Biosynthesis of Riboflavin

TOC

doi: 10.1128/ecosal.3.6.3.2

MARKUS FISCHER¹* AND ADELBERT BACHER²*

[SECTION EDITOR: T. BEGLEY]

Posted December 10, 2010

Institute of Food Chemistry, University of Hamburg, Grindelallee 117, D-20146 Hamburg,¹ and Lehrstuhl für Biochemie, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching,² Germany

^*Corresponding authors. Mailing address for Markus Fischer: Institute of Food Chemistry, University of Hamburg, Grindelallee 117, D-20146 Hamburg, Germany. Phone: 0049-40-428384359, Fax: 0049-40-428384342, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it . Mailing address for Adelbert Bacher: Lehrstuhl für Biochemie, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Germany. Phone: 0049-89-3204456, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

All cellular organisms require coenzymes derived from riboflavin (vitamin B₂) as cofactors for numerous redox enzymes (111). Flavocoenzymes are also required by certain enzymes catalyzing nonredox reactions, such as certain dehydration and isomerization reactions, the light-driven repair of DNA photodamage, and blue-light sensing in plants and certain fungi (147).

Riboflavin is biosynthesized by plants and by numerous microorganisms. Animals depend on nutritional sources that may include contributions by the gastrointestinal microbial flora, especially in ruminants.

The biosynthesis of the vitamin has been studied for more than five decades and has been reviewed previously in considerable detail. This review will focus on more recent studies of the enzymes of the biosynthesis pathway and their reaction mechanisms, some of which are unusually complex. Whereas Gram-negative bacteria will be at the center of this chapter, the numerous differences found in other microorganisms and in plants will be addressed at least briefly. For a more historical description of the work on the pathway, the reader is directed to earlier reviews in the field (9, 11, 12, 13, 14, 23, 24, 25, 50, 53, 126, 128, 130, 142, 168).

Over the past two decades, fermentation processes have replaced the chemical synthesis of riboflavin, which is required for use as a drug, food and feed additive, and food colorant (for a review, see references 150 and 164). This development started from early discoveries on naturally occurring riboflavin overproducers, such as certain ascomycetes, yeasts, and bacteria. The early work in the area has been reviewed elsewhere (40). Natural flavin overproducers were also preferred objects of study in the early days of riboflavin biosynthesis research. Thus, the observation that fermentation yields could be improved by purine supplements was conducive to the identification of GTP as the starting material for riboflavin biosynthesis (99).

The discovery of a green fluorescent compound, 6,7-dimethyl-8-ribityllumazine (compound 8, Fig. 1), in the fermentation broth of the flavinogenic ascomycetes Ashbya gossypii and Eremothecium ashbyii opened the way to the discovery of riboflavin synthase (specified in Escherichia coli by the ribC gene) (Fig. 1), which catalyzes the dismutation of that pteridine derivative, affording riboflavin (compound 9) and 5-amino-6-ribityllumazine-2,4(1H,3H)-pyrimidinedione (compound 5) (103, 104). The latter product was shown much later to serve as a biosynthetic precursor of compound 9 that can be recycled in the pathway (128, 131).

Fig. 1Biosynthesis of riboflavin. GTP (1); 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (2); 5-amino-6-ribosylamino-4(3H)-pyrimidine 5′-phosphate (3); 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate (4), 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (5); ribulose 5-phosphate (6); 3,4-dihydroxy-2-butanone 4-phosphate (7); 6,7-dimethyl-8-ribityllumazine (8); riboflavin (9); FMN (10); FAD (11). Enzymes involved in the riboflavin pathway of E. coli are encoded by the following genes: ribA, GTP cyclohydrolase II; ribD, pyrimidine deaminase/pyrimidine reductase; ribE, lumazine synthase; ribB, 3,4-dihydroxy-2-butanone 4-phosphate synthase; ribC, riboflavin synthase; ribF, riboflavin kinase/FAD synthetase.

