Biochemical and structural explorations of α-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 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.

Dual Activities of Oxidation and Oxidative Decarboxylation by Flavoenzymes

“…The mechanism of decarboxylation of α‐keto‐acids by Y128F of Hmo was thus proposed to occur via an attack of C4a−OOH intermediate on the α‐keto carbon and the oxidative decarboxylation mechanism is thought to be dependent on physiochemical effects and binding geometry of substrate or intermediate in the active site environment [27] . The absence of the p ‐OH group in the Y128F variant may introduce some space allowing access of O 2 to the C4a redox‐active center [27b] . However, Y152F in LaMO does not catalyze pure oxidative decarboxylation, but also 20–30 % increase of l ‐lactate oxidation [28b] while Y146 variants in LaOX cannot catalyze the oxidative decarboxylation.…”

Flavin Mononucleotide-Dependent l-Lactate Dehydrogenases: Expanding the Toolbox of Enzymes for l-Lactate Biosensors

“…Apart from LOx, the HAOx family contains glycolate oxidase (GLO), long- or medium-chain 2-hydroxy acid oxidase (HAO , ), flavocytochrome b 2 (FCB2 , ), L-lactate monooxygenase (LMO), mandelate dehydrogenase (MDH), 4-hydroxymandelate oxidase (HMO), glycolate dehydrogenase (GLDH), and membrane-bound L-lactate dehydrogenase (mLDH , ). Despite most of these different HAOx members being known since at least the 1990s, most of the individual groups are still poorly characterized with only 1–3 sequences studied experimentally.…”