Evolution of Vitamin B2 Biosynthesis. A Novel Class of Riboflavin Synthase in Archaea

Fischer, Markus; Schott, Anne-Kathrin; Römisch, Werner; Ramsperger, Arne; Augustin, Martin; Fidler, Alexander; Bacher, Adelbert; Richter, Gerald; Huber, Robert; Eisenreich, Wolfgang

doi:10.1016/j.jmb.2004.08.016

Cited by 36 publications

(43 citation statements)

References 67 publications

(58 reference statements)

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“…Recombinant RS of M. jannaschii (MjaRS) was expressed and purified as described (21). Crystals of MjaRS grew in space group P1, containing one pentamer in the asymmetric unit.…”

Section: Resultsmentioning

confidence: 99%

“…Completely sequenced archaeal genomes typically comprise sets of two similar genes, which specify a riboflavin synthase and a lumazine synthase. Sequence arguments also showed that the divergence between the paralogous lumazine synthases and riboflavin synthases occurred early in evolution (21).…”

Section: Discussionmentioning

confidence: 99%

“…In line with the sequence characteristics, RS from Methanocaldococcus jannaschii (MjaRS) has been shown to be a homopentamer in solution (21). This paper reports the crystal structure of that enzyme.…”

mentioning

confidence: 84%

“…Protein Preparation, Crystallization, and Data Processing-Cloning, expression, and purification of the protein have been described elsewhere (21). Crystals of wild-type MjaRS were grown at 18°C using the sitting drop vapor diffusion method by mixing equal amounts of protein (6 mg/ml) in 100 mM potassium phosphate, pH 7.0, containing 30 mM Tris and 2 mM dithiothreitol with a reservoir solution containing 0.1 M HEPES, pH 7.0, and 40% 2-methyl-2,4-pentanediol.…”

Section: Methodsmentioning

confidence: 99%

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Crystal Structure of an Archaeal Pentameric Riboflavin Synthase in Complex with a Substrate Analog Inhibitor

Ramsperger

Augustin

Schott

et al. 2006

Journal of Biological Chemistry

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Whereas eubacterial and eukaryotic riboflavin synthases form homotrimers, archaeal riboflavin synthases from Methanocaldococcus jannaschii and Methanothermobacter thermoautrophicus are homopentamers with sequence similarity to the 6,7-dimethyl-8-ribityllumazine synthase catalyzing the penultimate step in riboflavin biosynthesis. Recently it could be shown that the complex dismutation reaction catalyzed by the pentameric M. jannaschii riboflavin synthase generates riboflavin with the same regiochemistry as observed for trimeric riboflavin synthases. Here we present crystal structures of the pentameric riboflavin synthase from M. jannaschii and its complex with the substrate analog inhibitor, 6,7-dioxo-8-ribityllumazine. The complex structure shows five active sites located between adjacent monomers of the pentamer. Each active site can accommodate two substrate analog molecules in anti-parallel orientation. The topology of the two bound ligands at the active site is well in line with the known stereochemistry of a pentacyclic adduct of 6,7-dimethyl-8-ribityllumazine that has been shown to serve as a kinetically competent intermediate. The pentacyclic intermediates of trimeric and pentameric riboflavin synthases are diastereomers.Riboflavin (vitamin B 2 ) serves as the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential cofactors for several oxidoreductases that are indispensable in most living cells. The work on riboflavin biosynthesis in microorganisms has been covered extensively in recent reviews (1-4). Riboflavin is biosynthesized in plants, many bacteria, and in fungi but not in animals. Therefore, enzymes of this pathway have been proposed to be attractive targets for antimicrobial strategies (5-7).In the final steps of the biosynthetic pathway, lumazine synthase (LS) catalyzes the condensation of the pyrimidinedione (1) with 3,4-dihydroxy-2-butanone-4-phosphate (2) to release water, inorganic phosphate and 6,7-dimethyl-8-ribityllumazine (DMRL) 7 (8, 9), and riboflavin synthase (RS) catalyzes a dismutation of DMRL (3) affording riboflavin (4) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) (Fig. 1); more specifically, that reaction involves the transfer of a four-carbon moiety between two DMRL molecules serving as donor and acceptor, respectively (10 -13). Both reactions are thermodynamically irreversible (9, 14) and can proceed in the absence of a catalyst (11,(15)(16)(17).LS from fungi are c 5 -symmetric homopentamers, whereas the enzymes from plants, Archaea, and most eubacteria studied are 532 symmetric capsids of 60 identical subunits, which are best described as dodecamers of pentamers. The subunit folding patterns of pentameric and icosahedral LS are similar. In Bacillaceae, lumazine synthase and riboflavin synthase form a complex comprising an icosahedral capsid of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits; historically, these unusual enzyme complexes were designated heavy riboflavin synthase (18,19). Riboflavin syn...

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“…Recombinant RS of M. jannaschii (MjaRS) was expressed and purified as described (21). Crystals of MjaRS grew in space group P1, containing one pentamer in the asymmetric unit.…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Crystal Structure of an Archaeal Pentameric Riboflavin Synthase in Complex with a Substrate Analog Inhibitor

Ramsperger

Augustin

Schott

et al. 2006

Journal of Biological Chemistry

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“…All the enzymes mentioned are homotrimers. However, RF synthases from archaea are homopentamers, with unrelated amino acid sequences (121,362).…”

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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Biosynthesis of Vitamin B₂: A Unique Way to Assemble a Xylene Ring

Fischer

Bacher

2011

ChemBioChem

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The biosynthesis of one riboflavin (vitamin B(2)) molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. In the final step, the tricyclic isoalloxazine chromophore, which is the hallmark of flavocoenzymes, arises from a highly unusual dismutation of bicyclic 6,7-dimethyl-8-ribityllumazine that is catalyzed by riboflavin synthase but can also proceed without catalysis. The reaction proceeds via a pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazine molecules, whose cleavage into riboflavin and a pyrimidine derivative (by a sequence of two elimination steps) is mechanistically straightforward. Recently, the formation of the pentacyclic adduct has been proposed to involve a hydride transfer step followed by a [4+2] cycloaddition. Surprisingly, two different classes of riboflavin synthases utilize different diastereomers of the pentacyclic adduct, but the newly generated chiral centers are lost upon the intermediates' subsequent fragmentation.

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Evolution of Vitamin B2 Biosynthesis. A Novel Class of Riboflavin Synthase in Archaea

Cited by 36 publications

References 67 publications

Crystal Structure of an Archaeal Pentameric Riboflavin Synthase in Complex with a Substrate Analog Inhibitor

Crystal Structure of an Archaeal Pentameric Riboflavin Synthase in Complex with a Substrate Analog Inhibitor

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

Biosynthesis of Vitamin B₂: A Unique Way to Assemble a Xylene Ring

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Evolution of Vitamin B2 Biosynthesis. A Novel Class of Riboflavin Synthase in Archaea

Cited by 36 publications

References 67 publications

Crystal Structure of an Archaeal Pentameric Riboflavin Synthase in Complex with a Substrate Analog Inhibitor

Crystal Structure of an Archaeal Pentameric Riboflavin Synthase in Complex with a Substrate Analog Inhibitor

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

Biosynthesis of Vitamin B2: A Unique Way to Assemble a Xylene Ring

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Biosynthesis of Vitamin B₂: A Unique Way to Assemble a Xylene Ring