8, 757 (1969) Pollack, J. R., Neilands, J. B.: Biochem. Biophys. Res. Commun. 38, 989 (1970) O'Brien, E. G., Gibson, F.: Biochim. Biophys. Acta 215, 393 (1970) Tait, G. H.: Biochem. J. 146, 191 (1975) Ong, S. A., Peterson, T., Neilands, J. B.: J. Biol. Chem. 254, 1860 (1979) Peterson, T., Neilands, J. B.: Tetrahedron Lettr., 4804 (1979) Griffiths, G., Sigel, S. P., Payne, S. M., Neilands, J. B.: J. Biol. Chem., 259, 383 (1984) Corey, E. J., Bhattacharyya, S.: Tetrahedron Lettr., 3919 (1977) Rastetter, W. H., Erickson, T. J., Venuti, M. C.: J. Org. Chem. 45, 5011 (1980) Shanzer, A., Libman, J.: J. Chem. Soc. Chem. Commun. 15, 846 (1983)

96

A. Chimiak and J. B. Neilands

12. Bergeron, R. J., Kline, S. J.: J. Am. Chem. Soc. 104, 4489 (1982) 13. Eng-Wilmot, D. L., van der Helm, D.: ibid. 102, 7719 (1980) 14. Neilands, J. B., Peterson, T., Leong, S. A.: In Organic Chemistry in Biology and Medicine, (Martell, A. E. ed., Am. Chem. Soc., Washington, D.C., p. 263 (1980) 15. Neilands, J. B.: Structure and Bonding, in press, this volume 16. Monzyk, B., Crumbliss, A. L.: J. Inorganic Biochem. 19, 19 (1983) 17. Lindgren, B. O., Nilsson, T.: Acta Chem. Scan& 27, 888 (1973) 18. Merz, K. W., Fink, J.: Arch. Pharm. 289, 347 (1956) 19. Arnow, L. E.: J. Biol. Chem. 118, 531 (1937) 20. Fiss, E. H., Stanley-Samuelson, P., Neilands, J. B.: Biochemistry 21, 4517 (1982)

Mutational Analysis of Rhodotorulic Acid Synthesis in Rhodotorula pilimanae Alexander Liu* and J. B. Neilands Department of Biochemistry, University of California, Berkeley, CA 94720

The current status of research on the aerobactin gene complex of Escherichia coli has been reviewed from the standpoint of its organization and regulation. The reasons for selection of the basidiomycetous yeast, Rhodotorula pilimanae, as the most suitable eukaryotic species to which to extend similar studies have been enumerated. A convenient bio-assay is described for detection of mutants of Rhodotorula pilimanae blocked in the biosynthesis of rhodotorulic acid. The application of the bio-assay to a mutagenized culture of Rhodotorula pilimanae enabled collection of a series of mutants, one of which forms an altered version of the constituent amino acid of rhodotorulic acid, N6-acetyl-N~-hydroxyornithine.

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

98

II.

The Aerobactin Gene Complex of Escherichia coli

IIL

Rhodotorula pilimanae as a Eukaryotic Siderophore Producing Analogue of Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100

IV.

Detection of Mutants Defective in Biosynthesis of Rhodotorulic Acid . . . . . . . . .

102

V.

A New Hydroxamate From a Mutant of Rhodotorulapilimanae . . . . . . . . . . . .

104

VI.

Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105

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

98

VII. Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

106

VIII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

106

* Present address: Harvard Medical School, Boston, MA 02115 Structure and Bonding 58 © Springer-Verlag Berlin Heidelberg 1984

98

A. Liu and J. B. Neilands

I. Introduction Iron, which is an essential element for virtually all life forms, is toxic in excess1). As a consequence, the uptake of the metal is carefully regulated at the membrane level in microbes, plants and animals2). An understanding of the mechanism of this regulation at the molecular level would be of obvious importance to research on the physiological basis for health and disease. This information is most easily gained through application, in microbial species, of the techniques of molecular genetics. The means whereby microorganisms assimilate iron may be divided into a relatively inefficient process, called "low affinity" and a relatively efficient process, designated "high affinity''2). In the latter, Fe(III) specific carders, termed siderophores3) and matching membrane receptors are used for uptake of iron. The high affinity system is present in the vast majority of aerobic and facultative anaerobic microorganisms critically examined for its presence. This system, which is amenable to genetic analysis, is over-expressed (derepressed) under conditions where iron is limiting. Typical examples of iron poor environments are the tissues of living cells and certain soils. In tissues, iron is a trace element and is bound in heine or in the iron transport and storage proteins. In aerobic environments iron is often abundant but is unavailable owing to the small solubility product of Fe(OH)3, < 10-38 M. Microorganisms growing in such surroundings will have an obvious competitive "edge" if they can express the carrier-and-receptor mediated system for iron assimilation.

H. The Aerobactin Gene Complex of Escherichia coli Without any doubt the microorganism for which we have the greatest depth of knowledge regarding its genetic base is the enteric bacterium Escherichia coli. Considerable strides have been made in understanding certain of the siderophore mediated iron assimilation systems in this species. Thus the aerobactin system carded on the 90 kb ColV plasmid found in clinical isolates of E. coli has been cloned onto a small, multi-copy vector specifying resistance to ampicillin 4). The complete aerobactin gene complex is contained on about 6.5 kb of DNA which, in minicell preparations charged with 35Smethionine, has been shown to encode the synthesis of five proteins. These have been separated by electrophoresis in sodium dodecylsulfate on polyacrylamide gels and shown to have relative molecular weights (MW) of 27,000, 33,000, 53,000, 63,000 and 74,000, respectively5). The gene order appears to be 63,000 -* (27,000 + 33,000) ~ 53,000 74,000. Thus far the function of only the largest polypeptide has been determined unequivocally: it is the outer membrane receptor for ferric aerobactin6-a). Mutants lacking this component are completely resistant to the bacteriocin, cloacin, from Enterobacter cloacae. Cloacin, like several other lethal agents generated in the microbial world, penetrates the cells of E. coli carrying pColV via the 74,000 MW outer membrane receptor. This coincidence, not uncommon in the interaction of the lethal agents, consisting of bacteriophages, bacteriocins and antibiotics, with the cell membrane of bacteria has been referred to as the "iron Achilles heel". Iron is apparently so physiologically important to

Mutational Analysis of Rhodotorulic Acid Synthesis in Rhodotorulapilimanae

99

the bacteria that they maintain receptor systems for its harvesting even though such systems render the cell susceptible to attack by deadly agents which have adapted their structures so as to focus on this particular port of entry. The relationship is also of great practical importance for the cloning of the siderophore systems since a plasmid containing a vector and the cloned sequences will be both resistant to a specified antibiotic and susceptible to a lethal agent, the latter in the case of aerobactin being cloacin. The genes for aerobactin biosynthesis and transport may be organized into a single operon regulated by a single promoter-operator segment of DNA 9). The DNA sequence of this promoter shows that it is of the "strong" variety and is very closely related to the consensus sequence for E. coli promoters. The + 1 base is situated slightly closer to the 10 Pribnow box than in the consensus sequence but the intervening spacing to the - 35 region is perfectly optimal at 17 bases. The leader transcript is some 30 bases in length, the ribosome binding site is in the expected location, and the first codon is ATG, for methionine. Further downstream is an open reading frame through the 63,000 MW protein. The segment between this cistron and the next carries a Shine-Dalgarno sequence but no second promoter. It has not yet been established if there is one or more additional promoters somewhere within the gene complex, but it is entirely possible that there is but a single promoter-operator regulating the entire gene complex. Regulation by iron is observed in the cloned sequences, probably acting at the transcriptional level4). The cloned sequences of the aerobactin system indicate the presence of a convenient restriction site for insertion of the lacZ gene, minus its promoter. This gene, coding for ~-galactosidase, greatly facilitates detection of regulation of the operon. The [3-galactosidase levels can be easily and accurately determined by use of the chromogenic substrate analogue, Gal-X or, alternatively, derepressed (overproducing) colonies can be detected on galactose-pH indicator agar owing to the color change associated with the production of acid from fermentation of the sugar. Armed with this plasmid and detection system it is then only necessary to make mutations in the chromosome and test the effects on level of expression of [3-galactosidase. While the chromosome could be mutated with chemical reagents, transpositional mutagenesis is preferred since only a single transposon enters the chromosome and leads to "knockout" mutations. Applied to the aerobactin system, this approach had yielded a collection of mutants, some of which are derepressed in high iron, i.e., aerobactin synthesis is not impeded by the metal ~°). It is anticipated that at least some of these mutants will be defective in the synthesis of a repressor protein, the function of which is to complex iron and in the iron laden form bind to the operator region of the DNA to prevent read-through of RNA polymerase. As an alternative to this "negative" type of control, the regulatory protein could be held off the DNA in its iron complex form and only bind and activate the DNA promoteroperator when depleted of iron. In any event these regulatory proteins will constitute a new class of iron proteins. The elucidation of their structures, how they bind the iron (II or III) and how they complex with the DNA to inhibit or activate transcription constitute one of the most challenging arenas of research in bioinorganic chemistry. Within bacterial species cases of both positive and negative control are known. Four DNA binding proteins have been particularly well studied, namely, the lac repressor, the k Cro repressor, the k repressor and the CAP activator-repressorn). The last three have been crystallized and their structures determined. These three proteins contain two consecutive alpha helices believed to be an intrinsic part of their DNA binding domains. Recently, the bacteriophage ~ Cro repressor protein has been crystallized in a complex -

100

A. Liu and J. B. Neilands

with short duplex strands of oligo-deoxynucleotides12). The crystals diffract to approximately 3 A resolution and should afford detailed information on the mode of binding of the Cro repressor to the DNA. Once the aerobactin repressor has been defined genetically, it will then be necessary to determine if the same protein regulates formation of the chromosomally encoded siderophore, enterobactin. The cloning of this protein will provide it in quantities such that it can be treated by the standard methods of protein chemistry and in the event that it can be crystallized, its interaction with the operator can be examined by methods analogous to those used for the repressors just described. It appears unlikely that the siderophores themselves are involved, as their iron complexes, in regulation. This is because ent mutants of E. coli which are defective in synthesis of enterobactin still overproduce the 81,000 MW outer membrane receptor for ferric enterobactin 13). Similarly, heme is not involved since the repression and derepression of synthesis of siderophore receptors operate normally in hemA mutants blocked in porphyfin biosynthesis. Thus by elimination we are left with a relatively classical type of repressor mediated control which is common to several well studied bacterial systems. The molecular mechanics and mode of action of this system will soon be known in some detail and it is hence pertinent to initiate the move up to a eukaryotic species. It should be mentioned that the chromosomally encoded enterobactin in siderophore complex of E. coli has been cloned on phage M~t14' 15). This system is organized into several transcriptional units spread across some 26 Kb of DNA and is hence substantially more complicated than the aerobactin system just described.

