Kemp elimination catalysts by computational enzyme design

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination—a model reaction for proton transfer from carbon—with measured rate enhancements of up to 105 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in kcat/Km (kcat/Km of 2,600 M-1s-1 and kcat/kuncat of >106). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future. The design of enzymes able to catalyse re-actions that are not catalysed by natural biocatalysts is a tremendous challenge for computational protein design. Röthlisberger et al. now report using computational protein design to generate eight novel enzymes able to catalyse the Kemp elimination — a model reaction for proton transfer from carbon. The activity of the designed enzymes was enhanced by directed in vitro evolution, thereby demonstrating a powerful strategy for the creation of novel enzymes. A computational protein design was used to generate eight enzymes that were able to catalyse the Kemp elimination, a model reaction for proton transfer from carbon. Directed evolution was used to enhance the catalytic activity of the designed enzymes, demonstrating that the combination of computational protein design and directed evolution is a highly effective strategy to create novel enzymes.