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

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 K_m value of 3.50 mM and a k_cat of 99.75 s⁻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.