HI. Rhodotorulapilimanae as a Eukaryotic Siderophore Producing Analogue of Escherichia coli As siderophores are produced generally among the fungi, we have a catalogue of species from which to chose the system most ammenable to study by the tools of molecular genetics, bearing in mind that many of the genetic procedures and manipulations commonly employed in E. coli are not yet possible in the fungi, which are substantially more complicated as regards their genetic constitution. Without any doubt the most desirable eukaryotic species in which to launch an investigation of the molecular genetics of siderophore synthesis would be a Saccharomyces spp. This is because of the large reservoir of genetic information now available with this species. Unfortunately, however, we have never detected either an hydroxamate or a catechol type siderophore from brewer's or baker's yeast, and it is not known if this organism has a high affinity iron assimilation system. For these reasons we have had to turn to another eukaryotic spp. Several factors pointed to rhodotorulic16) acid as the prime choice for moleculargenetic studies of a siderophore from higher microorganisms. It is a relatively simple compound and consists of the diketopiperazine of NS-acetyl-NS-hydroxy-L-omithine(I). In fact, it is the simplest member of a family of related compounds in which the acyl groups are enlarged. These include dimerum (or dimerumic) acid, coprogen, triomicin and isotriornicin. The structure has not been confirmed by X-ray crystallography, but there is no doubt about its authenticity. Rhodotorulic acid has been synthesized in

Mutational Analysisof Rhodotorulic Acid Synthesisin Rhodotorulapilimanae

O

OH

101

O~w"N"~N_C/CH3

CH3 I, Rhodotorulic Acid Switzerland17) and by two laboratories in Japan is' 19), one of which also prepared the enantiomeric form of the siderophore. Rhodotorulic acid is made in truly outrageous quantities by certain basidiomycetous yeasts2°) belonging to the genera Leucosporidium, Rhodosporidium, Rhodotorula, Sporidiobolus and Sporobolomyces. Yields of several grams per liter can be achieved with relative ease. This fact, together with the sparing solubility in water, about 1%, means that a slightly concentrated cell supernatant will start to deposit crystals of rhodotorulic acid. The high yield coupled with poor water solubility has enabled a novel approach to its biosynthesis2x). Rhodotorulic acid forms a stable complex containing 2/3 atom iron (III) per mo116). It is highly potent, either as ligand or iron (III) complex, as a siderophore in such test organisms as Arthrobacter flavescens JG-9 or Escherichia coli RW 193. Rhodotorula pilimanae was trained by serial transfer to grow in media containing 99.9% deuterium oxide. Then, unlabelled, H-form amino acids were fed into the culture to test their effectiveness as precursors of rhodotorulic acid. The latter was scanned by proton NMR to ascertain the position of the label. These measurements, together with data from mass spectroscopy, revealed the route of biosynthesis to be:

NH2

NH2

OH

NH2 ol"

NH2

H CoK /

/

\\

HO

0

COOH

COOH

COOH

L-orthinine

II, Nr-hydroxyorthinine

N~-acetyl-Nr-hydroxyorthinine

= I

Thus it is anticipated that a relatively small number of steps are involved in this pathway. Ornithine is oxidized on the N~-atom to afford Nr-hydroxy-ornithine (II) by a novel oxygenase enzyme20. In view of the facile chemical synthesis of organic hydroxylamines by an oxaziridine route developed by Widner and Keller-Schierlein22), it is tempting to speculate that pyridoxal phosphate is involved as shown below.

NH2

~

NH2

COOH L-orthinine

+PALPO Pyridoxal Phosphate (PALPO)

NH2 ~A,~N=CH_~/N

COOH

\

C

o

,,

COOH

C

+ PALPO

/

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A. Liu and J. B. Neilands

While the next step, the acetylation of N%hydroxyornithine, will proceed without catalysis it is most likely that the acetyl moiety is transferred enzymatically from acetyl-coenzyme A. When R. pilimanae is cultured at low values of pH the biosynthetic route is interdicted and 1-hydroxy-3(S)-amino-2-piperidone is the product23). This may be due to OH

I

the difficulty of acetylation of the charged species R-NH~", the pKa of which is about 5. Finally, the demonstration of ATP-32pPi exchange activity in extracts of R. pilimanae fortified with N%acetyl-N6-hydroxyornithine indicates that the amino acid "monomer" goes through an amino acid adenylate intermediate21). R. pilimanae grows on a simple medium consisting of ammonium sulfate, phosphate, trace metals and a carbon source, such as sucrose. It forms discrete colonies arising from single cells, the latter beautifully colored by a carotenoid pigment. The chromophore, however, is not correlated with rhodotorulic acid biosynthesis since a white mutant forms just as much of the siderophore. As is characteristic of yeasts, the presence of a thick, chitinous wall, ca. 80/~ in R. pilimanae, the cells of the organism are not easily disrupted. This, in addition to the rudimentary state of knowledge of their molecular genetics, are two major disadvantages in working with this organism. However, in spite of the truly massive quantities of rhodotorulic acid formed in low iron media, the addition of as little as 1 or 2 mg of the metal per liter is sufficient to shut down synthesis of the siderophore to barely detectable levels. This indicates that the message may be unusually stable and that the system is tightly regulated by iron. On the "plus" side of the ledger, the fungus Penicillium lilacinum forms an extra-cellular lytic enzyme which hydrolyses the cell walls of Rhodotorula spp., thus affording easy access to the messenger RNA. In this laboratory a project is underway to define the genes required for rhodotorulic acid biosynthesis, to clone them and to examine the mechanism of their regulation by iron at the molecular level. The first efforts have been directed toward collection of a library of mutants blocked at various stages in the synthesis of rhodotorulic acid.

IV. Detection of Mutants Defective in Biosynthesis of Rhodotorulic Acid R. pilimanae UCD 67-64 is grown to stationary phase in the usual low iron medium 16), washed and resuspended to an absorbancy of 20 at 650 nm in 50 mM potassium phosphate buffer pH. A 5 ml aliquot is treated at room temperature with 150 Ixl of ethyl methane sulfonate and at 30 rain intervals 100 ~tl aliquots are removed, diluted with 6% sterile sodium thiosulfate solution and spread on minimal agar plates. The survival curve (Fig. 1) was determined after growth at 30 °C for 4 days. A 3% concentration of mutagen gave the most satisfactory results. Low iron nutrient agar plates are prepared by dissolving 8 g of Difco nutrient broth, 0.1 mmol deferriferrichrome A and 15 g agar per liter. Specially purified agar is available but appears unnecessary for the uses intended here. One ml of an overnight nutrient broth culture of E. coli RW 193 ATCC 33 475 was seeded into the 40 °C melted agar and the plates allowed to solidify. R. pilimanae cells from a culture mutagenized to ca. 1%

Mutational Analysis of Rhodotorulic Acid Synthesis in Rhodotorula pilimanae I001

i

~

103 l

I0 0

>=

0 /

I

I

I

I

I

2

3

Hours Fig. 1. Survival curve of Rhodotorula pilimanae UCD 67-64 after mutagenesis with ethyl methane sulfonate (see text)

survival were diluted and spread on standard minimal agar plates at a density of about 50 cells per plate. After the colonies had grown to 2-3 mm in diameter a substantial portion of each colony was picked and transferred to the low iron agar-E, coli seeded plates at a density, evenly spaced, of 30 patches per plate. After overnight growth at 37 °C colonies excreting rhodotorulic acid were easily identified by a halo of exhibition of growth. Colonies forming reduced or no halo were selected for further study. Out of a total of 5000 colonies screened, 12 (numbered ALN 1 through ALN 12) failed to produce a halo. These were grown up in minimal medium and the cell supernatants assayed with the perchlorate reagent for hydroxamate. Nine mutants (ALN 1-ALN9) produced low levels of rhodotorulic acid while the remaining 3 (ALN 10--ALN 12) formed undetectable amounts. Production of rhodotorulic acid in wild type cells begins in 3 to 5 days on minimal medium. But mutant ALN 10 made a new hydroxamate which differed from rhodotornlic acid in the color of its ferric derivative. In the assay2°) solution rhodotorulic acid gives a brownish-purple ferric complex; the compound from ALN 10 yielded a purple colored complex. The compounds could be further distinguished by their mobility on paper electrophoresis at pH 5. Rhodotorulic acid is neutral, while the mutant hydroxamate moves to the anode at a rate characteristic of a monovalent anion. Another distinction is in the rate of production, the mutant hydroxamate not appearing until 10 to 15 days of incubation.

104

A. Liu and J. B. Neilands I

I

I

) (4)

) (3) ) (2)

e-

r~ (,J

Fig. 2. Growth rate curves for

Rhodotorula spp on various media. (1) R. pilimanae AL 10 on minimal medium, (2) R.

pilimanae AL 10 on minimal

I

I

I

5

I0

15

Days

medium with 10 I~g/mliron salt, (3) R. pilimanae AL 10 on minimal mediumwith 10 Itg/ml rhodotorulie acid, and (4) R. pilimanae UCD 67-64on mini. mal medium

Evidence that mutation of the rhodotorulic acid pathway affects growth on low iron medium was obtained by studying the effect of these additives on the growth rate of ALN 10 in minimal medium. Supplementation with either 10 mg/l rhodotorulic acid or 10 mg/l iron improved growth to a substantial degree (Fig. 2).

V. A New Hydroxamate From a Mutant of Rhodotorula pilimanae In order to isolate the mutant hydroxamate, a 2 1culture of ALN 10 was centrifuged and the cell free supematant passed through a 2 x 50 cm column containing the anion exchanger AG-1 X2, 50-100 mesh. The column was washed with water and the anions eluted at a flow rate of 0.5 ml/min by application of a linear salt gradient of 0 to 0.5 NI-LC1, 2 1total volume. Each 0.5 1of eluate was adjusted to pH 2 with concentrated HCI, saturated with ammonium sulfate and extracted 5 times with 20 ml of benzyl alcohol. The pooled organic extracts were clarified by centrifugation and filtration and then diluted with 0.5 1 of diethyl ether. The hydroxamate was returned to water by extraction 5 times with 20 ml of water. Finally, the aqueous solution was lyophilized, dissolved in a small volume and passed through a 1 x 30 cm molecular sieve column containing P-2, 200-400 mesh, using a distilled water eluant at a flow rate of 0.2 ml/min for elution. The lyophilized eluate afforded about 6 mmol of hydroxamate, representing a yield of ca. 40% from the original 2 1 of cell supernatant. The mutant hydroxamate proved to be a colorless, hygroscopic solid soluble in water and insoluble in non-polar solvents. It gave negative tests for both ninhydrin and tetra-

Mutational Analysisof RhodotorulicAcid Synthesisin Rhodotorulapilimanae

105

zolium sprays thus indicating the absence of both amino and hydroxyamino groups. A total hydrolysate in 6 N HC1 yielded, NS-hydroxyornithine, identical to that liberated from rhodotorulic acid as found by paper electrophoresis at pH 6.5. The presence of N ~hydroxyornithine in the molecule was confirmed by proton NMR, all of the resonances expected for the amino acid being detected in the mutant hydroxamate. Attempts to further characterize the mutant hydroxamate are in progress.

