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

preprint
revised on 08.10.2020 and posted on 09.10.2020 by Michal Biler, Rory Crean, Anna K. Schweiger, Robert Kourist, Shina Caroline Lynn Kamerlin

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 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 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. 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.

Funding

Knut and Alice Wallenberg Foundation 2018.0140

Swedish Research Council 2019-03499

Swedish National Infrastructure for Computing 2017/12-11, 2018/2-3 and 2019/2-1

Carl Tryggers Foundation for Scientific Research CTS 19:172

Professorship for Molecular Biotechnology at TU Graz

History

Email Address of Submitting Author

lynn.kamerlin@kemi.uu.se

Institution

Uppsala University

Country

Sweden

ORCID For Submitting Author

0000-0002-3190-1173

Declaration of Conflict of Interest

No conflict of interest.

Version Notes

Version 2.0, revised from initial submission.

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