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The diverse roles of the \textit{Pseudomonas aeruginosa} phospholipase PlcH and the \textit{Toxoplasma gondii} serine palmitoyltransferase in sphingolipid biochemistry.

THYE, JULIE,KRISTINE (2014) The diverse roles of the \textit{Pseudomonas aeruginosa} phospholipase PlcH and the \textit{Toxoplasma gondii} serine palmitoyltransferase in sphingolipid biochemistry. Doctoral thesis, Durham University.

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Sphingolipids are essential for cell survival for almost all eukaryotic cells. Due to the large variety in sphingolipid structure, they possess a wide range of functions from acting as structural components of membranes to participating in cell signalling pathways. Sphingolipid metabolism and catabolism can occur via numerous routes and the disruption of sphingolipids or sphingolipid biosynthetic pathways is therefore detrimental to cells.
The vast majority of prokaryotes do not utilise sphingolipids, however certain prokaryotic enzymes which posses similar functionality to those involved in sphingolipid biosynthesis, are crucial to pathogen-host interactions.
For instance, the opportunistic human pathogen, \textit{Pseudomonas aeruginosa}, which represents a particular threat to cystic fibrosis patients, expresses a variety of virulence factors. One of these, the heterodimeric complex, PlcHR$_{2}$, is a phospholipase C. It also possesses sphingomyelinase properties which has been suggested to be responsible for the highly specific cytotoxic effects of the enzyme. The first aim of the thesis was to express and purify the PlcH part of the complex in order to functionally and biophysically characterise it, laying the foundation towards the crystal structure determination. Furthermore, the sphingomyelinase activity assay was established, utilising PlcHR$_{2}$, in order to screen several commercially available compounds and an in-house library of ceramide analogues for potential inhibitory effect on the enzyme. Five inhibitory and five activating compounds were identified.
Sphingolipids also play crucial roles in more complex human pathogens, including the apicomplexan protozoan parasite, \textit{Toxoplasma gondii}, which causes a life-threatening disease, toxoplasmosis, in animals and humans. The first step in the sphingolipid biosynthetic pathway is catalysed by serine palmitoyltransferase (SPT), making it an important drug target in several parasites as preventing this step from occurring in the biosynthesis will result in an incapability of the parasite to proliferate. Several constructs of the SPT from \textit{T. gondii} were designed in order to establish the smallest catalytically active domain. The constructs were expressed and purified in order to characterise the enzyme by a range of biochemical and biophysical methods, with the ultimate aim of obtaining a crystal structure for the enzyme.
The final part of the thesis focussed on the crystallisation and crystal structure determination of a triosephosphate isomerase (TPI) from a hyperthermophilic archaeon, \textit{Sulfolobus solfataricus}. The TPI protein assumes a ($\beta$ $\alpha$)$_{8}$-barrel fold, the most common protein fold. It is thus an ideal target to study to gain an understanding of the evolutionary process that enzymes undergo to stabilise their structure and function at the extreme conditions they are subjected to in extremophiles.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Faculty and Department:Faculty of Science > Chemistry, Department of
Thesis Date:2014
Copyright:Copyright of this thesis is held by the author
Deposited On:26 Nov 2014 13:21

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