Biochemical and structural explorations of α‐hydroxyacid oxidases reveal a four‐electron oxidative decarboxylation reaction

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...

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Bibliographic Details
Published in:Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 2019-08, Vol.75 (8), p.733-742
Main Authors: 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
Format: Article
Language:English
Subjects:
Alcohol Oxidoreductases - chemistry
Alcohol Oxidoreductases - genetics
Binding Sites
Biochemistry
Cloning, Molecular - methods
Crystal structure
Crystallography
Crystallography, X-Ray - methods
Decarboxylation
Electrons
Enantiomers
Escherichia coli - genetics
Flavin mononucleotide
Flavin Mononucleotide - metabolism
hydride transfer
Kinetics
mandelate oxidase
Mandelic Acids - metabolism
Mutagenesis, Site-Directed
Oxidation
Oxidation-Reduction
oxidative decarboxylation
Protein Binding
Reaction kinetics
Research Papers
Substrate inhibition
Substrate Specificity
α‐hydroxyacids
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Summary: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. Structural and enzymological explorations of α‐hydroxyacid oxidases uncover new flavin mononucleotide‐mediated reactions and intermediates.
ISSN:2059-7983
0907-4449
2059-7983
1399-0047
DOI:10.1107/S2059798319009574