Biosynthesis of riboflavin

VITAMINS AND HORMONES, VOL. 61

Biosynthesis of Riboflavin ADELBERT BACHER, SABINE EBERHARDT, WOLFGANG EISENREICH, MARKUS FISCHER, STEFAN HERZ, BORIS ILLARIONOV, 1 KLAUS KIS, AND GERALD RICHTER Lehrstuhl fitr Organisehe Chemie und Biochemie, Technische Universit~t Mi~nchen, D-85747 Garching, Germany

I. Introduction II. An Overview of the Riboflavin Biosynthetic Pathway III. Riboflavin Is Formed from a Purine Precursor via Diaminopyrimidine Intermediates IV. GTP Cyclohydrolase II V. Reductase and Deaminase VI. An Elusive Phosphatase VII. 3,4-Dihydroxy-2-butanone4-Phosphate Synthase VIII. Lumazine Synthase IX. Riboflavin Synthase X. Riboflavin Synthase Paralogs in Bioluminescence XI. Riboflavin Synthase of Archaebacteria XII. The Lumazine Synthase/Riboflavin Synthase Complex of Bacillaceae XIII. Flavokinase and FAD Synthetase XIV. Riboflavin Production by Fermentation XV. Inhibitors of Riboflavin Biosynthesis XVI. Biosynthesis of Riboflavin in Plants XVII. Regulation of Riboflavin Biosynthesis in Bacillus subtilis XVIII. Biosynthesis of 5-deaza-7,8-didemethyl-8-hydroxyriboflavin XIX. Biosynthesis of Molybdopterin XX. Conclusion References

The biosynthesis of one riboflavin molecule requires one molecule of GTP a n d two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 4,5-diaminopyrimidine t h a t is converted to 5-amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione by a sequence ofdeamination, side chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4phosphate obtained :from ribulose 5-phosphate affords 6,7-di1On leave of absence from the Institute for Biophysics, Krasnoyarsk 660036, Russia. Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0083-6729/01 $35.00

2

ADELBERTBACHERetal. methyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. Two reaction steps in the biosynthetic pathway catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase and riboflavin synthase are mechanistically very complex. The enzymes of the riboflavin pathway are potential targets for antibacterial agents, o 2oolA~ad~mic Press.

I. INTRODUCTION

The isolation and structure elucidation of riboflavin took place in the period 1920 to 1930 and culminated in the award of the Nobel Prize in chemistry to Paul Karrer and Richard Kuhn in 1937 and 1938. The role of the flavocoenzymes riboflavin 5-phosphate (FMN) and flavin adenine dinucleotide (FAD) as cofactors ofredox reactions began to unfold in the years following the structure elucidation. A large number of flavin-dependent redox enzymes have since been found, and many more remain to be discovered (Mfiller, 1991). Procedures for chemical synthesis of riboflavin were developed by the research groups of Karrer and Kuhn. Modifications of these methods have been used extensively for the bulk synthesis of the vitamin (Isler et al., 1988). However, it was also noted early on that riboflavin can be obtained relatively easily by fermentation processes using a variety of bacteria and fungi (Demain, 1972). In fact, certain microorganisms such as the cotton pathogen A s h b y a gossypii can generate amounts of riboflavin well above their apparent metabolic requirements. Initial studies on the biosynthesis of the vitamin were directly linked to practical concerns about enhancement of riboflavin yields in early biotechnological production methods. Notably, MacLaren reported in 1952 that riboflavin production in a culture of the flavinogenic ascomycete could be stimulated by the addition of purines to the culture medium. This suggested a biosynthetic relationship between purines and the vitamin, which was amply confirmed by subsequent work. The early work on the biosynthesis of the vitamin has been reviewed frequently (Bacher, 1991a; Bacher and Ladenstein, 1991; Bacher et al., 1993, 1996; Brown and Neims, 1982; Brown and Reynolds, 1963; Brown and Williamson, 1987; Demain, 1972; Plaut, 1961, 1971; Plaut et al., 1974; Schlee, 1969; Young, 1986), and the reader is directed to these articles for details.

BIOSYNTHESISOF RIBOFLAVIN

3

II. AN OVERVIEW OF THE RIBOFLAVIN BIOSYNTHETIC PATHWAY

The pathway of riboflavin biosynthesis is summarized in Fig. 1. Basically, a riboflavin molecule is assembled from one molecule of GTP and two molecules of ribulose phosphate (Bacher and Mailander, 1973, 1976; Baugh and Krumdiek, 1969; Foor and Brown, 1975, 1980; LeVan et al., 1985; Plaut, 1971). The first committed step is the opening of the imidazole ring of GTP (I) accompanied by the loss of a pyrophosphate moiety (Foor and Brown, 1975, 1980). The conversion of the reaction product II to V requires hydrolysis of the position 2 amino group and reduction of the ribosyl moiety, resulting in the formation of the polyol side chain. In eubacteria, the ring deamination reaction precedes the reduction of the sugar moiety (Burrows and Brown, 1978). In yeast, the opposite sequence of reaction steps has been found (Bacher and Lingens, 1970; Nielsen and Bacher, 1981). The sequence of events in plants and archaea remains to be determined. Indirect evidence indicates that the phosphoric acid residue of V must be hydrolyzed prior to the enzymatic formation of the terminal intermediate, 6,7-dimethyl-8-ribityllumazine (VIII) (Harzer et al., 1978; Neuberger and Bacher, 1986), which is obtained by condensation of dephosphorylated VI with VII, yielding the lumazine VIII (Kis et al., 1995; Neuberger and Bacher, 1986; Volk and Bacher, 1988). The carbohydrate precursor VII, which was discovered relatively recently, is biosynthesized by an unusual reaction from ribulose 5-phosphate (X) (Volk and Bacher, 1988, 1990). The lumazine intermediate VIII is subject to an unusual dismutation reaction yielding riboflavin (IX) and its own biosynthetic precursor VI, which can be recycled in the biosynthetic process (Fig. 2) (Harvey and Plaut, 1966; Plaut, 1960, 1963; Plaut and Harvey, 1971; Plaut et al., 1970; Wacker et al., 1964). The mechanistic details of these reactions will be described in more detail later. Flavocoenzymes appear to be absolutely indispensable in all cellular organisms. Animals and certain microorganisms depend on the uptake of riboflavin as a vitamin. Enzymes for the conversion of riboflavin to the coenzymes are required in all organisms, since riboflavin is an obligatory intermediate in the pathway to the flavocoenzymes. Work on the biosynthesis of riboflavin has been performed with eubacteria (predominantly Bacillus subtilis and Escherichia coli), yeasts (e.g., Saccharomyces cerevisiae and Candida guilliermondii), and ascomycetes (Ashbya gossypii, Eremothecium ashbyii). These studies have culminated in the development of efficient fermentation proce-

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III. RIBOFLAVIN

IS FORMED

FROM

A PURINE

PRECURSOR

VIA DIAMINOPYRIMIDINE INTERMEDIATES

Ample evidence obtained between 1950 and 1970 indicated a biosynthetic relationship between purines and riboflavin (Brown et al., 1958; Brown, 1960; Goodwin, 1963; Goodwin and McEvoy, 1959; Goodwin and Pendlington, 1954; MacLaren, 1952). All nitrogen atoms of the purine precursor and the carbon atoms of its pyrimidine ring were shown to be incorporated into the vitamin (Audley and Goodwin, 1962; Bacher and Mailander, 1973, 1976; Goodwin and Pendlington, 1954; Goodwin and Treble, 1958; Howells and Plaut, 1965; Mail~nder and Bacher, 1976; Miersch et al., 1978, 1980). It was also shown that the specific precursor of the vitamin is a nucleoside or nucleotide at the biosynthetic level of guanine and that the ribose moiety of that precursor is transformed into the ribityl moiety of the vitamin (Bacher and Mailander, 1973; Keller et al., 1988; Mail~nder and Bacher, 1976). Evidence for the involvement of pyrimidine intermediates in the pathway of riboflavin biosynthesis was first obtained by work with riboflavin deficient mutants. Several diaminopyrimidine derivatives (VI, XIII, XIV; Fig. 3) were detected in riboflavin-deficient mutants of Saccharomyces cerevisiae (Bacher and Lingens, 1968, 1970, 1971; Bacher et al., 1969; Lingens et al., 1967; Logvinenko et al., 1975; Oltmanns et

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B I O S Y N T H E S I S OF RIBOFLAVIN

9

al., 1969; Shavlovsky et al., 1979). Later studies showed these compounds to be formed by hydrolytic degradation of the actual biosynthetic intermediates.

IV. GTP CYCLOHYDROLASEII The first committed reaction in the biosynthetic pathway of riboflavin is catalyzed by GTP cyclohydrolase II, which was first isolated from Escherichia coli cell extract (Foor and Brown, 1975, 1980). The enzyme catalyzes the release of C-8 of GTP (I) as formate; this reaction is accompanied by the release of pyrophosphate from the triphosphoribosyl side chain. It has been proposed that the reaction could be initiated by the transfer of a phosphoguanosyl group to an acceptor amino acid such as serine or threonine under formation of pyrophosphate (Bacher et al., 1993). The imidazole ring of the covalently bound phosphoguanosyl moiety could then be opened with release of formate, and the reaction could be terminated by hydrolysis of the phosphodiester linkage between the enzyme and the substrate. This working hypothesis, however, remains to be tested experimentally. The reaction catalyzed by GTP cyclohydrolase II is similar to the early steps of the reaction catalyzed by GTP cyclohydrolase I (Fig. 4), the first committed enzyme in the biosynthetic pathways of tetrahydrofolate and of unconjugated pteridines (for review, see Green et al., 1996). The initial reaction step catalyzed by that enzyme is the hydrolytic opening of the C-8/N-7 bond of GTP, affording XV (Bracher et al., 1998; Shiota et al., 1969; Wolf and Brown, 1969). After hydrolytic release of formate from this intermediate, the ribose side chain is used to form the pyrazine ring of the enzyme product dihydroneopterin triphosphate (XVI; Fig. 4) in subsequent reaction steps (Bracher et al., 1998). The replacement ofhistidine 179 of the E. coli enzyme affords mutant proteins catalyzing the conversion of GTP to the formamide derivative XV, where the reaction is arrested (Bracher et al., 1999). It will be interesting to see whether GTP cyclohydrolases I and II use similar reaction chemistry despite the absence of any detectable sequence similarity. A large number of genes and putative genes specifying GTP cyclohydrolase II have been sequenced (Fuller and Mulks, 1995; Lee et al., 1994; Richter et al., 1993; van Bastelaere et al., 1995). Certain bacteria and plants form bifunctional enzymes with GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase activity (Herz et al., 1999; Ritz, 1999).

