Browsing Theses and Dissertations School of Pharmacy by Author "Lobo, Sandra"
Analysis of the role of an acetoacetyl CoA thiolase with a novel acetyl CoA:ACP transacylase activity in the initiation of straight chain fatty acid biosynthesis in Streptomyces collinusLobo, Sandra; Reynolds, Kevin A. (2000)Streptomycetes predominantly produce branched-chain fatty acids (BCFA) with a small proportion of straight-chain fatty acids (SCFA). In vivo studies with thiolactomycin, a type-II fatty acid synthase inhibitor suggest that beta-ketoacyl ACP synthase (KAS III) is the major enzyme initiating BCFA biosynthesis, while a KAS III-independent pathway might be partially responsible for initiating SCFA biosynthesis. An acetyl CoA:ACP transacylase (ACT), putatively responsible for initiating the SCFA biosynthetic pathway, has been separated from KASIII and purified to near homogeneity (85 fold) from the crude extract of Streptomyces collinus. This enzyme was found to be less sensitive to inhibition by thiolactomycin (IC50 0.34 +/- 0.04 mM) as compared to KAS III from S. glaucescens (IC50 20 muM). The N-terminal peptide sequence of this enzyme revealed high sequence identity to members of the thiolase superfamily of enzymes. The gene (fadA) encoding the enzyme (FadA) with ACT activity was sequenced, cloned and overexpressed in Escherichia coli. The native purified and the recombinant FadA were found to have both thiolase activity and a novel ACT activity, which has never been observed before. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for the 2-carbon precursor acetyl CoA, but did not have a specificity for the type of ACP available, using S. glaucescens FAS ACP, E. coli FAS ACP and frenolicin (type-II polyketide) ACP as substrates. Evidence indicates that the formation of the same covalent S-acetyl enzyme intermediate in the active site, and similar enzyme mechanisms may be involved in both ACT and thiolase activities. The overexpression of fadA in S. collinus, however, did not reveal a significant change in phenotype from the wild type strain, and studies with a fadA gene disruption strain of S. collinus revealed only a minor role for the ACT activity of FadA in the initiation of the SCFA biosynthetic pathway. KAS III, thus appears to be the major enzyme initiating both the SCFA and BCFA pathway, and its broad substrate specificity together with intracellular pool sizes of precursors is the major determinant of the type of fatty acids produced by S. collinus.