Please use this identifier to cite or link to this item:
http://hdl.handle.net/11375/27775
Title: | Genomic Enzymology Study of the Aminoglycoside Antibiotic Acetyltransferases |
Authors: | Bordeleau, Emily |
Advisor: | Wright, Gerry |
Department: | Biochemistry |
Keywords: | antibiotic resistance |
Publication Date: | 2022 |
Abstract: | Since their discovery over 40 years ago, considerable knowledge has been obtained on the diversity, and structure-function relationships of aminoglycoside acetyltransferases (AACs), responsible for antibiotic resistance among priority clinical pathogens. In recent years, investigations have expanded to biochemical characterizations of AACs found in environmental reservoirs. The successful design of next-generation aminoglycosides (AGs) depends on an up-to-date understanding of the broader AG resistome. Towards this goal, I present the first structural analysis for the unique apramycin modifying enzyme, ApmA. Apramycin is a veterinary antibiotic that is in development for clinical use. The atypical chemical scaffold provides inherent protection from many clinically relevant resistance mechanisms. Prior to the work presented herein, apmA was an uncharacterized apramycin resistance element among environmental species. I heterologously expressed and subsequently purified ApmA to characterize the nature of resistance towards this unique aminoglycoside. The results report the first acetyltransferase of the left-handed β-helix (LβH) superfamily involved in AG detoxification. Secondly, I completed a comprehensive characterization of ApmA utilizing a structurally diverse panel of AGs for susceptibility testing, protein engineering, steady-state kinetics, and x-ray crystallography. Through these approaches, I establish the structural and functional features that define ApmA’s place within the LβH superfamily and set it apart from other known AACs. The biochemical data presented describes a chemical mechanism dependent on the substrate specificity. Furthermore, I describe the molecular determinants behind AG-modification of clinically relevant AGs. Lastly, I describe the first comprehensive structural and functional study of clinical and environmental Antibiotic_NAT (A_NAT) inactivating enzymes. A pan-family antibiogram was obtained and mapped to the reconstructed phylogeny for the A_NAT family. Crystallographic analysis of representatives from each clade was completed with our collaborators from the University of Toronto. Through the analysis of several ligand-bound A_NAT complexes, I contributed to the elucidation of structural features responsible for substrate specificity. The collective findings from these chapters have extended the protein landscape involved in AG-acetylation from one commonly used fold to three distinct architectures, each unique in underlying chemical mechanism and dissemination. |
URI: | http://hdl.handle.net/11375/27775 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
bordeleau_emily_A_finalsubmission2022Aug_PhD.pdf | 16.2 MB | Adobe PDF | View/Open |
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.