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BIOSYNTHESIS OF RIBOFLAVIN V. REDUCTASE AND DEAMINASE

The accumulation of IV in its dephosphorylated form by certain yeast m u t a n t s had suggested that the ribosyl side chain of the product of GTP cyclohydrolase (II) is reduced under formation of the ribityl side chain of the vitamin (Mail/~nder and Bacher, 1976). The mechanism of the reduction has been investigated by in vivo experiments with the ascomycete Ashbya gossypii (Keller et al., 1988). Feeding of [l-2H]ribose afforded [1'-2Hi]riboflavin. These data suggested that the phosphoribosyl side chain is reduced via a Schiff base (XVII) as an intermediate (Fig. 5). The enzyme-catalyzed reduction is stereospecific, and the hydrogen supplied by the reducing agent is incorporated into the pro-(S) position. An enzyme catalyzing the reduction of II was partially purifled fromAshbya gossypii (Hollander and Brown, 1979). It was found to require NADPH as cofactor. Enzymes catalyzing the hydrolytic deamination of IV were partially purified from cell extracts of Ashbya gossypii (Hollander and Brown, 1979) and from S. cerevisiae (Nielsen and Bacher, 1981). They have not been studied in detail up to now. Sequence similarity arguments suggest that the deaminase of S. cerevisiae is specified by the RIB2 gene. In eubacteria, the deamination of the pyrimidine ring in II precedes the reduction of the side chain. Initial studies with cell extracts of E. coli had suggested separate enzymes catalyzing the two reaction steps (Burrows and Brown, 1978). More recently, it was found that the ribD gene ofE. coli and the ribG gene ofB. subtilis specify bifunctional proteins with a N-terminal deaminase domain and a C-terminal reductase domain. The reductase-catalyzed reaction requires NADH or NADPH as cofactor (Richter et al., 1997). A large number of putative orthologs of the bifunctional ribD gene have been found in eubacterial genomes. These genes and their cognate proteins have not been studied in detail.

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12

ADELBERT BACHER et al.

VI. AN ELUSIVE PHOSPHATASE In fungi as well as in eubacteria, it has been shown that the early steps of the riboflavin pathway afford V (Bacher, 1984; Bacher et al., 1982; Hollander and Brown, 1979; Klein and Bacher, 1980; Nielsen and Bacher, 1981, 1983, 1988). On the other hand, it has been shown that the committed substrate of 6,7-dimethyl-8-ribityllumazine synthase is unphosphorylated VI (Harzer et al., 1978; Neuberger and Bacher, 1986). It follows that V must be dephosphorylated prior to its conversion to riboflavin. However, the enzyme responsible for the dephosphorylation of V is unknown. Mutants with riboflavin deficiency caused by the absence of a specific dephosphorylating enzyme have not been described. Hence, it appears possible that a general phosphatase of low substrate specificity is involved. However, this raises the problem of how the dephosphorylation can be focused on the intermediate V rather than its structurally similar precursor III. The dephosphorylation of IV would result in the formation of XIII (Fig. 3), which cannot serve as substrate for the deaminase; hence, its production would be wasteful (Hollander and Brown, 1979). A recombinant strain ofB. subtilis is capable of producing 15 g of riboflavin per liter of culture medium during a fermentation period of 58 h (Perkins et al., 1991; Hfimbelin et al., 1999). The overproducing strain harbors multiple copies of the riboflavin operon. According to current knowledge, the riboflavin operon of B. subtilis does not specify a protein with phosphatase activity. Although the riboflavin production in that strain exceeds that of the wild strain by approximately four orders of magnitude, the elusive dephosphorylation step does not appear to represent a bottleneck that limits the riboflavin production.

VII. 3,4-DIHYDROXY-2-BUTANONE

4-PHOSPHATE SYNTHASE

Early studies by numerous authors had established the lumazine derivative VIII as the direct biosynthetic precursor of riboflavin (Katagiri et al., 1958a,b; Korte andAldag, 1958; Korte et al., 1958; Kuwada et al., 1958; Maley and Plaut, 1959; Masuda, 1956a,b; Plaut, 1960, 1963). It was obvious that the formation of that compound from a purine precursor required a biosynthetic precursor supplying carbon atoms 6~, 6, 7, and 7~ of VIII. The early stages of the quest for this elusive fourcarbon precursor have been reviewed repeatedly (Bacher, 1991a; Bacher et al., 1993, 1996). Briefly, a considerable number of compounds were claimed incorrectly to serve as the biosynthetic four-carbon precursor,

BIOSYNTHESIS OF RIBOFLAVIN

13

including acetoin, pentoses, tetroses, and the ribityl side chain of the pyrimidine VI. Bacher, Floss, and co-workers addressed the origin of the elusive four-carbon compound by a series of in vivo studies using a variety of singly or multiply 13C-labeled precursors such as carbohydrates, polyols, and carboxylic acids (Bacher et al., 1983, 1985; Floss et al., 1983; Le Van et al., 1985; Neuberger and Bacher, 1985; Volk and Bacher, 1991). The 13C distribution in the biosynthetic riboflavin samples was monitored by nuclear magnetic resonance (NMR) spectroscopy, and the label distribution in different parts of the molecule were compared. More specifically, systematic comparison between the labeling patterns of the ribityl side chain and the xylene ring of riboflavin, which was known to be formed by dismutation of the lumazine intermediate VIII, indicated that carbon atoms 6~, 6, and 7 of lumazine reflected the labeling pattern of C-1 to C-3 of the pentose pool. The labeling pattern of the 7~ methyl group of V I I I reflected that of C-5 of the pentose. Moreover, the in vivo studies suggested an intramolecular r e a r r a n g e m e n t resulting in a direct bond between C-3 and C-5 of a hypothetical pentose precursor.

Subsequent studies resulted in the isolation of the enzyme 3,4-dihydroxy-2-butanone 4-phosphate synthase from cell extracts of the weakly flavinogenic yeast Candida guilliermondii (Volk and Bacher, 1988, 1990). The enzyme was found to convert X to VII. Formate was established as the second enzyme product. The enzyme requires Mg 2÷ but no other cofactors. The reaction mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase was studied in some detail using 13C-labeled ribulose phosphate as substrate (Volk and Bacher, 1988, 1990, 1991). Specifically, it was confirmed that C-4 of X together with the attached hydrogen is eliminated as formate, that the recombination of the carbon fragment representing C-1 to C-3 of the substrate with C-5 of the substrate occurs by an intramolecular reaction, and that the hydrogen at C-3 of the enzyme product stems from solvent water. Moreover, it was found that a proton is introduced from solvent at the methyl group of VII. The hypothetical mechanism in Fig. 6 is based on these data. It is proposed that the reaction is initiated by the formation of the enediol intermediate XVIIL The elimination of water yields the enol XlX, which is converted to the methyldiketone XX by tautomerization. A sigmatropic migration of the terminal phosphoryl carbinol group is assumed to yield the branched carbohydrate XXI. Elimination of formate and keto-enol tautomerization of the resulting enediol under incorporation of a solvent proton terminates the reaction.

14

ADELBERT BACHERet

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An enediol (XXV) derived from ribulose bisphosphate (XXIV) with close structural similarity to XVIII is assumed to serve as the CO 2 acceptor in the reaction catalyzed by ribulose bisphosphate carboxylase (Fig. 7) (Peach et al., 1978; Pierce et al., 1980, 1986). A retroaldol reaction of the branched acid XXVI affords two molecules of 3-phosphoglycerate. The diketo compound XX, assumed to serve as intermediate in the reaction catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase, was proposed to be a side product formed from ribulose 1,5-

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15

BIOSYNTHESISOF RIBOFLAVIN

CH3 _.

