The Experts below are selected from a list of 276 Experts worldwide ranked by ideXlab platform
A. Bacher - One of the best experts on this subject based on the ideXlab platform.
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the lumazine Synthase Riboflavin Synthase complex shapes and functions of a highly variable enzyme system
FEBS Journal, 2013Co-Authors: Rudolf Ladenstein, A. Bacher, Markus FischerAbstract:The xylene ring of Riboflavin (vitamin B2) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine Synthase and Riboflavin Synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine Synthase subunits and a core of three Riboflavin Synthase subunits, whereas many other bacteria have empty lumazine Synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine Synthases, and the Riboflavin Synthases of Archaea are paralogs of lumazine Synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine Synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.
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O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase
The Journal of organic chemistry, 2012Co-Authors: Arindam Talukdar, A. Bacher, Markus Fischer, Boris Illarionov, Yujie Zhao, Mark CushmanAbstract:Lumazine Synthase catalyzes the penultimate step in the biosynthesis of Riboflavin, while Riboflavin Synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine Synthase and Riboflavin Synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine Synthase and Riboflavin Synthase inhibitors, while the C-nucl...
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mechanistic insights on Riboflavin Synthase inspired by selective binding of the 6 7 dimethyl 8 ribityllumazine exomethylene anion
Journal of the American Chemical Society, 2010Co-Authors: Ryuryun Kim, Markus Fischer, Wolfgang Eisenreich, Boris Illarionov, Mark Cushman, Chan Yong Lee, Monika Joshi, A. BacherAbstract:Riboflavin Synthase catalyzes the transfer of a four-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of Riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of Riboflavin Synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4 + 2 cycloaddition, affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.
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discovery and development of the covalent hydrates of trifluoromethylated pyrazoles as Riboflavin Synthase inhibitors with antibiotic activity against mycobacterium tuberculosis
Journal of Organic Chemistry, 2009Co-Authors: Yujie Zhao, A. Bacher, Markus Fischer, Boris Illarionov, Gunda I Georg, Phillip E Fanwick, Scott G Franzblau, Baojie Wan, Mark CushmanAbstract:A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli Riboflavin Synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](o-tolyl)methanone (18). The hit compound had a kis of 8.7 μM vs. M. tuberculosis Riboflavin Synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and nonreplicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 μM vs. M. tuberculosis replicating phenotype and 48.9 μM...
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biosynthesis of vitamin b2 structure and mechanism of Riboflavin Synthase
Archives of Biochemistry and Biophysics, 2008Co-Authors: Markus Fischer, A. BacherAbstract:The biosynthesis of one Riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. GTP is hydrolytically opened, converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate leads to 6,7-dimethyl-8-ribityllumazine. The final step in the biosynthesis of the vitamin involves the dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by Riboflavin Synthase. The mechanistically unusual reaction involves the transfer of a four-carbon fragment between two identical substrate molecules. The second product, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, is recycled in the biosynthetic pathway by 6,7-dimethyl-8-ribityllumazine Synthase. This article will review structures and reaction mechanisms of Riboflavin Synthases and related proteins up to 2007 and 122 references are cited.
Markus Fischer - One of the best experts on this subject based on the ideXlab platform.
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ral ssBioMed CentBMC Biochemistry Open AcceResearch article Riboflavin Synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments
2016Co-Authors: Markus Fischer, Ann-kathrin Schott, Stefan Gerhardt, Wolfgang Eisenreich, Boris Illarionov, Mark Cushman, Gerald Richter, Kristina Kemter, Richard Feicht, Stefan SteinbacherAbstract:Background: Riboflavin Synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into Riboflavin in the last step of the Riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate Riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin Synthase is therefore a potential target for the development of antiinfective drugs. Results: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to Riboflavin Synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of Riboflavin from 6,7
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the lumazine Synthase Riboflavin Synthase complex shapes and functions of a highly variable enzyme system
FEBS Journal, 2013Co-Authors: Rudolf Ladenstein, A. Bacher, Markus FischerAbstract:The xylene ring of Riboflavin (vitamin B2) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine Synthase and Riboflavin Synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine Synthase subunits and a core of three Riboflavin Synthase subunits, whereas many other bacteria have empty lumazine Synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine Synthases, and the Riboflavin Synthases of Archaea are paralogs of lumazine Synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine Synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.
