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Directed evolution of an enantiocomplementary SNArase reveals divergent catalytic features

Year: 2026
Type: article
Abstract: Enzymes that catalyze non-natural C–C bond-forming reactions are powerful tools in asymmetric synthesis, yet reprogramming their active sites to invert stereochemical outcome remains challenging. Building on our recently engineered S N Arase, S N Ar1.3, which performs enantioselective nucleophilic aromatic substitutions with carbon nucleophiles, we now report the evolution of an enantiocomplementary biocatalyst (eS N Ar1.3) that displays enhanced activity and expanded substrate scope. Structural and computational analyses uncover both conserved and divergent features between S N Ar1.3 and eS N Ar1.3. Despite retaining similar electrophile binding poses and a conserved catalytic arginine, the halide-binding pocket of S N Ar1.3 has been abandoned in eS N Ar1.3. Instead, His23 has emerged as a key motif that works with Arg124 to accurately position the nucleophilic substrate. Calculations reveal that Arg124 also plays a crucial role in facilitating halide release during catalysis. Our study demonstrates how evolution can reshape enzyme mechanisms in unforeseen ways, highlighting the importance of exploring diverse trajectories to access new functions.
DOI: 10.1038/s41467-026-76922-5
Authors: Thomas M. Lister, George W. Roberts, Cristina Duran, Guillem Casadevall, Fei Zhao, Alexander A. V. Millman, Igor Larrosa, Sílvia Osuna, Anthony P. Green
Language: English
Open Access status: gold
Estimated APC paid: $6,990
Funders: Diamond Light Source, UK Research and Innovation, Henry Royce Institute, Directorate for Biological Sciences, Engineering and Physical Sciences Research Council, H2020 European Research Council
Awards: G2072291230, G2158237762, Design and Evolution of Photoenzymes for Triplet Energy Transfer Catalysis, G2373750278, EPSRC Centre for Doctoral Training in Integrated Catalysis, G2703599441, G3119637445, G3305536412, Sir Henry Royce Institute - Manchester and NNL Equipment, G4630886971, G504327055, New Horizons in C–H Activation: the ‘Real-World Molecules’ Challenge, G5424142534, G576151491, G5820086322, G6148779978, G6907549827, The Royce: Capitalising on the investment, G708391930, International Centre for Enzyme Design (ICED), Future Biomanufacturing Research Hub, G7445816383, G7514479249, Sir Henry Royce InsStitute - recurrent grant
OpenAlex ID: W7203863016
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