Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release
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.
Authors
- Roberto A. Chica (ORCID: https://orcid.org/0000-0003-3789-9841)
- Rojo V. Rakotoharisoa (ORCID: https://orcid.org/0000-0002-6901-4529)
- Sílvia Osuna (ORCID: https://orcid.org/0000-0003-3657-6469)
- Cindy Klaus (ORCID: https://orcid.org/0000-0001-5977-1158)
- Janet Sánchez (ORCID: https://orcid.org/0009-0003-5258-4511)
- Pooja Asthana (ORCID: https://orcid.org/0000-0002-3417-9198)
- James S. Fraser (ORCID: https://orcid.org/0000-0002-5080-2859)
- Niayesh Zarifi (ORCID: https://orcid.org/0000-0003-4748-7082)
- Hiva Doustmohammadi (ORCID: https://orcid.org/0000-0003-4941-7782)
- Serena E. Hunt (ORCID: https://orcid.org/0000-0001-7258-6440)
Institutions
- Institució Catalana de Recerca i Estudis Avançats (ES)
- University of Ottawa (CA)
- University of California, San Francisco (US)
- Universitat de Girona (ES)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-09-30
- DOI
- https://doi.org/10.1038/s41467-025-63802-7
- Citations
- 6
- Primary Topic
- Biochemical and Molecular Research
- Type
- article
- Field-Weighted Citation Impact
- 4.84
Funders
- U.S. Department of Energy
- Alliance de recherche numérique du Canada
- National Institutes of Health
- Natural Sciences and Engineering Research Council of Canada
- National Institute of General Medical Sciences
- Basic Energy Sciences
- Biological and Environmental Research
- Lawrence Berkeley National Laboratory