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Tytuł pozycji:

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.

Tytuł :
Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.
Autorzy :
Broom A; Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, ON, K1N 6N5, Canada.
Rakotoharisoa RV; Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, ON, K1N 6N5, Canada.
Thompson MC; Department of Bioengineering and Therapeutic Science, University of California, San Francisco, San Francisco, CA, 94158, USA.; Department of Chemistry and Chemical Biology, University of California, Merced, Merced, CA, 95343, USA.
Zarifi N; Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, ON, K1N 6N5, Canada.
Nguyen E; Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, ON, K1N 6N5, Canada.
Mukhametzhanov N; Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, ON, K1N 6N5, Canada.
Liu L; Department of Bioengineering and Therapeutic Science, University of California, San Francisco, San Francisco, CA, 94158, USA.
Fraser JS; Department of Bioengineering and Therapeutic Science, University of California, San Francisco, San Francisco, CA, 94158, USA.
Chica RA; Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, ON, K1N 6N5, Canada. .
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Źródło :
Nature communications [Nat Commun] 2020 Sep 23; Vol. 11 (1), pp. 4808. Date of Electronic Publication: 2020 Sep 23.
Typ publikacji :
Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.
Język :
English
Imprint Name(s) :
Original Publication: [London] : Nature Pub. Group
MeSH Terms :
Computer Simulation*
Evolution, Chemical*
Directed Molecular Evolution/*methods
Enzymes/*chemistry
Lyases/*chemistry
Catalysis ; Catalytic Domain ; Crystallography, X-Ray ; Enzyme Stability ; Enzymes/genetics ; Enzymes/metabolism ; Kinetics ; Lyases/genetics ; Lyases/metabolism ; Molecular Dynamics Simulation ; Mutation ; Protein Conformation ; Protein Engineering
References :
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Grant Information :
R21 GM110580 United States GM NIGMS NIH HHS; P30 GM124169 United States GM NIGMS NIH HHS; F32 HL129989 United States HL NHLBI NIH HHS; R01 GM123159 United States GM NIGMS NIH HHS; R01 GM124149 United States GM NIGMS NIH HHS
Substance Nomenclature :
0 (Enzymes)
EC 4.- (Lyases)
Entry Date(s) :
Date Created: 20200924 Date Completed: 20201013 Latest Revision: 20201110
Update Code :
20201110
PubMed Central ID :
PMC7511930
DOI :
10.1038/s41467-020-18619-x
PMID :
32968058
Czasopismo naukowe
The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (k cat /K M 146 M -1 s -1 ). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (k cat /K M 103,000 M -1 s -1 ) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

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