Modification of Nitrile Hydratase by Substrate Channel Engineering Regulates Monocyanamide Synthesis

Cyanoacetamides(CAAM) are key precursors for the synthesis of agrochemicals and bioactive organic compounds. The activity of nitrile hydrolase (NHase) on nitrile substrates was greatly improved in our previous studies by optimizing the ribosome binding site and α/β subunit expression balance. However, during the NHase-catalyzed hydration of malononitrile, this increased catalytic activity greatly facilitated the further conversion of the intermediate cyanoacetamide to the by-product malonamide, thus preventing the enrichment of cyanoacetamide. To solve this problem, we designed a mutant library of NHase by substrate channel modification based on the strategy of semi-rational design. Among them, the βM40A mutant showed 7.68% higher regioselectivity than wild-type NHase. Structural and mechanistic analyses showed that the mutant (1) remodeled the substrate binding pocket, (2) narrowed the substrate channel, and (3) increased the metal ion-carbonitrile distance by 1.1 Å. Our results suggest that the enzyme’s regioselectivity for the product can be controlled by rational design of key amino acids in the NHase substrate channel, which provides an effective strategy for enrichment of the monocyanamide product. Graphical