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Title: Biochemical characterisation and exploitation of the enzyme RelA from biothreat agents
Author: Wilkinson, Rachael Claire
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2016
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Recent years have seen a rise in bacterial resistance to clinically available antibiotics in tandem with a decrease in the discovery of novel antimicrobials. Consequently this has renewed concern over pathogenic bacteria, for which treatment has previously been available. One method of overcoming antibacterial resistance is the identification of new inhibitors against novel targets within bacteria. The stringent response is essential to bacterial survival during unfavourable conditions and is underpinned by the actions of the signalling molecules guanosine penta- and tetra-phosphate. In ?- and ?-proteobacteria the synthesis of these molecules is predominantly catalysed by the enzyme RelA. Knockouts of genes encoding RelA result in reduced virulence within several pathogenic bacteria. The focus of this project was to characterise the RelA enzymes from the biothreat agents Francisella tularensis (F. tularensis), Yersinia pestis (Y. pestis) and Burkholderia pseudomallei (B. pseudomallei) and develop a method for identifying inhibitors of RelA. F. tularensis RelA purified as a stable dimer that could be concentrated to 10 mg/mL, which is higher than that reported for other RelA enzymes. F. tularensis RelA demonstrated strict specificity for GTP as a pyrophosphate acceptor, except in the presence of methanol. Substrate specificity was not observed for the other RelA enzymes investigated. Steady state kinetic characterisation of F. tularensis RelA consistently generated data that fitted a sigmoidal function (R2 = 0.95). Kinetic parameters calculated for F. tularensis RelA gave a Vmax of 12.27 (± 0.37) × 10-3 s-1 and 14.02 (± 0.90) × 10-3 s-1 and K1/2 values of 351.5 ± 15 ?M and 1072 ± 84 ?M for ATP and GTP respectively. F. tularensis RelA was shown to be activated by stalled ribosomal complexes from Escherichia coli (11 fold) and to a lesser extent those from F. philomiragia (1.39 fold). Activation of RelA was observed upon the addition of ppGpp, EC50 of 60 ± 1.9 ?M, which demonstrated that the ACT domain is not involved in this process. A high throughput method for the screening of inhibitors of F. tularensis RelA was developed. This method demonstrated a good screening window with a high Z? factor. The high throughput method was validated against a small library of focussed molecules.
Supervisor: Roach, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available