Six enzyme activities are known to be involved in the riboflavin pathway, which is summarized in Fig. 1. Briefly, GTP (compound 1) is converted into 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (compound 2), the first committed intermediate of the pathway, which is then converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5) by a sequence of ring deamination, side chain reduction, and dephosphorylation (Fig. 1). The details of the dephosphorylation are still unknown. Subsequently, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5) is converted into 6,7-dimethyl-8-ribityllumazine (compound 8) by condensation with 3,4-dihydroxy-2-butanone 4-phosphate (compound 7) but is then in part regenerated by the dismutation of the lumazine derivative 6,7-dimethyl-8-ribityllumazine (compound 8) by the action of riboflavin synthase (Fig. 1).

GTP CYCLOHYDROLASE II

The fermentation yield of riboflavin by Eremothecium ashbyii can be increased by the supplementation of cultures with certain purines and pyrimidines (99). Numerous tracer studies with isotope-labeled purines confirmed that all nitrogen atoms and all pyrimidine carbon atoms of the vitamin are derived from the purine skeleton. In vivo studies with purine mutants of Aerobacter aerogenes established a guanine derivative as the specific precursor (3). Further in vivo studies with a purine-deficient mutant of Salmonella enterica serovar Typhimurium established that the pyrimidine motif and the ribose side chain of a guanosine derivative serve as building blocks for the pyrimidine moiety and the ribityl side chain of riboflavin (4, 100).

An enzyme catalyzing the release of formate and of pyrophosphate from GTP, named GTP cyclohydrolase II, was found in E. coli (55, 56). GTP cyclohydrolase II of E. coli is specified by the ribA gene. Earlier studies had identified GTP cyclohydrolase I, which catalyzes the first committed step in the biosynthesis of tetrahydrofolate; more specifically, that enzyme also catalyzes the release of formate from the substrate, GTP, but continues to convert the heterocyclic intermediate formed in that process into dihydroneopterin 3′-triphosphate (for a review, see reference 25). Whereas the type II enzyme involved in the biosynthesis of riboflavin requires magnesium ions for activity, the type I enzyme involved in tetrahydrofolate biosynthesis does not. More recently, both type I and type II GTP cyclohydrolases were also found to use essential zinc ions for catalysis (2, 76, 133). A recently described type III GTP cyclohydrolase found in Archaea converts GTP into 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone monophosphate and is also believed to require zinc (62).

The type I, type II, and type III GTP cyclohydrolases are devoid of sequence similarity. Whereas the release of C-8 of the imidazole ring of GTP is the defining feature of all three enzyme types, other catalytic aspects are different.

A notable feature of the reaction catalyzed by GTP cyclohydrolase II is the hydrolytic release of fragments from two different motifs of the substrate, GTP. More specifically, the release of formate from the imidazole ring requires the cleavage of two carbon-nitrogen bonds; in addition to that mechanistically complex reaction, a pyrophosphate unit is removed from the triphosphate motif of GTP. Pyrophosphate acts as an inhibitor (55). The reaction sequence has been investigated in some detail by nuclear magnetic resonance (NMR) spectroscopy and by pre-steady-state kinetic analysis (136, 144). By using [¹³C]GTP in order to increase the sensitivity and specificity of NMR analysis, the product was unequivocally identified as 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-monophosphate (compound 2, Fig. 1). In aqueous solution, the glycosidic bond of that intermediate is subject to rapid anomerization, affording an equilibrium mixture of the α and β anomers (136). Not surprisingly, the β anomer could be shown by kinetic analysis to be the specific product of GTP cyclohydrolase II and the specific substrate of the pyrimidine deaminase, catalyzing the consecutive step in the biosynthetic pathway (48, 144).

Pre-steady-state kinetic analysis indicated that the first step in the reaction sequence catalyzed by GTP cyclohydrolase II involves the formation of a covalent phosphoguanyl derivative of the enzyme with the release of pyrophosphate. Studies with H₂¹⁸O showed that the enzyme-catalyzed hydrolysis reaction contributes solvent oxygen exclusively to the heterocyclic product 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate and not to pyrophosphate (136). X-ray structure analysis of E. coli GTP cyclohydrolase suggested arginine 128 as the acceptor for the phosphoguanyl moiety (133).

Following the formation of the covalent phosphoguanyl intermediate, the enzyme initiates the release of formate by a water molecule activated by the essential zinc ion that forms a complex with three cysteine residues (Cys54, Cys65, and Cys67), thus affording a covalently enzyme-bound formamide-type intermediate. The formamide motif is then hydrolyzed, again by the catalytic action of the zinc ion (76).

