Fluorine-19 NMR studies of the mechanism of riboflavin synthase. Synthesis of 6-(trifluoromethyl)-8-(D-ribityl)lumazine and derivatives

Cushman, Mark; Patel, Hemantkumar H.; Scheuring, Johannes; Bacher, Adelbert

doi:10.1021/jo00067a041

Cited by 23 publications

(29 citation statements)

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“…Cushman and his co-workers (Cushman et al, 1991(Cushman et al, , 1992(Cushman et al, , 1993 synthesized several trifluoromethyl lumazines with close structural similarity to presumed reaction pathway intermediates (Plaut et al, 1974;Paterson & Wood, 1972). Ligand perturbation studies monitored by 19 F NMR unexpectedly showed the existence of three different binding modes for the interaction of each respective compound with riboflavin synthase (Cushman et al, 1991(Cushman et al, , 1992(Cushman et al, , 1993). An approximate 1:1:1 stoichiometry for these different binding modes at saturating ligand concentrations was deduced from 19 F NMR signal integrals.…”

Section: Discussionmentioning

confidence: 99%

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NMR Analysis of Site-Specific Ligand Binding in Oligomeric Proteins. Dynamic Studies on the Interaction of Riboflavin Synthase with Trifluoromethyl-Substituted Intermediates

et al. 1996

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The binding of small ligands to symmetrical oligomeric proteins may lead to a number of different partially ligated intermediates but should finally yield a symmetrical fully ligated enzyme/ligand complex. In the case of the trimeric protein, riboflavin synthase, some ligands form an unexpected protein/ligand complex, even in the presence of a large excess of ligand. Three different bound forms were observed by 19F NMR spectroscopy, and Scatchard-type analysis suggested binding sites of similar affinities. NOESY analysis of the kinetic network revealed that the three bound states exchange with free ligand, but not with each other, thus suggesting that the trimeric enzyme could be asymmetrical. This information permits appropriate precautions to be taken during X-ray structure analysis of riboflavin synthase, which is in progress. Quantitative analysis of the NOESY spectra yielded different rate constants for the different binding sites. For comparison, the monomeric lumazine protein was investigated as an example of a case with simple two-site exchange. For such systems, all kinetic parameters including kon and the dissociation constant can be determined from the NOESY spectrum. The data show that NMR spectroscopy can produce qualitative and quantitative information in cases of nonequivalent binding sites in oligomeric proteins if isolated NMR signals of the different forms can be observed. The technique is not limited to 19F as reporter nucleus.

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Section: Discussionmentioning

confidence: 99%

“…The nature of the nucleophile is unknown. Indirect evidence suggests that it could be a water molecule (Cushman et al, 1993). After deprotonation of the 7-methyl group of the acceptor molecule, the resulting anion 2 could attack C-6 of 3.…”

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NMR Analysis of Site-Specific Ligand Binding in Oligomeric Proteins. Dynamic Studies on the Interaction of Riboflavin Synthase with Trifluoromethyl-Substituted Intermediates

et al. 1996

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“…Thus, 19 F and 13 C NMR studies on lumazine protein in complex with various ligands invariably show single signal sets for the bound ligand, 21,22,26 whereas similar studies with riboflavin synthase show multiple signal sets in line with the fact that the six ligandbinding domains of the enzyme are all topologically non-equivalent. 21,22,27,28 A recent study has also shown that bulky single-site replacements at the Nterminal ligand-binding site of lumazine protein result in major changes of ligand affinity and of the chemical shift values of bound ligands (riboflavin or 6,7-dimethyl-8-ribityllumazine). 18 On the other hand, similar replacements of topologically equivalent residues at the C-terminal domain had hardly any effect on ligand binding and chemical shift values of the bound ligands.…”

Section: The Bound Fluorophorementioning

confidence: 99%

Structure of Lumazine Protein, an Optical Transponder of Luminescent Bacteria

Chatwell

Illarionova

Illarionov

et al. 2008

Journal of Molecular Biology

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“…Cushman and his coworkers have prepared several trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine, which are thought to mimic reaction-pathway intermediates of riboflavin synthase (Cushman et al, 1991(Cushman et al, , 1992(Cushman et al, , 1993. "IF-NMR studies with these analogs suggested that the trimeric riboflavin synthase of B. subtilis has a non-symmetric structure conducive to somewhat different environments for ligands bound at the different subunits of the homotrimer.…”

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Cloning, Sequencing, Mapping and Hyperexpression of the ribC Gene Coding for Riboflavin Synthase of Escherichia coli

Eberhardt

Richter

Gimbel³

et al. 1996

European Journal of Biochemistry

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The gene coding for riboflavin synthase of Escherichia coli has been cloned by marker rescue on a 6-kb fragment that has been sequenced. The riboflavin synthase gene is identical to the ribC locus and codes for a protein of 21 3 amino acids with a m i s s of 23.4 kDa. It was mapped to a position at 37.5 min on the physical map of the E. coli chromosome. The 3' end of the ribC gene is directly adjacent to the cfa gene, which codes for cyclopropane-fatty-acid synthase. This gene is followed by two open reading frames designated ydhC and ydhB, which are predicted to code for putative proteins with 403 amino acids and 310 amino acids, respectively. The gene ydhhc is similar to genes coding for resistance against various antibiotics (cmlA, bcr) and probably codes for a transmembrane protein.The protein specified by ydhB shows sequence similarity to a large family of DNA-binding proteins and probably represents a helix-turn-helix protein. The ydhB gene is directly adjacent to the regulatory gene purR. A 288-bp segment of the cfa gene has earlier been mapped incorrectly to a position adjacent to greA at 67 min. The ribC gene was hyperexpressed in recombinant E. coli strains to a level of about 30 o/c of cellular protein.The protein was purified to homogeneity by chromatography. The specific activity was 26000 nmol . mg . h-'. The protein sediments at a velocity of . F~~) = 3.8 S. Sedimentation-equilibrium centrifugation indicated a molecular mass of 70 kDa, consistent with a trimer structure. The primary structure of riboflavin synthase is characterized by internal sequence similarity (25 identical amino acids in the Cterminal and N-terminal parts) suggesting two structurally similar folding domains.

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Fluorine-19 NMR studies of the mechanism of riboflavin synthase. Synthesis of 6-(trifluoromethyl)-8-(D-ribityl)lumazine and derivatives

Cited by 23 publications

References 0 publications

NMR Analysis of Site-Specific Ligand Binding in Oligomeric Proteins. Dynamic Studies on the Interaction of Riboflavin Synthase with Trifluoromethyl-Substituted Intermediates

NMR Analysis of Site-Specific Ligand Binding in Oligomeric Proteins. Dynamic Studies on the Interaction of Riboflavin Synthase with Trifluoromethyl-Substituted Intermediates

Structure of Lumazine Protein, an Optical Transponder of Luminescent Bacteria

Cloning, Sequencing, Mapping and Hyperexpression of the ribC Gene Coding for Riboflavin Synthase of Escherichia coli

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