Synthesis of unsaturated fatty acids and the lesion in fab B mutants.

Two classes of Escherichia coli unsaturated fatty acid auxotrophs, which are complementary both in vivo and in vitro, have been utilized to study unsaturated fatty acid synthesis. /3 Hydroxydecanoyl thioester dehydrase, which catalyzes the first committed reaction in the unsaturated fatty acid synthetic pathway, is defective in one class, fab A. The defect in the second class, fab B, was unknown. The unsaturated fatty acid synthetic activity defective in fab B extracts was isolated from fab A extracts on the basis of an in vitro complementation assay. Throughout a 586-fold purification procedure the fab A unsaturated fatty acid synthetic activity was associated with /3-ketoacyl acyl carrier protein (ACP) synthetase, suggesting that the two activities are catalyzed by the same protein. Further support for this suggestion derived from the findings that both activities were purified together during the isolation of homogeneous /3-ketoacyl-ACP synthetase from wild type E. coIi B, both were inhibited to the same extent by iodoacetamide, both were protected against iodoacetamide inhibition by acetyl-ACP, and both were inactivated at 43” at identical rates. The fact that wild type /3-ketoacyl-ACP synthetase stimulated unsaturated fatty acid’ synthesis by fab B extracts suggested that fab B fl-ketoacyl-ACP synthetase is defective in unsaturated fatty acid synthetic activity. Although partially purified fab B P-ketoacyl-ACP synthetase was shown to be defective in unsaturated fatty acid synthetic activity, the mutant enzyme catalyzed normally condensation reactions involving fatty acyl-ACPs that are intermediates in the synthesis of saturated and unsaturated fatty acids. Although the unsaturated fatty acid synthetic activity of /3-ketoacylACP synthetase is not understood, these studies demonstrate that this enzyme catalyzes a unique reaction in the synthesis of unsaturated fatty acids.