The cleavage of the phosphate bond motif that links the intermediates covalently to the enzyme can occur either before or after the hydrolytic opening of the imidazole ring (76, 133). Hence, the E. coli enzyme converts about 10% of the substrate into GMP (136). It was also shown previously that the 5′-triphosphates of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 8-oxo-7,8-dihydroguanosine can be converted into the respective monophosphates, although the enzyme is unable to open the imidazole ring of the structurally modified guanine residues of these nucleotides (86). Surprisingly, the formation of the covalent phosphoguanosyl derivative of the enzyme is the rate-determining step of the overall reaction sequence (136).

GTP cyclohydrolase II of E. coli is a c₂-symmetric homodimer. The two equivalent active sites are located in the interface region between the two subunits (133). The enzyme has a specific activity of 182 nmol mg⁻¹ min⁻¹ (76). The reaction rates of orthologs from various organisms are generally low, with only a few catalytic cycles per minute and per subunit. With the exception of pyrimidine reductase, all enzymes of the pathway have similarly low rates (Table 1). Since riboflavin is required in only trace amounts, these enzymes may have specifically evolved to operate at low rates.

TABLE 1.Specific activities of enzymes involved in the biosynthesis of riboflavin in *E. coli*
Enzyme(s)	Gene	Activity (nmol mg⁻¹ min⁻¹) at 37°C	Reference
GTP cyclohydrolase II	ribA	182	76
Pyrimidine deaminase	ribD	360	134
Pyrimidine reductase	ribD	110 (NADPH), 125 (NADH)	134
3,4-Dihydroxy-2-butanone 4-phosphate synthase	ribB	283	135
Lumazine synthase	ribE	197	110
Riboflavin synthase	ribC	430	41

CONVERSION OF 2,5-DIAMINO-6-RIBOSYLAMINO-4(3H)-PYRIMIDINONE 5'-MONOPHOSPHATE INTO 5-AMINO-6-RIBITYLAMINO-2,4(1H,3H)-PYRIMIDINEDIONE

In eubacteria, the product of GTP cyclohydrolase II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate, is further converted by a sequence of deamination followed by side chain reduction (Fig. 1) (27, 134). The bifunctional deaminase-reductase of E. coli is specified by the ribD gene. The protein is a homodimer of 39.5 kDa subunits. Sequence comparison identified the deaminase domain as a member of the cytidine deaminase family (48, 134). In line with that family relationship, the enzyme requires zinc for catalytic activity. Whereas the substrate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (compound 2), is subject to spontaneous anomerization in aqueous solution, the β anomer has been shown to be the specific substrate of the deaminase (48, 136).

X-ray structure analysis of E. coli RibD protein revealed a c₂-symmetric homodimer (153). The catalytic zinc ion of the deaminase domain is bound by an absolutely conserved motif of His50, Cys75, and Cys84. The deamination reaction catalyzed by the N-terminal deaminase domain is believed to involve the addition of water to C-2 of the substrate compound 2 under formation of a tetragonal intermediate that is followed by the release of ammonia, affording compound 3.

The deaminase product is converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate (compound 4) by the C-terminal reductase domain (Fig. 1) of the RibD protein. NADPH serves as the second substrate. The reductase domain is a member of the dihydrofolate reductase superfamily. The structure of RibG protein of Bacillus subtilis, a homotetrameric ortholog of the bifunctional RibD protein of E. coli, has also been determined by X-ray crystallography (153).

Plants use the same pathway as eubacteria for the transformation of compound 2 into compound 3. However, the plant enzyme does not catalyze the reduction of compound 3 to compound 4 (48).

In Archaea and in fungi, the sequence of reactions is inverse relative to that in eubacteria, with side chain reduction preceding the deamination step; these respective reactions are catalyzed by separate proteins (27, 28, 63).

