Molecular and structural studies on proteins involved in lipid biosynthesis in plants and bacteria
Fatty acid synthesis in plants and most bacteria is catalysed by a type II, dissociable fatty acid synthetase, critically involving the participation of acyl-carrier protein (ACP) during synthesis and elongation reactions. This provides the substrate for glycerolipid biosynthesis, the first reaction of which in plants, is catalysed by a soluble glycerol-3-phosphate acyltransferase (G3PAT).A candidate gene for malonyl CoA:ACP transacylase (MCAT) from plants has been identified using homology to known MCAT sequences. A full-length cDNA was isolated from a Brassica napus embryo library and used to complement an E.coli mutant defective in MCAT activity, so providing proof of function. E.coli ACP has been investigated in attempts to obtain crystals which can be used in structural investigations to solve its structure via X-ray crystallography. Wild type butryl-ACP can be successfully crystallised and defracts to 2.0 Ǻ resolution, and is thus a candidate for solving the structure of the protein. The complete structure of butryl-ACP was elucidated following the introduction of new methionine residues into the protein using site directed mutagenesis, and the production of recombinant proteins containing selenomethionine as heavy metal derivatives. Squash recombinant G3PAT was over-expressed and used to obtain crystals which defract to1.8 Ǻ. In order to solve the phasing problem selected cysteine residues were removed from the protein, resulting in the identification of histidine 279 as an additional heavy metal binding site. The structure of the enzyme was solved to 1.8 Ǻ resolution and potential substrate binding sites have been modelled into it on the basis of the conserved H(X)(_4)D acyltransferase domain. The site for G3P binding has been confirmed following site directed mutagenesis and novel substrate selectivity has been introduced into the protein by PCR mediated mutagenesis. These studies have resulted in the first X-ray structure of two components of lipid biosynthesis, increased our understanding of the reactions they catalyse and successfully identified an authenticated cDNA for MCAT from plants.