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

Autor: Liao H; School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, China., Pan H; Huzhou College, Huzhou, 313000, China., Yao J; School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, China., Zhu R; School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, China., Bao W; School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, China. wennabao@163.com.; Zhejiang Provincial Key Laboratory for Chemical and Biological Processing Technology of Farm Products, Hangzhou, 310023, China. wennabao@163.com.
Jazyk: angličtina
Zdroj: Biotechnology letters [Biotechnol Lett] 2024 Oct; Vol. 46 (5), pp. 739-749. Date of Electronic Publication: 2024 May 13.
DOI: 10.1007/s10529-024-03490-3
Abstrakt: 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 Zn 2+ , Mn 2+ 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 18 O-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.
(© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)
Databáze: MEDLINE
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