Since the terminal enzymes of the riboflavin pathway are unable to deal with phosphorylated substrates, the position 5′ phosphoester motif must be hydrolytically removed at the level of compound 4 as a prerequisite for the formation of 6,7-dimethyl-8-ribityllumazine (compound 8). However, the details of phosphate release remain elusive. More specifically, it is unknown whether a specific enzyme of the riboflavin pathway or a general phosphatase takes charge. There is not even formal proof for the requirement of any enzyme for that reaction step, although a nonenzymatic reaction appears to be unlikely.

In any case, one would intuitively expect the dephosphorylation to have some degree of substrate specificity, since the premature dephosphorylation of the intermediate compound 2 or 3 would be wasteful and could generate potentially deleterious materials in view of the reactivity of diaminopyrimidine-type compounds with molecular oxygen. The situation is reminiscent of the biosynthesis of tetrahydrofolate, in which a triphosphate unit must be removed from the product of GTP cyclohydrolase I, dihydroneopterin 3′-triphosphate. Recently, a nudix enzyme has been shown to remove pyrophosphate from dihydroneopterin 3′-triphosphate, the second step in the pterin branch of the folate synthesis pathway of bacteria and plants (83).

Strains of Bacillus subtilis that have been genetically engineered to hyperexpress the genes of the riboflavin operon can produce riboflavin in the range of at least 15 g per liter (119). Since this result was apparently obtained without intentional genetic modification of any phosphatase genes (the rib operon does not specify a phosphatase), the putative phosphatase must be endowed with remarkably high dephosphorylation capacity since the specific riboflavin productivity of the recombinant production strains is several orders of magnitude above the level of nonflavinogenic bacteria.

3,4-DIHYDROXY-2-BUTANONE 4-PHOSPHATE SYNTHASE

The origin of the four-carbon unit that is required for the conversion of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5) to 6,7-dimethyl-8-ribityllumazine (compound 8) was a controversial topic for a long period. The early studies on this problem have been reviewed repeatedly (cf. references 9, 11, and 135).

The more recent developments were based on the findings of in vivo studies using various ¹³C-labeled precursors, such as glucose and glycerol, which indicated that the four-C moiety originates from the pentose phosphate pool by an unusual process involving the elimination of C-4 via an intramolecular rearrangement (7, 8, 54, 92, 113, 159, 160, 161).

Subsequent work with riboflavin-deficient mutants of the yeast Candida guilliermondii culminated in the isolation of an enzyme which converts ribulose 5-phosphate (compound 6) into 3,4-dihydroxy-2-butanone 4-phosphate (compound 7) (159, 160, 161). The enzyme requires magnesium ions for catalytic activity. The enzymatic transformation involves the release of C-4 of ribulose 5-phosphate (compound 6) as formate. The reaction is believed to start with the formation of an endiol intermediate from ribulose 5-phosphate. The subsequent elimination of water, followed by tautomerization, generates a dione that can be converted to a branched aldose derivative by sigmatropic rearrangement (45, 151, 161). The elimination of formate generates yet another endiol-type intermediate that affords 3,4-dihydroxy-2-butanone 4-phosphate (compound 7) by tautomerization. Only the L-(S)-enantiomer of compound 7 is formed (160, 161). The stereochemistry of the rearrangement reaction has been studied in some detail (61). The rate constants of 3,4-dihydroxy-2-butanone 4-phosphate synthases from various microbial organisms are generally low (Table 1) (43, 45, 93, 135).

3,4-Dihydroxy-2-butanone 4-phosphate synthase specified by the ribB gene of E. coli (Fig. 1) is a c₂-symmetric homodimer of 47-kDa subunits (79, 94). The structure of the E. coli enzyme has been elucidated by X-ray crystallography and by NMR spectroscopy (79, 94). X-ray structures were also reported for the enzymes from Magnaporthe grisea, Methanocaldococcus jannaschii, and Candida albicans (43, 96, 151, 152). A flexible loop comprising several highly conserved glutamate and aspartate residues is believed to fold over the substrate once it has bound to the active site. The replacement of any of the conserved acidic amino acid residues in that loop inactivates the enzyme. Several other charged amino acid residues at the active site are likewise essential for enzymatic activity (45).

In plants and in many microorganisms (but not in E. coli), 3,4-dihydroxy-2-butanone-4-phosphate synthase and GTP cyclohydrolase II form fusion proteins with GTP cyclohydrolase II as the C-terminal domain (69). The rates of product formation by the two initial reactions of the convergent biosynthetic pathway are thus rigidly coupled.

