biostudies-literatureOtten RETH Zürich FoundationU.S. Department of EnergySwiss National Science FoundationHoward Hughes Medical InstituteBioXFEL Science and Technology CenterDamon Runyon Cancer Research FoundationNIH HHSNIGMS NIH HHS1442-1446https://www.ebi.ac.uk/biostudies/studies/S-EPMC9616100biostudies-literatureUnknown370(6523)The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of nuclear magnetic resonance, crystallography, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and accelerate this transformation by nearly nine orders of magnitude. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-staScience (New York, N.Y.)How directed evolution reshapes the energy landscape in an enzyme to boost catalysis.PMC9616100P41 GM1033931231306DE-FG02-05ER15699P30 GM124169S10 OD021832DRG-2114-12T32 GM135126Padua RAPNguyen VSui SCohen AEBunzel HAOtten RKern DPitsawong WPerry SLHilvert DPatterson MfalseHow directed evolution reshapes the energy landscape in an enzyme to boost catalysis.The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of nuclear magnetic resonance, crystallography, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and accelerate this transformation by nearly nine orders of magnitude. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-sta2020-01-01T00:00:00Z2020 Dec2026-05-28T00:37:10.12Z2025-04-06T21:03:48.735ZS-EPMC96161003321428910.1126/science.abd3623