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Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase

The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA...

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Detalles Bibliográficos
Autores principales: Coulther, Timothy A., Pott, Moritz, Zeymer, Cathleen, Hilvert, Donald, Ondrechen, Mary Jo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons, Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284568/
https://www.ncbi.nlm.nih.gov/pubmed/33938058
http://dx.doi.org/10.1002/pro.4099
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author Coulther, Timothy A.
Pott, Moritz
Zeymer, Cathleen
Hilvert, Donald
Ondrechen, Mary Jo
author_facet Coulther, Timothy A.
Pott, Moritz
Zeymer, Cathleen
Hilvert, Donald
Ondrechen, Mary Jo
author_sort Coulther, Timothy A.
collection PubMed
description The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5–5, RA95.5–8, and RA95.5–8F, representing progress along an evolutionary trajectory with increasing activity, is examined. Computed measures of coupling between charged states of residues show that, as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues is increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, are tested using site‐directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants show decreases in k (cat) relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibit significant decreases in k (cat)/K (M) across the series. Only modest decreases in k (cat)/K (M) are observed for the E85Q variants with little effect on k (cat). Computed metrics of the degree of coupling between protonation states rise significantly as evolution proceeds and catalytic turnover rate increases. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst.
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spelling pubmed-82845682021-07-21 Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase Coulther, Timothy A. Pott, Moritz Zeymer, Cathleen Hilvert, Donald Ondrechen, Mary Jo Protein Sci Full‐Length Papers The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5–5, RA95.5–8, and RA95.5–8F, representing progress along an evolutionary trajectory with increasing activity, is examined. Computed measures of coupling between charged states of residues show that, as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues is increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, are tested using site‐directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants show decreases in k (cat) relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibit significant decreases in k (cat)/K (M) across the series. Only modest decreases in k (cat)/K (M) are observed for the E85Q variants with little effect on k (cat). Computed metrics of the degree of coupling between protonation states rise significantly as evolution proceeds and catalytic turnover rate increases. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst. John Wiley & Sons, Inc. 2021-05-24 2021-08 /pmc/articles/PMC8284568/ /pubmed/33938058 http://dx.doi.org/10.1002/pro.4099 Text en © 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Full‐Length Papers
Coulther, Timothy A.
Pott, Moritz
Zeymer, Cathleen
Hilvert, Donald
Ondrechen, Mary Jo
Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase
title Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase
title_full Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase
title_fullStr Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase
title_full_unstemmed Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase
title_short Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase
title_sort analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase
topic Full‐Length Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284568/
https://www.ncbi.nlm.nih.gov/pubmed/33938058
http://dx.doi.org/10.1002/pro.4099
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