6,7-DIMETHYL-8-RIBITYLLUMAZINE SYNTHASE

6,7-Dimethyl-8-ribityllumazine synthase specified by the ribE gene of E. coli (Fig. 1) catalyzes the condensation of 3,4-dihydroxy-2-butanone 4-phosphate (compound 7) with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5) under the release of inorganic phosphate. The biosynthetic pathway involves the L-(S)-enantiomer of compound 7, which is the product of 3,4-dihydroxy-2-butanone 4-phosphate synthase, but 6,7-dimethyl-8-ribityllumazine synthase has a remarkably low level of substrate specificity; the velocity observed with the naturally occurring L-(S)-enantiomer exceeds that observed with the D-(R)-enantiomer only by a factor of 5 (81).

The regiospecificity of the reaction suggests that the first reaction step is a nucleophilic attack of the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate (compound 7) by the position 5 amino group of the heterocyclic cosubstrate 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5). The resulting Schiff base is believed to eliminate inorganic phosphate in the formation of an enamine-type intermediate (81, 114). It is unknown whether the subsequent ring closure proceeds with or without prior tautomerization. Direct evidence for the Schiff base intermediate was obtained by pre-steady-state kinetic analysis (65, 145). The enzyme requires no cofactors. The rate constants of lumazine synthases are generally modest, in the same range as those of several other riboflavin biosynthetic enzymes (Table 1).

The 6,7-dimethyl-8-ribityllumazine synthase of E. coli is a 60-mer with icosahedral 532 symmetry (110). The capsid-like structure is best described as a dodecamer of pentamers. The 60 equivalent active sites are all located at the respective interfaces of adjacent subunits in the pentamer motifs, where they form relatively large cavities. Since the outer surface of the capsid-like enzyme represents a relatively dense structure, the exchange of substrates and products between the active sites and the bulk solvent is believed to require major conformational fluctuations of the capsid.

Like the enzyme from E. coli, the representatives from Bacillus subtilis, Aquifex aeolicus, Methanocaldococcus jannaschii, and spinach (Spinacia oleracea) form capsids with icosahedral 532 symmetry and a mass of about 1 MDa (66, 89, 90, 121, 169). The icosahedral lumazine synthases of Bacillaceae contain homotrimeric riboflavin synthase in the central core of the icosahedral capsid (5, 6). Lumazine synthases of Candida albicans, Magnaporthe grisea, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Mycobacterium tuberculosis are homopentamers (58, 87, 106, 108, 109, 121). More recently, dimers of pentamers have been observed in Rhizobiales (84, 85, 172).

Systematic mutagenesis of lumazine synthase failed to reveal any amino acid residues that are indispensable for catalysis (46). This result is surprising, since proton transfer reactions appear to be necessary for the complex reaction trajectory. It has been proposed that lumazine synthases exert their catalytic effect predominantly via control of the reaction entropy (46). Their function may in fact be limited to establishing close physical contact between the reactants. It is relevant that the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5) with 3,4-dihydroxy-2-butanone 4-phosphate (compound 7) can proceed under very mild conditions in the absence of any catalyst and that the rate acceleration by the enzyme is really quite modest (82).

RIBOFLAVIN SYNTHASE

Riboflavin synthase specified by the ribC gene of E. coli (Fig. 1) was discovered long before any other enzyme of the riboflavin biosynthetic pathway (67, 101, 116, 126, 131). Most notably, the reaction catalyzed by this enzyme involves a highly unusual dismutation conducive to the exchange of a four-carbon unit which generates one molecule each of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5) from two molecules of 6,7-dimethyl-8-ribityllumazine (compound 8) (125, 129).

Riboflavin synthase of E. coli requires no cofactors.

