Bioorganic and biochemical studies of cyclopropane fatty acid synthase from Escherichia coli



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Cyclopropane fatty acids (CFAs), which represent an important class of modified lipids widely distributed in microorganisms and plants, are synthesized by cyclopropane fatty acid synthase (CFA synthase). CFAs are formed by the addition of a methylene group, derived from S-adenosyl-L-methionine, to the double bond of unsaturated fatty acids. The physiological role of CFAs in the membrane is uncertain, however their occurrence has been linked to pathogenicity in certain microorganisms. Both the unique chemistry of the reaction and the possible role of CFAs in cell membranes have motivated our study of CFA synthase. An optimized purification protocol and efforts towards an improved activity assay method are presented. Secondly, biochemical experiments are described that specifically investigate the role(s) of cysteine in catalysis. Also, the functions of three conserved tyrosine residues were explored using mutagenesis experiments. Results of enzyme incubations with a selection of phospholipid mechanistic probes, including an oxirane, two allylic ether, a vinylfluorine, and a difluorocyclopropane derivative, are presented. In addition to the mechanistic information elicited from experiments with these substrate analogs, each of the analogs was found to be a reversible inhibitor of CFA synthase. Using newly developed methodology, their inhibition activities were characterized. The information garnered from the experiments with CFA synthase described in this dissertation, do not provide evidence for the presence of a catalytic nucleophile. In contrast, support is presented with respect to a cationic mechanism. By providing a better understanding of CFA synthase catalysis, this work is a crucial step towards the development of more potent inhibitors as antimicrobial drug and herbicide candidates.