Search

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver for the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of the lactonase Sso Pox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being > 1,000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, ~1,000,000-fold and beyond, since some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops, as revealed by a suite of crystal structures. This suggests that specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining an enzyme activity profile., Competing Interests: CONFLICT OF INTEREST STATEMENT DD is a shareholder and CEO of Gene&GreenTK. EC is a shareholder and a co-founder of Gene&GreenTK. PJ, RB and EC report receiving personal fees from Gene&GreenTK during the study. MHE, DD and EC have filed the patent EP3941206. MHE and CB have a patent WO2020185861A1. MHE is a co-founder, a former CEO and equity holder of Gene&GreenTK, a company that holds the license to WO2014167140 A1, FR 3068989 A1, FR 19/02834. MHE received fees from Gene&GreenTK. MHE interests with Gene&GreenTK have been reviewed and managed by the University of Minnesota in accordance with its Conflict-of-Interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.