The regiochemistry of the enzyme-catalyzed reaction requires an antiparallel orientation of the two substrate molecules at the active site (Fig. 2) (18, 49, 117, 118, 127, 146). The enzyme substrate, 6,7-dimethyl-8-ribityllumazine, has an unexpectedly low pK of 8.4 (124), and deprotonation affords a complex equilibrium of anionic species, including an exomethylene-type species as well as several tricyclic species which result from the addition of the position 3′ or position 4′ hydroxy group of the ribityl side chain to C-7 of the lumazine moiety (20, 22, 123). The exomethylene anion was proposed to be an early intermediate of the complex reaction trajectory (127). A reaction intermediate characterized by an absorption maximum at 412 nm was first observed by stopped-flow analysis and was later trapped and isolated by a rapid quenching approach (71, 72, 74). By using ¹³C-labelled 6,7-dimethyl-8-ribityllumazines as precursors, the structure of that intermediate was identified as a pentacyclic compound referred to herein as compound 12 (Fig. 2) (71, 73, 132). The cleavage of that intermediate by a sequence of two elimination reactions appears to be quite plausible, whereas its formation is less easily explained.

Fig. 2Hypothetical mechanism of riboflavin formation by riboflavin synthase by means of the pentacyclic intermediate (12); R, ribityl. Numbers correspond to the compounds identified in the legend to Fig. 1.

Riboflavin synthase of E. coli is a nonsymmetrical homotrimer (95). The three subunits are joined by a triple-helix motif formed by the C-terminal segments. Each subunit comprises two closely similar folding domains, and each of these domains can bind one molecule of 6,7-dimethyl-8-ribityllumazine (compound 8) in a shallow surface cavity (59, 95). An active site can be formed at the interface between the N-terminal domain of one subunit and the C-terminal domain of an adjacent subunit. The two domains that jointly form an active site are related by pseudo-c₂-symmetry, and the two bound substrate molecules obey the same pseudosymmetry that sets the stage for the exchange of a four-carbon moiety, with the antiparallel regiochemistry that had been unravelled much earlier by isotope-labeling experiments (19, 117, 118, 146). Whereas apparently only two of the six folding domains can participate in catalysis at a given time, it is likely that different active sites involving different domains can be formed sequentially by way of conformational fluctuations, but this hypothesis has not been confirmed yet by experimental evidence (47, 59, 95, 141).

The N-terminal domain of E. coli riboflavin synthase can be expressed as a stable protein whose ligand binding properties are similar to those of the holoprotein (42). Surprisingly, the artificial protein domain forms a c₂-symmetric dimer. The location of the dimer axis is different from that of the axis relating the two domains of the active site, and the dimer is devoid of catalytic activity (42, 107, 156).

Recent studies have shown that the riboflavin synthases of Archaea are paralogs of lumazine synthase that are devoid of sequence similarity to the eubacterial riboflavin synthases. The reaction catalyzed by the enzyme from Methanocaldococcus jannaschii proceeds via a pentacyclic intermediate that is a diastereomer of the eubacterial reaction intermediate 12 (52, 71, 73).

RIBOFLAVIN SYNTHASE PARALOGS IN BIOLUMINESCENCE

Monomeric paralogs of riboflavin synthase are present in certain luminescent bacteria, including Photobacterium and Vibrio spp. (88, 91, 115, 148). They are devoid of catalytic activity and are believed to serve as optical transponders in the photoemission of bacterial luciferase. Specifically, lumazine protein of Photobacterium leiognathi binds 6,7-dimethyl-8-ribityllumazine (compound 8), yellow fluorescent protein of Vibrio fischeri Y1 binds flavin mononucleotide (FMN) (compound 10), and blue fluorescent protein of Vibrio fischeri binds 6-methyl-7-oxo-8-ribityllumazine (97, 122). The single ligand binding site of lumazine protein from Photobacterium leiognathi is located at the N-terminal domain (75).

RIBOFLAVIN KINASE/FAD SYNTHETASE

Organisms of all taxonomic kingdoms are faced with the necessity to convert endogenous or exogenous riboflavin into FMN (riboflavin 5′-phosphate, compound 10) and flavin adenine dinucleotide (FAD) (compound 11), the molecular species serving as cofactors for flavoproteins. The phosphorylation of the 5´-hydroxy group of riboflavin, yielding FMN, is catalyzed by riboflavin kinase. The enzyme requires ATP as the cosubstrate. FMN can be further converted to FAD by FAD synthetase. ATP serves as the cosubstrate, and the reaction involves the release of inorganic pyrophosphate. Both reaction steps require magnesium ions as cofactors. For a review of the early work in the area, see reference 10.

