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

Abstract

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)- dependent enzyme that oxidizes mandelate to benzoylformate. How the FMNdependent 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 activesite geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a fourelectron 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.