The biosynthesis of pteridines. Part V. The synthesis of riboflavin from pteridine precursors

Rowan, T; Wood, H. C. S.

doi:10.1039/j39680000452

Cited by 45 publications

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“…Appropriate conditions for the nonenzymatic conversion of 2 molecules of 6,7-dimethyl-8-ribityllumazine to RF and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione are boiling water solutions at acidic or neutral pH (28,29,380).…”

Section: Riboflavin Synthasementioning

confidence: 99%

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

Abbas

Sibirny

2011

Microbiol Mol Biol Rev

316

272

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SUMMARY Riboflavin [7,8-dimethyl-10-(1′- d -ribityl)isoalloxazine, vitamin B 2 ] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis , Ashbya gossypii , and Candida famata . Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

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Section: Riboflavin Synthasementioning

confidence: 99%

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

Abbas

Sibirny

2011

Microbiol Mol Biol Rev

316

272

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“…Their elegant work has been reviewed repeatedly (2,23,24). Wood, Plaut, and their coworkers have found that the dismutation of the lumazine that leads to the formation of riboflavin can also proceed nonenzymatically in boiling aqueous solution over a wide pH range (8,22,27).The sequences of riboflavin synthases from several eubacterial species and from yeast have been reported (10,13,14,(18)(19)(20) and are characterized by sequence homology between the N-terminal and C-terminal halves, suggesting that the peptides form two homologous folding domains (29). Riboflavin synthases of Bacillus subtilis and Escherichia coli are trimers of identical subunits.…”

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Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum

et al. 1997

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Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg ؊1 h ؊1 at 65°C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B 12 ). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg ؊1 h ؊1 at 65°C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5 phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria.The direct biosynthetic precursor of riboflavin, 6,7-dimethyl-8-ribityllumazine (Fig. 1), was discovered by Masuda and by Plaut and his coworkers (for a review, see reference 23) in studies with flavinogenic fungi. The lumazine derivative compound 4 is converted to riboflavin (5) by an unusual dismutation reaction involving the exchange of a four-carbon unit between two identical substrate molecules which is catalyzed by the enzyme riboflavin synthase. The second product of the dismutation reaction is 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (2), which can be returned to the riboflavin pathway via the enzyme 6,7-dimethyl-8-ribityllumazine synthase (for a review, see reference 4).The complex reaction mechanism of riboflavin synthase has been studied in considerable detail by Plaut and his coworkers. Their elegant work has been reviewed repeatedly (2,23,24). Wood, Plaut, and their coworkers have found that the dismutation of the lumazine that leads to the formation of riboflavin can also proceed nonenzymatically in boiling aqueous solution over a wide pH range (8,22,27).The sequences of riboflavin s...

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“…Thus, riboflavin is obtained on boiling of neutral or acidic aqueous solutions of 6,7-dimethyl-8-ribityllumazine (2,3). The enzyme-catalyzed and the uncatalyzed reactions proceed with identical regiochemistry involving a head-to-tail arrangement of the two 4-carbon moieties from which the xylene ring of riboflavin is assembled (4)(5)(6).…”

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“…The exomethylene type lumazine anion (1a) was proposed to attack the adduct of a second substrate molecule and a nucleophile (2) resulting in the formation of a lumazine dimer (3) as an initial reaction step ( Fig. 1; refs.…”

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A pentacyclic reaction intermediate of riboflavin synthase

Illarionov

Eisenreich

Bacher

2001

Proc. Natl. Acad. Sci. U.S.A.

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The S41A mutant of riboflavin synthase from Escherichia coli catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a very low rate. Quenching of presteady-state reaction mixtures with trifluoroacetic acid afforded a compound with an absorption maximum at 412 nm (pH 1.0) that can be converted to a mixture of riboflavin and 6,7-dimethyl-8-ribityllumazine by treatment with wild-type riboflavin synthase. The compound was shown to qualify as a kinetically competent intermediate of the riboflavin synthase-catalyzed reaction. Multinuclear NMR spectroscopy, using various 13 C-and 15 N-labeled samples, revealed a pentacyclic structure arising by dimerization of 6,7-dimethyl-8-ribityllumazine. Enzyme-catalyzed fragmentation of this compound under formation of riboflavin can occur easily by a sequence of two elimination reactions.T he final step in the biosynthesis of riboflavin is the dismutation of 6,7-dimethyl-8-ribityllumazine (1) affording riboflavin (5) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (6) in stoichiometric amounts (Fig. 1). The reaction involves the transfer of a 4-carbon moiety from a donor substrate molecule to an acceptor molecule.Rowan and Wood (1) showed that the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine can proceed in the absence of any catalyst under relatively mild experimental conditions. Thus, riboflavin is obtained on boiling of neutral or acidic aqueous solutions of 6,7-dimethyl-8-ribityllumazine (2, 3). The enzyme-catalyzed and the uncatalyzed reactions proceed with identical regiochemistry involving a head-to-tail arrangement of the two 4-carbon moieties from which the xylene ring of riboflavin is assembled (4-6).The enzyme-catalyzed reaction requires the simultaneous presence of two substrate molecules with an essentially antiparallel orientation at the active site of the enzyme (4). Ligandbinding studies confirmed that each subunit of the homotrimeric riboflavin synthase can bind two lumazine molecules (7).More recently, it was shown that each riboflavin synthase subunit comprises two tandem domains with similar sequence and folding topology. Each of these domains can accommodate one lumazine molecule, and the active sites are located at the interfaces of the N-and C-terminal domains, respectively (8, 9).The position 7 methyl group of 6,7-dimethyl-8-ribityllumazine (1) is unusually acidic with a pK value of 8.5. The exomethylene type lumazine anion (1a) was proposed to attack the adduct of a second substrate molecule and a nucleophile (2) resulting in the formation of a lumazine dimer (3) as an initial reaction step ( Fig. 1; refs. 4, 10, and 11). A ring-opening reaction was suggested to afford the intermediate 4.The crystal structure of riboflavin synthase is still unknown despite considerable efforts. Recently, mutants of riboflavin synthase from Escherichia coli have been identified with reduced rates of riboflavin formation (12). Specifically, an S41A mutant catalyzed the formation of riboflavin at a rate that was lower by several orde...

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The biosynthesis of pteridines. Part V. The synthesis of riboflavin from pteridine precursors

Cited by 45 publications

References 0 publications

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum

A pentacyclic reaction intermediate of riboflavin synthase

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