VanS and VanR form the two-component regulatory system that controls inducible glycopeptide resistance in Enterococci. Upon induction VanS, a sensor histidine kinase, phosphorylates itself on a conserved histidine residue. This phosphate is transferred to a conserved aspartate residue on VanR. The phosphorylated form of VanR is the transcriptional activator of the vanAHX genes which, when expressed, directly confer vancomycin resistance. VanS also possesses VanR phosphatase activity, providing a mechanism by which to repress vanAHX transcription. This thesis describes approaches used towards the crystallisation of VanS and VanR. These are based upon previous crystallisation studies resulting in full-length VanS crystals which diffracted to 8Å, as well as a cytoplasmic structure of an analogous histidine kinase from Thermatoga maritima (Marina et al., 2005). Full-length and truncated forms of VanSA and full-length VanRA were cloned, expressed in E.coli and purified for crystallisation studies. Autokinase activity was biochemically characterised using radiolabelling and spectrophotomic assays, in tandem with a novel application of mass spectrometry. Site-directed mutagenesis of VanSA, led to the observation that ATP hydrolysis may occur independently of the autokinase function of VanSA. Adenosine 5’ tetraphosphate was also discovered, as a novel product of VanSA. Based upon these data an expanded model for VanS autokinase activity has been proposed. This may be expanded to include the phosphotransfer and phosphatase mechanisms and validated through the measurement of associated product formation. Finally a new mechanism for the control of the VanRS two component system has been proposed. Future studies will validate and expand this model. This work has significantly increased our knowledge of this system providing the tools and foundations that will lead to the elucidation of the way that this two component system functions. This has the potential for the development of novel inhibitors that either complement or supersede existing therapies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:549496 |
| Date | January 2010 |
| Creators | Quigley, Andrew Michael |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/3190/ |
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