Bidirectional Regulation of Nitrilase Reaction Specificity by Tuning the Characteristic Distances Between Key Residues and Substrate

Nitrilases are a class of enzymesthat hydrolyze nitriles to carboxylicacids and ammonia. However, as research has progressed, the hydrationactivities that convert nitriles to amides are also found in nitrilasesfrom different sources, which result in difficulties for high-purityproduction of carboxylic acids and meanwhile endow the enzyme withpotential for valuable amides biosynthesis. In this study, the distancebetween the residues Cys and Glu in the catalytic triad (D (C-E)), as well as that between the cyano group ofnitrile substrates and Glu (D (CN-E)) were determined to coaffect the reaction pathway. A strategy ofswitching the characteristic distance "D (C-E)-D (CN-E)"of nitrilase was proposed to regulate its reaction specificity. Bycomputer-aided in silico analysis and mutagenesis of hotspot residues,a triple mutant K200R/R224W/N246V and a hexamutant A87M/I91P/I136Q/M164V/R224S/V226Rwere obtained with strict hydrolysis and hydration activity, respectively.With phenylacetonitrile as a substrate, the content of phenylaceticacid produced by the triple mutant was increased from 50.9% to 98.5%,while that of phenylacetamide was increased from 49.1% to 96.4% bythe hexamutant. The established computational design strategy providedimportant guidance to engineer nitrilases with inverse reaction specificityand meanwhile broadened their application fields.