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O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase
The Journal of organic chemistry, 2012Co-Authors: Arindam Talukdar, A. Bacher, Markus Fischer, Boris Illarionov, Yujie Zhao, Mark CushmanAbstract:Lumazine Synthase catalyzes the penultimate step in the biosynthesis of Riboflavin, while Riboflavin Synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine Synthase and Riboflavin Synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine Synthase and Riboflavin Synthase inhibitors, while the C-nucl...
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mechanistic insights on Riboflavin Synthase inspired by selective binding of the 6 7 dimethyl 8 ribityllumazine exomethylene anion
Journal of the American Chemical Society, 2010Co-Authors: Ryuryun Kim, Markus Fischer, Wolfgang Eisenreich, Boris Illarionov, Mark Cushman, Chan Yong Lee, Monika Joshi, A. BacherAbstract:Riboflavin Synthase catalyzes the transfer of a four-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of Riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of Riboflavin Synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4 + 2 cycloaddition, affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.
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discovery and development of the covalent hydrates of trifluoromethylated pyrazoles as Riboflavin Synthase inhibitors with antibiotic activity against mycobacterium tuberculosis
Journal of Organic Chemistry, 2009Co-Authors: Yujie Zhao, A. Bacher, Markus Fischer, Boris Illarionov, Gunda I Georg, Phillip E Fanwick, Scott G Franzblau, Baojie Wan, Mark CushmanAbstract:A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli Riboflavin Synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](o-tolyl)methanone (18). The hit compound had a kis of 8.7 μM vs. M. tuberculosis Riboflavin Synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and nonreplicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 μM vs. M. tuberculosis replicating phenotype and 48.9 μM...
Mark Cushman - One of the best experts on this subject based on the ideXlab platform.
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ral ssBioMed CentBMC Biochemistry Open AcceResearch article Riboflavin Synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments
2016Co-Authors: Markus Fischer, Ann-kathrin Schott, Stefan Gerhardt, Wolfgang Eisenreich, Boris Illarionov, Mark Cushman, Gerald Richter, Kristina Kemter, Richard Feicht, Stefan SteinbacherAbstract:Background: Riboflavin Synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into Riboflavin in the last step of the Riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate Riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin Synthase is therefore a potential target for the development of antiinfective drugs. Results: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to Riboflavin Synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of Riboflavin from 6,7
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O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase
The Journal of organic chemistry, 2012Co-Authors: Arindam Talukdar, A. Bacher, Markus Fischer, Boris Illarionov, Yujie Zhao, Mark CushmanAbstract:Lumazine Synthase catalyzes the penultimate step in the biosynthesis of Riboflavin, while Riboflavin Synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine Synthase and Riboflavin Synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine Synthase and Riboflavin Synthase inhibitors, while the C-nucl...
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mechanistic insights on Riboflavin Synthase inspired by selective binding of the 6 7 dimethyl 8 ribityllumazine exomethylene anion
Journal of the American Chemical Society, 2010Co-Authors: Ryuryun Kim, Markus Fischer, Wolfgang Eisenreich, Boris Illarionov, Mark Cushman, Chan Yong Lee, Monika Joshi, A. BacherAbstract:Riboflavin Synthase catalyzes the transfer of a four-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of Riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of Riboflavin Synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4 + 2 cycloaddition, affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.
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discovery and development of the covalent hydrates of trifluoromethylated pyrazoles as Riboflavin Synthase inhibitors with antibiotic activity against mycobacterium tuberculosis
Journal of Organic Chemistry, 2009Co-Authors: Yujie Zhao, A. Bacher, Markus Fischer, Boris Illarionov, Gunda I Georg, Phillip E Fanwick, Scott G Franzblau, Baojie Wan, Mark CushmanAbstract:A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli Riboflavin Synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](o-tolyl)methanone (18). The hit compound had a kis of 8.7 μM vs. M. tuberculosis Riboflavin Synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and nonreplicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 μM vs. M. tuberculosis replicating phenotype and 48.9 μM...
