Structure-activity relationship between Klebsiella pneumoniae β- Lactamase CTX-M-15 and selected β-lactam antibiotics: Evaluating the binding site promiscuity
| dc.contributor.author | Esau, Veruschka Chloe-Zaan | |
| dc.contributor.co-supervisor | Sayed, Yasien | |
| dc.contributor.supervisor | Achilonu, Ikechukwu Anthony | |
| dc.date.accessioned | 2024-10-22T10:39:34Z | |
| dc.date.available | 2024-10-22T10:39:34Z | |
| dc.date.issued | 2023-08 | |
| dc.description | Research thesis submitted in fulfilment of the requirements for the degree Master of Science, to the Faculty of Science, School of Molecular and Cell Biology, at the University of the Witwatersrand, Johannesburg, 2023. | |
| dc.description.abstract | Background: Nosocomial infections have become a major concern in Sub-Saharan Africa. Bacteria predominantly cause these infections due to resistance development attributed to lack of novel therapeutics. These resistant bacteria are classed as ESKAPE pathogens that gained resistance to most known antimicrobials; one such is the Gram-negative Klebsiella pneumoniae. K. pneumoniae developed resistant strategies against most known β-lactam antibiotics through the development of the enzyme, β-lactamase. Beta-lactamases hydrolyse the β-lactam ring of β-lactam antibiotics rendering them ineffective towards K. pneumoniae. This research aimed to investigate the structural and functional characteristics of K. pneumoniae β-lactamase and asses the conformational stability with penicillin and cefoperazone (CPZ). Methods: Overexpression of recombinant wild-type and mutant-(S70A) K. pneumoniae β-lactamase in E. coli T7 cells using a pET-28a vector and protein purification using Immobilised Metal Affinity Chromatography (IMAC), and enzyme activity assessed with nitrocefin. Secondary, tertiary, and quaternary structure studies were conducted with Far-UV CD, tryptophan fluorescence, ANS fluorescence and size exclusion HPLC respectively. The thermal stability and binding mechanisms were assessed with thermal shift assay and isothermal titration calorimetry (ITC). Results: The wild-type and mutant-(S70A) K. pneumoniae β-lactamase was successfully expressed and purified. S70A-KpBlac-1 showed no activity towards nitrocefin, and WT-KpBlac-1 was highly activity towards nitrocefin. The native structures were determined to be alpha-helical, but alpha-helical content is lost upon penicillin and CPZ binding to unorder and β-stranded conformations. The binding site was determined to be solvent exposed with one hydrophobic active site. The proteins were monomeric. CPZ induce thermal stability on S70A-KpBlac-1, whereas as penicillin binding had no effect on the thermal stability. Penicillin binding to S70A-KpBlac-1 was endothermic and the protein had low binding affinity for penicillin. CPZ binding was exothermic, and the protein had higher binding affinity for the substrate. Conclusion: Potential novel inhibitor design should be focused on CPZ. Through substrate-based drug discovery, potential drugs should confer a similar shape, size, or stereochemistry as CPZ. This would change the conformation of the protein, be tightly bound to the active site, and lower inhibitor concentration would be required. Therefore, these findings contribute, and provide insights on potential novel inhibitors against highly antibiotic resistant bacteria. | |
| dc.description.sponsorship | National Research Foundation (NRF). | |
| dc.description.submitter | MM2024 | |
| dc.faculty | Faculty of Science | |
| dc.identifier | 0000-0002-3547-595X | |
| dc.identifier.citation | Esau, Veruschka Chloe-Zaan. (2023). Structure-activity relationship between Klebsiella pneumoniae β- Lactamase CTX-M-15 and selected β-lactam antibiotics: Evaluating the binding site promiscuity. [Master's dissertation, University of the Witwatersrand, Johannesburg]. https://hdl.handle.net/10539/41816 | |
| dc.identifier.uri | https://hdl.handle.net/10539/41816 | |
| dc.language.iso | en | |
| dc.publisher | University of the Witwatersrand, Johannesburg | |
| dc.rights | ©2023 University of the Witwatersrand, Johannesburg. All rights reserved. The copyright in this work vests in the University of the Witwatersrand, Johannesburg. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of University of the Witwatersrand, Johannesburg. | |
| dc.rights.holder | University of the Witwatersrand, Johannesburg | |
| dc.school | School of Molecular and Cell Biology | |
| dc.subject | Nosocomial infections | |
| dc.subject | Antibiotic resistance | |
| dc.subject | ESKAPE pathogens | |
| dc.subject | Klebsiella pneumoniae | |
| dc.subject | Beta-Lactamase CTX-M-15 | |
| dc.subject | Penicillin | |
| dc.subject | Cefoperazone | |
| dc.subject | Nitrocefin | |
| dc.subject | Immobilised Metal Affinity Chromatography | |
| dc.subject | Thermal shift assay | |
| dc.subject | Issothermal titration calorimetry | |
| dc.subject | UCTD | |
| dc.subject.other | SDG-3: Good health and well-being | |
| dc.title | Structure-activity relationship between Klebsiella pneumoniae β- Lactamase CTX-M-15 and selected β-lactam antibiotics: Evaluating the binding site promiscuity | |
| dc.type | Dissertation |