Significantly enhancing the stereoselectivity of a regioselective nitrilase for the production of (S)-3-cyano-5-methylhexanoic acid using an MM/PBSA method.

The present investigation describes the successful molecular modification of a regio- and stereo-specific nitrilase toward rac-ISBN to (S)-CMHA, a critical intermediate in the preparation of optically pure pregabalin. Two hotspots of Trp57 and Val134 were identified based on the classical binding free energy molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation method. Mutants W57F/V134M and W57Y/V134M were successfully obtained with high enantioselectivity (E >300). Furthermore, these two mutants were efficiently capable of kinetic resolution of rac-ISBN to (S)-CMHA, with both exhibiting a high e.e. (>99.9%), as well as conversion ratios of 43.8% and 40.9%, respectively. Docking and molecular dynamics simulation analysis clarified that the underlying mechanisms were related to a DC-S switch and the formation of a hydrogen bond in the active center of nitrilase. The successful utilization of the MM/PBSA method for identifying hotspots that modulate the stereoselectivity in our study could provide guidelines for the molecular modification of nitrilases, and the mutants obtained could be potentially utilized for the industrial preparation of optically pure pregabalin.