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FIG. 8. Formation of 2-C-methyl-D-erythritol4-phosphate (XXIX)from 1-deoxy-D-xylulose 5-phosphate (XXVII) by the enzyme 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Isopentenyl pyrophosphate (XXX), demethytallyl pyrophosphate (XXXI).

bisphosphate in the presence of nonactivated ribulose bisphosphate carboxylase. The hypothetical intermediate XX of the 3,4-dihydroxy-2-butanone 4phosphate synthase reaction is structurally similar to ~ O : I I (Fig. 8), which was found to serve as an intermediate in the recently discovered nonmevalonate pathway of isoprenoid biosynthesis. In that pathway, a sigmatropic migration is supposed to yield the branched carbohydrate XXVIII, which is subsequently reduced under formation of XXlX (for review, see Eisenreich et al., 1998; Rohmer, 1999). Apparently, the final reprotonation of the enediol XXII! in the 3,4-dihydroxy-2-butanone 4-phosphate synthase reaction (Fig. 6) proceeds under enzymatic catalysis since only the L(S-enantiomer of VII is obtained (Volk and Bacher, 1988, 1990). Recently, the stereochemistry of the rearrangement reaction was studied in more detail (GStze et al., 1998). Stereospecifically labeled 5-[2H1]ribulose 5-phosphates were enzymatically converted to VII. The study showed that the rearrangement proceeds under retention of the configuration at C-4 of v i i , which is well in line with a 1,2-sigmatropic rearrangement (Mickon and Weglein, 1975; Woodward and Hoffmann, 1970). Strupp and Eschenmoser obtained VIII by reaction of VI with XXIV (Fig. 7) in the absence of an enzyme catalyst (Eschenmoser and Loewenthal, 1992; Strupp, 1992). Compound XXIV was used in these experiments because the l-phosphate group is superior as a leaving group to the position 1 hydroxyl group of X. Using 13C-labeled starting materials, Strupp and Eschenmoser found that the uncatalyzed reaction proceeds with elimination of C-3 of the carbohydrate precursor as opposed to the elimination of C-4 in the enzyme-catalyzed reaction. The ribB gene specifying the 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli was cloned by marker rescue using a riboflavin-deficient mutant ofE. coli (Richter et al., 1992). Genes and pu-

16

ADELBERTBACHERetal.

tative genes specifying 3,4-dihydroxy-2-butanone 4-phosphate synthase were cloned from numerous microorganisms and from plants (Herz et al., 1999; Lee and Meighen, 1992; Lee et al., 1994; Richter et al., 1992). In certain bacterial species and in plants, 3,4-dihydroxy-2butanone 4-phosphate synthase forms part of a bifunctional fusion protein with GTP cyclohydrolase II (Richter et al., 1993). The joint expression of these enzyme activities from both branches of the convergent riboflavin pathway may be relevant for regulatory aspects and may facilitate the formation of the committed intermediate in the required stoichiometric amounts, thus avoiding wasteful oversynthesis of one committed precursor. The monofunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase of E. coli is a homodimer with a mass of about 47 kDa. Despite this large size, the enzyme has been studied extensively by NMR spectroscopy (Kelly et al., 1999; Richter et al., 1999).

VIII. LUMAZINE SYNTHASE

6,7-Dimethyl-8-ribityllumazine synthase catalyzes the condensation of VI with VII yielding VIII. The enzyme was first obtained from B. subtilis as a complex with riboflavin synthase, which will be described in more detail later. Initially, only the enzymatic function of the riboflavin synthase module of that enzyme complex could be detected. Hence, the high molecular weight enzyme complex was originally designated "heavy riboflavin synthase" (Bacher and Mailfinder, 1978; Bacher et al., 1976, 1980). This earlier designation of the lumazine synthase/riboflavin synthase complex has caused some confusion, resulting in the incorrect annotation of newly found lumazine synthase genes in databases. The functional analysis of the lumazine synthase module became possible after the identification of the second enzyme substrate VII (Kis et al., 1995; Neuberger and Bacher, 1986; Volk and Bacher, 1988, 1990, 1991). The reaction appears mechanistically straightforward. The condensation of the substrates involves the release of inorganic phosphate and water. The regiochemistry of the enzyme-catalyzed reaction has been determined by studies with aaC-labeled VII (Kis et al., 1995; Nielsen et al., 1986). The data suggest that the reaction is initiated by the formation ofa Schiffbase between the position 5 amino group of VI and the carbonyl group of VII, which is not hydrated to an appreciable degree in aqueous solution (Fig. 9). The hydrogen atom in position 3 of the imine intermediate is activated by the imine bond, which is conju-

17

BIOSYNTHESIS OF RIBOFLAVIN

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N

HN"JL'N'ALo I OH2 H H-C-OH I H-C-OH XXXIII I H-C-OH I CH2OH

O "'~, N~ l J " NH

NH

"

0 NI ~ ' - N H

"

Ho,,N" £,N" O ~-~CH 2 H-C-OH ' H-C-OH XXXlV I H-C-OH I CH2OH

OtH2 H H-G-OH ' H-C-OH I H-C-OH I CH2OH

XXXV

CH 2 H-C-OH i H-C-OH I H-C-OH I CH2OH

VIII

FIG. 9. Mechanism for the formation of 6,7-dimethyl-8-ribityllumazine (VIII).

gated to the pyrimidine ring. Elimination of phosphate from XXXII yields the enol type intermediate XXXIII, which is supposed to cyclize by intramolecular addition of the ribityl-substituted amino group in the 6 position of the pyrimidine. Tautomerization of the enol type structure XXXIII may precede the ring closure, but the details are unknown. The stereoselectivity of lumazine synthase with respect to the carbohydrate VII is very low. The velocity observed with the naturally occurring L(S)-enantiomer exceeds that observed with the D(R)-enantiomer only by a factor of 5. The carbohydrate substrate VII of lumazine synthase is a rather unstable compound that decomposes spontaneously in neutral aqueous solutions at room temperature by elimination of phosphate to give butanedione (Kis et al., 1995). Moreover, VII reacts spontaneously with VI at room temperature and neutral pH to give VIII (Kis et al., 2000). This spontaneous condensation reaction is incompletely regioselective, as shown by studies with 1-13C-labeled VII. These data suggest partitioning of this nonenzymatic reaction. The regiospecific product component was likely to be formed via the pathway shown in Fig. 9. The nonregiospecific pathway may proceed via elimination of phosphate

18

ADELBERT BACHER et al.

from VII and subsequent reaction of the resulting butanedione with the pyrimidine VI, yielding the product VIII (Kugelbrey, 1997). The structure of the lumazine synthase/riboflavin synthase complex of B. subtilis has been studied in considerable detail by X-ray crystallography and electron microscopy (Bacher et al., 1986, 1994; Ladenstein et al., 1983, 1994; Ritsert et al., 1995; Schott et al., 1990b). The bifunctional enzyme has a molecular mass of about 1 MDa. Sixty lumazine synthase subunits form a capsid with icosahedral 532 symmetry. A homotrimer of riboflavin synthase is enclosed in the core space of the capsid. Initial X-ray crystallography work established the structure of the icosahedral lumazine synthase capsid at 3.3 A resolution but gave no information on the riboflavin synthase module located in the core space of the icosahedral shell (Ladenstein et al., 1988a,b). An improved resolution of 2.4 A was achieved with empty lumazine synthase capsids obtained by in vitro reconstitution (Ritsert et al., 1995). These artifactual molecules were prepared by dissociation of the enzyme complex at slightly elevated pH followed by chromatographic isolation of the lumazine synthase subunits, which were then reassembled by a liganddriven renaturation process using the substrate analog 5-nitro-6-ribitylamino-2,4( 1H, 3H)-pyrimidinedione. The lumazine synthase of E. coli is also an icosahedral capsid but does not enclose riboflavin synthase (MSrtl et al., 1996). The available data suggest tentatively that the lumazine/riboflavin synthase complex is a peculiarity of Bacillaceae. Recombinant lumazine synthase of spinach also forms an icosahedral capsid (Jordan et al., 1999; Persson et al., 1999). The lumazine synthases ofS. cerevisiae and of Mycobacterium tuberculosis form pentamers (MSrtl et al., 1996). The peptide fold and the pentamer topology are closely similar to those of the icosahedral proteins, which are best described as dodecamers of pentamers. The structure of the pentameric lumazine syonthase from S. cerevisiae has been determined to a resolution of 1.85 A (Meining et al., 2000). The 60 equivalent active sites of lumazine synthases (5 per pentamer) are located at the interface regions of adjacent subunits in the pentamer modules. Both adjacent subunits contribute to the surface of the active site cavity. The hydroxyl groups of the ribityl side chain of the substrate VI form hydrogen bonds with the peptide backbone and with amino acid side chains (Goetz et al., 1999). An aromatic amino acid residue (tryptophan or phenylalanine) is in close contact with the pyrimidine ring of the substrate. A phosphonate analog XLVII (Fig. 15) of the hypothetical intermedi-

BIOSYNTHESIS OF RIBOFLAVIN

19

6 "'''HN

.

.

.

.

.

.

-~jl'~v ~NH~

.

FIG. 10. A representation of the substrate molecule VI showing the active site of lumazine synthase and solvent molecules t h a t interact with adjacent amino acids. Hydrogen bonds are shown as dashed lines. Phe-113' belongs to the neighboring subunit.

ate XXXI! has been cocrystallized with the lumazine synthase of S. cerevisiae (Meining et al., 2000). The phosphonate group formed an ion pair with the guanidinium group of Arg-136. The catalytic activity of" lumazine synthase from B. subtilis is remarkably inert to the exchange of amino acid residues forming part of the catalytic cavity (Fischer, 1997). The replacement of the aromatic amino acid residue adjacent to the pyrimidine ring of VI by other amino acids has little effect on catalytic activity. It has not been possible to identify amino acid residues specifically involved in proton transfer reactions, although the hypothetical reaction mechanism in Fig. 9 would suggest that appropriate acidic or basic amino acids could specifically assist the various hypothetical proton transfer steps involved in the reaction sequence (Fig. 10). The condensation reaction between VI and VII can proceed spontaneously, without enzyme catalysis, at neutral pH and room temperature and at millimolar concentration of both reactants. In other words, the activation barrier of the reaction is quite low (Kugelbrey, 1997). Mutagenesis data suggest that the lumazine synthase may act predominantly by reducing the activation entropy of the condensation reaction. Holding the two reactants in close proximity may suffice to achieve the required catalytic acceleration. The apparent simplicity of the reaction mechanism and the intricacy of the three-dimensional structure of lumazine synthase form a striking contrast.