A significant step in the research on riboflavin kinase and FAD synthetase was the discovery of a bifunctional flavokinase/FAD synthetase in Brevibacterium ammoniagenes and Corynebacterium ammoniagenes (102, 112). The rapid deployment of whole genome sequences during the past decade has shown that the bifunctional enzyme type is widespread in eubacteria, including E. coli, whereas monofunctional FAD synthetases and monofunctional flavokinases are typical in yeasts and animals (140). The bifunctional flavokinase/FAD synthetase of E. coli is specified by the ribF gene.

X-ray structures have been reported for riboflavin kinases of Schizosaccharomyces pombe and humans and for FAD synthetase of Thermotoga maritima (16, 78, 162, 163).

BIOMIMETIC FORMATION OF RIBOFLAVIN AND EVOLUTION OF THE PATHWAY

Some reactions of the riboflavin biosynthesis pathway can proceed without catalysis under relatively mild conditions, despite their considerable mechanistic complexity. Thus, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5) reacts with 3,4-dihydroxy-2-butanone 4-phosphate (compound 7) at room temperature and neutral pH with a velocity that is high enough to require the inclusion of blank reaction mixtures without enzyme in order to avoid faulty results of enzyme assays (82). The regiospecificity of the uncatalyzed reaction is reduced compared to that of the enzyme-catalyzed reaction (81, 82). Most probably, the uncatalyzed reaction can proceed via two different pathways, either via the pathway catalyzed by the enzyme or via the formation of diacetyl as an intermediate by spontaneous phosphate elimination (82).

6,7-Dimethyl-8-ribityllumazine (compound 8) has also been obtained nonenzymatically by boiling an alkaline solution containing 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 5) and ribulose 1,5-bisphosphate (44, 154). The uncatalyzed reaction involves the loss of C-3 of ribulose 1,5-bisphosphate, whereas the enzymatic formation of the biosynthetic intermediate, 3,4-dihydroxy-2-butanone 4-phosphate, involves the loss of C-4 of the substrate, ribulose 5-phosphate (compound 6). Hence, the reaction mechanism of the uncatalyzed reaction is not identical to that of the natural biosynthetic pathway (82).

The mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine (compound 8) can proceed without catalysis in boiling aqueous solution at neutral or acidic pH (17, 117, 138, 139). The regiospecificity is the same as that of the enzyme-catalyzed reaction (18, 138, 139). In light of these findings, it appears that riboflavin or structural analogs of riboflavin may have originated through uncatalyzed reactions, prior to the evolution of some or all of the riboflavin biosynthetic enzymes.

Archaea have riboflavin synthases that are paralogs of lumazine synthase. Sequence comparisons suggest that the divergence of the two paralogous families (lumazine synthases and archaeal riboflavin synthases) must have occurred at an early time in evolution (49, 132).

The riboflavin biosynthesis enzymes of plants are more reminiscent of those of eubacteria than of those of Archaea. Thus, plants use the bacterial pathway via 5-amino-6-ribosylamino-4(3H)-pyrimidine 5′-phosphate (intermediate 3). However, the deaminase and reductase are separate proteins. The riboflavin biosynthesis enzymes of plants carry putative plastid-targeting sequences, and the entire pathway is believed to proceed inside chloroplasts (48, 51, 69, 121). The similarity of the plant and eubacterial pathways may reflect an endosymbiont origin of the plant riboflavin pathway.

REGULATION OF THE RIBOFLAVIN PATHWAY

The regulation of riboflavin biosynthesis in Gram-positive bacteria has been studied in some detail. In Bacillus subtilis, all genes specifying enzymes of the riboflavin pathway are part of a single operon that carries an RFN motif upstream of the promoter. The RFN segment (FMN riboswitch) of the mRNA binds FMN (compound 10) and functions as an antiterminator that stabilizes the mRNA in the absence of bound FMN (57, 64, 80, 157, 158, 165).