VI. Summary and Conclusions Rhodotorulic acid appears to be the hydroxamate siderophore of choice for study of the molecular genetics of its biosynthesis and regulation, being a good deal simpler in its constitution than the ferrichromes and higher members of the rhodotorulic acid family, such as coprogen. We have described a simple system for mutagenesis of Rhodotorula pilimanae and for detection of mutants defective in biosynthesis of rhodotorulic acid. Application of the new tools of molecular genetics should produce a body of information for the iron assimilation system of R. pilimanae which can be compared with that accumulated in E. coli. The mode of regulation of the rhodotorulic acid gene complex will be of particular interest since it may serve as a model for corresponding processes in plants and animals. It has already been established the N6-acetyl-N6-hydroxyornithine synthesis is not feed-back regulatedz3). The same conclusion applies to rhodotorulic acid since high levels of exogenously added siderophore did not inhibit its synthesis. Iron had no effect on conversion of hydroxyaminopiperidone to rhodotorulic acid but severely repressed formation of N6-hydroxyornithine from ornithine. Iron had a similar effect on conversion of N~-acetyl-N~-hydroxyornithine to rhodotorulic acid and no ATp-32pPi exchange could be measured in extracts of high iron grown cells supplemented with the amino acid. Thus whatever the mechanism of regulation by iron, it appears to affect coordinately all segments of the biosynthetic pathway. The mutant hydroxamate uncovered in this study must be substituted on the alphaamino group. This conclusion is supported by the observation that the molecule is negatively charged at neutral pH. In R. pilimanae ALN 10 the mutagenic lesion probably occurs late in the biosynthetic pathway, possibly at the cyclization step. This would lead to accumulation of N~-acetyl-N~-hydroxyornithine which is subsequently modified at the alpha amino group. Failure completely to synthesize rhodotorulic acid results in diminished capacity of R. pilimanae to fluorish in an environment low in iron. The characterization of this compound and the metabolic products of other mutants in the pathway should establish the number and order of genes in the rhodotorulic acid complex. In parallel with the mutational analysis of rhodotorulic acid biosynthetic pathway a separate study is underway to obtain directly the DNA involved in this process. Cells grown at low iron should have a substantially augmented level of mRNA, which probably can be recovered owing to the expected presence of the usual 3' polyA tail. Treatment with reverse transcriptase in the presence of an oligo-T primer should yield crude cDNA. Digestion with S 1 nuclease would remove the single stranded hairpin loop to give double stranded cDNA. The latter can be inserted into pBR322 by use of either terminal transferase or linker oligo-nucleotides with suitable restriction sites. The resulting plas-

106

A. Liu and J. B. Neilands

mids can be used to transform E. coli RW 193 ATCC 33 475 cultured under conditions where only siderophore synthesizing cells will propagate. Fortunately, E. coli responds to rhodotorulic acid as a siderophore 24). As a variation to this approach, the plaque hybridization assay may be employed. High molecular weight D N A may be extracted from R. pilimanae, restricted and ligated into bacteriophage 2. Cells of E. coli could then be infected, plated and probed with 32p-cDNA. In addition, a cosmid packaging system may prove to be a very efficient means of getting rhodotorulic acid genes into E. coli. Once this is achieved, the D N A sequence can be determined and the nature of its regulation examined in studies comparable to those now in progress on aerobactin in

E. coli.

VII. Note Added in Proof The molecular weight of the third protein in the aerobaction operon is now believed to be 32,000 rather than 27,000 (Bindereif, A., Paw, B. H., Neilands, J. B.: J. Biol. Chem., submitted).

VIII. References 1. Jacobs, A., Worwood, A. (eds.): Iron in Biochemistry and Medicine, Academic Press, London 1980 2. Neilands, J.: Ann. Rev. Nutr. 1, 27 (1981) 3. Neilands, J. B.: Ann. Rev. Biochem. 50, 715 (1981) 4. Bindereif, A., Neflands, J. B.: J. Bacteriol. 153, 111 (1983) 5. Bindereif, A., Thorsness, P. E., Neilands, J. B.: Inorganica Chemica Acta - Bioinorganic Chemistry 79, 78 (1983) 6. Bindereif, A., Baun, V., Hantke, K.: J. Bacteriol. 150, 1472 (1982) 7. Grewal, K. K., Warner, P. J., Williams, P. H.: FEBS Lettr. 140, 27 (1982) 8. Van Tiel-Menkveld, G. J., Mentjox-Vervunrt, J. M., Oudega, B., DeGraff, F. K.: J. Bacteriol. 150, 490 (1982) 9. Bindereif, A., Neilands, J. B.: J. Biol. Chem., submitted 10. Bagg, A., Neilands, J. B.: Abstr. 84th Ann. Meeting, Am. Soc. Microbiol., St. Louis, MO, Mar. 4-9 (t984) 11. Takeda, Y., Ohlendorf, D. H., Anderson, W. F., Matthews, B. W.: Science 221, 1020 (1983) 12. Anderson, W. F., Cygler, M., Vandonselaar, M., Ohlendorf, D. H., Matthews, B. W., Kim, J., Takeda, Y.: J. Mol. Biol. 168, 903 (1983) 13. Klebba, P. E., Mclntosh, M. A., Neilands, J. B.: J. Bacteriol. 149, 880 (1982) 14. Laird, A. J., Ribbons, D. W., Woodrow, G. C., Young, I. G.: Gene 11, 347 (1980) 15. Laird, A. J., Young, I. G.: ibid. 11, 359 (1980) 16. Atkin, C. L., Neilands, J. B.: Biochemistry 7, 3734 (1968) 17. Widmer, J., Keller-Schierlein, W.: Helv. Chim. Aeta 52, 388 (1969) 18. Isowa, Y., Takashima, T., Ohmori, M., Kurita, H., Sato, M., Mori, K.: Bull. Chem. Soc. Japan 45, 1467 (1972) 19. Fujii, T., Hatanaka, Y.: Tetrahedron 29, 3825 (1973) 20. Atkin, C. L., Neilands, J. B., Phaff, H. J.: J. Bacteriol. 103, 722 (1970) 21. Akers, H. A., Neilands. J. B4 In Biological Oxidation of Nitrogen, (Gorrod, J. W., ed.), Elsevier North Holland, Amsterdam, p. 429 (1978) 22. Widmer, J., Keller-Schierlein, W.: Heir. China. Acta 57, 1904 (1974) 23. Akers, H. A.: Doctoral Dissertation, Univ. of California, Berkeley 1974 24. Hantke, K.: Mol. Gen. Cenet. 191, 301 (1983)

Phytosiderophores Structures and Properties of Mugineic Acids and Their Metal Complexes Yukio Sugiura I and Kyosuke Nomoto 2 1 Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan 2 Suntory Institute for Bioorganic Research, Shimamoto-cho, Mishimagun, Osaka 618, Japan

In graminaceous plants such as barley, oats, and wheat, novel iron-chelating amino acids are secreted from the roots. A typical example is mugineic acid. A phytosiderophore, mugineic acid significantly stimulates iron-uptake and chlorophyll synthesis in rice plants. Most microbial siderophores have hydroxamate or phenolate groups as Fe(III)-coordination donors, while phytosiderophores consist of carboxyl, amine, and hydroxyl groups as the ligand functional groups. The mugineic acid-Fe(III) complex and its structurally analogous Co(III) complex have been characterized by some spectroscopic and X-ray diffraction methods. The coordination of mugineic acid to Co(III) and Fe(III) ions involves the azetidine nitrogen, secondary amine nitrogen, both terminal carbox'ylate oxygens as basal planar donors, and the hydroxyl oxygen and intermediate carboxylate oxygen as axial donors in nearly octahedral configuration. The M6ssbauer (AEo = 0.24 and 6vc = + 0.39 ram/see) and ESR (g = 9.4, 4.51, 4.44, and 4.31) parameters of the mugineic acid-Fe(III) complex are characteristic of high-spin (S = 5/2) ferric type. Of special interest is the apparent high reduction potential (E~a --- - 102 mV vs. NHE) of the mugineic acid-Fe(III) complex, as compared to those of the microbial hydroxamates and ferric enterobactin. The mechanism of iron-absorption and -transport in gramineous plants probably includes Fe(III)-solubilization by mugineic acid and reduction from the thermodynamically stable ferric mugineic acid complex (log K~L = 18.1) to the weakly bound ferrous complex (log K ~ = 8.1).

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

II.

Plant Culture and Isolation of Mugineic Acid . . . . . . . . . . . . . . . . . . . . .

110

III.

Structural Elucidation of Mugineic Acids . . . . . . . . . . . . . . . . . . . . . . . 1. Mugineic Acid (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Avenic Acid A (2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. 2'-Deoxymugineic Acid (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. 3-Hydroxymugineic Acid (4) . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Distichonic Acid A (5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Other Similar Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112 112 116 117 117 118 118

IV.

Chemical Synthesis of Mugineic Acids . . . . . . . . . . . . . . . . . . . . . . . . 1. Synthesis of 2'-Deoxymugineic Acid (3) . . . . . . . . . . . . . . . . . . . . . 2. Synthesis of Avenic Acid A (2) . . . . . . . . . . . . . . . . . . . . . . . . . .

118 118 120

V.

Is Mugineic Acid a Phytosiderophore? . . . . . . . . . . . . . . . . . . . . . . . .

121

VI.

Some Properties of Mugineic Acid-Iron(III) Complexes . . . . . . . . . . . . . . .

122

Structure and Bonding 58 © Springer-Verlag Berlin Heidelberg 1984

108 VII.

Y. Sugiura and K. Nomoto Structure and Properties of Structurally Analogous Mugineic Acid-Cobalt(III) Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124

VIII. Structure and Properties of Mugineic Acid-Copper(II) Complex . . . . . . . . . . .

127

IX.

Solution Structures of Muginei¢ Acid and Its Metal Complexes . . . . . . . . . . . .

130

X.

Probable Mechanism of Iron Transport in Gramineous Plants . . . . . . . . . . . .

132

XI.

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

134

Phytosiderophores

109

I. Introduction Most aerobic and facultative anaerobic bacteria, as well as fungi, excrete low-molecular weight compounds, generically termed "siderophores", for the solubilization and transport of Fe(III) 1-3). On the other hand, plants also require a continuing supply of a number of metal elements to maintain proper growth. Above all, iron is one of essential elements for plants in terms of chlorophyll biosynthesis. Although green plants contain a substantial quantity of iron, the metal ion is not transported from the old to new leaves. Thus, a ceaseless supply of iron is necessary for the plants 4' 5). Insufficient iron supplementation leads to the development of iron chlorosis which turns the young leaves to yellow or white. As a result, the plants cease to grow and eventually they wither up. In general, iron exists sufficiently in soil6). Nevertheless, the amount of Fe(III) ion available in the aqueous solution of most calcareous soils is insufficient for plant growth, and iron chlorosis develops in plants 7). The solubility of Fe(III) ion at pH 4 or more approximately decreases to one thousandth for each rise in the pH values). Some plants do not succumb to iron stress and grow well in alkaline soil. The phenomenon suggests that such plants are endowed with the biochemical function which turns insoluble iron into useful Fe(III) ion in soil. It is possible to classify the plants susceptible to iron stress (iron-efficient plants) separately from the ones insusceptible to iron stress (iron-inefficient plants). For the purpose of the availability of iron, four biochemical factors appear to be involved in iron absorption and transport in iron-efficient plants9): 1) release of hydrogen ions from the roots, 2) release of reducing compounds from the roots, 3) reduction of ferric ion to ferrous ion at the roots, and 4) increase of organic acid (particularly citrate) in the roots. A possible phytosiderophre with an essential iron-uptake and -transport function in plants had not been discovered, although iron nutrition problems were centered in the roots and on chemical substances that affect absorption and translocation of iron from the growth medium. It is well-known that rice is a typical cultured plant susceptible to iron stress, and that some graminaceous plants such as barley and oat are also considerably susceptible to iron stress. Takagi 1°) found that (1) when the roots of water-cultured rice and oats were soaked in deionized water, the root-washings contained an ironchelating substance, (2) the plants secreted more of the substance in iron-deficient condition, and (3) the secretion of chelating compound in barley was far more than that in rice. In view of these facts, we firstly picked on barley (Hordeum velgareL. var. minorimugi), isolated a new amino acid named mugineic acid(l) u), and elucidated the chemical structure. Similar iron-chelating amino acids were also isolated from some species of gramineous plants: avenic acid A(2) from oats (Avena sativa L.) 12), 2'-deoxymugineic acid(3) from wheat (Triticum aestivumL.) 13), 3-hydroxymugineic acid(4) from rye (Secalecereale L.) 14), and distichonic acid(5) from beer barley (Hordeum vulgate L. var. distichum) 14). Mugineic acid and pseudo-mugineic acid appear to be of general occurrence in graminaceous plants. New iron-chelating substances isolated from the roots of gramineous plants, phytosiderophores, are described here by contrasting them with bacterial siderophores and plant siderophores (see Chart 1).