20

ADELBERTBACHERetal.

IX. RIBOFLAVINSYNTHASE The direct precursor of riboflavin, 6,7-dimethyl-8-ribityllumazine, was originally observed by Masuda as a green fluorescent spot on paper chromatograms from cultures of the flavinogenic fungus Erem o t h e c i u m ashbyii (Masuda, 1956a,b, 1958). Early work by several research groups established that the compound could be converted to riboflavin by the action of riboflavin synthase (Goodwin and Horton, 1961; Kuwada et al., 1958; Maley and Plaut, 1959). Plaut and co-workers found that the enzyme reaction generates the second product VI (Wacker et al., 1964), and they showed unequivocally that the formation of riboflavin occurs by transfer of a four-carbon unit via dismutation of two identical substrate molecules. Surprisingly, it was also found that this dismutation could occur in boiling aqueous solutions of VIII without enzyme catalysis (Beach and Plaut, 1969; Rowan and Wood, 1963, 1968). The enzyme-catalyzed and the uncatalyzed reaction appear to proceed with the same regiospecificity (Beach and Plaut, 1970a; Paterson and Wood, 1969, 1972; Sedlmaier et al., 1987). The two fourcarbon units forming the xylene moiety of the vitamin are assembled with head to tail orientation (Beach and Plaut, 1970b; Paterson and Wood, 1969, 1972; Sedlmaier et al., 1987). 6,7-Dimethyl-8-ribityllumazine has a pK value of about 8.4 (Pfleiderer and Hutzenlaub, 1973). The monoanion forms a complex equilibrium mixture involving various tricyclic structures resulting from the nucleophilic attack of side chain hydroxyl groups at C-7. However, the equilibrium mixture was also shown to contain a small amount of the exomethylene structure XXXVI (Beach and Plaut, 1970a, 1971; Bown et al., 1986; Pfleiderer et al., 1971). Plaut et al. showed that the acidic protons of the position 7 methyl group are easily exchanged with solvent water (Beach and Plaut, 1970a; Paterson and Wood, 1969, 1972; Plaut et al., 1970), and this exchange is accelerated by riboflavin synthase (Plaut et al., 1970). On the basis of these findings, the involvement of anionic molecular species in the formation (both enzymatic and nonenzymatic) of riboflavin from VIII was proposed (Fig. 11). In support of the proposed mechanism, Plaut and coauthors could show that substrate analogs carrying a position 7 oxo group can be interpreted as intermediate analogs and are indeed potent inhibitors of riboflavin synthase (Winestocket al., 1963). Ligand binding studies also showed that each subunit of riboflavin synthase from B. subtilis can bind two substrate molecules (Harvey and Plaut, 1966; Otto and Bacher, 1981; Plaut, 1971). On the other hand, riboflavin and analogs of VI

21

BIOSYNTHESIS OF RIBOFLAVIN

0 HN.,,,g,,.,,~N -CH3

RI H3CyN--T~N~.~O

)~:

+

R

Nu

O HN,,~/N~ CH3

JR I H3C~I~ N~ +

N~. O"T

O

VIII

VIII

XXXVI

XXXVII

Nu

o

NUR

H

i R

HN.~/N- CH3 H3C.I~N. N~ O-

HN~,,~/N

O'J',,.N~.,~N~ I~

O~N/~[~N~

~

N I ~ N~H H" ~''-:- H3C H C)

0 HN~ N

o

,..i~N ~TNH

XXXIX H3C H HI R ~ J l ' / / N ' ~ l I N~--~ / O-

O.~ ' ,.,N/~,... . N. ~ XL

H3C~I-N N O ~I H3C H2N O

I~

XXXVlII O R H=,,,'~N'.,,.~CH2 _H3C'-..~'I~IvN~/O-

.1.CH 2

, ..~NH

H3C H2N O XLI

R

HN,~N~_.]~CH 3

HhL N~ O-

O,~L..N/.~...N,j..,.~CH3 + ,2N~N~'H R IX

O VI

FTc. 11. Proposed mechanism of enzyme-catalyzed riboflavin formation (R = N u is a potential active site nucleophile.

n-ribityl).

could only bind at a ratio of one molecule per subunit. These findings are well in line with the view that the dismutation reaction requires the simultaneous binding of two identical substrate molecules at the active site. The sequence of riboflavin synthase from B. subtilis shows considerable similarity between the N-terminal and C-terminal portions (Fig. 12) (Schott et al., 1990a). This suggests that the enzyme subunit folds into two domains with closely similar folding topology. The active site of the enzyme may be located at the interface of two domains, each of which could contribute a half-site for the binding of one substrate molecule. Together, the two half-sites would constitute the active site (Cushman et al., 1993). In agreement with this hypothesis, the putative domains can be expressed as recombinant proteins that can bind analogs of VIII stereospecifically. The recombinant N-terminal domain

22

ADELBERT BACHER et al.

20

1

M F T

I

F V SlGJH 97

I E E

G T

V D G!TJA

E

E S M K K A G H

MALT

T R

V Y Y D L

I E E K S N

100

I

C S K

-

E D V

T K T L

120

40

60

H L G D

A Y N

I

V T D F

K N O F

V D V M

V

T

V

I F G L

E D T V

I S L

K G

D 140

Y

K,~v SE~I

96 L~R

FSEK

E

II~H~I 160

80

SE

IlL

M D P S

I GSKVN

A~A

A N G R F G G H

I E ~ C D ~M~/ G K Y M Y R F L H K A N E N K T O 180

202

FIG. 12. Internal homology in the amino acid sequence of riboflavin synthase ofB. s u b tilis.

comprising amino acid residues 1-97 of E. coli riboflavin synthase forms a homodimer (Eberhardt et al., 2000). This is surprising in light of the trimeric structure of the full-length protein. Preliminary crystallographic analysis of riboflavin synthase from E. coli shows that the protein has pseudo-D 3 symmetry (Meining et al., 1998). This is well in line with the hypothetical domain structures described. Indirect information on the substrate binding sites of riboflavin synthase was obtained by 19F-NMR monitored ligand perturbation experiments using various trifluoromethyl derivatives of VIII (Fig. 13). These experiments showed an unexpectedly large signal multiplicity for the enzyme-bound ligands (Cushman et al., 1992). For example, at least four different enzyme-bound states had to be assumed on basis of experiments with XLIII. Extensive ligand binding studies were also performed with the covalent hydrates of XLII (Cushman et al., 1991, 1993) (Fig. 13). The two stereoisomers differ by their configurations at C-7. They are not subject to racemization and can be separated by high-performance liquid chromatography (HPLC). The configurations shown in Fig. 13 were suggested on basis of solid-state NMR studies of lumazine with one of the epimers (Goetz et al., 1999). Only epimer A binds to riboflavin synthase.

23

BIOSYNTHESIS OF RIBOFLAVIN O

O F3C~ N~ "y'7 ~ FaC~'N f I R

F3C~ ..N.~ "..,,~.1 ~

~NH X N f "O

HO~%',N /

I

H

R

~NH ~,N / H

"O

XLII

XLII

Epimer B

Epimer A

O F3C~ N~ ~r Fj ~ I

R

"NH H

XLIII

FI(;. 13. Trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine (VII) (R = Dribityl). 6,7-Bis(trifluoromethyl)-8-D-ribityllumazine hydrate (XLII), 6-trifluoromethyl7-oxo-8-D-ribityllumazine (XLIII).

X. RIBOFLAVIN SYNTHASE PARALOGS IN BIOLUMINESCENCE

Several proteins with remarkable fluorescent properties and with sequence similarity to riboflavin synthase have been found in marine luminous bacteria, where they are assumed to serve as optical transducers ofbioluminescence. The isolation and properties oflumazine protein from Photobacterium phosphoreum have been described (Gast and Lee, 1978; Koka and Lee, 1979; Small et al., 1980). This 21-kDa protein contained VIII as noncovalently bound prosthetic group. After addition of the lumazine protein to the purified luciferase from the same organism, a shift of the maximal emission wavelength and an increase of the q u a n t u m yield were observed. Similar fluorescent proteins have been discovered in other luminous bacteria, for example, lumazine protein from Photobacterium leiognathi (Lee et al., 1985), yellow fluorescence protein from Vibrio fischeri Y1 (Daubner et al., 1987; Macheroux et al., 1987), and blue fluorescence protein from Vibrio fischeri Y1 (Karatani et al., 1992; Petushkov and Lee, 1997). It has also been shown that all of them can occur in complex with different ligands: lumazine protein with VIII, XLVI, riboflavin, and FMN (Petushkov et al., 1995); yellow fluorescence protein with VIII, riboflavin, and FMN; and blue fluores-

24

ADELBERTBACHERetal.

cence protein with XLVI and riboflavin (Petushkov and Lee, 1997). In contrast to riboflavin synthase, fluorescent proteins appear to bind a single ligand molecule per protein monomer. Fluorolumazines bound to lumazine protein display only a single 19F-NMR signal per CF 3 group (Scheuring et al., 1994a,b), whereas multiple 19F signals were observed in the case of riboflavin synthase (Cushman et al., 1992, 1993). Lee and co-workers proposed that energy transfer from luciferase to the fluorophores of the optical transducers occurs by weak dipoledipole interaction (Lee, 1993; Lee et al., 1991; Petushkov et al., 1996). On the other hand, Hastings and co-workers have noticed that the energy transfer alone cannot account for the increase of onset kinetics of bioluminescence in the presence of fluorescent proteins, and therefore these proteins should be involved in the luciferase reaction in one of the early stages prior to formation of the excited state of the reaction intermediate (Eckstein et al., 1990; Wilson and Hastings, 1998).

