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.
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Funding
This study was funded by the Huzhou Scientific and Technological Project (2022GZ56) and the Education of Zhejiang Province of China (Y202248484).
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WNB and HFP contributed to the study conception and design. Material preparation, data collection and analysis were performed by HXL, JFY and RLZ. The first draft of the manuscript was written by HXL and all authors commented on previous version of the manuscript. All authors read and approved the final manuscript.
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Liao, H., Pan, H., Yao, J. et al. Essential amino acid residues and catalytic mechanism of trans-epoxysuccinate hydrolase for production of meso-tartaric acid. Biotechnol Lett (2024). https://doi.org/10.1007/s10529-024-03490-3
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DOI: https://doi.org/10.1007/s10529-024-03490-3