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a new series of n 2 4 dioxo 6 d ribitylamino 1 2 3 4 tetrahydropyrimidin 5 yl oxalamic acid derivatives as inhibitors of lumazine Synthase and Riboflavin Synthase design synthesis biochemical evaluation crystallography and mechanistic implications
Journal of Organic Chemistry, 2008Co-Authors: Yanlei Zhang, A. Bacher, Markus Fischer, Rudolf Ladenstein, Boris Illarionov, Guangyi Jin, Ekaterina Morgunova, Mark CushmanAbstract:The penultimate step in the biosynthesis of Riboflavin is catalyzed by lumazine Synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine Synthase-catalyzed reaction were synthesized and evaluated as lumazine Synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine Synthase, Bacillus subtilis lumazine Synthase, and Schizosaccharomyces pombe lumazine Synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli Riboflavin Synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine Synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.
Boris Illarionov - One of the best experts on this subject based on the ideXlab platform.
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ral ssBioMed CentBMC Biochemistry Open AcceResearch article Riboflavin Synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments
2016Co-Authors: Markus Fischer, Ann-kathrin Schott, Stefan Gerhardt, Wolfgang Eisenreich, Boris Illarionov, Mark Cushman, Gerald Richter, Kristina Kemter, Richard Feicht, Stefan SteinbacherAbstract:Background: Riboflavin Synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into Riboflavin in the last step of the Riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate Riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin Synthase is therefore a potential target for the development of antiinfective drugs. Results: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to Riboflavin Synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of Riboflavin from 6,7
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O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine Synthase and Riboflavin Synthase
The Journal of organic chemistry, 2012Co-Authors: Arindam Talukdar, A. Bacher, Markus Fischer, Boris Illarionov, Yujie Zhao, Mark CushmanAbstract:Lumazine Synthase catalyzes the penultimate step in the biosynthesis of Riboflavin, while Riboflavin Synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine Synthase and Riboflavin Synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine Synthase and Riboflavin Synthase inhibitors, while the C-nucl...
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mechanistic insights on Riboflavin Synthase inspired by selective binding of the 6 7 dimethyl 8 ribityllumazine exomethylene anion
Journal of the American Chemical Society, 2010Co-Authors: Ryuryun Kim, Markus Fischer, Wolfgang Eisenreich, Boris Illarionov, Mark Cushman, Chan Yong Lee, Monika Joshi, A. BacherAbstract:Riboflavin Synthase catalyzes the transfer of a four-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of Riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of Riboflavin Synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4 + 2 cycloaddition, affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.
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discovery and development of the covalent hydrates of trifluoromethylated pyrazoles as Riboflavin Synthase inhibitors with antibiotic activity against mycobacterium tuberculosis
Journal of Organic Chemistry, 2009Co-Authors: Yujie Zhao, A. Bacher, Markus Fischer, Boris Illarionov, Gunda I Georg, Phillip E Fanwick, Scott G Franzblau, Baojie Wan, Mark CushmanAbstract:A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli Riboflavin Synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](o-tolyl)methanone (18). The hit compound had a kis of 8.7 μM vs. M. tuberculosis Riboflavin Synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and nonreplicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 μM vs. M. tuberculosis replicating phenotype and 48.9 μM...
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a new series of n 2 4 dioxo 6 d ribitylamino 1 2 3 4 tetrahydropyrimidin 5 yl oxalamic acid derivatives as inhibitors of lumazine Synthase and Riboflavin Synthase design synthesis biochemical evaluation crystallography and mechanistic implications
Journal of Organic Chemistry, 2008Co-Authors: Yanlei Zhang, A. Bacher, Markus Fischer, Rudolf Ladenstein, Boris Illarionov, Guangyi Jin, Ekaterina Morgunova, Mark CushmanAbstract:The penultimate step in the biosynthesis of Riboflavin is catalyzed by lumazine Synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine Synthase-catalyzed reaction were synthesized and evaluated as lumazine Synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine Synthase, Bacillus subtilis lumazine Synthase, and Schizosaccharomyces pombe lumazine Synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli Riboflavin Synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine Synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.
Rudolf Ladenstein - One of the best experts on this subject based on the ideXlab platform.