XI. RIBOFLAVIN SYNTHASE OF ARCHAEBACTERIA A riboflavin synthase gene was cloned from Methanobacterium thermoautotrophicum by marker rescue using a ribC m u t a n t of E. coli (Eberhardt et al., 1997). The gene specifies a peptide of 153 amino acid residues. The length of that sequence is only about 70% as compared to riboflavin synthase from bacteria and fungi. The internal sequence similarity characteristic for the riboflavin synthase of eubacteria and fungi is missing. The protein from M. thermoautotrophicum has no sequence similarity with those of eubacterial and fungal origin. Putative orthologs of the M. thermoautotrophicum riboflavin synthase gene are present in the genomes of several archaebacteria. None of these genomes comprises a homolog of the eubacterial/fungal riboflavin synthase. However, Pyrococcus furiosus, which has been classifted as an archaeon, carries a putative ortholog of the eubacterial/fungal riboflavin synthase gene but no homolog of the methanobacterial riboflavin synthase gene. This m a y be due to horizontal gene transfer. The ribC gene specifying the riboflavin synthase of M. thermoautotrophicum has been expressed in a recombinant E. coli strain. The catalytic activity is low by comparison with that of eubacterial riboflavin synthase, even at a relatively high temperature. The enzyme requires magnesium ions for activity, whereas the eubacterial enzyme requires no cofactors whatsoever. The recombinant M. thermoautotrophicum enzyme forms an oligomer, possibly a hexamer.

BIOSYNTHESISOF RIBOFLAVIN

25

XII. THE LUMAZINE SYNTHASE/RIBOFLAVIN SYNTHASE COMPLEX OF BACILLACEAE

In all Bacillus and Clostridium strains studied so far, lumazine synthase and riboflavin synthase can form an enzyme complex with a relative mass of about 1 MDa (Bacher et al., 1980). In these complex proteins, a trimeric riboflavin synthase module is enclosed in the central core of the icosahedral capsid consisting of 60 lumazine synthase subunits. In buffers of low ionic strength and slightly elevated pH, the enzyme complex dissociates (Bacher et al., 1986). More specifically, the riboflavin synthase trimer (~ subunits) is released, and the lumazine synthase (~ subunits) assembles under formation of even larger aggregates with molecular weights of several megadaltons. In contrast to the strictly symmetrical structure of the native capsid of 60 lumazine synthase subunits, these larger aggregates do not obey strict symmetry and form a heterogeneous mixture. In the native lumazine synthase/riboflavin synthase, the lumazine synthase subunits exceed the number of the enclosed riboflavin synthase by a factor of 20. However, riboflavin synthase trimers not associated to lumazine synthase are also present to a variable extent in the cell extracts of different Bacillus and Clostridium strains (Bacher et al., 1980). The reaction steps catalyzed jointly by lumazine synthase and riboflavin synthase are summarized in Fig. 2. The overall stoichiometry requires two molecules of VII and one molecule of VI for the formation of one molecule of riboflavin. Notably, the pyrimidine substrate of lumazine synthase is regenerated by riboflavin synthase. On average, pyrimidine molecules must pass the cycle twice for conversion to riboflavin. The lumazine synthase/riboflavin synthase complex catalyzing this complex reaction sequence shows unexpected properties that can be attributed to channeling of intermediates between the active sites of the lumazine synthase and riboflavin synthase subunits (Kis and Bacher, 1995). The substrate channeling is conducive to an increased rate ofriboflavin formation under conditions of low substrate concentration.

XIII. FLAVOKINASE AND F A D SYNTHETASE

The flavocoenzymes FMN (XI) and FAD (XII) are absolutely required in all cellular organisms. Whereas plants and certain microorganisms obtain riboflavin biosynthetically, other microorganisms and animals

26

ADELBERTBACHERetal.

must obtain the vitamin from extraneous sources. In both cases, riboflavin must be enzymatically converted to the coenzyme forms. Riboflavin kinase converts riboflavin to its 5'-phosphate, flavin mononucleotide (FMN). The reaction requires ATP. FMN is converted to flavin adenine dinucleotide (FAD) by FAD synthetase. Flavokinase and FAD synthetase activity have been documented in numerous organisms. The earlier literature has been reviewed in considerable detail (Bacher, 1991b). Flavokinases have been isolated from rat liver (Merril et al., 1980), Pichia guilliermondii (Kashenko and Shavlovsky, 1976), and mung bean (Sobhanaditya and Appaji Rao, 1981). FAD synthetases have been obtained from rat liver (Oka and McCormick, 1987), beans (Giri et al., 1960), and S. cerevisiae (Wu et al., 1995). The enzyme from S. cerevisiae has already been cloned. A bifunctional flavokinase/FAD synthetase was obtained from Corynebacteriurn amrnoniagenes (Manstein and Pai, 1986; Hagihara et al., 1995; Nakagawa et al., 1995). A similar bifunctional enzyme was subsequently shown to be formed by E. coli (Kitatsuji et al., 1993; Kamio et al., 1985). Genetic information obtained via genome sequencing projects suggest that the occurrence bifunctional flavokinase/FAD synthetase may be a universal factor in eubacteria. The enzyme of B. subtilis has been implicated in the regulation of riboflavin biosynthesis in that microorganism (Kearney et al., 1979; Mack et al., 1998; Coquard et al., 1997; Kreneva and Perumov, 1990).

XIV. RIBOFLAVIN PRODUCTION BY FERMENTATION

More than 106 kg of riboflavin are produced per year for use in human and animal nutrition and as a food colorant. Until recently, the vitamin was predominantly manufactured by chemical synthesis. It has long been known that the vitamin can be produced by fermentation procedures (Demain, 1972). In fact, a considerable number of microorganisms (bacteria, yeasts, ascomycetes) are capable of producing relatively large amounts of the vitamin. Strains of the ascomycete Ashbya gossypii and the yeast C a n d i d a f a m a t a producing more than 10 g of product per liter have been obtained by classic mutagenesis and selection procedures (Heefner et al., 1992). A recombinant Bacillus subtilis strain carrying multiple copies of the B. subtilis riboflavin operon (Hfimbelin et al., 1999; Perkins et al., 1991; Sauer et al., 1996) has been reported to produce 15 g of riboflavin per liter during a fermentation period of 58 h. The riboflavin yield was further increased by integrat-

27

BIOSYNTHESIS OF RIBOFLAVIN

ing one additional copy of the ribA gene specifying GTP cyclohydrolase and 3,4-dihydroxy-2-butanone 4-phosphate synthase into an independent locus on the B a c i l l u s chromosome (Hfimbelin et al., 1999; Ritz, 1999).

XV. INHIBITORS OF RIBOFLAVIN BIOSYNTHESIS

Whereas animals depend on a nutritional source of riboflavin, certain bacteria are absolutely dependent on endogenous biosynthesis because they lack an uptake system for the vitamin. Inhibitors of riboflavin biosynthetic enzymes could therefore be used for chemotherapy of infections with gram-negative bacteria and possibly yeasts. Certain parallels between the pathways of folate and riboflavin biosynthesis are worth noting. GTP is the committed precursor for both pathways. Certain microorganisms are unable to acquire folate from their environment and are therefore absolutely dependent on its synthesis. Sulfonamide type inhibitors of folate biosynthesis have a long and successful history in the chemotherapy of bacterial and parasite infections. A large number of substrate analogs of riboflavin synthase have been reported by the research groups of Plaut, Wood, and Cushman (A1-Hassan et al., 1980; Cushman et al., 1998; Ginger et al., 1984; Wood et al., 1974; Wrigglesworth et al., 1984). Some of these inhibitors (i.e., X L I V XLVI; Fig. 14) have K i values below 100 nM. More recently, a number of compounds has been tested as inhibitors of lumazine synthase. Certain compounds can inhibit both riboflavin synthase and lumazine synthase. Notably, a hydrate of VIII has been proposed as an intermediate in the reaction mechanisms of both en-

o

R

XLIV

H

o

R

H

XLV

o

R

H

XLVI

FIG. 14. Potent inhibitors for riboflavin synthase (R = D-ribityl). 6,7-Dioxo-8-D-ribityllumazine (X]LIV), 6-carboxy-7-oxo-8-Dribityllumazine (XLV), 6-methyl-7-oxo-8-D-ribityllumazine (XLVI).

28

ADELBERTBACHERetal.

zymes, and the covalent hydrate XLII can bind to both enzymes (Cushman et al., 1991, 1992, 1993). Ribitylpyrimidine phosphonates and sulfonates, which are analogs of the Schiff base intermediate XXXII of the lumazine synthase reaction, inhibit lumazine synthase in the range o f K i = 100-500 pM (Cushman et al., 1999a). Ribitylpyrimidines attached to a sulfonylphenyl group (such as XLVIII and XLIX) inhibited both lumazine synthase and riboflavin synthase (Fig. 15). The inhibition type of these dual function inhibitors is best described as mixed type inhibition, which indicates a complex two-substrate, two-products inhibition mechanism for lumazine synthase. Bislumazines were recently prepared as dual function inhibitors for riboflavin synthase. In the bislumazines two lumazine molecules are connected by a polymethylene linker chain, which should bring both lumazine moieties in close proximity, analogous to the proposed transition state in the riboflavin synthase mechanism. The best inhibitor was obtained with a tetramethylene linker (Ki = 37 pM) (Cushman et al., 1999b). None of the enzyme inhibitors developed so far has antibacterial activity in vivo. This may be due to insufficient penetration, which appears likely in light of the rather close structural similarity of these compounds with riboflavin.

0

HN ~

~'NH~

O

I

R XLVII

0

i l ~ i i

Na" - O - - S ~

(

0

0

J)---C--NH

Jut..

