Synthesis, properties, and base-catalyzed interactions of 8-substituted 6,7-dimethyllumazines

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

doi:10.1021/jo00824a020

Cited by 29 publications

(19 citation statements)

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“…The aromatic methine protons at positions 6 and 9 of the benzenoid ring of flavins exhibit singlets in the region 7.5-8.5 ppm (18,19); these resonances were not observed with the isolated compound. Though several flavins exhibit absorption spectra with maxima in the region around 400 nm (20)(21)(22)(23)(24), their absorption spectra are otherwise quite different from those of the lumazines (11). Moreover, their behavior in acid and base is different from that of lumazines as seen from their absorption spectra.…”

Section: Resultsmentioning

confidence: 99%

Separation and structure of the prosthetic group of the blue fluorescence protein from the bioluminescent bacterium Photobacterium phosphoreum

Koka

Lee²

1979

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

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ABSTRACr The highly fluorescent prosthetic group of the blue fluorescence protein purified from the bioluminescent bacterium Photobacterium phosphoreum has been dissociated and separated from its apoprotein by affinity chromatography on Cibacron Blue-Sepharose. It has been identified as 6,7-dimethyl-8(1'-D-ribityl)lumazine by several methods of characterization, all of which gave results identical to those for an authentic sample. In neutral solution, absorption maxima are at 407, 275 (shoulder), and 256 nm, with a single fluorescence maximum at 491 nm. The proton magnetic resonance spectrum exhibits a singlet at 2.66 ppm corresponding to the methyl substituted at the 6 position of lumazine and a multiplet centered at 3.85 ppm corresponding to the C-2'-5' protons of the ribityl group. A Raman spectrum was obtained by the technique of coherent anti-Stokes Raman scattering and the RF values by paper chromatography were determined in four solvent systems. The isolated compound was readily transformed into riboflavin by riboflavin synthetase. Fifty grams (dry weight) of P. phosphoreum contains at least 20 mg of this lumazine derivative, an amount comparable to that found in other microorganisms classified as riboflavin overproducers. The overproduction of this lumazine in this case apparently has to do with its function in the generation of bioluminescence.A blue fluorescence protein was isolated from extracts of the bioluminescent bacterium Photobacterium phosphoreum by Gast and Lee (1); the protein was shown to meet the requirements of the in vivo bioluminescence emitter by three significant observations: (i) its fluorescence emission is identical to the bioluminescence spectrum both in position of the maximum, 476 nm, and in spectral distribution; (ii) the in vitro bioluminescence reaction of reduced flavin mononucleotide, luciferase, oxygen, and long-chain aliphatic aldehyde is stimulated by the addition of this protein both in terms of the decay rate of light emission and the total light yield; and (iii) the in vitro bioluminescence spectrum is shifted to match that of the in vivo when this protein is included in the in vitro reaction (1). The blue fluorescence protein is associated with luciferase and has a molecular weight of 22,000 (1). The protein-bound prosthetic group is highly fluorescent, with a quantum yield of 0.45 (1). Alteration of solution conditions perturbs this fluorescence to match either the in vitro or the in vivo bioluminescence of P. phosphoreum (1).The blue fluorescence protein was further purified by affinity chromatography on a Cibacron Blue-Sepharose column, which removed all traces of flavin and other fluorescent impurities (2, 3). The purified protein showed absorption maxima at 412 and 274 nm with a single absorption band in the visible region (3).The finding of a blue fluorescence protein also in another species of bioluminescent bacterium, Photobacterium fischeri (4), suggests that the fluorophore functions in some way in the bioluminescence and therefore its chemical structure sho...

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Section: Resultsmentioning

confidence: 99%

Separation and structure of the prosthetic group of the blue fluorescence protein from the bioluminescent bacterium Photobacterium phosphoreum

Koka

Lee²

1979

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

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“…Early work by Plaut, Wood, Pfeiderer, and their respective groups had established that the position 7 methyl group of DMRL is acidic with a pK a around 9 (41). This unusually high CH acidity has been attributed to the resonance stabilization of the lumazine anion (13,(42)(43)(44). The authors quoted above have proposed several variations on a common theme for the transformation of DMRL into riboflavin (11,39,40,45,46).…”

Section: Discussionmentioning

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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“…The methylene group 7␣ of Compound Q is structurally equivalent to the acidic 7␣ methyl group of 6,7-dimethyl-8-ribityllumazine (16). In keeping with this, the hydrogen atoms bound to 7␣ were exchanged rapidly with deuterium atoms from the solvent D 2 O.…”

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

confidence: 96%

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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Synthesis, properties, and base-catalyzed interactions of 8-substituted 6,7-dimethyllumazines

Cited by 29 publications

References 0 publications

Separation and structure of the prosthetic group of the blue fluorescence protein from the bioluminescent bacterium Photobacterium phosphoreum

Separation and structure of the prosthetic group of the blue fluorescence protein from the bioluminescent bacterium Photobacterium phosphoreum

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

A pentacyclic reaction intermediate of riboflavin synthase

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