Structural insight into the stereoselectivity of R-selective nitrilase: reshaping the substrate pocket with an “extend-and-lock” strategy for efficient brivaracetam precursor synthesis

Abstract

Nitrilases capable of stereoselective hydrolysis of 2-substituted succinonitriles are of considerable value for the synthesis of chiral γ-aminobutyric acid derivatives. However, their stereoselective control toward 2-substituted succinonitriles remains poorly understood and represents a significant challenge. Here, we report nitrilase (PgNIT)-catalyzed desymmetrizing hydrolysis of 2-propylsuccinonitrile for production of the brivaracetam precursor (R)-3-cyanohexanoic acid with high conversion (45.05%) and excellent enantioselectivity (99.01% ee) at a high substrate loading (100 g L⁻¹). Guided by structural analysis, an “extend-and-lock” strategy was devised to reconfigure the substrate-binding conformation in two sub-pockets. Comparative binding-region analysis combined with energy-decomposition profiling identified F135 and R199 as key residues regulating the differential binding of (S)- and (R)-enantiomers. Subsequent engineering of the C-interface generated a triple mutant T59D/F135L/R199W that exhibited markedly improved catalytic activity and excellent enantioselectivity towards 2-alkyl substituted succinonitriles. Molecular dynamics simulations revealed that these mutations remodeled pocket topology and conformational dynamics, and uncovered an unexpected role of the C-interface in governing substrate ingress and binding orientation. This work provides mechanistic insight into nitrilase stereocontrol and establishes a generalizable strategy for developing stereoselective nitrilases for the efficient synthesis of chiral γ-aminobutyric acid derivatives.