Fatty acyl-ACP Research Articles

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. β-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 β-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 β-ketoacyl-ACP synthetase from wild type E. coli 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 β-ketoacyl-ACP synthetase stimulated unsaturated fatty acid synthesis by fab B extracts suggested that fab B β-ketoacyl-ACP synthetase is defective in unsaturated fatty acid synthetic activity. Although partially purified fab B β-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 β-ketoacyl-ACP synthetase is not understood, these studies demonstrate that this enzyme catalyzes a unique reaction in the synthesis of unsaturated fatty acids.

Acyl Carrier Protein: XV. STUDIES OF β-KETOACYL-ACYL CARRIER PROTEIN SYNTHETASE

β-Ketoacyl-acyl carrier protein (ACP) synthetase catalyzes the chain elongation step of fatty acid biosynthesis, the condensation of fatty acyl-ACP with malonyl-ACP yielding β-ketoacyl-ACP, CO2, and ACP. This enzyme catalyzes two additional reactions, fatty acyl-CoA-ACP transacylation and the decarboxylation of malonyl-ACP to form acetyl-ACP and CO2. Both the β-ketoacyl-ACP synthetase and the fatty acyl-CoA-ACP transacylase reactions are inhibited by low levels of iodoacetamide, and this inhibition can be prevented by a prior incubation of the enzyme with fatty acyl-CoA compounds that are substrates in the transacylase reaction. These inhibition experiments suggest that the fatty acyl-ACP and fatty acyl-CoA may have a common site on the enzyme. The malonyl-ACP decarboxylase reaction is only partially inhibited by iodoacetamide. The findings presented help to explain the fatty acyl-CoA-dependent malonyl-CoA-14CO2 exchange reaction catalyzed by β-ketoacyl-ACP synthetase.

Acyl Carrier Protein: XIV. FURTHER STUDIES ON β-KETOACYL ACYL CARRIER PROTEIN SYNTHETASE FROM ESCHERICHIA COLI

Abstract β-Ketoacyl acyl carrier protein (ACP) synthetase, which catalyzes the condensation of fatty acyl-ACP with malonyl-ACP to form β-ketoacyl-ACP, has been crystallized in a highly active form which is stable for several months. Phosphate has been shown to cause enzyme activation which may be due to a conformational change of the protein. Guanidine hydrochloride and sodium dodecyl sulfate have been shown to cause dissociation of the active enzyme into inactive subunits. By several methods the subunit molecular weight was found to be approximately 34,000. This and other data suggest that the subunits are similar and perhaps identical. Enzymatic activity can be partially recovered from guanidine hydrochloride-denatured enzyme.

Catalysis of Water Oxygen and of Acetate Incorporation into Fatty Acids by Escherichia coli Fatty Acid Synthetase

During synthesis of long chain fatty acids by an Escherichia coli fatty acid synthetase preparation in the presence of acyl carrier protein (ACP), fatty acyl-ACP is cleaved. The cleavage in H18OH resulted in the incorporation of about 1.7 atoms of water oxygen into each molecule of fatty acid synthesized. A portion of these water oxygens entered the fatty acid as a result of an exchange reaction. Addition of acetate and malonate decreased the number of water oxygens incorporated per fatty acid synthesized to 1.2 and inhibited the exchange of oxygen between the fatty acid carboxyl group and water. Free acetate and to a much lesser extent free malonate were incorporated into fatty acid. The synthesis of fatty acid and the incorporation of acetate were differentially inhibited by high levels of ACP fraction. No transfer of oxygen from the carboxyl group of acetate to the carboxyl group of long chain fatty acids was noted. These data support the proposal that fatty acyl-ACP is hydrolytically cleaved by the E. coli fatty acid synthetase preparation. The hydrolysis reaction was not rate-limiting as shown by the lack of detectable fatty acyl-ACP levels at any time during the course of the incubation.

Involvement of Acyl Carrier Protein in Acylation of Glycerol 3-Phosphate in Clostridium butyricum: I. PURIFICATION OF CLOSTRIDIUM BUTYRICUM ACYL CARRIER PROTEIN AND SYNTHESIS OF LONG CHAIN ACYL DERIVATIVES OF ACYL CARRIER PROTEIN

Abstract Clostridium butyricum grown in medium supplemented with radioactive pantothenic acid incorporates this compound into both protein and nonprotein fractions of the cell. Acyl carrier protein (ACP) is the major protein into which pantothenic acid is incorporated. C. butyricum ACP was isolated as a homogeneous protein, as judged by column chromatography on DEAE-Sephadex, disc gel electrophoresis, and analytical ultracentrifugation. This protein contains a single sulfhydryl residue, 2-mercaptoethylamine, which is a component of the prosthetic group, 4'-phosphopantetheine. The prosthetic group is cleaved from the protein when the protein is incubated at pH 12, 70°, for 1 hour. Although this protein has properties similar to those of ACP of Escherichia coli, the amino acid compositions of the two proteins are different. The chemical synthesis of long chain fatty acyl-ACP derivatives is described. In this procedure other unidentified groups of the protein are acylated as well as the sulfhydryl group. The effects of acylation or acylation and alkylation of groups other than the sulfhydryl group of ACP were studied by testing ACP regenerated from such derivatives in the malonyl coenzyme A-CO2 exchange reaction.