The hottest structure in the PDB. Literally!

Combining in silico design of enzymes with laboratory directed evolution opens new horizons to enhance catalytic function. In this study, we utilized nuclear magnetic resonance, cryogenic and high-temperature X-ray crystallography, and stopped-flow techniques to investigate, at the atomic level, the evolution of an enzyme designed for a simple proton transfer reaction. We demonstrate that directed evolution progressively selected for a narrower transition state ensemble compatible with catalysis, boosting the reaction rate by an astonishing nine orders of magnitude. The mutations acquired during the directed evolution process enabled considerable conformational adjustments, such as high-energy backbone shifts. These coordinated changes effectively organized critical elements of the enzyme, such as the catalytic base and the oxyanion hole, increasing the stabilization of the transition state. Our findings suggest that efficient enzymes can be obtained for various chemical processes by sampling conformational substates and deliberately stabilizing the active conformations over the unproductive ones.

This work was supported by the Howard Hughes Medical Institute and published in Science 370, 1442–1446 (2020).