110

Y. Sugiura and K. Nomoto

COO

COOH

CO0-

R2 1

RI= H,

R2= OH

3

RI= R2= H

4

R1 = R2= OH

Co0-

CO0-

COOH

coo-

coo-

COOH

NH~ y

~'NH~ V

~'OH

OH

II. Plant Culture and Isolation of Mugineic Acid Plant culture was carded out in a glass house. Young seedlings of barley were cultivated in nutrient solution (pH 6) with the composition as shown in Table 1. The solution was aerated continuously. When barley plants fully expanded their 2rid and 3rd leaves, after one week or more, they were transported to iron-deficient solution, and then the culture was continued for another one week or more. The composition of iron-deficient solution was the same as described in Table 1, except for the absence of ferric chloride. When the yellowing of the leaves was developed moderately, the root-washings were collected by the soakage of the root in deionized water once a day for two weeks. The root-washings were first chromatographed on~Amberlite IR-120 B (H + form) followed by the separation on Dowex 50W X2 (pH 2.5-3.1, ammonia-formate buffer). Gel-filtration (Sephadex G-10) of the fractions containing the iron-chelating activity yielded pure mugineic acid(l) as fine crystals. The iron-chelating activity was determined by the o -

111

Phytosiderophores Table 1. Composition of nutrient solution

Salt concentration (raM)

Element concentration (ppm)

KNO3

5.0

K, 195.5 x 104 NO3 - N, 70.0 x 104

Ca(NO3)24H20

5.0

Ca, 200.0 x 104 NO3 - N , 140.0 x 104

MgSO47H20 NI-hH2PO3

2.0 1.0

Mg, 48.6 x 10a P, 3.1 x 104 NI-h - N , 1.4 x 104

H3BO3 MnCI24H20

3.0 x 10-3 5.0 x 10-4

B, 33.0 Mn, 27.5

CuSO45H20 ZnSO47HzO

2.0 x 10-4 4.0 x 10-4

Cu, 12.8 Zn, 26.0

(NH4)6Mo70244H20 NaCI

1.0 x 10-3 5.0 x 10-2

Mo, 672.0 Na, 1150 C1, 1775

HC1 FeC136H20

0.1 1.1 x 10-2

CI, 3550 Fe, 0.61 CI, 1.17

Root-washings

of w a t e r - c u l t u r e d

barley, I

Hordeum vuZ@ave L. vat. M i n o r i m u g i Amberlite

i adsorbed

IR-120B

( H+ )

effluent

IN_NH4OH

'

I

adsorbed

eluate

I

D o w e x 50W x 4 I.SN-HCI

( H+ )

eluate

D o w e x 50W x 4 ( pH 2.5 a m m o n i a formate ) gradient eluates with a m m o n i a - f o r m a t e b u f f e r ( pH 2.5 - 3.1

I fr. 45 - 62

l fr.

65 - 76

I Sephadex G-IO amorphous powder (mugineic acid B )

)

Sephadex G~IO amorphous

powder

I recrystallization from water Fig. 1. Isolation procedure of mugineic acid

mugineic

acid

phenanthroline method. Figure I shows the experimental procedures for the isolation of mugineic acid u). The cultivation of other gramineous plant species and the isolation of analogous iron-chelators were also performed by similar procedures.

112

Y. Sugiura and K. Nomoto

III. Structural Elucidation of Mugineie Adds

1. Mugineic Acid (1) Mugineic acid(l) isolated from barley was obtained as fine platelets from water (mp 210-212 *C (decomp) and [ a ] o - 70.7°) and showed a weak positive ninhydrin reaction. The mass spectrum (FD-MS; m/z 321 (M + H) ÷) and the elemental analysis of 1 gave the molecular formula Ct2H20OsN2. The IR spectrum exhibited characteristic bands at 3450-3200 (OH and NH) and 1605 cm -1 (CO0-). In the 1H NMR spectrum, all the proton signals were analyzed thoroughly with the aid of double resonance experiments (see Fig. 2 and Table 2). The results indicate the presence of two -CHz--CHz-CH- and one-CHz-CH-CH- moieties in the molecule. The 13C-NMR spectrum demonstrated the presence of three carbonyl carbons, five methylene carbons, and four methine carbons (see Table 3). The compound(l) contains multiple water-solubilizing functional groups. In order to investigate the nature of these functional groups, certain chemical modifications of 1, for example acetylation and methylation, were attempted, but valuable structural information was not obtained. Consequently, the complete structure and stereochemistry of 1 were determined by X-ray crystallography. Figure 3 shows a stereo view and structural formula of mugineic acid(l). The absolute configuration of 1 was clarified on the basis of the following evidence. The hydrolysis of 1 with 6 N HCI at

DHO

1 coo-

4 'coo"

4

3'

2m3 j°

l

4 "coo.

OH

a

I 2 JB

I

I 5

I 4

i 3

Fig. 2. IH-NMR spectrum of mugineic acid (in D20, 100 MHz)

I

2

ppm

2.95 b t, 7.6

4.75 t, 9.5

4.98 t, 6.5

3.86 s

4.76 t, 9.5

avenic acid A '

2'-deoxymugineie acid

3-hydroxymugineic acid

distichonic acid A a

nicotianamine

2.62 m

4.80 ddd 4,6,6.5

2.62 m

1.53-1.81 m

2.57 m

C-3

4.02 m

4.02 dd 6,11

4.04 m

3.52 t, 7.6

4.01 m

C-4

4.28 dd 4,11

3.41 m

3.30 d, 7

3.47 m

3.45 m

2.24--2.68 m

3~45 m

C-I'

2.22 m

4.49 td, 7, 3

4.42 m

2.17 m

1.53-1.81 m

4.42 m

C-2'

3.80 dd, 6, 8

4.13 d, 3

3.84 d, 3.5

3.84 dd, 6, 8

3.02 b t, 7.6

3.82 d, 3.5

C-Y

3.27 t, 7.5

3.15 t, 7.5

3.23 m

3.27 t, 7.5

2.24-2.68 m

3.22 m

C-I"

2.22 m

1.98 m

2.13 m

2.17 m

1.53-1.81 m

2.12 m

C-2"

3.89 dd, 4, 7.5

4.23 dd, 4.5, 8

4.34 dd, 5, 7.5

4.36 dd, 5, 7.5

3.92 dd, 5.4, 8.1

4.33 dd, 5, 7.5

C-3"

Chemical shifts were expressed by ppm downfield from TPS (sodium 2,2,3,3-tetradeutero-3-(trimethylsilyl)propionicacid) used as an external standard and coupling constants by Hz. Abbreviations: s = singlet, d -- doublet, dd = doublet of doublets, t = triplet, m = multiplet. ' Avenic acid A and distichonie acid A are slightly soluble in water, ~H-NMR spectra of avenic acid A and distichonic acid A were measured in 1 NNaOD and 1 N-DCI, respectively b The assignment may be interchanged

4.85 t, 9.5

mugineic acid

C-2

Table 2. 1H-NMR Spectral Data (in D 2 0 , 100 MHz)

"O ~r O

¢:h

170.1 s

170.0

s

3-hydroxymugineie acid

distichonic acid A a 68.9 d

175.1

s

76.3 d

68.7 d

t

23.0

65.3 d

23.0 t

33.9 c t

23.8 t

C-3

t

53.0

62.8 t

53.2 t

60.2 t

53.1 t

C-4

t

52.5

t

51.0

57.7 t

52.6 t

45.4 a t

58.0 t

C-I'

t

26.7

d

66.2

67.2 d

26.4 t

35.0~ t

66.6 d

C-2'

d

61.4

d

64.2

66.8 d

61.3 d

62.2 b d

66.6 d

C-3'

Chemical Shifts are given in ppm downfield from TPS as an external standard. " Avenic acid A and distichonie acid A were measured in 1 N-NaOD and 1 N-DCi, respectively These assignments may be reversed

nicotianamine

49.3 t

174.7 s

2'-deoxymugineic acid

63.3 b d

183.1 s

avenic acid A '

69.7 d

179.4 s

C-2

mugineic acid

C-1

Table 3, 13C-NMR Spectral Data (in D20, 25 MHz)

s

173.8

s

168.9

170.8 s

173.6 s

183.1 s

171.0 s

C-4'

t

46.0

t

46.2

46.8 t

45.8 t

45.1 d t

46.9 t

C-I"

t

29.0

t

31.0

31.5 t

31.7 t

36.3 c t

31.6 t

C-2"

d

54.6

d

69.7

70.7 d

70.4 d

72.1 d

71.0 d

C-3"

s

175.0

s

177.7

179.0 s

178.9 s

182.3 s

179.5 s

C-4"

o

o

e,

|l

e6

II

o

¢

Fig. 3. Structure and a stereo view of mugineic acid

cb

w

o(e).....o(~,

Crystal Data of mugineic acid rap. 210-212 ° (decomp.) CuH20OsN:, orthorhombic space group p 212121 a = 5.1042(5), b = 25.458(9), c = 10.979(4).~ D m = 1.49 g • cm -3 (by floatation in CCl4/n-hexane mixture), Dc= 1.48g.cm -3,Z=4

°(2L-.c(o--°~° o(,.L ..o~3)

t.~

8"

Z:r

116

Y. Sugiura and K. Nomoto

6N HCI

CO2H HO

CO2H

NH2

H2N

6

OH 7

Chart 2

•/•O 2H

H2N

NH2

CuCO3 PhCH2OCOCi

,,,

C~v~2H phCH202CN

L--=, Y.-d.tamino-n-b~y~.c acid ( 8 )

NC / ~ 2 H phCH202C OH 10

NH2

HN02 ACOH

9

H2 Pd-C

_,--

/~Oo~ H H2N r.-'t-amiaxr-a-~n-butyric acid ( 7 )

Chart 3

120 °C gave two products, the compounds (6) and (7) (see Chart 2). The compound(6) was confirmed to be homoserine by comparison of the spectral data with those of an anthentic sample. The compound(7) was assumed to be y-amino-a-hydroxybutyric acid on the basis of the ~H and 13C NMR spectral data and the behavior on paper electrophoresis, and was identical in all respects with L-y-amino-a-hydroxybutyric acid derived from L-a,y-diaminobutyric acid as shown in Chart 3, except for the optical rotation 15-17). Craig and Roy is) have reported that the knowledge of ORD curves between 200 and 225 nm permits the direct assignment of the absolute configuration of ahydroxy acids, a-substituted alcohols, and their derivatives; namely, the ORD curves of the L- and D-series gave positive and negative Cotton effects, respectively. Comparison of the CD curves of 7 (Ae216nm = + 0.084) and L-y-amino-a-hydroxy-butyricacid (AE216n m = + 0.083) derived from L-a,?-diaminobutyric acid, clearly demonstrated that the chiral center in 7 is S configuration. The oxidation of 1 with KMnO4 yielded L-(-)-azetidine-2carboxylic acid(13) ([a]D -- 96.90)19). On the basis of these results, it was concluded that mugineic acid is 2(S),2' (S),3'(S),Y(S)-N-[3-carboxy-(3-carboxy-3-hydroxypropylamino)2-hydroxypropyl]azetidine-2-carboxylicacid.

