Identification of key residues modulating the stereoselectivity of nitrile hydratase toward rac‐mandelonitrile by semi‐rational engineering

Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high‐value chiral chemicals is often time‐, money‐, and resource‐intensive; traditional semi‐rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot‐spot residues. In this study, through ligand‐protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity toward rac‐mandelonitrile (MAN). All the four selected residues had numerous collisions with rac‐MAN. Five mutants significantly shifting stereoselectivity towards (S)‐MAN were obtained from site‐saturation mutagenesis, one of them, at position βPhe37, exhibiting efficient production of (S)‐MAN with 96.8% eep, was isolated and further analyzed. The increased pulling force observed during SMD simulation was found to be in good coincidence with the formation of hydrogen bonds between (R)‐MAN and residue βHis37. (R)‐MAN had to break these barriers to enter the active site of nitrile hydratase and S selectivity was thus improved. The results indicated that combining steered molecular dynamics simulation with a traditional semi‐rational design significantly reduced the select range of hot‐spot residues for the evolution of NHase stereoselectivity, which could serve as an alternative for the modulation of enzyme stereoselectivity.