المؤلفون: Lin, Kuan‐Hung, Lyu, Syue‐Yi, Yeh, Hsien‐Wei, Li, Yi‐Shan, Hsu, Ning‐Shian, Huang, Chun‐Man, Wang, Yung‐Lin, Shih, Hao‐Wei, Wang, Zhe‐Chong, Wu, Chang‐Jer, Li, Tsung‐Lin

المصدر: Protein Science. 29(7)

مصطلحات موضوعية: Inorganic Chemistry, Chemical Sciences, Amycolatopsis, Bacterial Proteins, Catalytic Domain, Flavins, Mixed Function Oxygenases, Oxidation-Reduction, Baeyer-Villiger oxidation, flavin mononucleotide, mandelate oxidase, monooxygenase, oxidative decarboxylation, Biochemistry and Cell Biology, Computation Theory and Mathematics, Other Information and Computing Sciences, Biophysics, Biochemistry and cell biology, Medicinal and biomolecular chemistry

الوصف: Though reactive flavin-N5/C4α-oxide intermediates can be spectroscopically profiled for some flavin-assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition-like complexes, the α-ketoacid…N5-FMNox complex (I), the FMNox -N5-aloxyl-C'α- -C4α+ zwitterion (II), and the FMN-N5-ethenol-N5-C4α-epoxide (III), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMNox an alternative reaction center can polarize to an ylide-like mesomer in the active site. In contrast, four distinct flavin-C4α-oxide adducts (IV-VII) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer-Villiger/peroxide-assisted decarboxylation, and epoxidation reactions. In conjunction with stopped-flow kinetics, the multifaceted flavin-dependent reaction continuum is physically dissected at molecular level for the first time.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/8xw3d695Test

المؤلفون: Lyu, Syue-Yi, Lin, Kuan-Hung, Yeh, Hsien-Wei, Li, Yi-Shan, Huang, Chun-Man, Wang, Yung-Lin, Shih, Hao-Wei, Hsu, Ning-Shian, Wu, Chang-Jer, Li, Tsung-Lin

المصدر: Acta Crystallographica Section D, Structural Biology. 75(10)

مصطلحات موضوعية: Actinobacteria, Alcohol Oxidoreductases, Amycolatopsis, Binding Sites, Cloning, Molecular, Escherichia coli, Flavin Mononucleotide, Kinetics, Mutation, Oxidation-Reduction, Substrate Specificity, electrophilic, nucleophilic duality, alpha-hydroxyacid oxidases, flavin mononucleotide, oxidative decarboxylation, monooxygenase, p-hydroxymandelate oxidase, electrophilic/nucleophilic duality, α-hydroxyacid oxidases, Physical Sciences, Chemical Sciences, Biological Sciences, Biophysics

الوصف: The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/5jx70520Test

المؤلفون: Yeh, Hsien-Wei, Lin, Kuan-Hung, Lyu, Syue-Yi, Li, Yi-Shan, Huang, Chun-Man, Wang, Yung-Lin, Shih, Hao-Wei, Hsu, Ning-Shian, Wu, Chang-Jer, Li, Tsung-Lin

المصدر: Acta Crystallographica Section D, Structural Biology. 75(8)

مصطلحات موضوعية: Alcohol Oxidoreductases, Binding Sites, Cloning, Molecular, Crystallography, X-Ray, Decarboxylation, Escherichia coli, Flavin Mononucleotide, Kinetics, Mandelic Acids, Mutagenesis, Site-Directed, Oxidation-Reduction, Protein Binding, Substrate Specificity, mandelate oxidase, flavin mononucleotide, oxidative decarboxylation, hydride transfer, alpha-hydroxyacids, α-hydroxyacids, Physical Sciences, Chemical Sciences, Biological Sciences, Biophysics

الوصف: p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallography, were exploited to reach these conclusions and provide additional insights.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/0ts55916Test

المؤلفون: Lin, Kuan-Hung, Lyu, Syue-Yi, Yeh, Hsien-Wei, Li, Yi-Shan, Hsu, Ning-Shian, Huang, Chun-Man, Wang, Yung-Lin, Shih, Hao-Wei, Wang, Zhe-Chong, Wu, Chang-Jer, Li, Tsung-Lin

المصدر: Protein science : a publication of the Protein Society, vol 29, iss 7

مصطلحات موضوعية: Biophysics, Computation Theory and Mathematics, Baeyer-Villiger oxidation, Mixed Function Oxygenases, flavin mononucleotide, Bacterial Proteins, Flavins, Catalytic Domain, oxidative decarboxylation, Biochemistry and Cell Biology, monooxygenase, Other Information and Computing Sciences, Oxidation-Reduction, Amycolatopsis, mandelate oxidase

الوصف: Though reactive flavin-N5/C4α-oxide intermediates can be spectroscopically profiled for some flavin-assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition-like complexes, the α-ketoacid…N5-FMNox complex (I), the FMNox -N5-aloxyl-C'α- -C4α+ zwitterion (II), and the FMN-N5-ethenol-N5-C4α-epoxide (III), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMNox an alternative reaction center can polarize to an ylide-like mesomer in the active site. In contrast, four distinct flavin-C4α-oxide adducts (IV-VII) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer-Villiger/peroxide-assisted decarboxylation, and epoxidation reactions. In conjunction with stopped-flow kinetics, the multifaceted flavin-dependent reaction continuum is physically dissected at molecular level for the first time.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=od_______325::a85afd46ca1a2bb0e25b61a56b7e6e61Test
https://escholarship.org/uc/item/8xw3d695Test