2. Avenic Acid A (2) Avenic acid A(2) isolated from oats, CnH22N2Os (FD-MS; m/z 345 (M + Na)+), mp > 300 °C, and [a]D + 16.4 °, showed positive color with ninhydrin reagent. The IR spectrum gave the absorption bands at 3450 (NH and OH), 1710 (COOH), 1580 and 1395 (COO-) and 2850 cm -1 (> +NH2). The 1H and 13C NMR data of 2 revealed the presence

Phytosiderophores

117

of three -CHz--CHz--CH- systems and three carbonyl carbons in the molecule (see Tables 2 and 3). The oxidation of 2 with KMnO4 yielded aspartic acid, homoserine, and malic acid. The CD curves of all the products obtained by the KMnOa-oxidation exhibited a positive Cotton effect (aspartic acid: [0]201nm= + 13 370, homoserine: [0h97nm = + 8384, and malic acid: [01206m = + 10 340), indicating that the absolute configurations of all asymmetric centers in 2 are S15). Thus, the structure of avenic acid A is 2(S)•3•(S)•y'(S)-N-[3-(3-hydr•xy-3-carb•xypr•py•amin•)-3-carb•xypr•py•]-h•m•serine as shown in the formula 2.

3. 2'-Deoxymugineic Acid (3) 2'-Deoxymugineic acid(3) isolated from wheat, C~2H20OTN2 (El-MS; m/z 286 (M ÷ - H20), mp 198.4--200.5°, and [a]D -- 70.5 °, was positive for ninhydrin reaction. The IR spectrum showed the absorption bands at 3450 (NH and OH), 1715 (COOH), and 1610 and 1395 cm -1 (CO0-). The 1H and t3c NMR spectral data indicated the presence of three -CH2-CH2-CH- systems and three carbonyl carbons in the molecule (see Tables 2 and 3). Detailed comparison of the NMR information among 3, 1, and nicotianamine(ll) afforded the evidence for the presence of azetidine-2-carboxylic acid(C-I-C-4), 3-carboxypropyl(C-l'-C-4'), and 3-hydroxy-3-carboxypropylamino (C1"-C-4") moieties in 3. The oxidation of 3 with KMnO4 gave L-azetidine-2-carboxylicacid ([aiD -- 129.3°), a slight amount of L-aspartic acid (positive CD Cotton effect at 205 nm), and 2(S),3'(S)-N-[(3-amino-3-carboxypropyl)]azetidine-2-carboxylicacid ([a]o -72.5°). Therefore, it is concluded that 2'-deoxymugineic acid is 2(S),3'(S),Y'(S)-N-[(3-carboxy3-hydroxypropylamino)-3-carboxypropyl]azetidine-2-carboxylic acid.

4. 3-Hydroxymugineic Acid (4) 3-Hydroxymugineic acid(4) isolated from rye, C12H2009N2(FD-MS; 337 (M + Na)+), mp 205-213 °C (decomp), and [a]D -- 52.4°, showed positive color with ninhydrin reagent. The IR spectrum exhibited the absorption bands at 3400, 3230, and 3050 (NH and OH) and 1620 cm -1 (COO-). The 1H and 13C NMR spectral data of 4 were similar to those of 1, except for the proton and carbon signals of C-1-C-4 in the azetidine-2carboxylic acid moiety. The methylene signal at 2.57 ppm due to C-3 in 1 is absent in 4, and instead the methine signal at 4.80 ppm is observed. The methine signal attributed to C-2 in 4 was detected as doublet (J = 6.5 Hz) at 4.98 ppm. Furthermore, the splitting pattern of the methylene signal to C-4 is simplified, compared with the corresponding signal of 1 (see Table 2). In the comparative t3C NMR spectra of 4 and 1, conspicuous differences were the presence of a methylene carbon signal at 23.8 ppm due to C-3 in 1 and of a methine carbon signal at 65.3 ppm in 4. The downfield shifts of the carbon signals at a-position (C-2 and C-4) and the slight upfield shift of the carbon signal at r position (C-1) are also observed in 4 as compared with 1. These findings indicate that an hydroxyl group is situated at C-3. The configurations at C-2', C-3', and C-3" in 4 were the same as those in 1, since the chemical shifts and the splitting patterns of C-2', C-3', and C-3" proton signals of 4 coincide with those of the corresponding signals of 1 (see Table 2). The CD curve of y-amino-a-hydroxybutyric acid obtained by 6 N HC1 hydro-

118

Y. Sugiuraand K. Nomoto

lysis of 4 gave a positive Cotton effect (Ae216,m = + 0.083), suggesting that the chiral center at C-3" in 4 is S configuration18). Thus, the structure including the streochemistry of C-2', C-3', and C-3" of 3-hydroxymugineic acid is represented by the formula 4.

5. Distichonic Acid A (5) Distichonic acid A(5) isolated from beer barley, C10HasOsN2 (FD-MS; m/z 259 (M + 1 - 2 H20)+), mp 220-221 °C (decomp), and Ae207~ = + 3.25, was positive for ninhydrin reaction. The IR spectrum exhibited the absorption bands at 3450, 3350, 3170 (NH and OH), 1720 (COOH), and 1605 and 1380 cm -1 (CO0-). In the 1H NMR spectrum of 5, all the proton signals were assigned thoroughly with the aid of the double resonance technique. Table 2 shows the presence of one -CH2--CH2--CH-, one --CH2--CH--CH-, and one -CH2- system in the molecule 5. The 13C NMR data also revealed the signals due to three carbonyl carbons, four methylene carbons, and three methine carbons (see Table 3). Since this compound contains no unsaturation rings, the formula 5 is concluded to be the structure for 5.

6. Other Similar Amino Acids Nicotianamine, 2(S),3'(S),3"(S)-N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]-azetidine-2-carboxylic acid(//), has been isolated from the leaves of Nicotiana tobacum L. (Solanaceae)2°' 21) and seeds of Fagus silvatica L. (Fagaceae)22,23). Another amino acid, 2(S),3'(S)-N-(3-amino-3-carboxypropyl)-azetidine-2-carboxylicacid(12), has also been obtained from the latter. Further, azetidine-2-carboxylic acid(13) which is the constituent of mugineic acid(I) has been found in various species of Liliaceae24-27), Agaraceae 19), Chenopodiaceae 28), and Leguminosae 29), and this compound has been proposed as a precursor of mugineic acid and nicotianamine23). Recently, Budensinsky suggested that nicotianamine(ll) is a possible phytosiderophore which functions in iron transport and/or metabolism in the vascular plants 3°). Of special interest is the fact that mugineic acid(/) and nicotianamine(ll) have remarkably similar structures (see Charts 1 and 4).

IV. Chemical Synthesis of Mugineic Acids

1. Synthesis of 2'-Deoxymugineic Acid (3) 31) The synthesis of 2'-deoxymugineic acid(3) was achieved through reductive coupling of three optically active units, L-a-hydroxy-y-butyrolactone(14), L-homoserine(6), and the unique L-azetidine-2-carboxylic acid(13) as shown in Scheme 1. L-a-hydroxy-y-butyrolactone(14)32) was prepared from L-malic acid in three steps. The compound(14) was converted into a diasteromeric mixture of tetrahydropyranylated derivatives(15). The diasteroisomers were separated by chromatography. The hydrolysis of 15 with 2.5%

119

Phytosiderophores

coo-

coo-

coo-

11

coo-

CO0-

H "

~t3 12

coo-

13 Chart 4

C02CH2Ph ~R

R%,/~THP

90%

14 R=H

0 0 +

~NH2.1.FA

16 R=CH2OH

18

17 R=CHO

CM.I~

~ N/~THP O

CO2CH2ph

C02CH2Ph NH-TFA

I R

13

22

19 R=H

51%

20 R=CO2-t-Bu

CO.[H2r~ C0:H:~

co2c..P, co2a.~

co~..p., L

I

C02-t-Bu 21 R=CH2OH 23

R=CHO

Scheme 1

59%

C02"VSu 24

100%

120

Y. Sugiuraand K. Nomoto

KOH-tetrahydrofuran followed by benzylation afforded y-hydroxybenzylester(16). The oxidation of 16 with pyridinium chlorochromate (PPC) yielded L-malic halfaldehyde(17). The coupling of 17 and the homosefine moiety(18) was achieved via reducfive amination33) by using of NaBHaCN, and the procedure gave the desired lactone amine(19). The protection of 19 with di-tert-butyldicarbonate yielded the tert-butoxycarbonyl derivative(20) and the subsequent benzylation gave rise to the dibenzylester(21). Azetidine-2-carboxylic acid(13) was transformed into the trifluoroacetic acid salt of 2benzyloxycarboxylazetidine(22) in three steps. The PPC oxidation of 21 afforded the aldehyde(23), which upon reductive amination with 22 yielded the protected 2'-deoxymugineic acid(24). Removal of all the protecting groups under mild acidic condition followed by chromatographic separation on Dowex 50W X4 and Sephadex G-10 afforded optically pure 2'-deoxymugineic acid(3) as white crystals, mp 196-199 °C and [a]D - 66.6°.

