Essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase for production of meso-tartaric acid.

Abstract

This study aimed to discuss the essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase from Pseudomonas koreensis for the production of meso-tartaric acid. The optimum conditions of the enzyme were 45°C and pH 9.0, respectively. It was strongly inhibited by Zn2+, Mn2+ and SDS. Michaelis-Menten enzyme kinetics analysis gave a Km value of 3.50mM and a kcat of 99.75s-1, with an exceptional EE value exceeding 99.9%. Multiple sequence alignment and homology modeling revealed that the enzyme belonged to MhpC superfamily and possessed a typical α/β hydrolase folding structure. Site-directed mutagenesis indicated H34, D104, R105, R108, D128, Y147, H149, W150, Y211, and H272 were important catalytic residues. The 18O-labeling study suggested the enzyme acted via two-step catalytic mechanism. The structure and catalytic mechanism of trans-epoxysuccinate hydrolase were first reported. Ten residues were critical for its catalysis and a two-step mechanism by an Asp-His-Asp catalytic triad was proposed.