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Title: Mechanistic and structural analysis of the enzymes polyphosphate kinase and exopolyphosphatase from biothreat agents
Author: Parnell, Alice Elizabeth
ISNI:       0000 0004 6347 4246
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2016
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There is an urgent need for the discovery of novel antibiotics as the past few decades have seen a worrying increase in the number of resistant bacteria and the number of new compounds released on the market diminishing. One way of discovering new compounds is through identification of inhibitors of novel targets in bacteria. Polyphosphate metabolism is controlled by two types of enzyme in bacteria: polyphosphate kinase (PPK) and exopolyphosphatase (PPX). PPK enzymes can degrade polyphosphate to synthesise nucleotide phosphates and can also catalyse the reverse reaction. PPX breaks down inorganic polyphosphate chains into individual phosphate residues using water. The stringent response is a form of stress response that occurs in response to nutritional deprivation and is characterised by the accumulation of pppGpp and ppGpp. E. coli PPX has been shown to be inhibited by accumulation of ppGpp during the stringent response, thus providing one of the links between the stringent response and polyphosphate metabolism. Knockout of genes encoding PPK and PPX have been shown to reduce virulence in several pathogenic bacteria. The structure of F. tularensis PPK2 has been solved as an apo-structure, with polyphosphate and with both polyphosphate and AMPPCH2PPP. These structures, combined with site-directed mutagenesis, kinetic studies and binding studies have enabled determination of a possible catalytic mechanism for this family of enzymes. A crystal form has been identified for B. pseudomallei PPX but the solved structure is lacking electron density for hundreds of amino acid residues and it was not possible to refine it to an acceptable standard for publication. A high throughput method for the screening of B. pseudomallei PPX, with a good screening window identified by a high Z' factor, was developed. This was used to screen B. pseudomallei PPX against a fragment library of molecules.
Supervisor: Roach, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available