QM/MM study of the catalytic reaction of potato epoxide hydrolase

Epoxide hydrolases are essential enzymes that convert epoxides into 1,2-diols, contributing to detoxification, metabolism, and signaling in a wide range of organisms. In this study, we employed hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to investigate the catalytic mechanism of potato epoxide hydrolase 1 (StEH1), specifically focusing on the hydrolysis of trans-stilbene oxide into chiral diols. Based on the crystal structure of StEH1 (PDB ID: 2CJP), we modeled enzyme-substrate interactions and examined the roles of the catalytic triad (Asp105, His300, and Asp265), two tyrosine residues (Tyr154 and Tyr235) involved in substrate polarization, and a crystallographic water molecule acting as the hydrolytic nucleophile. Our QM/MM calculations revealed a three-step reaction mechanism: alkylation, dealkylation, and proton relay. We also determined the optimal protonation states of several active-site residues, particularly His104 and His300, to ensure an accurate mechanistic picture. These results clarify previously debated aspects of the mechanism, such as the protonation state of His300 and the function of the tyrosine residues, and provide new insights into substrate specificity and offer valuable information for future efforts in inhibitor design and enzyme engineering.