2. Synthesis of Avenic Acid A(2) 34) The synthesis of avenic acid A(2) was performed by reductive coupling of the protected aldehyde(23), an intermediate employed in the synthesis of 2'-deoxymugineic acid(3), and L-homoserine lactone trifluoroacetic acid(18) (see Scheme 2). Thus, L-tert-butoxyearbonyl-N-(3-O-tetrahydropyranyl-3-carboxybenzylpropyl)homoserine aldehyde benzylester(23) and homoserine lactone trifluoroacetic acid(18) were treated with NaBH3CN to yield the protected avenic acid A lactone(25). The hydrogenation with H2/Pd-C, followed by treatment with trifluoroacetic acid gave the lactone trifluoroacetate(26) in quantitative yield. The hydrolysis of 26 with 2.5% KOH afforded the potassium salt of 2, which upon treatment with Dowex 50W X4 resin (H + form), and subsequently Sephadex G-10, gave optically pure avenic acid A(2), mp > 300 °C and Aezo~m = + 2.60.

coza.~ cozc.z~ OHC-

o~O

N,

89%

N H

C02-t- Bu

23

lip CO2-I-8u

25

,

86%

coz~

,

H

TFA

L-I

TFA

26 Scheme 2

CO~¢~hc~c~

.C02H

OH

100%

121

Phytosiderophores

V. Is Mugineic Acid a Phytosiderophore? In price plants, the effect of mugineic acid on the 59Fe-uptake and the synthesis of chlorophyll was investigated35). Figure 4 shows significant stimulation of mugineic acid for both the iron-uptake and the chlorophyll synthesis. In contrast, the effects of nicotianamine, EDTA, desferrioxamine, and citric acid were remarkably small and comparable to the control. 2'-Deoxymugineic acid and HEDTA gave an appreciably positive effect, although their abilities were lower than that of mugineic acid. It is of interest to note that mugineic acid and HEDTA are structurally analogous. Indeed, the formation constants of the HEDTA-Fe(III) complex (log K~L = 19.8)36) and the HEDTA-Fe(II) complex (log K~th = 11.6) are close to those (18.1 and 8.1) 37) of the corresponding mugineic acid-iron complexes, and the HEDTA ligand has the same kind of hydroxyl coordination for Fe(III). The structural similarity of 2'-deoxymugineic acid and HEDTA to mugineic acid probably contributes to the positive effect on the 59Fe-uptake and the chlorophyll synthesis. Several iron-solubilizing amino acids isolated from other gramineous plants all have similar structures with six coordination groups (see Chart 1). The large difference in the biological activity between 2'-deoxymugineic acid and nicotianamine strongly suggests the important contribution of the terminal alcoholic

Chlorophyll contents, %(wet wt.)

?

?

|

!

contEol

~///////////////////~

mugi..ic acid

! !

[

2'-d,ox~uqineic acid

~//////~/~]

nicotianamine

~

I I

~

desferrioxamine

'

: chlorophyll : 59iron

citric acid mugineic acid(-Fe)

o

•

°

59Iron uptake(~g/ a plant)

Fig. 4. Effect of some iron-chelatorson iron-uptake and chlorophyllsynthesisin water-culturedrice plant

122

Y. Sugiuraand K. Nomoto

hydroxyl group in the mugineic acid ligands for the iron-uptake. In spite of the high solubilizing ability of desferrioxamine, this compound did not stimulate iron-uptake by the rice plant. Presumably, the phenomenon is due to little permeability of the desferrioxamine-Fe(III) complex into membranes of the root. The experimental results of the 59Fe-uptake and the chlorophyll synthesis demonstrate that mugineic acid is really a phytosiderophore in rice plant.

VI. Some Properties of Mugineic Acid-Iron(IH) Complexes37) The electronic absorption spectrum of the golden yellow-colored mugineic acid-Fe(III) complex exhibited peaks at 350(e 1520), 250(8350), and 204 nm(35 000). In the CD spectrum, three Cotton peaks were also detected at 380(he -2.39), 270(+2.22), and 235 nm(+ 9.11). The visible absorption band of 350 nm must be due to a ligand - , metal charge-transfer transition, because high-spin ferric ion has no spin-allowed d-d transition. Figure 5 shows the Mfssbauer spectra of the mugineic acidJTFe(III) complex under the ,

,

,

,

,

"

i

,

,

i

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Phytosiderophores

123

condition of zero and 600-G magnetic fields. The obtained Mtssbauer parameters (AEQ = 0.24 and 6Fe = + 0.39 mm/s) at 194 K are characteristic of those for high-spin ferric (S = 5/2) complexes. The present high-spin ferric assignment was also supported by the large magnetic hyperfine interaction induced at low temperature by the applied magnetic field. The hyperfine field, Hhf = ca. - 500 KOe, is the usual value for high-spin ferric ion. Indeed, typical low-spin Fe(III) (S = 1/2) complexes have large quadrupole splitting (AEQ = 2-3 mm/s). As clearly shown in Fig. 6, the X-band ESR spectrum of the mugineic aeid-Fe(III) complex exhibited the signals at g = 9.4, 4.51, 4.44, and 4.31 which are typical of high-spin Fe(III). On the other hand, the reduced mugineic acid57Fe(II) complex showed the M6ssbauer spectrum characterized by a single quadrupole

(A) g'4.44

,H

200 G

(B) ,4.45

,H 200 G

Fig. 6A, B. ESR spectra for Fe(III) complexesof mugineicacid (A) and nicotianamine(B) at pH 7.0 and 77K

124

Y. Sugiuraand K. Nomoto

doublet at 110 K. The quadrupole splitting (AE o = 2.88 mm/s) and isomer shift (rye = + 1.16 mrn/s) are typical of high-spin ferrous ion (S = 2), and these values fall in the range of values observed for ionic ferrous complexes. The pale yellow-colored mugineic aeid-Fe(II) complex formed by reduction of the corresponding Fe(III) complex with sodium dithionite or NAD(P)H was ESR negative at 77 K, and the reduced complex was reoxidized by molecular oxygen to give the original ESR spectrum of the mugineic acidFe(III) complex. In general, an S = 2 spin state has a relative short-lattice time, and such an ESR spectrum of high-spin Fe(II) complex is difficult to obtain. The g-tensor anisotropy of the nicotianamine-Fe(III) complex (g = 9.5, 4.56, 4.45, and 4.02) is larger than that of the mugineic acid-Fe(III) complex (g = 9.4, 4.51, 4.44, and 4.31). The difference in these spectra is probably attributed to the coordination of the terminal amino nitrogen in nicotianamine ligand instead of the alcoholic hydroxyl oxygen in mugineic acid ligand to Fe(III) ion. The same g-values of the Fe(III) complexes between mugineic acid and 2'deoxymugineic acid strongly indicate that the intermediate alcoholic O(7)H group is not participating to the complexation with ferric ion.

VII. Structure and Properties of Structurally Analogous Mugineic Acid-Cobalt(IH) Complex35'37) Figure 7 shows the stereoscopic drawing of the mugineic acid-Co(III) complex and the coordination about the Co(III) center determined by X-ray crystallographic analysis. The two molecules (A and B) exist in an asymmetric unit and the structures of both the molecules are remarkably similar to each other. The mugineic acid-Co(III) complex forms a nearly octahedral configuration in which the azetidine ring nitrogen N(1), secondary amine nitrogen N(2), and both terminal carboxylate oxygens O(1) and O(5), coordinate to Co(III) ion as basal planar donor atoms, and the hydroxyl oxygen 0(8) and intermediate carboxylate oxygen O(3) bind as axial donors. The valency angles N(2)-CoO(1)(174 °) and N(1)-Co-O(5)(1740) suggest that the coordination geometry around the cobalt atom slightly distorts from square-planar toward tetrahedral geometry. The axial bonds O(3)-Co-O(5)(166 °) also somewhat deviate from the normal of the basal plane. Probably, these distortions can be accounted for by the steric constrains of both a sixmembered chelate ring Co-N(1)-C(5)-C(6)-C(7)-N(2)and a seven-membered ring Co-N(2)-C(9)-C(10)-C(11)-C(12)-O(5). However, these bond angles are larger than those (168.8, 168.8, and 144.3°) in the mugineic acid-Cu(II) complex 3a) which has a distorted tetragonal configuration (see the following section). The results of the X-ray structural determination and atomic absorption spectrometry revealed that one sodium ion and four water molecules are involved in an asymmetric unit of the mugineic acidCo(III) complex. Figure 8 shows the arrangement of the hydrated sodium ion. The sodium ion coordinates two carboxyl oxygens, two hydroxyl oxygens, and two water oxygen atoms. Several hydrogen bonds between the polyhedra and the complex stabilize the crystal structure. We also note that a hydrogen bond is formed between both the alcoholic hydroxyl oxygens O(8) of the A and B molecules. Figure 9 represents the electronic absorption and CD spectra of the mugineic acid-Co(III) complex, together

125

Phytosiderophores

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(A)

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0(31 (B)

Fig. 7. Molecular structures of the complexes (molecules A and B) and coordination about the cobalt ion in molecules A and B of the mugineic acid-Co(III) complex. Bond lengths and angles are shown in/~, unit and in degrees

with those of the corresponding Fe(III) complex. On the basis of analogy with electronic spectra of well-known low-spin Co(III) complexes 39), the band at 18 000 cm -1 is assigned to the 1A1 --~ XE1 transition and that at 25 400 cm -1 to the ~Az -* 1A2 transition. A calculation of the in-plane crystal field strength yielded a value of 2920 cm -~ for Dqxy. The calculated out-of-plane crystal field parameter Dqz(D4h), 1990 cm -1, indicates that weaker ligands occupy the axial sites of Co(III). The crystal field stabilization for d 6 cobalt complex (24 Dq) is considerably greater than that for high-spin ferric complex (0 Dq). Thus, the Co(III)-substituted complex of mugineic acid should be kinetically inert. However, the similarity in the coordination chemistries between Fe(III) and Co(III) makes the present Co~III) complex a useful vehicle for probing the structure of the biological important mugineic acid-Fe(III) complex, and the coordination structure of the ferric complex should be sufficient alike to that of the demonstrated cobaltic complex.

Y. Sugiura and K. Nomoto

126

*: Occupancy = 1/2 Fig. 8. Arrangement of molecules in the crystal. Hydrogen bonds are shown by broken lines, and water molecules are represented by W

Phytosiderophores

127 V&-Fe(III)

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Fig. 9. Electronic ( ~ ) and CD (...... ) spectra for Fe(III) and Co(III) complexes of mugineic acid

VIII. Structure and Properties of Mugineic Acid-Copper(II) Complex3s) Of interest is the fact that the iron-solubilizing action of mugineic acid is strongly inhibited by the presence of transition metal ions, especially Cu(II) ion 1°'4°). The X-ray crystallographic analysis of the single crystal mugineic acid-Cu(II) complex showed that (1) the mugineic acid ligand acts as a hexadentate ligand and (2) the Cu(II) site is coordinated by the azetidine ring nitrogen N(1), secondary amine nitrogen N(2), and both terminal carboxylate oxygens O(1) and 0(5) in an approximate planar arrangement, while the hydroxyl oxygen 0(8) and intermediate carboxylate oxygen 0(4) are bonded axially (see Fig. 10). The main differences between the present Cu(II) and

