Abstract Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design. Using this method, we redesigned the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation, and thereby increase catalytic efficiency with a non-native substrate to alter substrate selectivity. Steady-state kinetics of designed variants revealed selectivity switches of up to 1900-fold, and structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirmed that conformational equilibria favoured the target conformation. Our computational approach opens the door to the fine-tuning of enzyme conformational landscapes to create designer biocatalysts with tailored functionality.

Computational remodeling of an enzyme conformational landscape for altered substrate selectivity