Elucidation of the bacterial sphingolipid biosynthetic pathway in Sphingomonas wittichii.

Cells are made of membranes which are composed of chemicals called 'lipids' - these contain hydrophobic (water hating) and hydrophilic (water loving) parts. Membranes have to be strong to keep cell contents in but also be able to let molecules in (nutrients, metals, salts) - as well as keeping toxic materials out and expelling waste. They must also stop water flooding in and bursting the cell by increased osmotic pressure. Cells have evolved different membranes with different chemical composition. Mammals have complicated membranes and they generate 100s of different lipids. Similarly, yeast, plants and viruses have species-specific lipids. Bacteria too have unique and unusual lipids - they also play important roles in the immune response and inflammation. Mammals have evolved to recognise their own lipids as 'self' but can expertly detect foreign lipids from pathogenic bacteria, fungi and viruses. Once detected, the mammalian cell can mount an effective immune response to kill the invader. This then begs the question, if a bacterium has evolved to have lipids similar to a human's - how do we tell them apart? Looking more closely at the lipids themselves our project will focus on a special branch of interesting lipids called 'sphingolipids'. They were discovered >100 years ago in human brains by John Thudichum who knew that they played an important role in brain chemistry. It took until the 1930s for Herbert Carter to work out the chemistry of the sphingolipids - a polar, water soluble head and a fatty acid non-polar tail. They were found to be made from the common amino acid L-serine and a long carbon (>C16) chain. Scientists have long wondered about how sphingolipids are made inside the cell from common building blocks and then transported to the outside - this must happen very quickly when the cells are rapidly growing and dividing. Also, sphingolipids are dangerous - too many or too little in one cell can be lethal so the amounts are delicately controlled in a way we still don't fully understand. To uncover the chemical details and explore the enzymes involved we and other scientists are studying sphingolipid biosynthesis in humans, plants, yeast and bacteria. We have chosen an interesting bacterium Sphingomonas wittichii because it is not harmful to man - in fact it can degrade toxins to harmless molecules. These Sphingomonas are highly unusual because they make sphingolipids that resemble our own to some extent. We will explore how Sphingomonas makes sphingolipids by carefully characterising the genes that encode the enzymes that carry out the initial conversion of serine and the fatty acid, through the complex 2nd and 3rd steps, and beyond. We are helped because the Department of Energy (USA) have already sequenced the Sphingomonas wittichii genome and predict it to have >5000 genes. However, we do not know which ones are involved in sphingolipid biosynthesis. We will use chemical, biochemical, genetic and molecular biology methods to help us understand each step. We have already made a start and found an unusual small protein (~80 amino acids long) that we think links sphingolipid and fatty acid biosynthesis. Most of the work will be carried out in Edinburgh but we will also work with Jim Naismith in St.Andrews who can determine the 3D structure of a protein, as well as a genetics expert in the USA, Teresa Dunn. Our teamwork will put us ahead of our competitors. By the end of the grant we will have determined the basic roadmap of bacterial sphingolipid biosynthesis and be able to begin to compare it with the map in humans, plants and yeast. We'll obtain insight into how these species evolved to make the same sphingolipid and begin to understand how each controls the amount in each cell. Whilst we carry out the work we will make sure we give seminars to experts and the general public telling them what we've found out and will also publish in highly-rated international journals that will benefit UK science.

Sphingolipids (SLs) and their glycosylated forms (GSLs) are essential components of eukaryotic membranes and are also potent signalling molecules. They are composed of a long chain base and a polar head group. They are found in humans, plants, yeasts and some bacteria. The first step in SL biosynthesis in all organisms is the coupling the amino acid L-serine with a long chain fatty acid thioester substrate. This is catalysed by a pyridoxal 5'-phosphate (PLP) cofactor dependent enzyme, serine palmitoyltransferase (SPT). It produces the 1st intermediate ketodihydrosphingosine (KDS), CO2 and CoASH. SPTs from eukaryotes are heterodimeric membrane bound enzymes but in bacteria such as Sphingomonas they are soluble cytoplasmic enzymes. We were the first to characterise the high resolution x-ray structure of a bacterial SPT and we have used it to model the human enzyme. The SL biosynthetic pathways in various organisms are complicated and the enzymes tightly controlled but detailed structural analysis is lacking. In this project we will continue our breakthrough SL biosynthesis work on a model bacterial system - Sphingomonas wittichii whose genome sequence is known. We will use chemical, biochemical and genetic tools to identify and characterise the early steps in the pathway. We have already identified a potential novel link between SL and fatty acid synthesis - we discovered a small acyl carrier protein (ACP) that may complex with the SPT to make KDS. Thereafter the KDS product is streospecifically reduced by a reductase using a NAD(P) cofactor then N-acylated to form a ceramide. We will complement these methods with tools from yeast genetics to search for bacterial homologs. We will conclude with a preliminary study of how sugars are attached to the SL core to generate GSLs and identify any kinases. Our work will provide the first insight into SL biosynthesis in this kingdom of life and allow a comparison with the SL pathway in higher organisms.

