Computational analysis of hydride and proton transfer steps in l-lactate oxidase based on QM and QM–MM methods

Abstract

L-Lactate oxidase (LOX) is a member of α‑hydroxy acid-oxidizing enzymes, and it catalyzes oxidation of l-lactate into pyruvate using flavin mononucleotide (FMN) as cofactor. Based on the proposed mechanism, l-lactate is oxidized as a result of deprotonation of its α-C by flavin through hydride transfer in the reductive half-reaction. In the subsequent oxidative half-reaction, the reduced flavin is reoxidized by molecular oxygen. The crystal structures of the enzyme complexed with l-lactate and pyruvate provided further clues related to binding and catalysis. LOX is involved in the metabolism of l-lactate which is used as carbon and energy source in some pathogenic bacterial species. Elucidation of the enzyme mechanism of LOX is important since novel pharmaceuticals can be developed against bacterial infections. In this computational study, we analyzed the mechanism of the reductive half-reaction of LOX using QM–MM (ONIOM) and QM methods though model enzyme-reactant, transition state, and enzyme-product complexes. The models were formulated using the crystal structure of LOX complexed with l-lactate. Our calculations showed that the reductive half-reaction proceeds first with a barrierless proton transfer process from α‑hydroxyl group of l-lactate to His265, then a hydride ion transfers from α-C of l-lactate to N5 position of isoalloxazine ring of flavin. It was also found that the active site residues such as Asp174, Tyr146, Tyr40, Arg268, and Arg181 have important functions in the binding and catalysis of substrate through H-bonding interactions.