Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase

Michal Biler, Rory M Crean, Anna K Schweiger, Robert Kourist, Shina Caroline Lynn Kamerlin

Research output: Contribution to journalArticle

Abstract

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 date, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond and metadynamics 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 nonconverted 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. Finally, our results indicate a switch from preferential cleavage of the pro-(R) to the pro-(S) carboxylate group in the CLG-IPL variant of AMDase for all substrates studied. This appears to be due to the emergence of a new hydrophobic pocket generated by the insertion of the six amino acid substitutions, into which the pro-(S) carboxylate binds. Our results 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.

Original languageEnglish
JournalJournal of the American Chemical Society
DOIs
Publication statusE-pub ahead of print - 12 Nov 2020
Externally publishedYes

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