Rational design of the substrate binding pocket in epoxide hydrolase for enhanced enantioselectivity towards chiral ortho-methylstyrene oxide

Epoxide hydrolases are crucial enzymes for the stereospecific hydrolysis of racemic epoxides, serving as biocatalysts for synthesizing optically pure epoxides. The (S)-ortho-methylstyrene oxide (oMSO) is a valuable chiral building block in asymmetric organic transformations. Our lab previously elucidated the crystal structure of AuEH2, an epoxide hydrolase from Aspergillus usamii. Here, we conducted saturation mutagenesis on hotspots in the AuEH2 substrate binding pocket (SBP), creating a library of 181 single-site mutations. Screening with racoMSO revealed a mutational landscape of enzymatic activity and E value. Using an innovative sequence-activity relationship (innov'SAR) machine learning approach, a double mutant AuEH2A214V/S247Q was identified with obviously improvements enantiomeric ratio (E value= 28.3) for (R)-oMSO that was increased 7.9-fold over wild type. Using E. coli/Aueh2A214V/S247Q at 10 mg/mL, we dynamically resolved 20 mmol/L rac-oMSO at 25 degrees C, yielding (S)-oMSO with 96.7 % ees and 1,2-diol (R)-oMPED with 81.0 % eep. Molecular dynamics (MD) simulations indicated that changes in SBP residue interactions and non-covalent interactions with (R)-oMSO contributed to the increased activity and enantioselectivity of AuEH2A214V/S247Q.