HN

I

li

NH

0

II

,/'~---~

F~S---( (

0

0

It

} )~C~NH

HN

I

R XLVlII

II

~u~

NH

O

R XLIX

FIG. 15. Inhibitors for lumazine synthase and riboflavin synthase (R = D-ribityl). 5-(6D-Ribitylamino-2,4-dihydroxypyrimidin-5-yl)-l-pentylphosphonic acid (XLVII), 5-{benzolamino[4'-(sodium sulfonate)]}-6-ribitylaminouracil (X]LVIII), 5-[4-(fluorosulfonyl)benzoylamino]-6-ribitylaminouracil (XLIX).

BIOSYNTHESISOF RIBOFLAVIN

29

XVI. BIOSYNTHESIS OF RIBOFLAVIN IN PLANTS

Arabidopsis thaliana and tomato produce bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II proteins similar to the bifunctional enzymes from eubacteria. The N terminus of these proteins contains a large number of serines and threonines. This segment is not required for catalytic activity. It is assumed to serve as a signal sequence for the import of the protein into plastids (Herz et al., 1999). Lumazine synthase of spinach is located in chloroplasts. The chloroplast import is accompanied by removal of an N-terminal leader sequence of 66 amino acid residues. Expression of amino acid residues 67-222 in a bacterial host yields a multimeric enzyme with icosahedral 532 symmetry (Jordan et al., 1999; Persson et al., 1999). Riboflavin synthase has been purified 600-fold from spinach (Mitsuda et al., 1971). The enzyme has not been characterized in detail.

XVII. REGULATION OF RIBOFLAVIN BIOSYNTHESIS IN B A C I L L U S S U B T I L I S

All riboflavin biosynthetic genes form part of an operon in the Bacillaceae. In B. subtilis, this operon also contains an unannotated gene designated ribT It is not required for the biosynthesis of riboflavin, and its function is unknown. The 5' end of the operon is preceded by a complex regulatory sequence. It was proposed that expression of the genes of the operon might be regulated by a transcription termination/antitermination mechanism (Perkins and Pero, 1993). A regulatory gene, ribC, was found to specify a bifunctional riboflavin kinase/FAD synthetase catalyzing the biosynthesis of the flavocoenzymes FMN and FAD from riboflavin, which have been shown to downregulate the transcription of the riboflavin operon (Mack et al., 1998). Riboflavin biosynthesis can be modulated over a 30-fold range (Bacher and Mail~inder, 1978). The details of this regulatory mechanism are still unknown.

X V I I I . BIOSYNTHESIS OF 5-DEAZA-7,8-DIDEMETHYL8-HYDROXYRIBOFLAVIN

Cofactor F42o (L, Fig. 16), found in methanogenic bacteria, is structurally similar to flavins (Cheeseman et al., 1972). More specifically, the chromophore of that coenzyme is 5-deaza-7,8-didemethyl-8-hydroxyri-

30

ADELBERTBACHERetal. O

HO

I CH2 I H--C--OH I H--C~OH I

H--C--OH

O

O

CH 3

HzC~O~P--O-'--k,

I

CO0-

O

N

y

0-

H

0

coo COO-

H

O

I H--C--OH

I I H--C~OH I

H--C ~OH

LI

HzC--OH

FIG. 16. Structure of coenzyme F42 o (L) and 5-deaza-7,8-didemethyl-8-hydroxyriboflavin (LI).

boflavin (LI) (Eirich et al., 1978). The cofactor plays a central role in the formation of m e t h a n e and energy generation by methanogenic bacteria (for review, see DiMarco et al., 1990). Coenzyme F42 o has also been found to serve as second chromophore in DNA photolyases of cyanobacteria and mycobacteria (Daniels et al., 1985). The heterocyclic deazaflavin chromophore is biosynthesized from the riboflavin precursor VI. The phenolic ring and carbon atom 5 are derived from L I I (Fig. 17) (Reuke et al., 1992). The details of the assembly of the two biosynthetic precursors, which must involve the removal of the amino substituent of VI, are unknown.

31

BIOSYNTHESIS OF RIBOFLAVIN

~ HO

v

,~COOH O

+

I

CH2

LII

I

CHOH

I

v,

CHOH

I

CHOH

I

CH2OR

O

HO

0

I

CH2

I

CHOH

LI

I

CHOH

I

CHOH

I

CH=OH

F~c. 17. S t a r t i n g substrates for the biosynthesis of 5-deaza-7,8-didemethyl-8-hydroxyriboflavin. 4-Hydroxyphenylpyruvate (LII).

XIX. BIOSYNTHESIS OF MOLYBDOPTERIN Molybdopterin (LIII) is a cofactor of several redox enzymes such as sulfite oxidase, nitrate reductase, aldehyde oxidoreductase, and xanthine dehydrogenase (for review, see Kisker et al., 1997). The elucidation of its molecular structure extended over a period of m a n y decades. The tricyclic structure shown in Fig. 18 was derived from X-ray crystal structure analysis of several molybdopterin enzymes (Boyington et al.,

32

ADELBERT BACHERetal. 0 HN

~L~..,;1~98~

H2N~ "~Nf

I

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1997; Chan et al., 1995; Hille, 1996; Romeo et al., 1995; Schindelin et al., 1996). The structure is best described as a pteridine with a fourcarbon side chain in position 6 that forms a third ring by addition of a side chain hydroxyl group to the dihydropyrazine ring. Molybdopterin is found in bacteria, plants, and animals. A relatively large number of gene products are involved in its biosynthesis. The following discussion is limited to the early steps of the biosynthetic pathway leading to formation of the pteridine chromophore, which has been shown to involve a highly unusual rearrangement reaction similar to that involved in the formation of the riboflavin precursor VII. Also in parallel with the biosynthesis of riboflavin, the pyrimidine ring of molybdopterin is derived from that of a guanine precursor. However, many details of the molybdopterin biosynthetic pathway are still unknown. The early steps of molybdopterin biosynthesis are believed to involve proteins specified by the genes moaA, moaB, and m o a C (for review, see Rajagopalan, 1996). The joint action of these genes is conducive to the formation of a compound, designated precursor Z, whose structure remains to be determined, moeA m u t a n t s of E. coli are unable to transform precursor Z to molybdopterin. The unstable precursor Z accumulated by moeA mutants can be oxidized, affording the stable compound Z (LIV) (Fig. 18). Wuebbens and Rajagopalan studied the formation of the artifactual compound Z using various 14C-labeled guanosine samples and concluded that a part of the ribose side chain of a guanosine nucleoside or nucleotide becomes part of the four-carbon side chain of compound Z

BIOSYNTHESIS OF RIBOFLAVIN

33

(carbon atoms 2'-4'). Moreover, these data suggested that C-8 of the imidazole ring of guanine becomes incorporated into carbon atom 1' of compound Z (Wuebbens and Rajagopalan, 1995). A more detailed analysis of the complex reaction involved studies with a moeA mutant ofE. coli carrying a plasmid directing the expression of the m o a A B C genes (Rieder et al., 1998). This recombinant strain afforded increased amounts of compound Z. Experiments with uniformly 13C5-1abeled X and [8-13C,7-15N]guanine afforded multiple stable, isotopically labeled compound Z samples that were analyzed by NMR spectroscopy. These experiments confirmed that the four-carbon side chain of compound Z is indeed a mosaic obtained from parts of a pentose with an interspaced carbon atom derived from C-8 of guanine. The data showed that the reshuffling of the molecular fragments occurs by intramolecular rearrangements. On the basis of these data, it has been proposed that the imidazole ring of GTP is opened in a reaction reminiscent of that catalyzed by GTP cyclohydrolase II (Fig. 19). It is then assumed that a branched carbohydrate moiety is formed by the transfer of a formyl group from the formamide group in the postulated intermediate LV. The proposed reaction sequence, resulting in the insertion of a formyl group between the fragments of a carbohydrate moiety, is surprisingly similar to the reaction catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase. The crucial steps of both reactions are shown with similar molecular topology for ease of comparison of the reaction steps (Fig. 19). An entirely different sequence of reactions has been proposed on basis of work with yeast (Irby and Adair, 1994). These studies suggested that the biosynthesis of molybdopterin branches from the biosynthetic pathway of folic acid, where GTP is converted to dihydroneopterin triphosphate by the action of GTP cyclohydrolase I (Fig. 20). A retroaldol cleavage following the hydrolysis of the phosphate residues affords 6-hydroxymethyldihydropterin(LVI). Aldol addition of a triose such as glyceraldehyde 3-phosphate was then supposed to yield the four-carbon side chain of molybdopterin (LIII). Further studies are required to establish unequivocally whether different pathways for molybdopterin biosynthesis operate in yeast and bacteria.

XX.

CONCLUSION

Flavin coenzymes are essential redox cofactors that must be obtained by enzymatic conversion of riboflavin in all cellular organisms. The ri-

34

ADELBERT BACHER et al.

A Enzyme

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FIG. 19. (A) Proposed mechanism for the formation of precursor Z. (B) The similarity of reaction A with the reaction catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase is shown for comparison.

