Inverting the stereoselectivity of nitrilase toward racemic isobutylsuccinonitrile via modulating the key residues in the substrate binding pocket

A "polarity scanning" strategy was successfully performed in the present study to identify the critical residues for inverting the stereoselectivity of R-selective nitrilase as the product with S-type was the expected one. Mutant W187N exhibiting distinct stereoselectivity was generated with S-configuration (92.3 %, e.e). A combined mutant W187N/S189N demonstrated a further stereoselectivity improvement with S-selectivity (98.1 %, e.e) was finally acquired when the combination mutation was carried out regarding to site Trp187 and Ser189. Steered molecular dynamic (SMD) stimulation results indicated that the orientation of reacted beta-cyano group in the substrate with distinct configurations switched dramatically, which resulted in the inverted stereoselectivity of nitrilase. The present study would not only deepen the theoretical basis for the kinetic resolution of dinitrile substrates by nitrilase, but also provide a case for other enzymes to synthesize pharmaceutical and chemical intermediates with enantio-complementarity.