Award details

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

Reference	BB/F009739/1
Principal Investigator / Supervisor	Professor Dominic Campopiano
Co-Investigators / Co-Supervisors
Institution	University of Edinburgh
Department	Sch of Chemistry
Funding type	Research
Value (£)	303,812
Status	Completed
Type	Research Grant
Start date	31/05/2008
End date	30/05/2011
Duration	36 months

Abstract

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 Å 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.

Summary

All cell walls have to be tough and durable and provide a physical barrier to protect the cell from external factors. They have to keep the cell contents inside but also allow molecules (e.g. nutrients) to pass into the cell and waste to leave. Humans, plants and bacteria have different cell wall components and they are made up of numerous complex building blocks called sphingolipids. It was recently discovered that the chemical reaction at the start of the sphingolipid synthetic pathway in all species is the same and begins by connecting an amino acid to a fatty acid. This reaction is catalysed by an enzyme called serine palmitoyltransferase (SPT) and is dependent on a B vitamin cofactor. We'd like to understand in molecular detail how sphingolipid membranes are made. Studying sphingolipid chemistry and the enzymes that make them in humans and plants is difficult because the building blocks and enzymes are embedded in membranes. To make it easier to study them, it helps to extract them into water and to do this we have to use detergents. Unfortunately, the enzymes often stop working in water. However, a bacterium (called Sphingomonas) was discovered that makes only one type of sphingolipid and its SPT enzyme is soluble in water. We can purify milligram amounts of this SPT and we have recently determined its 3D molecular structure to atomic resolution. We'd now like to understand how it catalyses the chemical reaction and how it can be inhibited. It turns out that sphingolipids not only play structural roles in cells, but also regulate and control the way the cell works. Inhibitors of sphingolipid production might turn out to be new anti-cancer or anti-inflammatory drugs. We can use our bacterial water-soluble SPT structure as a model for the human membrane-bound enzyme. Also, it has been discovered that some people have a neurological disease where their SPT enzyme is mutated so we can also use our model to understand how these mutations can cause problems with spingolipid chemistry in the brain.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Industrial Biotechnology, Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/F008503/1 Serine palmitoyltransferase / structure and function of the first enzyme in sphingolipid biosynthesis

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