Abstract
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.
Highlights
Introduction pHydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate
It has been hypothesized that the H2O2 generated at the active site of lactate monooxygenase (LMO) acts on pyruvate to form acetate by H2O2-mediated oxidative decarboxylation because the dissociation of pyruvate is a slow step (Giegel et al, 1990; Lopalco et al, 2016)
To investigate the mechanism of the four-electron oxidative decarboxylation reaction catalyzed by the hydroxymandelate oxidase (Hmo) single mutant Y128F, we first solved crystal structures of the Y128F mutant in complex with different ligands such as (S)-mandelate, (S)-2-phenylpropionate, benzoylformate, benzaldehyde and benzoate
Summary
Introduction pHydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. To the best of our knowledge, this is the second example after lactate monooxygenase (LMO) of an enzyme that performs a ThDP/PLP/NADPHindependent oxidative decarboxylation reaction at the expense of one molecule of O2 with the concomitant production of CO2 and H2O (Ghisla & Massey, 1989). It has been hypothesized that the H2O2 generated at the active site of LMO acts on pyruvate to form acetate by H2O2-mediated oxidative decarboxylation because the dissociation of pyruvate is a slow step (Giegel et al, 1990; Lopalco et al, 2016). Aside from this non-ping-pong kinetic description, how H2O2
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