BIOSYNTHESIS OF RIBOFLAVIN

B

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BIOSYNTHESIS OF RIBOFLAVIN

37

boflavin required as precursor is obtained by de novo biosynthesis or from nutritional sources. Certain microorganisms such as the Bacilliaceae can obtain riboflavin in both ways. Others are absolutely dependent on a nutritional source (owing to the absence of the riboflavin biosynthesis enzymes) or on endogenous biosynthesis (owing to the absence of an uptake system). Pathogenic microorganisms in the latter group should be susceptible to inhibition of riboflavin biosynthetic enzymes by appropriate enzyme inhibitors. Thus, the enzymes of the riboflavin pathway appear as potential targets for novel antiinfective agents. A detailed understanding of the mechanism of the riboflavin pathway could serve as a basis for the development of antiinfective agents of this novel type. It is noteworthy that several of these enzymes catalyze reactions of extraordinary mechanistic complexity, thus providing numerous opportunities for the design of transition state type inhibitors. In striking paradox with this mechanistic complexity, some of these reactions can proceed in the absence of any catalyst under relatively mild conditions. These findings appear relevant for the evolution of coenzymes and are highly compatible with the hypothesis that flavocoenzymes arose early in prebiotic evolution. REFERENCES A1-Hassan, S. S., Kulick, R. J., Livingstone, D. B., Suckling, C., and Wood, H. C. S. (1980). Specific enzyme inhibitors in vitamin biosynthesis. Part 3. The synthesis and inhibitory properties of some substrates and transition state analogues of riboflavin synthase. J. Chem. Soc., Perkin Trans.1 12, 2645-2656. Audley, B. G., and Goodwin, T. W. (1962). Studies on the biosynthesis of riboflavin. VII. The incorporation of adenine and guanine into riboflavin and into nucleic acid purines in Eremothecium ashbyii and Candida flavori. Biochem. J. 84, 587-592. Bacher, A. (1984). Biosynthesis of riboflavin. Preparation of phosphorylated pyrimidine intermediates. Z. Naturforsch. B 39B, 252-258. Bacher, A. (1986). Heavy riboflavin synthase from Bacillus subtilis. In "Methods in Enzymology (F. Chytid and D. B. McCormick, eds.), Vol. 122, pp. 192-199. Academic Press, New York. Bacher, A. (1991a). Biosynthesis of flavins. In "Chemistry and Biochemistry of Flavoproteins" (F. Mtiller, ed.), Vol. 1, pp. 215-259. CRC Press, Boca Raton, FL. Bacher, A. (1991b). Riboflavin kinase and FAD-synthetase. In "Chemistry and Biochemistry of Flavoenzymes" (F. Mtiller, ed.), Vol. 1, pp. 349-370. CRC Press, Boca Raton, FL. Bacher, A., and Ladenstein, R. (1991). The lumazine synthase/riboflavin synthase complex of Bacillus subtilis. In "Chemistry and Biochemistry of Flavoproteins" (F. Mtiller, ed.), Vol. 1, pp. 293--316. CRC Press, Boca Raton, FL. Bacher, A., and Lingens, F. (1968). Nachweis von 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidin als Akkumulat bei einer Riboflavinmangelmutante von Saccharomyces cerevisiae. Angew. Chem. 80, 237-238; Angew. Chem., Int. Ed. Engl. 7, 219220.

38

ADELBERTBACHERetal.

Bacher, A., and Lingens, F. (1970). Biosynthesis of riboflavin. Formation of 2,5-diamino6-hydroxy-4-(l'-D-ribitylamino)pyrimidinein a riboflavin auxotroph. J. Biol. Chem. 245, 4647-4652. Bacher, A., and Lingens, F. (1971). Biosynthesis of riboflavin. Formation of 6-hydroxy2,4,5-triaminopyrimidine in rib7 mutants of Saccharomyces cerevisiae. J. Biol. Chem. 246, 7018-7022. Bacher, A., and Mail~inder, B. (1973). Biosynthesis of riboflavin. The structure of the purine precursor. J. Biol. Chem. 248, 6227-6231. Bacher, A., and Mail~nder, B. (1976). Biosynthesis of riboflavin. Structure of the purine precursor and origin of the ribityl side chain. Ia "Flavins and Flavinproteins" (T. P. Singer, ed.), pp. 733-736. Biological and Medical Press, Amsterdam. BacheI, A., and Mail~inder, B. (1978). Biosynthesis of riboflavin in Bacillus subtilis: Function and genetic control of the riboflavin synthase complex. J. Bacteriol. 134, 476482 (1978). Bacher, A., Banr, R., Oltmanns, O., and Lingens, F. (1969). Biosynthesis of riboflavin. Mutants accumulating 6-hydroxy-2,4,5-triaminopyrimidine.F E B S Lett. 5, 316-318. Bacher, A., Baur, R., Eggers, U., Harders, H., and Schnepple, H. (1976). Riboflavin synthases of Bacillus subtilis. In "Flavins and Flavoproteins" (T. P. Singer, ed.), pp. 729732. Biological and Medical Press, Amsterdam. Bacher, A., Baur, R., Eggers, U., Harders, H., Otto, M. K., and Schnepple, H. (1980). Riboflavin synthases of Bacillus subtilis. Purification and properties. J. Biol. Chem. 255, 632-637. Bacher, A., Nielsen, P., Rauschenbach, P., and Klein, G. (1982). Biosynthesis of riboflavin. Preparation and enzymatic conversion of phosphorylated pyrimidine intermediates. In "Flavins and Flavoproteins" (V. Massey, ed.), pp. 495-499. Elsevier, Amsterdam. Bacher, A., Le Van, Q., Keller, P. J., and Floss, H. G. (1983). Biosynthesis of riboflavin. Incorporation of 13C-labeled precursors into the xylene ring. J. Biol. Chem. 258, 1343113437. Bacher, A., Le Van, Q., Keller, P. J., and Floss, H. G. (1985). Biosynthesis of riboflavin. Incorporation of multiply 13C-labeled precursors into the xylene ring. J. Am. Chem. Soc. 107, 6380-6385. Bacher, A., Ludwig, H. C., Schnepple, H., and Ben-Shaul, Y. (1986). Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation. J. Mol. Biol. 187, 75-86. Bacher, A., Eisenreich, W., Kis, K., Ladenstein, R., Richter, G., Scheuring, J., and Weinkauf, S. (1993). Biosynthesis of flavins. In "Bioorganic Chemistry Frontiers" (H. Dugas and F. P. Schmidtchen, eds.), pp. 147-192. Springer-Verlag, Berlin. Bacher, A., Ritsert, K., Kis, K., Schmidt-B~se, K., Huber, R., Ladenstein, R., Scheuring, J., Weinkauf, S., and Cushman, M. (1994). Studies on the biosynthesis of flavins. Structure and mechanism of 6,7-dimethyl-8-ribityllumazinesynthase. In "Flavins and Flavoproteins" (K. Yagi, ed.), pp. 53-62. de Gruyter, Berlin. Bacher, A., Eberhardt, S., and Richter, G. (1996). Biosynthesis of riboflavin. In "Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology" (F. C. Neidhardt et al., eds.), 2nd ed., Vol. 2, pp. 657-664. American Society for Microbiology, Washington, DC. Baugh, G. M., and Krumdiek, C. L. (1969). Biosynthesis of riboflavine in Corynebacterium species: The purine precursor. J. Bacteriol. 98, 1114-1119. Beach, R., and Plaut, G. W. E. (1969). The formation of riboflavin from 6,7-dimethyl-8ribityllumazine in acid media. Tetrahedron Lett. 40, 3489-3492.

BIOSYNTHESISOF RIBOFLAVIN

39

Beach R., and Plaut, G. W. E. (1970a). Investigations of structures of substituted lumazines by deuterium exchange and nuclear magnetic resonance spectroscopy. Biochemistry 9, 760-770. Beach R., and Plaut, G. W. E. (1970b). Stereospecificity of the enzymic synthesis of the oxylene ring of riboflavin. J. Am. Chem. Soc. 92, 2913-2916. Beach, R., and Plaut, G. W. E. (1971). The synthesis, properties and base-catalyzed interactions of 8-substituted 6,7-dimethyllumazines. J. Org. Chem. 36, 3937-3943. Bown, D. H., Keller, P. J., Floss, H. G., Sedlmaier, H., and Bacher. A. (1986). Solution structures of 6,7-dimethyl-8-substituted lumazines. 13C-NMR evidence for intramolecular ether formation. J. Org. Chem. 51, 2461-2467. Boyington, J. C., Gladyshev, V. N., Khangulov, S. K., Stadtman, T. C., and Sun, P. D. (1997). Crystal structure of formate dehydrogenase H: Catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster. Science 275, 1305-1308. Bracher, A, Eisenreich, W., Schramek, N., Ritz, H., Ghtze, E., Herrmann, A., Gfitlich, M., and Bacher, A. (1998). Biosynthesis of pteridines. NMR studies on the reaction mechanism of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase. J. Biol. Chem. 273, 28132-28141. Bracher, A., Fischer, M., Eisenreich, W., Ritz, H., Schramek, N., Boyle, P., Gentili, P., Huber, R., Nar, H., Auerbach, G., and Bacher, A. (1999). Histidine 179 mutants of GTP cyclohydrolase I catalyze the formation of 2-amino-5-formylamino-6-ribosylamino4(3H)-pyrimidinone triphosphate. J. Biol. Chem. 274, 16727-16735. Brown, E. G., Goodwin, T. W., and Jones, O. T. G. (1958). Studies on the biosynthesis of riboflavin. IV. Purine metabolism and riboflavin synthesis in Eremothetium ashbyii. Biochem. J. 68, 40-49. Brown, G. M. (1960). Biosynthesis of water-soluble vitamins and derived coenzymes. Physiol. Rev. 40, 331-368. Brown, G. M., and Neims, A. (1982). Adv. Enzymol. 53, 345. Brown, G. M., and Reynolds, J. J. (1963). Biogenesis of the water-soluble vitamins. Annu. Rev. Biochem. 32, 419-462. Brown, G. M., and Williamson, J. M. (1987). Biosynthesis of folic acid, riboflavin, thiamine and pantothenic acid. In "Escherichia coli and Salmonella t h y p h i m u r i u m " (F. C, Neidhardt et al., eds.)~ Vol 1., pp. 521-538. American Society for Microbiology, Washington, DC. Burrows, R. B., and Brown, G. M. (1978). Presence in Escherichia coli of a deaminase and a reductase involved in biosynthesis of riboflavin. J. Bacteriol. 136, 657-667. Chan, M. K., Mukund, S., Kletzin, A., Adams, M. W. W., and Rees, D. C. (1995). Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase. Science 267, 1463-1469. Cheeseman, P., Toms-Wood,A., and Wolfe, R. S. (1972). Isolation and properties of a fluorescent compound, factor 420, from Methanobacterium strain. J. Bacteriol. 112, 527-531. Coquard, D., Huecas, M., Ott, M., van Dijl, J. M., van Loon, A. P. G. M., and Hohmann, H.-P. (1997). Molecular cloning and characterization of the ribC gene from Bacillus subtilis: A point mutation in ribC results in riboflavin overproduction. Mol. Gen. Genet. 254, 81-84. Cushman, M., Patrick, D. A., Bacher, A., and Scheuring, J. (1991). Synthesis of epimeric 6,7-bis(trifluoromethyl)-8-ribityllumazinehydrates. Stereoselective interaction with the light riboflavin synthase of Bacillus subtilis. J. Org. Chem. 56, 4603-4608. Cushman, M., Patel, H. H., Scheuring, J., and Bacher, A. (1992). 19F NMR Studies on the

