Biophysical and Biochemical characterisation of Serine Palmitoyltransferase in Apicomplexan Parasites and Escherichia coli

Sphingolipids are essential molecules for the survival of all eukaryotic cells. Given their large structural variety, their functions span across a multitude of cellular processes, from maintaining cell membranes, to signalling, and apoptosis. The disruption of sphingolipid metabolism is therefore highly detrimental for eukaryotes. This includes the Apicomplexan parasites such as Toxoplasma gondii, one of the most widespread parasites worldwide, which poses great risk to immunocompromised individuals, in particular pregnant women and those affected by HIV. This thesis aimed at the characterisation of the first and rate limiting enzyme of the sphingolipid biosynthetic pathway, serine palmitoyltransferase (SPT) in Toxoplasma gondii and the related parasite Cryptosporidium through the use of computational tools such as protein structure prediction methods, evolutionary analysis, and biochemical and biophysical methods such as protein expression and purification, thermal shift assays, circular dichroism spectroscopy, small-angle x-ray scattering (SAXS), and crystallography.
The majority of prokaryotes are not known to make use of sphingolipids; however, some prokaryotes such as Sphingomonas spp. are famously known to synthesise them as specialised membrane components that contribute to unique cell-surface properties and stress tolerance. This thesis highlights the discovery of a presumed SPT identified in pathogenically relevant strains of E. coli (EcSPT). This thesis aimed at the characterisation of EcSPT by means of SAXS and crystallography, as well as evolutionary analysis, thermal shift assays, and activity assays. In this work, the structure of EcSPT was solved to 1.9 Å resolution, and it provides insight into its function, including the identification of flexible conserved active site loops, and the active site stabilisation of a transition state.
The last section of this work focuses on ligand-docking experiments as orthogonal methods to structural and biochemical work, as well as ligand-docking experiments in the field of rational drug design, in particular for antiparasitic drugs targeting a variety of serine hydrolases in Leishmania spp.
The work presented in this thesis has resulted in significant outcomes that advance the structural and biochemical understanding of SPT across clinically relevant organisms and provide a molecular rationale to guide the future development of selective inhibitors of this essential enzyme and the associated biosynthetic pathway.