Modulation of Nitrile Hydratase Regioselectivity towards Dinitriles by Tailoring the Substrate Binding Pocket Residues

Abstract

The regioselective hydration of dinitriles is one of the most attractive approaches to prepare ω‐cyanocarboxamides or diamides and such regioselectivity is often beyond the capability of chemical catalysts. The use of nitrile hydratase to biotransform dinitriles selectively would be highly desirable. Molecular docking of two aliphatic dinitriles and two aromatic dinitriles into the active site of a nitrile hydratase (NHase) from Rhodococcus rhodochrous J1 allowed the identification of proximal NHase substrate binding pocket residues. Four residues (βLeu48, βPhe51, βTyr68, and βTrp72) were selected for single‐ and double‐point mutations to modulate the NHase regioselectivity towards dinitriles. Several NHase mutants with an altered regioselectivity were obtained, and the best one was Y68T/W72Y. Docking experiments further indicated that the poor binding affinity of aliphatic and aromatic ω‐cyanocarboxamides to the NHase variants resulted in distinct regioselectivity between wild‐type and mutated NHases.