40

ADELBERTBACHERet al.

mechanism of riboflavin synthase. Synthesis of 6-(trifluoromethyl)-7-oxo-8-(Dribityl)lumazine and 6-(trifluoromethyl)-7-methyl-8-(D-ribityl)lumazine. J. Org. Chem. 57, 5630-5643. Cushman, M., Patel, H. H., Scheuring, J., and Bacher, A. (1993). 19F NMR studies of the mechanism of riboflavin synthase. Synthesis of 6-(trifluoromethyl)-8-(Dribityl)lumazine and derivatives. J. Org. Chem. 58, 4033-4042. Cushman, M., Mavandadi, F., Kugelbrey, K., and Bacher, A. (1998). Synthesis of 2,6dioxo-(1H,3H)-9-N-ribitylpurine and 2,6-dioxo-(1H,3H)-8-aza-9-N-ribitylpurine as inhibitors of lumazine synthase and riboflavin synthase. Bioorg. Med. Chem. 6, 409415. Cushman, M., Mihalic, J. T., Kis, K., and Bacher, A. (1999a). Design, synthesis, and biological evaluation of homologous phosphonic acids and sulfonic acids as inhibitors of lumazine synthase. J. Org. Chem. 64, 3838-3845. Cushman, M., Mavandadi, F., Young, D., Kugelbrey, K., Kis, K., and Bacher, A. (1999b). Synthesis and biochemical evaluation of bis(6,7-dimethyl-8-D-ribityllumazines)as potential bissubstrate analogue inhibitors of riboflavin synthase. J. Org. Chem. 64, 4635-4642. Daniels, L., Bakhiet, N., and Harmon, K. (1985). Widespread distribution of a 5deazaflavin cofactor in actinomycetes and related bacteria. Syst. Appl. Microbiol. 6, 12-17. Daubner, S. C., Astorga, A. M., Leisman, G. B., and Baldwin, T. O. (1987). Yellow light emission of Vibrio fischeri strain Y-l: Purification and characterization of the energy-accepting yellow fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 84, 89128916. Demain, A. L. (1972). Riboflavin oversynthesis. Annu. Rev. Microbiol. 26, 369-388. DiMarco, A., Bobick, T. A., and Wolfe, R. S. (1990). Unusual coenzymes of methanogenesis. Annu. Rev. Biochem. 59, 355-394. Eberhardt, S., Korn, S., Lottspeich, F., and Bacber, A. (1997). Biosynthesis of riboflavin. An unusual riboflavin synthase of Methanobacterium thermoautotrophicum. J. Bacteriol. 179, 2938-2943. Eberhardt, S., Zingier, N., Kemter, K., Cushman, M., and Bacher, A. (2000). Ligand binding properties of recombinant single domains of riboflavin synthase. Submitted for publication. Eckstein, J. W., Cho, K. W., Colepicolo, P., Ghisla, S., Hastings, J. W., and Wilson, T. (1990). A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system: Energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1. Proc. Natl. Acad. Sci. U.S.A. 87, 1466-1470. Eirich, L. D., Vogels, G. D., and Wolfe, R. S. (1978). Proposed structure for coenzyme F420 from Methanobacterium. Biochemistry 17, 4583-4593. Eisenreich, W., Schwarz, M., Cartayrade, A., Arigoni, D., Zenk, M. H., and Bacher, A. (1998). The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms. Chem. Biol. 5, R221-R233. Eschenmoser, A., and Loewenthal, E. (1992). Chemistry of potentially prebiological natural products. Chem. Soc. Rev. 23, 1-16. Fischer, M. (1997). Effiziente Methoden zur in vitro Mutagenese: Untersuchungen zum Mechanismus yon Lumazinsynthase und GTP-Cyclohydrolase I. Thesis, Technische Universit~t Mtinchen. Floss, H. G., Le Van, Q., Keller, P. J., and Bacher, A. (1983). Biosynthesis of riboflavin. An unusual rearrangement in the formation of 6,7-dimethyl-8-ribityllumazine.J. Am. Chem. Soc. 105, 2493-2494.

BIOSYNTHESISOF RIBOFLAVIN

41

Foor, F., and Brown, G. M. (1975). Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli. J. Biol. Chem. 250, 3545-3551. Foor, F., and Brown, G. M. (1980). GTP-cyclohydrolase II from Escherichia coli. In "Methods in Enzymology" (D. B. McCormick and L. D. Wright, eds.), Vol. 66, pp. 303-307. Academic Press, New York. Fuller, T, E., and Mulks, M. H. (1995). Characterization ofActinobacillus pleuropneumoniae riboflavin biosynthesis genes. J. Bacteriol. 177, 7265-7270. Gast, R., and Lee, J. (1978). Isolation of the in vivo emitter in bacterial luminescence. Proc. Natl. Acad. Sci. U.S.A. 75, 833-837. Ginger, C. D., Wrigglesworth, R., Inglis, W. D., Kulick, R. J., Suckling, C., and Wood, H. C. S. (1984). Specific enzyme inhibitors in vitamin biosynthesis. Part 5. Purification of riboflavin synthase by affinity chromatography using 7-oxolumazines. J. Chem. Soc., Perkin Trans. 1 5, 953-958. Giri, K. V., Rao, R. N., Cama, H., and Kumar, S. A. (1960). Studies on flavinadenine dinucleotide-synthesizingenzyme in plants. Biochem. J. 75, 381. Goetz, J. M., Poliks, B., Studelska, D. R., Fischer, M., Kugelbrey, K., Bacher, A., Cushman, M., and Sch~ifer, J. (1999). Investigation of the binding of fluorolumazines to the 1-MDa capsid oflumazine synthase by I~N{1~FIREDOR NMR. J. Am. Chem. Soc. 121, 7500-7508. GStze, E., Kis, K., Eisenreich, W., Yamauchi, N., Kakinuma, K., and Bacher, A. (1998). Biosynthesis of riboflavin. Stereochemistry of 3,4-dihydroxy-2-butanone 4-phosphate synthase reaction. J. Org. Chem. 63, 6456-6457. Goodwin, T. W. (1963). Riboflavin and related compounds. "The Biosynthesis of Vitamins and Related Compounds," p. 24. Academic Press, London. Goodwin, T. W., and Horton, A. A. (1961). Biosynthesis of riboflavin in cell-free systems. Nature (London) 191,772-774. Goodwin, T. W., and McEvoy, D. (1959). Studies on the biosynthesis of riboflavin. 5. General factors controlling flavinogenesis in the yeast Candida flavori. Biochem. J. 71, 742-748. Goodwin, T. W., and Pendlington, U. S. (1954). Studies on the biosynthesis of riboflavin. Nitrogen metabolism and flavinogenesis in Eremothetium ashbyii. Biochem. J. 57, 631-641. Goodwin, T. W., and Treble, D. H. (1958). The incorporation of acetoin into ring A of riboflavin by E. ashbyii: A new route for the biosynthesis of an aromatic ring. Biochem. J. 70, 14-15. Green, J. M., Nichols, B. P., and Matthews, R. G. (1996). Folate biosynthesis, reduction and polyglutamylation. In "Escherichia coli and Salmonella typhirimurium: Cellular and Molecular Biology" (F. C. Neidhardt et al., eds.), 2nd ed., Vol. 2, pp. 665-673. American Society for Microbiology, Washington, DC. Hagihara, T., Fulio, T., and Aisaka, K. (1995). Cloning of FAD synthetase gene from Corynebacterium ammoniagensis and its application to FAD and FMN production. Appl. Microbiol. Biotechnol. 42~ 724-729. Harvey, R. A., and Plaut, G. W. E. (1966). Riboflavin synthase from yeast: Properties of complexes of the enzyme with lumazine derivatives and riboflavin. J. Biol. Chem. 241, 2120-2136. Harzer, G., Rokos, H., Otto, M. K., Bacher A., and Ghisla, S. (1978). Biosynthesis ofribofiavin. 6,7-Dimethyl-8-ribityllumazine 5'-phosphate is not a substrate for riboflavin synthase. Biochim. Biophys. Acta 540, 48-54. Heefner, D. L., Weaver, C. A., Yarus, M. J., and Burdzinski, LA. (1992). Method for producting riboflavin with Candida famata. U.S. Pat. US1990000480267.

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