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Directed evolution provides insight into conformational substrate sampling by SrtA
The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578623/ https://www.ncbi.nlm.nih.gov/pubmed/28859178 http://dx.doi.org/10.1371/journal.pone.0184271 |
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author | Suliman, Muna Santosh, Vishaka Seegar, Tom C. M. Dalton, Annamarie C. Schultz, Kathryn M. Klug, Candice S. Barton, William A. |
author_facet | Suliman, Muna Santosh, Vishaka Seegar, Tom C. M. Dalton, Annamarie C. Schultz, Kathryn M. Klug, Candice S. Barton, William A. |
author_sort | Suliman, Muna |
collection | PubMed |
description | The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme’s inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts. |
format | Online Article Text |
id | pubmed-5578623 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-55786232017-09-15 Directed evolution provides insight into conformational substrate sampling by SrtA Suliman, Muna Santosh, Vishaka Seegar, Tom C. M. Dalton, Annamarie C. Schultz, Kathryn M. Klug, Candice S. Barton, William A. PLoS One Research Article The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme’s inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts. Public Library of Science 2017-08-31 /pmc/articles/PMC5578623/ /pubmed/28859178 http://dx.doi.org/10.1371/journal.pone.0184271 Text en © 2017 Suliman et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Suliman, Muna Santosh, Vishaka Seegar, Tom C. M. Dalton, Annamarie C. Schultz, Kathryn M. Klug, Candice S. Barton, William A. Directed evolution provides insight into conformational substrate sampling by SrtA |
title | Directed evolution provides insight into conformational substrate sampling by SrtA |
title_full | Directed evolution provides insight into conformational substrate sampling by SrtA |
title_fullStr | Directed evolution provides insight into conformational substrate sampling by SrtA |
title_full_unstemmed | Directed evolution provides insight into conformational substrate sampling by SrtA |
title_short | Directed evolution provides insight into conformational substrate sampling by SrtA |
title_sort | directed evolution provides insight into conformational substrate sampling by srta |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578623/ https://www.ncbi.nlm.nih.gov/pubmed/28859178 http://dx.doi.org/10.1371/journal.pone.0184271 |
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