Serine palmitoyltransferase / structure and function of the first enzyme in sphingolipid biosynthesis

See proposal from Edinburgh,

Sphingolipid biosynthesis commences with the condensation of L-serine and palmitoyl-CoA to produce 3- ketodihydrosphingosine (KDS). This reaction is catalysed by the PLP-dependent enzyme serine palmitoyltransferase (SPT) which is a member of a larger family of enzyme catalysing Claisen condensation reactions. SPT is a membrane-bound heterodimer (SPT1/SPT2) in eukaryotes such as humans and yeast and a cytoplasmic homodimer in the Gram-negative bacterium Sphingomonas paucimobilis. Unusually, the outer membrane of S. paucimobilis contains glycosphingolipid (GSL) instead of lipopolysaccharide (LPS), and SPT catalyses the first step of the GSL biosynthetic pathway in this organism. We have determined the crystal structure of the holo-form of S. paucimobilis SPT at 1.3 resolution. The enzyme is a symmetrical homodimer with two active sites and a monomeric tertiary structure consisting of three domains. The PLP cofactor is bound covalently to a lysine residue (Lys265) as an internal aldimine/Schiff base and the active site is composed of residues from both subunits, located at the bottom of a deep cleft. We have generated models of the human SPT1/SPT2 heterodimer from the bacterial structure by bioinformatic analysis. Mutations in the human SPT1-encoding subunit have been shown to cause a neuropathological disease known as hereditary sensory and autonomic neuropathy type I (HSAN1). In the proposed study, we will build from this platform to explore the substrate specificity and catalytic mechanism of this enzyme with a range of substrates, products and inhibitors using a combination of spectroscopy, crystallography, chemical analysis and informatics. We will use the bacterial SPT as a model to study mutations in SPT1 that cause HSAN1 disorders and make SPT fusions to investigate recently discovered viral fused SPTs.