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Title: Rescue of conformational dynamics in enzyme catalysis by directed evolution

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.

Authors:
ORCiD logo [1];  [2];  [2];  [1];  [2];  [3];  [1]; ORCiD logo [2]
  1. Brandeis Univ., Waltham, MA (United States)
  2. Univ. of California, San Francisco, CA (United States)
  3. Weizmann Inst. of Science, Rehovot (Israel)
Publication Date:
Research Org.:
Brandeis Univ., Waltham, MA (United States); Univ. of California, San Francisco, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1499927
Grant/Contract Number:  
FG02-05ER15699; AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Otten, Renee, Liu, Lin, Kenner, Lillian R., Clarkson, Michael W., Mavor, David, Tawfik, Dan S., Kern, Dorothee, and Fraser, James S.. Rescue of conformational dynamics in enzyme catalysis by directed evolution. United States: N. p., 2018. Web. doi:10.1038/s41467-018-03562-9.
Otten, Renee, Liu, Lin, Kenner, Lillian R., Clarkson, Michael W., Mavor, David, Tawfik, Dan S., Kern, Dorothee, & Fraser, James S.. Rescue of conformational dynamics in enzyme catalysis by directed evolution. United States. doi:10.1038/s41467-018-03562-9.
Otten, Renee, Liu, Lin, Kenner, Lillian R., Clarkson, Michael W., Mavor, David, Tawfik, Dan S., Kern, Dorothee, and Fraser, James S.. Tue . "Rescue of conformational dynamics in enzyme catalysis by directed evolution". United States. doi:10.1038/s41467-018-03562-9. https://www.osti.gov/servlets/purl/1499927.
@article{osti_1499927,
title = {Rescue of conformational dynamics in enzyme catalysis by directed evolution},
author = {Otten, Renee and Liu, Lin and Kenner, Lillian R. and Clarkson, Michael W. and Mavor, David and Tawfik, Dan S. and Kern, Dorothee and Fraser, James S.},
abstractNote = {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.},
doi = {10.1038/s41467-018-03562-9},
journal = {Nature Communications},
issn = {2041-1723},
number = 1,
volume = 9,
place = {United States},
year = {2018},
month = {4}
}

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