Complete computational design of high-efficiency Kemp elimination enzymes.

Until now, computationally designed enzymes exhibited low catalytic rates(1-5) and required intensive experimental optimization to reach activity levels observed in comparable natural enzymes(5-9). 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(1-5). Furthermore, designing a residue considered essential in all previous Kemp eliminase designs increases efficiency to more than 10(5) 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 methodology(11), our strategy enables programming stable, high-efficiency, new-to-nature enzymes through a minimal experimental effort.