Rescue of conformational dynamics in enzyme catalysis by directed evolution

Renee Otten,Dorothee Kern,James S Fraser,Lillian R Kenner,Michael W Clarkson,Dan S Tawfik,Lin Liu,David Mavor

doi:10.1038/s41467-018-03562-9

Abstract

Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes. Protein dynamics is 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 use directed evolution on an impaired mutant of the proline isomerase 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 is correlated with an accelerated interconversion between the two catalytically essential conformational sub-states, which are both captured in the high-resolution X-ray ensembles. Our data provide a glimpse of an evolutionary trajectory and show how subtle changes can fine-tune enzyme function.

Highlights

Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes
We use directed evolution on an impaired mutant of the proline isomerase 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
We find a direct correspondence of increased protein dynamics and faster catalysis with rate constants mirroring the catalytic turnover numbers along an evolutionary trajectory

Summary

Introduction

Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes. We use directed evolution on an impaired mutant of the proline isomerase 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. As evolutionary selection acts on function, a new challenge is to understand how evolution shapes these energy landscapes[9] This challenge is best exemplified by the common implication of protein dynamics as speculative explanation for the impressive functional improvements achieved via directed evolution, to improve enzyme activity[10,11,12], where often only minimal structural changes are observed[10,13]. The rescued variant displays increased conformational exchange between two catalytically essential sub-states that are revealed directly by the X-ray measurements

Methods

Results

Conclusion