Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release

Niayesh Zarifi, Pooja Asthana, Hiva Doustmohammadi, Cindy Klaus, Janet Sanchez, Serena E. Hunt, Rojo V. Rakotoharisoa, Sílvia Osuna, James S. Fraser and Roberto A. Chica ()
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Niayesh Zarifi: University of Ottawa
Pooja Asthana: University of California, San Francisco
Hiva Doustmohammadi: Universitat de Girona
Cindy Klaus: University of Ottawa
Janet Sanchez: Universitat de Girona
Serena E. Hunt: University of Ottawa
Rojo V. Rakotoharisoa: University of Ottawa
Sílvia Osuna: Universitat de Girona
James S. Fraser: University of California, San Francisco
Roberto A. Chica: University of Ottawa

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract The role of amino-acid residues distant from an enzyme’s active site in facilitating the complete catalytic cycle—including substrate binding, chemical transformation, and product release—remains poorly understood. Here, we investigate how distal mutations promote the catalytic cycle by engineering mutants of three de novo Kemp eliminases containing either active-site or distal mutations identified through directed evolution. Kinetic analyses, X-ray crystallography, and molecular dynamics simulations reveal that while active-site mutations create preorganized catalytic sites for efficient chemical transformation, distal mutations enhance catalysis by facilitating substrate binding and product release through tuning structural dynamics to widen the active-site entrance and reorganize surface loops. These distinct contributions work together to improve overall activity, demonstrating that a well-organized active site, though necessary, is not sufficient for optimal catalysis. Our findings reveal critical roles that distal residues play in shaping the catalytic cycle to enhance efficiency, yielding valuable insights for enzyme design.

Date: 2025
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Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-63802-7

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DOI: 10.1038/s41467-025-63802-7

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