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The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level

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 react...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778850/
https://www.ncbi.nlm.nih.gov/pubmed/31588923
http://dx.doi.org/10.1107/S2059798319011938
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author 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
author_facet 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
author_sort Lyu, Syue-Yi
collection PubMed
description 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 (FMN(ox)) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMN(ox) to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMN(red) dead-end adduct. In four-electron oxidation, an intramolecular disproportion­ation reaction via an N5-alkanol-FMN(red) C′α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMN(ox) cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.
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spelling pubmed-67788502019-10-21 The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level 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 Acta Crystallogr D Struct Biol Research Papers 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 (FMN(ox)) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMN(ox) to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMN(red) dead-end adduct. In four-electron oxidation, an intramolecular disproportion­ation reaction via an N5-alkanol-FMN(red) C′α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMN(ox) cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism. International Union of Crystallography 2019-09-24 /pmc/articles/PMC6778850/ /pubmed/31588923 http://dx.doi.org/10.1107/S2059798319011938 Text en © Lyu et al. 2019 http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.http://creativecommons.org/licenses/by/4.0/
spellingShingle Research Papers
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
The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level
title The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level
title_full The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level
title_fullStr The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level
title_full_unstemmed The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level
title_short The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level
title_sort flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778850/
https://www.ncbi.nlm.nih.gov/pubmed/31588923
http://dx.doi.org/10.1107/S2059798319011938
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