International competitiveness and opportunity for UK scientists to lead the field: We will endeavour to conduct research and publish papers that will bring UK-government funded research to the attention of the international scientific community and general public. Specifically we will aim for at least 4 papers from this grant. We will aim for high impact journals of the type - Nature Chemical Biology, Structure, J. Biol. Chem., JACS and flagship RSC journals such as Chem. Comm. In 2009/10 DJC has published in Nature Chemistry, JBC, Structure and Molecular Biosystems (Emerging Investigators Issue). DJC will publish 'Sphingolipid Biosynthesis in Man and Microbes' in Natural Product Reports for 2010/11 and a review of 'The a-oxoamine synthases' by ChemBiochem, 2010. The sphingolipid field is a mature but expanding area covering natural product biosynthesis, chemical biology, immunology, inflammation, and cancer biology but has recently moved into the modern 'omics' arena where combined genomic and proteomic data, coupled to detailed chemical biology information could lead to breakthroughs in synthetic biology. A detailed knowledge of how a 'simple' bacterium generates the key sphingolipid in its outer membrane will underpin future studies that will inform these exciting fields. The PI spoke at the IUPAC meeting in Glasgow, 2009 and will present at the 2010 Directing Biosynthesis meeting. The PI was invited to present their BBSRC-funded work at the flagship 2010 GRC symposium (Ventura) to an international audience (~200 participants) and we aim to present again at the 2012 GRC. The 'Lipidmaps' consortium (funded from 2003 by 2 consecutive NIH grants totalling >$70M) also presented results of their lipid profiling in human cells but not of the biosynthetic enzymes involved. They and others have not considered the potential of Sphingomonas and they encouraged us to explore this model organism. We have a window of opportunity for a UK-based group (in collaboration with an acknowledged lipid expert from the USA), using data from a DoE (USA) funded project, to lead an exciting field which will have broad appeal. Encouraging the next generation: The PI has a strong record in hosting younger scientists (5th or 6th year) in his laboratory funded by the Nuffield Foundation Science Bursary scheme. Joseph Coulson published a research paper (Clarke DJ, Coulson J, Baillie R & Campopiano DJ, Eur. J. Biochem., 2003), went on to study Chemistry at Oxford and has since become a chemistry secondary school teacher. DJC is part of a team funded by a Wellcome Trust People Award (WT087933MA) on 'Superbugs' that has generated a schools workshop that is fully hands-on and interactive. This has received great feedback from school kids, parents and teachers. We also presented our Superbug research at an ASCUS (Art Science Collaborative) event and a Science in the Parliament event (March 2010).In collaboration with Prof. John Govan, the PI is involved in Cystic Fibrosis research. We were awarded the largest grant (£564,769) by The Big Lottery Fund and CF Trust to study - 'Superbugs and life-threatening lung infection in cystic fibrosis: An investment in the future.' A consortium of six UK universities with 4PhDs and 1 PDRA were funded 2005-2008. Each PhD student still works in science and the PDRA Dr. Alan Brown is now a PI at Exeter. Public Awareness That scientists can now build organisms from scratch (re: Dr. Craig Venter's recent work) that can be engineered to make designer molecules or do specific biotechnology jobs has captured the public's imagination but also raised their fears. NESTA & RSC has followed the spirit of this to inform the public, and the PI, an engineer (Dr. Alistair Elfick) and fellow chemist (Dr. Scott Cockroft), were awarded a £10k grant 'Connections in Synthetic Biology' to explore label free detection of biomolecules. This innovative programme leads discussions through workshops, live video feeds and on Facebook.