Rescue of Conformational Dynamics in Enzyme Catalysis by Directed Evolution
Prof. James Fraser, Dr. Renee Otten, Dr. Lin Liu, Lillian Kenner, Dr. Michael Clarkson, Mr. David Mavor, Prof. Dan Tawfik, Dr. Dorothee Kern
Received: 18th September 17
Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes. However, a comprehensive understanding of how evolution shapes the energy landscape of catalysis remains a fundamental challenge. Protein dynamics is widely recognized as important, but due to the inherent flexibility of biological macromolecules it is often difficult to distinguish which conformational changes are directly related to function. Here, we used directed evolution on an impaired mutant of the human proline isomerase cyclophilin A (CypA) and identify two second-shell mutations that partially restore its catalytic activity. We show both kinetically, using NMR spectroscopy, and structurally, by room-temperature X-ray crystallography, how local perturbations propagate through a large allosteric network to facilitate conformational dynamics. The increased catalysis selected for in the evolutionary screen could be rationalized entirely by accelerated interconversion between the two catalytically essential conformational sub-states, which are both captured in the high-resolution X-ray ensembles at room temperature. Our data provide a glimpse of the evolutionary trajectory of an enzyme’s energy landscape and shows how subtle changes can fine-tune its function.
This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.