Ground-State Destabilization Controls the Selectivity of a Cofactor-Free Decarboxylase

Bacterial arylmalonate decarboxylase (AMDase) and evolved variants have become a valuable tool with which to access both enantiomers of a broad range of chiral arylaliphatic acids with high optical purity. Yet, the molecular principles responsible for the substrate scope, activity and selectivity of this enzyme are only poorly understood to this day, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond simulations were performed on wild-type AMDase and variants thereof, to obtain a better understanding of the underlying molecular processes determining reaction outcome. Our results clearly reproduce the experimentally observed substrate scope, and support a mechanism driven by ground-state destabilization of the carboxylate group being cleaved by the enzyme. In addition, our results indicate that, in the case of the non-converted or poorly-converted substrates studied in this work, increased solvent exposure of the active site upon binding of these substrates can disturb the vulnerable network of interactions responsible for facilitating the AMDase-catalyzed cleavage of CO2. Our results thus allow insight into the tight interaction network determining AMDase selectivity, which in turn provides guidance for the identification of target residues for future enzyme engineering.