MD and QM/MM studies on long-chain L-α-hydroxy acid oxidase: substrate binding features and oxidation mechanism

Long-chain L-α-hydroxy acid oxidase (LCHAO) is a flavin mononucleotide (FMN)-dependent oxidase that dehydrogenates l-α-hydroxy acids to keto acids. There were two different mechanisms, named as hydride transfer (HT) mechanism and carbanion (CA) mechanism, respectively, proposed about the catalytic process for the FMN-dependent L-α-hydroxy acid oxidases on the basis of biochemical data. However, crystallographic and kinetic studies could not provide enough evidence to prove one of the mechanisms or eliminate the alternative. In the present studies, theoretical computations were carried out to study the molecular mechanism for LCHAO-catalyzed dehydrogenation of L-lactate. Our molecular dynamics (MD) simulations indicated that L-lactate prefers to bind with LCHAO in a hydride transfer mode rather than a carbanion mode. Quantum mechanics/molecular mechanics (QM/MM) calculations were further carried out to obtain the optimized structures of reactants, transition states, and products at the level of ONIOM-EE (B3LYP/6-311++G(d,p)//B3LYP/6-31G(d,p):AMBER). Quantum chemical studies indicated that LCHAO-catalyzed dehydrogenation of L-lactate would be a stepwise catalytic reaction in a hydride transfer mechanism but not a carbanion mechanism. MD simulations, binding free energy calculations, and QM/MM computations were also implemented on the complex between L-lactate and Y129F mutant LCHAO. By comparing the Y129F mutant system with the wild-type system, it was further confirmed that the key residue Tyr129 in the active site of LCHAO would not affect L-lactate's binding to LCHAO but play an important role on the catalytic reaction process through an H-bond interaction.