Complete computational design of high-efficiency Kemp elimination enzymes

Summary, in English

Until now, computationally designed enzymes exhibited low catalytic rates^{1, 2, 3, 4–5} and required intensive experimental optimization to reach activity levels observed in comparable natural enzymes^{5, 6, 7, 8–9}. These results exposed limitations in design methodology and suggested critical gaps in our understanding of the fundamentals of biocatalysis^10,11. We present a fully computational workflow for designing efficient enzymes in TIM-barrel folds using backbone fragments from natural proteins and without requiring optimization by mutant-library screening. Three Kemp eliminase designs exhibit efficiencies greater than 2,000 M⁻¹ s⁻¹. The most efficient shows more than 140 mutations from any natural protein, including a novel active site. It exhibits high stability (greater than 85 °C) and remarkable catalytic efficiency (12,700 M⁻¹ s⁻¹) and rate (2.8 s⁻¹), surpassing previous computational designs by two orders of magnitude^{1, 2, 3, 4–5}. Furthermore, designing a residue considered essential in all previous Kemp eliminase designs increases efficiency to more than 10⁵ M⁻¹ s⁻¹ and rate to 30 s⁻¹, achieving catalytic parameters comparable to natural enzymes and challenging fundamental biocatalytic assumptions. By overcoming limitations in design methodology¹¹, our strategy enables programming stable, high-efficiency, new-to-nature enzymes through a minimal experimental effort.

• Author: Dina Listov; Eva Vos; Gyula Hoffka; Shlomo Yakir Hoch; Andrej Berg; Shelly Hamer-Rogotner; Orly Dym; Shina Caroline Lynn Kamerlin; Sarel J. Fleishman
• Department/s: Department of Chemistry
• Publishing year: 2025-07-31
• Language: English
• Pages: 1421-1427
• Publication/Series: Nature
• Volume: 643
• Issue: 8074
• Links: http://dx.doi.org/10.1038/s41586-025-09136-2
• Document type: Journal article
• Publisher: Nature Publishing Group
• Topic: Molecular Biology; Theoretical Chemistry (including Computational Chemistry)
• Status: Published
• ISBN/ISSN/Other: ISSN: 0028-0836