Protein Engineering for Enhanced Acyltransferase Activity, Substrate Scope, and Selectivity of the Mycobacterium smegmatis Acyltransferase MsAcT

The highly efficient and versatile acyltransferase MsAcT from Mycobacterium smegmatis catalyzes aqueous acyl transfer reactions, enabling applications in environmentally friendly processes and enzyme cascades. We rationally designed several variants with up to 30-fold increased acyl transfer to hydrolysis ratios while mostly retaining initial activity. Variants exhibiting broader acyl-donor substrate scope and higher or inverted enantioselectivity were also designed. Alterations of the catalytic His-Asp pair decreased the activation of hydrolytic water, thereby increasing acyl transfer to hydrolysis ratios. This study demonstrates that targeting the disruption of water networks and manipulating the activation of nucleophiles are promising strategies for engineering promiscuous acyltransferase activities.