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. 2024 Oct;46(5):739-749.
doi: 10.1007/s10529-024-03490-3. Epub 2024 May 13.

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

Hongxiu Liao #  1 , Haifeng Pan #  2 , Jinfeng Yao  1 , Ronglin Zhu  1 , Wenna Bao  3   4
Affiliations

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

Hongxiu Liao et al. Biotechnol Lett. 2024 Oct.

Abstract

Objectives: 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.

Results: 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 3.50 mM and a kcat 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.

Conclusions: 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.

Keywords: Meso-tartaric acid; Pseudomonas koreensis; Trans-epoxysuccinate hydrolase; Catalytic mechanism.

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