128

Y. Sugiuraand K. Nomoto

O(4}

Fig. 10. Bond lengths and angles of the Cu(II) coordinationsite in mugineicacid-Cu(II) complex previous Co(Ill) complexes of mugineic acid are as follows: (1) the axial bond lengths, Cu-O(8) (2.477/~) and Cu-O(3) (2.557/~,), are considerably longer than those in the Co(III) complex, (2) the deviations of the donor atoms from the basal plane in the mugineic acid-Co(III) complex are smaller than those (0.101, 0.184, -0.110, and 0.160/~, for N(1), O(5), N(2), and O(1)) in the corresponding Cu(II) complex, and (3) the intermediate carboxylate oxygen 0(4) coordinates to the Cu(II) ion in the place of the carboxylate oxygen O(3) in the Co(III) complex. Indeed, the coordination geometry around the copper atom distorts from square-planar toward tetrahedral, and the inclination angle of 22.2° for O(8) corresponds well to that (22*) of the axial Cu(II)-S bond in the violet glutathione-Cu(II) complex with a distorted square-pyramidal configuration41). The ESR spectrum of the mugineic acid-Cu(II) complex at pH 7 and 77 K is shown in Fig. 11, together with that of the nicotianamine-Cu(II) complex. Both ESR spectra exhibited a typical copper hyperfine pattern with approximately axial symmetry. Table 4 lists the ESR parameters estimated for the 1 : 1 Cu(II) complexes of mugineic acid, 2'deoxymugineic acid, distichonic acid A (glycine-type mugineic acid), and nicotianamine. Except for the nicotianamine-Cu(II) complex, the three Cu(II) complexes have remarkably similar ESR parameters, indicating the similarity of Cu(II)-coordination structure for these three ligands. The electronic absorption and CD spectra of the mugineic acidCu(H) complex revealed an absorption maximum at 14 600 cm -1 (e 65) and CD extrema at 16950 (Ae + 0.09) and 13 330 crn-t ( - 0.32), respectively. The visible bands at 16950 (dxz,yz"-~ dxz_y2)and 13 330 cm -1 (d~y~ dx2_y2)seen in the Cu(II) complex are characteristic of d-d bands for tetragonaUy distorted octahedral Cu(II)-type complexes. A band in the ultraviolet region occurred at 40300 crn-1 (e4500), which can be assigned to a N(tT) Cu(II) charge-transfer transition. The nicotianamine-Cu(II) complex showed an absorption maximum at 16 050 cm -I (e 95). In comparison with that of the mugineic acidCu(II) complex, the shift of the 2m~ to higher wavenumber is attributed to the replacement of one hydroxyl oxygen with amino nitrogen toward the Cu(II) coordination. Therefore, the Cu(II)-inhibition for iron-solubilizing action of mugineic acid can be explained by blockage of the Fe(III)-binding to mugineic acid by Cu(II) ion. Indeed, the mugineic acid-Cu(II) complex has a high formation constant (log KMr~L= 18.3)37). -

Phytosiderophores

129 g,=2.277

CA)

A

G~m

1

200H

I

---'----t

200 G H

>

J

Fig. 11 A, B. ESR spectra for the Cu(II) complexes of mugineic acid (A) and nicotianamine (B) in 1 : 3 ethylene glycol-water solution

Table 4. ESR parameters for Cu(II) complexes of mugineic acid and its related amino acids Ligand

gll

g~

104 All, cm -1

N-hfs (line)

Mugineic acid (MA)

2.277

2.060

175.9

5

2'-Deoxy mugineic acid (Deoxy MA)

2.277

2.065

172.9

5

Distichonic acid A (Glycine-type MA)

2.277

2.063

178.9

5

Nicotianaraine

2.236

2.054

190.4

7

130

Y. Sugiura and K. Nomoto

IX. Solution Structures of Mugineic Acid and Its Metal Complexes42) In order to clarify the structures in aqueous solution, 1H NMR spectral studies at 360 MHz have been conducted on mugineic acid and its metal complexes. Figure 12 shows the 1H NMR spectra of metal-free mugineic acid and its Fe(III), Co(Ill), and Zn(II) complexes at pD 4.5. In the mugineic acid-Fe(III) complex, no detectable proton signals were observed in the regions of + 100 ppm because of remarkable line broadening. This phenomenon is due to paramaguetism of high-spin Fe(III) ion. In contrast, mugineic acid and its Co(Ill) and Zn(II) complexes gave sharp and numerous proton signals, indicating the presence of diamagnetic ion in these complexes. Spin decoupling and pH titration experiments confirmed the signal assignments as shown in Fig. 12. The pD-dependent chemical shifts for all proton resonances have been investigated. The protonation at pD 3.5 clearly affected only the chemical shift of C3" methine proton, while the protonation of ca. pD 2.5 influenced both the chemical shifts of C 2 and C 3' methine protons. The data are consistent with the result of the potentiometric pH titra,~. + tion: pKI(COOH) = 2.39, pK2(COOH) = 2.76, pK3(COOH) = 3.40, piG( -- NH-) = 7.78, and pKs(-l~lHr-) = 9.5537). As a result of the deprotonation of the amine groups, the proton chemical shifts and the coupling constants of C 1' methylene protons changed from 3.4 (Jgem = 13.4 and J~ic = 9.4 Hz) and 3.5 ppm (Jgcm = 13.4 and J,~c = 2.6 Hz) to 2.7 ppm(doublet), suggesting that the deprotonations facilitate the rotation around C 1'C2' bond. Table 5 compares the proton chemical shifts for mugineic acid and the corresponding Co(HI) and Zn(II) complexes at pD 4.5. Upon metal-complexation, large changes in the chemical shifts are evidently observed in the proton resonances adjacent to six functional groups. All the proton resonances of the Co(IlI) complex were unchanged in the pD range 4-10, clearly indicating that the complex is very stable and

Table 5. Chemical shifts of mugineic acid and its metal complexes

Mugineic acid

Zn(II) complex

Co(III) complex

C-2 H

4.88

3.88

4.40

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2.71 2.57 4.03 4.09 3.54 3.42 4.44 3.84 3.20 3.28 2.04 2.18 4.18

2.55 2.07 3.56 3.81 3.00 2.80 4.10 3.36 2.59 2.72 2.02 2.22 4.52

2.73 2.31 3.57 3.89 2.88 2.88 4.37 3.23 2.53 2.91 1.97 1.97 3.10

C-4 H2 C-I'H 2

C-2'H C-3'H C-I"I-I2 C-2'~rI2

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132 '

Y. Sugiura and K. Nomoto

undergoes no structural alteration. The conformational analysis using chemical shifts and vicinal coupling constants showed that (1) mugineic acid coordinates to Co(III) and Zn(II) ions in hexadentate fashion by the six functional groups, (2) the three C 1'-C2', C 1"-C2", and C2"-C 3" bonds change from mixtures of rotamer populations in free mugineic acid to predominantly gaush-gaush populations 43) in the metal complexes, and (3) the structural conformation of the mugineic acid-Co(III) complex in aqueous solution corresponds well to that of its crystal structure determined by X-ray diffraction techniques.

X. Probable Mechanism of Iron Transport in Gramineous Plants Figure 13 shows a cyclic voltammogram for the mugineic acid-Fe(III) complex at pH 7.0. The present ferric complex exhibited quasi-reversible one electron redox wave with E~/2 value of - 102 mV vs. the normal hydrogen electrode (NHE). The value ip,c/ip,a ~ 1 for

I

0

I

I

I

-0.2

I

-0.4 E

vs.

i

I

-0.6

I

I

-0.8

SCE 9 V

Fig. 13. Cyclicvoltammogram of mugineic add-Fe(III) complex at pH 7.0. Experimental condition was as follows: 0.1 M KCI. 0.05 M sodium borate/0.05 M sodium phosphate buffer, and 1.0 mM mugineic acid-Fe(III) complex. The electrochemistry was performed on a hanging mercury drop electrode with 100 mV/s scan rate

Phytosiderophores

133

Table 6. Reduction potentials of some iron-transport compounds Complex

E(pH 7.0) vs. NHE, mV

Mugineic acid-Fe(III) Nicotianamine-Fe(III) Ferric aerobactin Ferric rhodotorulic acid Ferrichrome A Ferrioxamine B Enterobactin

- 102 - 181 - 336 -359 - 448 - 468 - - 750

the redox event also suggests effective chemical reversibility, whereas the nicotianamineFe(III) complex exhibited irreversible one electron redox wave at Ela = - 181 mV vs. NHE. As shown in Table 6, the salient feature of the electrochemical results is clearly high reduction potential of the mugineic acid-Fe(III) complex compared to the microbial hydroxamates (E1;z = - 350 - - 450 mV vs. NHE) and ferric enterobactin chelates ( - 7 5 0 mV vs. NHE) 44). Although it has been demonstrated that the iron of ferric enterobactin can be reduced in spite of the very negative potentials of the complex45~,the mugineic acid-Fe(III) complex has E(pH 7.0) = - 102 mV vs. NHE as the redox potential and is readily reducible by physiologically available reductants such as NAD(P)H ( - 3 2 0 mV vs. NHE) and glutathione ( - 2 3 0 mV vs. NHE). Therefore, the result strongly indicates that the mechanism of iron transport in gramineous plants probably includes iron reduction from the thermodynamically stable ferric mugineic acid complex (log K~m = 18.1) to the weakly bound ferrous complex (log Kr~ = 8.1). It has been proposed that intercellular iron release of microbial siderophores occurs by iron reduction in hydroxamates and by hydrolytic depolymerization in ferric enterobactin4~). The formation constant of the mugineic acid-Fe(III) complex is closer to that of N-(2-hydroxyethyl)ethylene-dinitrillo-dinitro-N,N',N'-triacetic acid(HEDTA)-Fe(III) complex (log Kr~ = 19.8) 36) rather than those of ferrichrome(29.1)47), ferrioxamine B(30.6), and ferric enterobactin ( - 52)481. However, direct comparison of the formation constants of these siderophores with the mugineic acid complex is not very informative because of the very different acidities of these ligands. Mugineic acid is a much better complexing agent for ferrous ion than ligands which contain hydroxamate or catecholate. Thus, the great difference in redox potentials would be explained by the relative stability of the ferrous complex of mugineic acid. It is known that heavy metal ions such as Cu(II) and Zn(II) induce typical iron chlorosis in rice plants, and that the iron-solubilizing action of mugineic acid is strongly inhibited by the presence of these heavy metals. The phenomenon is reasonably explained by competitive blocking of the mugineic acid-binding sites of Fe(III) by Cu(II) and/or Zn(II). On the basis of these results and Neilands's proposal for enterobacteria49), therefore, we would like to postulate the iron-absorption and -transport mechanism depicted in Fig. 14 for gramineous plants. In order to confirm this mechanism, we must demonstrate recycling of the ligand by using t4C-labeled mugineic acid in rice plants. Most microbial siderophores have hydroxamate or phenolate groups as Fe(III)-ligand donors. These coordination atoms are oxygen, except for mycobactin, agrobactin, and parabactin where a single tertiary nitrogen atom of oxazoline ring participates in bonding

134

Y. Sugiura and K. Nomoto

External ~ ! •

insoluble

Internal

Synthesis_of Chlorophylls

0 L • : Ferric Ion ~ ~ O: Ferrous Ion U R F

~

OA OC

OE

° Q ~

iron Enz~

t

Solublllzatlon 0

T

l

Enz

t

~ RegulatoryI Mechanl~ [

~uglnelc acid

Fig. 14. Scheme for absorption and transport of iron in gramineous plants

to the iron 5°). In contrast, phytosiderophores have carboxyl, amine, and hydroxyl groups as iron-ligand functional groups. The experimental results of 59Fe-uptake and chlorophyll synthesis clearly demonstrate that mugineic acid is a new phytosiderophore. The mechanism for absorption and transport of iron in gramineous plants involves the excretion of mugineic acids from the roots which aid Fe(III)-solubilization and reduction of Fe(III) to Fe(II).