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the lumazine Synthase Riboflavin Synthase complex shapes and functions of a highly variable enzyme system
FEBS Journal, 2013Co-Authors: Rudolf Ladenstein, A. Bacher, Markus FischerAbstract:The xylene ring of Riboflavin (vitamin B2) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine Synthase and Riboflavin Synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine Synthase subunits and a core of three Riboflavin Synthase subunits, whereas many other bacteria have empty lumazine Synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine Synthases, and the Riboflavin Synthases of Archaea are paralogs of lumazine Synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine Synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.
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a new series of n 2 4 dioxo 6 d ribitylamino 1 2 3 4 tetrahydropyrimidin 5 yl oxalamic acid derivatives as inhibitors of lumazine Synthase and Riboflavin Synthase design synthesis biochemical evaluation crystallography and mechanistic implications
Journal of Organic Chemistry, 2008Co-Authors: Yanlei Zhang, A. Bacher, Markus Fischer, Rudolf Ladenstein, Boris Illarionov, Guangyi Jin, Ekaterina Morgunova, Mark CushmanAbstract:The penultimate step in the biosynthesis of Riboflavin is catalyzed by lumazine Synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine Synthase-catalyzed reaction were synthesized and evaluated as lumazine Synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine Synthase, Bacillus subtilis lumazine Synthase, and Schizosaccharomyces pombe lumazine Synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli Riboflavin Synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine Synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.
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design synthesis and biochemical evaluation of 1 5 6 7 tetrahydro 6 7 dioxo 9 d ribitylaminolumazines bearing alkyl phosphate substituents as inhibitors of lumazine Synthase and Riboflavin Synthase
ChemInform, 2005Co-Authors: Mark Cushman, Markus Fischer, Rudolf Ladenstein, Boris Illarionov, Guangyi Jin, A. BacherAbstract:The last two steps in the biosynthesis of Riboflavin, an essential metabolite that is involved in electron transport, are catalyzed by lumazine Synthase and Riboflavin Synthase. To obtain structural probes and inhibitors of these two enzymes, two ribityllumazinediones bearing alkyl phosphate substituents were synthesized. The synthesis involved the generation of the ribityl side chain, the phosphate side chain, and the lumazine system in protected form, followed by the simultaneous removal of three different types of protecting groups. The products were designed as intermediate analogue inhibitors of lumazine Synthase that would bind to its phosphate-binding site as well as its lumazine binding site. Both compounds were found to be effective inhibitors of Bacillus subtilis lumazine Synthase as well as Escherichia coli Riboflavin Synthase. Molecular modeling of the binding of one of the two compounds provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. In ...
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The structure of the N-terminal domain of Riboflavin Synthase in complex with Riboflavin at 2.6 A resolution
Journal of molecular biology, 2003Co-Authors: Winfried Meining, Sabine Eberhardt, Adelbert Bacher, Rudolf LadensteinAbstract:Riboflavin Synthase of Escherichia coli is a homotrimer with a molecular mass of 70 kDa. The enzyme catalyzes the dismutation of 6,7-dimethyl-8(1'-D-ribityl)-lumazine, affording Riboflavin and 5-am ...
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crystallization and preliminary crystallographic analysis of the recombinant n terminal domain of Riboflavin Synthase
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: A. Bacher, Sabine Eberhardt, Winfried Meining, Rudolf LadensteinAbstract:Riboflavin Synthase catalyzes the final step in the biosynthesis of Riboflavin. Animals and humans lack this enzyme, whereas many bacteria and certain yeasts are absolutely dependent on endogenous Riboflavin synthesis. Riboflavin Synthase is therefore an attractive target for chemotherapy. The N-terminal domain of Riboflavin Synthase forms a dimer in solution and is capable of strongly binding Riboflavin. It can serve as a model for the binding site of the native enzyme. Structural information obtained from this domain at high resolution will be helpful in the determination of the binding mode of Riboflavin and thus for the development of antimicrobial drugs. Here, the crystallization and preliminary crystallographic analysis of the N-terminal domain of Riboflavin Synthase are reported. The crystals belong to the space group C2221, with unit-cell parameters a = 50.3, b = 104.7, c = 85.3 A, α = β = γ = 90°, and diffract to 2.6 A resolution.