Characterisation, inhibition, and structural analysis of mycobacterial DNA gyrase

Henderson, Sara (2018) Characterisation, inhibition, and structural analysis of mycobacterial DNA gyrase. Doctoral thesis, University of East Anglia.

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Tuberculosis is primarily a pulmonary condition caused by Mycobacterium tuberculosis, which causes a serious threat to human health. In 2016 more than 1.3 million people died of tuberculosis, meanwhile there were 10.4 million new infections worldwide. Treatment for tuberculosis currently takes a minimum of 6 months with a combination of four different antibiotics, but there is increasing resistance to the first line antibiotics. The second line antibiotics include the fluoroquinolones (moxifloxacin, levofloxacin and gatifloxacin), which target DNA gyrase, the only Type IIA topoisomerase in M. tuberculosis, of which there is limited structural and biochemical information available within the literature.

To address these problems, we sought to increase our knowledge of mycobacterial DNA gyrase through biochemical mechanistic studies, inhibition studies of known and novel inhibitors of DNA gyrase alongside structural studies by X-ray crystallography. This led us to determine that a fusion of the GyrB and GyrA subunits was fully active and, in some cases, potentially more active than using the individual subunits. In particular, we determined that a detectable and greater rate of ATPase activity was present in Mtb DNA gyrase than previously suggested. We found this rate to be highly DNA-dependent requiring a topologically unconstrained DNA substrate (linear or nicked plasmid) for the highest rates. In addition, we cloned, expressed, purified and partially characterised DNA gyrase from the thermophilic M. thermoresistibile for use in structural studies.

In investigating the novel tricyclic group of compounds optimised by Redx AntiInfectives, we determined that they act in a similar mechanism of action to novobiocin through biochemical studies. They differ in their binding pocket, as determined by X-ray crystallography, and mechanism of resistance, as determined by bacterial mutagenesis.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 9280 not found.
Date Deposited: 24 May 2019 10:30
Last Modified: 24 May 2019 10:30


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