Structure-guided protein design of fluoroacetate dehalogenase for kinetic resolution of rac-2-bromobutyric acid

Abstract R-2-Bromobutyric acid is a very important intermediate for the synthesis of agrochemicals and pharmaceuticals. Bioresolution of rac-2-bromobutyric acid (rac-2-BBA) provides a promising process for R-2-bromobutyric acid (R-2-BBA) production. The fluoroacetate dehalogenase (FAcD) has been always studied in the defluorination process. We found that FAcD RPA1163 showed detectable activity but no enantioselectivity towards rac-2-BBA. The iterative saturation mutagenesis (ISM) of FAcD RPA1163 resulted in a mutant H155V/W156R/Y219M, which catalyzed the kinetic resolution of rac-2-BBA to produce R-2-BBA with enhanced activity and enantioselectivity (99.3% ee). The high preference for S-2-bromobutyric acid (S-2-BBA) is of synthetic value. Molecular docking analysis indicated that the H155V/W156R/Y219M mutation reduced steric hindrance and broadened the halide pocket. It is not only the steric hindrance but also the electrostatic environment that has an effect on the activity and enantioselectivity.