Acknowledgment. We are grateful to Professor J. B. Neilands for kind invitation of the review and pertinent advice with the manuscript.

XI. References Snow, G. A.: Bacteriol. Rev. 34, 99 (1970) Neilands, J. B.: Annu. Rev. Biochem. 50, 715 (1981) Leong, S. A., Neilands, L B.: Arch. Biochem. Biophys. 218, 351 (1982) Brown, J. C., Holmes, R. H., Tiffin, L. O.: Soil. Sci. 86, 75 (1958) Brown, J. C.: Adv. Agron. 13, 329 (1961) Krauskopf, K. B.: Micronutrients in Agriculture. Proc. of a Symposium. Madison, Wisc. (Mortvedt, J. J., Giordano, P. C., Lindsay, W. L., Eds.), Soil Sci. Sac. of America, Inc., 1972, pp. 7--40 7. Oertli, J. C., Jacobson, L.: Plant Physiol. 35, 683 (1960) 8. Wallace, A., Lunt, O. R.: Am. Sac. Hart. Sci. 75, 819 (1960) 1. 2. 3. 4. 5. 6.

Phytosiderophores

135

9. Brown, J. C.: Bioinorganic Chemistry-II (Raymond, K. N., Ed.), Am. Chem. Soc. 1978, pp. 93-103 10. Takagi, S.: Soil. Sci. Plant Nutr. 22, 423 (1976) 11. Takemoto, T., Nomoto, K., Fushiya, S., Ouchi, R., Kusano, G., Hikino, H., Takagi, S., Matsuura, Y., Kakudo, M.: Proc. Japan Acad. 54B, 469 (1978) 12. Fushiya, S., Sato, Y., Nozoe, S., Nomoto, K., Takemoto, T., Takagi, S.: Tetrahedron Lett. 21, 3071 (1980) 13. Nomoto, K., Yoshioka, H., Arima, M., Takemoto, T., Fushiya, S., Takagi, S.: Chimia 35, 249 (1981) 14. Nomoto, K., OhFane, Y.: J. Syn. Org. Chem. Japan 40, 401 (1982) 15. Yamada, S., Kitagawa, T., Achiwa, A.: Tetrahedron Lett. 3007 (1967) 16.. Koga, K., Wu. C. C., Yamada, A.: Chem. Pharm. Bull. (Tokyo) 20, 1282 (1972) 17. Taniguchi, M., Koga, K., Yamada, S.: ibid. 20, 1438 (1972) 18. Craig, J. C., Roy, S. K.: Tetrahedron 21, 1847 (1965) 19. Fowden, L.: Biochem. J. 64, 323 (1956) 20. Noma, M., Noguchi, M., Tamaki, E.: Tetrahedron Lett. 2017 (1971) 21. Noma, M., Noguchi, M.: Phytochem. 15, 1701 (1976) 22. Kristensen, I., Larsen, P. O., Sorensen, H.: ibid. 13, 2803 (1974) 23. Kristensen, I., Larsen, P. O.: ibid. 13, 2791 (1974) 24. Virtanen, A. I., Linko, P.: Acta Chem. Scand. 9, 551 (1955) 25. Virtanen, A. I.: Angew. Chem. 67, 619 (1955) 26. Fowden, L.: Nature 176, 347 (1955) 27. Fowden, L., Bryant, M.: Biochem. J. 70, 626 (1958) 28. Fowden, L.: Phytochem. 11, 2271 (1972) 29. Sung, M.-L., Fowden, L.: ibid. 8, 2095 (1969) 30. Budensinsky, M., Budzikiewicz, H., Prokazka, A., Ripperger, H., Romer, A., Scholz, G., Schreiber, K.: ibid. 19, 2295 (1980) 31. Oh~ne, Y., Tomita, M., Nomoto, K.: J. Am. Chem. Soc. 103, 2401 (1981) 32. Collum, D. B., McDonald, J. H., III, Still, W. C.: ibid. 102, 2118 (1980) 33. Botch, R. F., Bemstein, M. D., Durst, H. D.: ibid. 93, 2897 (1971) 34. Ohfune, Y., Nomoto, K.: Chem. Lett. 827 (1981) 35. Mino, Y., Ishida, T., Ota, N., Inoue, M., Nomoto, K., Takemoto, T., Tanaka, H., Sugiura, Y.: J. Am. Chem. Soc. (in press) 36. Gustafson, R. L., Martell, A. E.: J. Phys. Chem. 67, 576 (1963) 37. Sugiura, Y., Tanaka, H., Mino, Y., Ishida, T., Ota, N., Inoue, M., Nomoto, K., Yoshioka, H., Takemoto, T.:.J. Am. Chem; Soc. 103, 6979 (1981) 38. Mino, Y., Ishida, T., Ota, N., Inoue, M., Nomoto, K., Yoshioka, H., Takemoto, T., Sugiura, Y., Tanaka, H.: Inorg. Chem. 20, 3440 (1981) 39. Wentworth, R. A. D., Pip~r, T. S.: ibid. 4, 709 (1965) 40. Hunter, J. G., Vergnano, O.: Ann. Appl. Biol. 40, 761 (1953) 41. Miyoshi, K., Sugiura, Y., Ishizu, K., Iitaka, Y., Nakamura, H.: J. Am. Chem. Soc. 102, 6130 (1980) 42. Iwashita, T., Mino, Y., Naoki, H., Sugiura, Y., Nomoto, K.: Biochemistry (in press) 43. Karplus, M.: J. Am. Chem. Soc. 85, 2870 (1963) 44. Harris, W. R., Carrano, C. J., Raymond, K. N.: ibid. 101, 2722 (1979) 45. Lodge, J. S., Gaines, C. G., Arceneaux, J. E. L., Byers, B. R.: Biochem. Biophys. Res. Commun. 97, 1291 (1980) 46. Cooper, S. R., McArdle, J. V., Raymond, K. N.: Proc. Natl. Acad. Sci. USA 75, 3551 (1978) 47. Schwarzenbach, G., Schwarzenbach, K.: Helv. Chim. Acta 46, 1390 (1963) 48. Harris, W. R., Weitl, F. L., Raymond, K. N.: J. Chem. Soc. Chem. Commun. 177 (1979) 49. Neilands, J. B.: Bioinorganic Chemistry-II (Raymond, K. N., Ed.), Am. Chem. Soc., 1977, pp. 3-32 50. Neilands, J. B., Peterson, T., Leony, S. A.: Inorganic Chemistry in Biology and Medicine (MarteN, A. E., Ed.), Am. Chem. Soc., 1979, pp. 263-278

Reactivity and Structure

Volume 9 J. ILBlackbomw, D. Young

Concepts in Organic Chemistry Editors: K.Hafner, J.-M.Lehn, C.W.Rees, P. v. R. Schleyer, B. M. Trost, ILZahradm'k

Metal Vapour Synthesis in Organometallic Chemistry

Volume 1 J.Tsuji

Volume 10 J.Tsuji

Organic Synthesis by Means of Transition Metal Complexes A Systematic Approach 1975. 4 tables. IX, 199 pages ISBN 3-540-07227-6 Volume 2 K.Fulmi

1979. 36 figures, 32 tables.XIII, 202 pages ISBN 3-540-09330-3

Organic Synthesiswith Palladium Compounds 1980. 9 tables. XII, 207 pages ISBN 3-540-09767-8 Volume 11

New Syntheses with Carbon Monoxide

1975. 72 figures, 2 tables. VII, 134 pages ISBN 3-540-07426-0

Editor: J. Falbe With contributions by H. Bahrmann, B. Cornils, C. D. Frohning, A. Mullen 1980. 118 figures, 127 tables. XIV, 465 pages ISBN 3-540-09674-4

Volume 3 I-LKwart, K. King

Volume 12 J. Fabian, H. Harlmann

Theory of Orientation and Stereoselection

d-Orbitals in the Chemistry of Silicon, Phosphorus and Sulfur 1977. 4 figures, 10 tables. VIII, 220 pages ISBN 3-540-07953-X Volume 4 W. P. Weber, G.W.Gokel

Phase Transfer Catalysis in Organi~ Synthesis 1977. Out of print. New edition in preparation Volume 5 N. D. Epiotis Theory of Organic Reactions 1978. 69 figures, 47 tables. XIV, 290 pages ISBN 3-540-08551-3 Volume 6 M. L. Bender, M. Komiyama

Cyclodextrin Chemistry 1978. 14 figures, 37 tables. X, 96 pages ISBN 3-540-08577-7

Light Absorption of Organic Colorants Theoretical Treatment and Empirical Rules 1980. 76 figures, 48 tables. VIII, 245 pages ISBN 3-540-09914-X Volume 13 G.W. Gokel, S. H. Korzeniowski

Macrocyclic Polyether Syntheses 1982. 89 tables. XVIII, 410 pages ISBN 3-540-11317-7 Volume 14 W. P. Weber

Silicon Reagents for Organic Synthesis 1983. XVIII, 430 pages ISBN 3-540-11675-3 Volume 15 A.J.Kirby

The Anomeric Effect and Related Stereoelectronic Effects at Oxygen 1983. 20 figures, 24 tables. VIII, 149 pages ISBN 3-540-11684-2

Volume 7 D. I. Davies, M. J. Parrott

Free Radicals in Organic Synthesis 1978. 1 figure. XII, 169 pages ISBN 3-540-08723-0 Volume 8 C.Birr

Aspects of the Merrifield Peptide Synthesis 1978. 62 figures, 6 tables. VIII, 102 pages ISBN 3-540-08872-5

Springer-Verlag Berlin Heidelberg NewYork

Tokyo

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P

Reactivity and Structure

Volume 16

Concepts in

Contents: Introduction. - Activation and Coupling. Reversible Blocking of Amino and Carboxyl Groups. - Semipermanent Protection of Side Chain Functions. - Side Reactions in Peptide Synthesis. - Tactics and Strategy in Peptide Synthesis. - Techniques for the Facilitation of Peptide Synthesis. - Recent Developments and Perspectives. - A u t h o r Index. - Subject Index.

Organic Chemistry Edtitors: K.Hafner, J.-M.Lehn, C.W.Rees,

P.v.R. Schleyer, B.M. Trost, R.Zahradnik

M.Bodanszky

Principles of Peptide Synthesis 1984. XVI, 307 pages. ISBN 3-540-12395-4

Volume 17

R.B. Bates, C.A. Ogle

Carbanion Chemistry 1983. VIII, 117 pages. ISBN 3-540-12345-8

Contents: Introduction. - Structures. - Preparations. Reactions of o Carbanions with Electrophiles. - Reactions ofrr Carbanions with Electrophiles. - Eliminations. - Oxidations. - Rearrangements. - Carbanion Equivalents. - Summary. - References. - Subject Index.

Volume 18

D.F.Taber

Intramolecular Diels-Alder and Alder Ene Reactions Springer-Verlag Berlin Heidelberg NewYork Tokyo

1984. VIII, 97 pages. ISBN 3-540-12602-3

Contents: T h e Intramolecular Diels-Alder Reaction: Variations and Scope. - The Intramolecular DielsAlder Reaction: Reacitivity and Stereocontrol. - The Intramolecular Alder Ene Reaction. - Subject Index.