Stereospecificity of the enzymic synthesis of the o-xylene ring of riboflavin

Beach, R. L.; Plaut, G.W.E.

doi:10.1021/ja00712a052

Cited by 67 publications

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“…1) that had been proposed earlier. A nucleophilic attack of one molecule of 6,7-dimethyl-8-ribityllumazine (exomethylene form, 1a) at C-6 of a second molecule of 6,7-dimethyl-8-ribityllumazine covalently bound at the acceptor site of the protein (2) could yield intermediate 3 (4,10,11). Deprotonation of 3 at C-7a and protonation at N-1 could afford 7.…”

Section: Isolation Of a Transient Molecular Species (Compound Q)mentioning

confidence: 99%

“…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).…”

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confidence: 95%

“…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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confidence: 99%

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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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Section: Isolation Of a Transient Molecular Species (Compound Q)mentioning

confidence: 99%

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confidence: 95%

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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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“…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.…”

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confidence: 99%

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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“…Later, Rowan and Wood (13) and Plaut and Beach (14) extended these observations and showed that the uncatalyzed reaction can proceed in neutral as well as in acidic aqueous solution. The uncatalyzed and the enzymecatalyzed reactions were shown to proceed with the same regiospecificity (5,19).…”

Section: Resultsmentioning

confidence: 99%

Presteady State Kinetic Analysis of Riboflavin Synthase

Illarionov

Haase

Bacher

et al. 2003

Journal of Biological Chemistry

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Riboflavin synthase catalyzes a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. The kinetics of the enzyme from Escherichia coli were studied under single turnover conditions. Stopped flow as well as quenched flow experiments documented the transient formation of a pentacyclic reaction intermediate. No other transient species were sufficiently populated to allow detection. The data are best described by a sequence of one second order and one first order reaction.Riboflavin synthase catalyzes a complex dismutation affording riboflavin (8) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (9) from 6,7-dimethyl-8-ribityllumazine (1) (1). The enzyme-catalyzed dismutation can be described as an exchange of a 4-carbon unit between one substrate molecule acting as donor and a second substrate molecule acting as acceptor (typically, however, dismutation reactions involve the exchange of simple particles, e.g. hydride anions) (1, 2). The regiochemical features of the reaction require an antiparallel arrangement of the two identical substrate molecules at the active site of the enzyme (3-5).Riboflavin synthases of Bacillus subtilis and Escherichia coli are homotrimers of 23.4-kDa subunits (6, 7). Each subunit folds into two closely similar domains (7,8). The homotrimeric enzymes have six substrate-binding sites (one binding site each on each N-terminal and C-terminal domain, respectively, of each subunit) (9 -11). Surprisingly, the crystal structure of riboflavin synthase from E. coli shows an inherently asymmetric molecule where only a single N-terminal domain and a single C-terminal domain are appropriately oriented for catalysis of a two-substrate reaction (8).Recently, we reported the formation of the pentacyclic lumazine dimer 6 from 6,7-dimethyl-8-ribityllumazine by the S41A mutant of E. coli riboflavin synthase (12). The native enzyme can generate 6,7-dimethyl-8-ribityllumazine (backward reaction) as well as a mixture of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (forward reaction) from that adduct, which fulfills the criteria for a kinetically competent reaction intermediate. The hypothetical reaction mechanism shown in Fig. 1 combines the earlier mechanistic suggestions of Rowan and Wood (13) and Beach and Plaut (14) with the more recent findings.This paper describes presteady state kinetic studies on this mechanistically complex enzyme-catalyzed reaction. and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione were synthesized by published procedures (15). A recombinant strain of E. coli engineered for overexpression of the ribC gene specifying riboflavin synthase of E. coli has been described elsewhere (16). EXPERIMENTAL PROCEDURES MaterialsProtein Purification-All procedures were performed at 4°C unless otherwise stated. Frozen cell mass (5 g) was thawed in 25 ml of 50 mM Tris hydrochloride, pH 7.2, containing 0.5 mM EDTA and 0.5 mM dithiothreitol (buffer A). The suspension was subjecte...

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Stereospecificity of the enzymic synthesis of the o-xylene ring of riboflavin

Abstract: Cryptates, a new type of metal cation complexes, have been described recently.12 It was found that the

Cited by 67 publications

References 1 publication

A pentacyclic reaction intermediate of riboflavin synthase

A pentacyclic reaction intermediate of riboflavin synthase

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

Presteady State Kinetic Analysis of Riboflavin Synthase

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