Yu_2021_Angew.Chem.Int.Ed.Engl_60_3679

A mirror-image strategy, that is, symmetry analysis of the substrate-binding pocket, was applied to identify two key amino acid residues W170 and V198 that possibly modulate the enantiopreference of a nitrilase from Synechocystis sp. PCC6803 towards 3-isobutyl glutaronitrile (1a). Exchange of these two residues resulted in the enantiopreference inversion (S, 90% ee to R, 47% ee). By further reshaping the substrate-binding pocket via routine site-saturation and combinatorial mutagenesis, variant E8 with higher activity and stereoselectivity (99% ee, R) was obtained. The mutant enzyme was applied in the preparation of optically pure (R)-3-isobutyl-4-cyanobutanoic acid ((R)-2a) and showed similar stereopreference inversion towards a series of 3-substituted glutaronitriles. This study may offer a general strategy to switch the stereopreference of other nitrilases and other enzymes toward the desymmetric reactions of prochiral substrates with two identical reactive functional groups.