The regulation of riboflavin biosynthesis in Gram-negative bacteria has not been analyzed in detail. In E. coli and in various Salmonella spp., the ribD gene specifying the bifunctional deaminase-reductase and the ribE gene specifying the 6,7-dimethyl-8-ribityllumazine synthase are part of a gene cluster that also comprises the antitermination factor nusB and an unidentified putative open reading frame (110). These genes may all be under common transcriptional control. Similar clusters are present in a wide variety of Gram-negative bacteria (173). The potential functional significance of this association is unknown.

In certain luminescent bacteria, enzymes of the riboflavin pathway are present in luciferase operons (105). Since reduced FMN serves as a cosubstrate for bacterial luminescence, the activation of the luciferase pathway may generate a requirement for additional riboflavin. Notably, these organisms also carry standard-type riboflavin operons.

RIBOFLAVIN TRANSPORT

Riboflavin auxotrophs of E. coli require very high concentrations of riboflavin (about 200 mg/liter) for growth (15). This finding suggests that E. coli and probably other members of the Enterobacteriaceae are devoid of a transport system for the vitamin.

Other bacteria, such as Bacillus subtilis and Lactobacillus casei, can utilize riboflavin efficiently at low concentrations. Recently, a riboflavin transport protein, RibU, has been studied in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000 (26). RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and FMN, presumably by means of the structurally conserved RFN element located between the transcription start site and the start codon. Riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain.

BIOTECHNOLOGY

The world production of riboflavin is assumed to exceed 3,000 metric tons per year for use as a nutritional supplement, animal feed additive, and food colorant. Until recently, the vitamin was manufactured predominantly by chemical synthesis using glucose as a chiral precursor.

Certain microorganisms had been known for a long period to produce excess amounts of riboflavin. Specifically, the flavinogenic ascomycetes Ashbya gossypii and Eremothecium ashbyii, as well as some flavinogenic yeasts such as Candida guilliermondii and Candida famata, have been studied in considerable detail. The early history of microbial vitamin B₂ production has been reviewed elsewhere (40).

Fermentation processes based on the use of Bacillus subtilis, Ashbya gossypii, and Candida famata have now replaced the chemical synthesis of the vitamin (150). Briefly, the production of riboflavin with Bacillus subtilis could be increased by the deregulation of purine synthesis and a mutation in the gene coding for the riboflavin kinase. The Bacillus subtilis process is further based on the overexpression of multiple recombinant, homologous chromosomal copies of the riboflavin operon under the control of a strong synthetic promoter. The introduction of an additional copy of the ribA gene (Bacillus subtilis nomenclature) has also been shown to increase the productivity of the strain (31, 70, 98, 149). In the process of production strain development, purine analogs (8-azaguanine and decoyinine), methionine sulfoxide, and roseoflavin were used to select resistant mutants (120). Roseoflavin resistance mutations were located in the 5′ region of the rib operon.

Riboflavin production strains of Ashbya gossypii and Candida famata have been obtained by multiple rounds of mutagenic treatment. Metabolic inhibitors, including itaconate, which inhibits the isocitrate lyase (143), and tubercidin (7-deaza-adenosine) (68), which inhibits purine biosynthesis, have been applied for mutant selection.

RIBOFLAVIN BIOSYNTHESIS GENES AS POTENTIAL DRUG TARGETS

Gram-negative bacteria are devoid of efficient transport systems for riboflavin or flavocoenzymes and are therefore absolutely dependent on the endogenous synthesis of the vitamin. Consequently, the pathway enzymes appear to be potential anti-infective drug targets. Notably, several riboflavin biosynthesis genes of S. enterica were recently shown to be essential for virulence (21, 137).

A considerable number of inhibitors for lumazine synthase and riboflavin synthase have been reported over an extended period (1, 29, 32, 33, 34, 35, 36, 37, 38, 39, 60, 155, 166, 167, 170, 171). Many of these compounds are structural analogs of enzyme substrates or reaction intermediates that carry a ribitol side chain that is crucial for substrate binding. The reported compounds have no in vivo activity. This lack of activity may be due to their failure to enter bacterial cells. However, inhibitors with more drug-like structures may be able to serve as lead compounds for the development of novel antibiotic agents, which are urgently required given the progressive attrition of the efficacy of all currently used antibiotics through resistance development. Recently, several robotic assays for high-throughput screening of enzymes of the riboflavin pathway have been developed (30, 77).

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