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.
Highlights
IntroductionGiven that the dissociation of pyruvate from LMO is a slow step, the H2O2 generated at the active site acts on pyruvate to form acetate via oxidative decarboxylation (Giegel et al, 1990; Lopalco et al, 2016)
Biochemical analysis showed that hydroxymandelate oxidase (Hmo) efficiently transforms (S)-mandelate to benzoylformate (Hubbard et al, 2000; Li et al, 2001), while a small peak that emerged at 17.5 min on LC traces in a dose-dependent manner drew our attention [Fig. 2(a)]
In the two-electron oxidation reaction, FMNox is reduced by a hydride directly from an -hydroxyacid; FMNred is instantly oxidized by O2 to yield H2O2
Summary
Given that the dissociation of pyruvate from LMO is a slow step, the H2O2 generated at the active site acts on pyruvate to form acetate via oxidative decarboxylation (Giegel et al, 1990; Lopalco et al, 2016). Aside from this non-ping-pong kinetic description, how H2O2 mediates the oxidative decarboxylation of -ketoacids remains elusive (Choong & Massey, 1980; Ghisla & Massey, 1977; Lockridge et al, 1972; Walsh et al, 1973). A biochemical/structural biology approach renders various liganded structures of the proteins, which in combination deconvolve reactive/unstable Hmo and its mutant-mediated reactions at the molecular level
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