Rational identification of a catalytically promiscuous nitrilase by predicting a unique catalytic triad motif feature through an in silico strategy

A catalytically promiscuous nitrilase (CP-NLase) with unnatural hydration activity is regarded as a promising alternative for use in benzylic amide production compared to the classic nitrile hydratase pathway. However, most nitrilases are high-fidelity enzymes; CP-NLases are rare, and their unnatural activity is relatively low. In this study, a rationally designed in silico strategy was developed to accurately identify CP-NLases with significant hydration activity. A key motif, LNCXE, responsible for the catalytic promiscuity in nitrilases was rationally identified by mimicking active site architectural features from related enzymes in combination with structural analysis. Utilizing it as a molecular probe, six CP-NLases with significant hydration activity were discovered from a collection of 652 putative nitrilases. Among them, a nitrilase (NitPC) from Phytophthora cactorum exhibited the highest promiscuous activity ever reported, producing significant amounts as high as 89.7% mandelamide. By further tuning the steric hindrance in the active pocket, a mutant (NitPC-W167A) with strict hydration activity was successfully obtained, and the amide ratio was improved to 99.8%. NitPC-W167A showed strong substrate tolerance and could completely convert up to 100 mM mandelonitrile into mandelamide. Moreover, NitPC-W167A exhibited significant promiscuous activity toward a series of arylacetonitriles and produced the corresponding amide as the major product. This study provides a practical strategy for the rational identification of CP-NLases and a promising biocatalyst for the efficient synthesis of benzylic amides.