Structural studies of enzymes in polyketide and lipid biosynthesis : Trans-AT polyketide synthases and eukaryotic lipins

Lipids are essential constituants for living organisms. They play various roles as energetic molecules, chemical messengers, membrane constituants and hormones. Several cascades of enzymatic reactions allow the conversion of glucose to acetyl-CoA, a substrate of the fatty acid synthase and the diversification of fatty acid to triacylglycerol, glycerophospholipids, sphingolipids and saccharolipids.
The first part of the thesis focuses on a homologue of the fatty acid synthase, which possesses the same structure and catalyses similar chemical reaction: the polyketide synthase (PKS). Polyketide are predominantly from microbial and plants origin. They are structurally complex compounds compared to the linear hydrocarbon chain of fatty acid. These molecules are interesting for their anti-microbial, anti-parasitic and anti-cancer properties. Understanding the synthesis mechanism of PKS is of main interest for the discovery of new therapeutical molecules. The family of trans-acyltransferase (AT) PKS are particularly attractive as they use an external AT for loading the acetyl-CoA to the PKS. This AT, which is not integrated into the assembly line of the PKS, may be easily switched to another AT. Incorporation of non-natural substrates to the polyketide backbone may generate new biologically active polyketides. The research project presented in this thesis investigates the mechanism of substrate delivery from the external AT to the trans-AT PKS. In canonical (called cis-AT) PKS, AT is attached by a rigid linker domain to the ketosynthase (KS) domain responsible for the chain elongation reaction. Here, the crystal structure of the ketosynthase domain from a Brevibacillus brevis (B.brevis) trans-AT PKS shows the conservation of the linker domain with a potential docking interface at the corresponding position of the AT in cis-AT PKS. From structure analysis and sequence alignment of 236 C-terminal linkers of KS domain from trans-AT PKS, four classes of docking interface were identified. The docking interface of the B. brevis KS-LD belonging to the class I presents a central hydrophobic groove and surrounding charge residues which form contact with neighbouring KS-LD in the crystal structure suggesting that this docking interface could be potentially used by a trans-AT. The structural organization of the trans-AT protein was further investigated in collaboration with the Dr. Roman Jakob. The trans-AT can be coupled to an enoyl-reductase (ER) in certain trans-AT PKS. The crystal structure of a trans-ER and the small-angle X-ray scattering (SAXS) analysis of a trans-AT-ER reveal the dimerization of the trans-AT-ER protein at the ER interface. From those results, a model of the organization of the trans-AT PKS could be suggested. In the trans-AT-ER proteins, the ER dimerizes and the AT may bind to linker domain of the KS.
The second part focuses on another protein of lipid synthesis cascade. The lipin catalyses the penultimate step of triacylglycerol synthesis by dephosphorylating the phosphatidate into diacylglycerol. The lipin is also transcription coactivator of lipid metabolism regulating genes. Role of lipin in the development of fatty liver dystrophy in mice has been shown and there are growing evidences that mutations and polymorphisms are responsible for metabolic and inflammatory disorders in human. The X-ray crystallographic and biochemical studies are of main interest to explore the effect of mutations on the lipin function and structure. Here, the bioinformatics analysis of lipin from several organisms helps mapping the structurally conserved and highly disordered regions and allows the selection of lipin candidate susceptible to crystallise. This thesis reports a protocol optimized for the expression and purification of the Kluyveromyces lactis lipin homologue PAH1 construct (Klarg, Met1 to Arg527). Initial biochemical studies reveal the monomeric and oligomeric states of Klarg. These states are not in equilibrium, independent of phosphorylation and, for the oligomeric form, resistant to reducing conditions. Activity assay and crystallisation test have been started but they require further optimization.