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...
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2017-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0184271&type=printable |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850231086002470912 |
|---|---|
| author | Muna Suliman Vishaka Santosh Tom C M Seegar Annamarie C Dalton Kathryn M Schultz Candice S Klug William A Barton |
| author_facet | Muna Suliman Vishaka Santosh Tom C M Seegar Annamarie C Dalton Kathryn M Schultz Candice S Klug William A Barton |
| author_sort | Muna Suliman |
| collection | DOAJ |
| 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 | Article |
| id | doaj-art-76e6f14461e446219b8a4145c14c57ae |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-76e6f14461e446219b8a4145c14c57ae2025-08-20T02:03:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01128e018427110.1371/journal.pone.0184271Directed evolution provides insight into conformational substrate sampling by SrtA.Muna SulimanVishaka SantoshTom C M SeegarAnnamarie C DaltonKathryn M SchultzCandice S KlugWilliam A BartonThe 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0184271&type=printable |
| spellingShingle | Muna Suliman Vishaka Santosh Tom C M Seegar Annamarie C Dalton Kathryn M Schultz Candice S Klug William A Barton Directed evolution provides insight into conformational substrate sampling by SrtA. PLoS ONE |
| 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 |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0184271&type=printable |
| work_keys_str_mv | AT munasuliman directedevolutionprovidesinsightintoconformationalsubstratesamplingbysrta AT vishakasantosh directedevolutionprovidesinsightintoconformationalsubstratesamplingbysrta AT tomcmseegar directedevolutionprovidesinsightintoconformationalsubstratesamplingbysrta AT annamariecdalton directedevolutionprovidesinsightintoconformationalsubstratesamplingbysrta AT kathrynmschultz directedevolutionprovidesinsightintoconformationalsubstratesamplingbysrta AT candicesklug directedevolutionprovidesinsightintoconformationalsubstratesamplingbysrta AT williamabarton directedevolutionprovidesinsightintoconformationalsubstratesamplingbysrta |