Computational Mechanistic Study of l-Aspartate Oxidase by ONIOM Method

l-Aspartateoxidase (Laspo) is responsible for the oxidationof l-aspartate into iminoaspartate using flavin as a cofactor.During this process flavin is reduced, and it can be reoxidized byeither molecular oxygen or fumarate. The overall fold and the catalyticresidues of Laspo are similar to succinate dehydrogenase and fumaratereductase. On the basis of deuterium kinetic isotope effects as wellas other kinetic and structural data, it is proposed that the enzymecan catalyze the oxidation of l-aspartate through a mechanismsimilar to amino acid oxidases. It is suggested that a proton is removedfrom the alpha-amino group, while a hydride is transferred fromC2 to flavin. It is also suggested that the hydride transfer is arate-limiting step. However, there is still an ambiguity about thestepwise or concerted mechanism of hydride- and proton-transfer steps.In this study, we formulated some computational models to study thehydride-transfer mechanism using the crystal structure of Escherichia coli l-aspartate oxidase incomplexes with succinate. The calculations involved our own N-layeredintegrated molecular orbital and molecular mechanics method, and weevaluated the geometry and energetics of the hydride/proton-transferprocesses while probing the roles of active site residues. Based onthe calculations, it is concluded that proton- and hydride-transfersteps are decoupled, and a stepwise mechanism might be operative asopposed to the concerted one.

Automated summary

l-Aspartateoxidase (Laspo) is an enzyme responsible for oxidizing l-aspartate into iminoaspartate using flavin as a cofactor. The enzyme has a similar fold and catalytic residues to succinate dehydrogenase and fumarate reductase. Based on kinetic and structural data, it is proposed that the enzyme catalyzes the oxidation of l-aspartate through a mechanism similar to amino acid oxidases. Computational models were formulated in this study to investigate the hydride-transfer mechanism, and the calculations suggest a stepwise mechanism rather than a concerted one.