[Semi-rational design of a nitrilase for efficient synthesis of 2-chloronicotinic acid]

2-chloronicotinic acid as an important building block has been widely used in pharmaceutical and pesticide synthesis. Chemical synthesis of 2-chloronicotinic acid suffers from poor specificity, high waste salt, and low yields. The synthesis of 2-chloronicotinic acid from 2-chloronicotinonitrile via nitrilase-catalyzed hydrolysis demonstrates high atom economy and environmental friendliness, representing a promising route for industrial production of 2-chloronicotinic acid. However, the o-substituted Cl of the pyridine ring causes a strong electronic effect and steric hindrance, resulting in low activity and catalytic efficiency. Focusing on the nitrilase GiNIT_M11, we employed semi-rational design to modify distal amino acid residues from the active center for efficient synthesis of 2-chloronicotinic acid. Two distal residues affecting activity, W66 and T107, were identified. A double mutant GiNIT_M11^W66F/T107S was constructed by iterative mutation, demonstrating 78.6% higher activity and 146.1% higher catalytic efficiency than GiNIT_M11. Molecular dynamics simulations revealed that the enhanced catalytic performance of GiNIT_M11^W66F/T107S resulted from synergistic effects of shortened nucleophilic attack, enhanced hydrogen bonding networks, and reduced hydrophobic environment and steric hindrance. This study is of great significance for elucidating the structure-function relationship of nitrilase and developing the industrial production of 2-chloronicotinic acid via nitrilase.