Molecular studies on plant glycerol-s-phosphate acyltransferases
The main objective of this research is to advance our understanding of the biochemical properties and the structure-function relationships of the chloroplast glycerol-3-phosphate acyltransferases in plants. De novo synthesised fatty acyl chains are diverted into the prokaryotic pathway of plant lipid biosynthesis by a soluble glycerol-3-phosphate acyltransferase (GPAT [EC. 184.108.40.206]) in the chloroplast. GPAT catalyses acylation at the sn- 1 position of sn-glycerol-3-phosphate to form lysophosphatidic acid. Recombinant GPAT from squash and Arabidopsis were overproduced in Escherichia coli, purified to about 23- fold and 90% pure enzyme using a procedure developed in this study. Antibodies were raised in rabbits against these denatured recombinant GPAT preparations and four peptide antigens, and preliminary experiments were performed to test their suitability for use in Western blotting. In collaboration with the University of Sheffield, squash GPAT was successfully crystallised, isomorphous heavy metal derivatives prepared and the complete 3-dimensional structure of the protein at 2.3 Angstrom resolution determined. The cloning, functional expression and characterisation of a novel GPAT from oil palm, 'domainswap' chimeric recombinant proteins of Arabidopsis and squash GPAT, and spinach and squash GPAT respectively, and the influence of the N-terminal domain and amino acid substitutions in the C-terminal domain of the squash GPAT, was described. By determining the apparent kinetic constants for acyl-ACP substrates of most of the enzymes and by in vitro assays using mixtures of two acyl-ACP substrates under physiologically relevant conditions, it was found that their substrate selectivities could be dramatically altered. The development of ribozyme- technology as a molecular tool to down-regulate the gene expression of one out of multiple GPATs, was initiated. The strategy would allow for a phenotypic indication of ribozyme- efficacy in vivo and may help further contribute to the role of glycerol-3-phosphate acyltransferase in processes determining the phenomenon of chilling-sensitivity of plants.