Coenzyme A-acyl Carrier Protein Transacylase Research Articles

Developmental specific expression and organelle targeting of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds.

In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.

Crystallization of the Malonyl Coenzyme A-Acyl Carrier Protein Transacylase from Escherichia coli

The malonyl coenzyme A-acyl protein transacylase, a single polypeptide chain of 358 amino acid residues and a molecular mass of 32 kDa, is a key component of the fatty acid synthase multienzyme complex. The elucidation of its three-dimensional structure will help in the understanding of the molecular basis of the biosynthesis of fatty acids, as well as of polyketides and related biologically active molecules. Three X-ray-quality crystal forms of the Escherichia coli fabD gene product encoding for malonyl coenzyme A-acyl carrier protein transacylase have been obtained using the hanging-drop method and ammonium sulfate as precipitant. Two are tetragonal and each contains two molecules in the asymmetric unit (form I: space group P 43(1)212 with a = b = 83?9 Å, c = 166?5 Å and form II: space group P4 with a = b = 132?64 Å, c = 38?85 Å), whereas the third form belongs to the hexagonal system and contains one molecule in the asymmetric unit (space group P61(5) with a = b = 68?52 Å, c = 117?71 Å). In each case, the diffraction pattern extends to approximately 2?0 Å resolution using CuKα radiation from a rotating anode source.

Molecular characterization of an Escherichia coli mutant with a temperature-sensitive malonyl coenzyme A-acyl carrier protein transacylase

The temperature-sensitive malonyl CoA-ACP transacylase found in the Escherichia coli strain LA2-89, carrying the fabD89 allele, was shown to result from the presence of an amber mutation in the fabD gene, at codon position 257, in combination with the supE44 genotype of this strain. The truncated form of the protein produced as the result of the amber mutation was demonstrated to be enzymatically inactive, whereas amber suppression rendered the resulting enzyme temperature labile. Site-directed mutagenesis of codon 257 revealed a requirement for an aromatic amino acid at this position in the polypeptide chain, to assure temperature stability of the enzyme.

Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli

We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli, Malonyl transacylase has been overexpressed 155-fold compared to a wild-type strain, Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of cis-vaccenate are incorporated into the membrane phospholipids.

Mutants of Escherichia coli with Temperature-sensitive Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

Abstract We have characterized two mutants of Escherichia coli in which fatty acid biosynthesis is adversely affected by elevated temperature. Under conditions which do not alter the activity of wild type cell-free extracts, virtually all of the malonyl coenzyme A-acyl carrier protein (ACP) transacylase and fatty acid synthetase activities present in mutant extracts are thermolabile. Malonyl transacylase from wild type cells restores normal fatty acid synthetase activity to inactivated mutant extracts. Furthermore, fatty acid synthesis in mutant extracts is corrected by malonyl-ACP. Malonyl transacylase and fatty acid synthetase activities are also restored in revertants which were selected for their ability to grow at elevated temperatures. These experiments furnish evidence that the mutants are altered in the structural gene for malonyl transacylase and that this is their only genetic defect. When these strains, defective in the synthesis of a substrate vital for fatty acid elongation, are grown at elevated temperatures, their phospholipids become deficient in long chain acyl groups. Supplementation of these cultures with long chain fatty acids permits sustained growth and the maintenance of a normal fatty acid composition.

The Isolation and General Properties of Escherichia coli Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

Abstract Malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli was purified 4800-fold by procedures which included chromatography on DEAE-cellulose, Sephadex G-100, Sephadex G-75, DEAE-Sephadex, and preparative polyacrylamide gel electrophoresis. The purified enzyme was shown to be homogeneous by electrophoresis on polyacrylamide gels, by sedimentation equilibrium centrifugation, and by amino acid analysis. A molecular weight of 36,660, obtained with carboxymethylated enzyme by sedimentation equilibrium measurements, agreed with determinations on the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (36,500) and by Sephadex G-100 column chromatography (37,000). An s20,w = 2.31 S was determined for the enzyme by sedimentation velocity measurements. Amino acid analysis and determination of the isoelectric point (pH 4.65) both showed the enzyme to be acidic. The purified enzyme was inhibited by N-ethylmaleimide and to a lesser extent by iodoacetamide, but inhibition by both reagents was significantly increased at pH values above 7.3. Preincubation of the enzyme with malonyl-CoA protected against inactivation by both sulfhydryl reagents. Reduction of aged enzyme preparations by incubation with dithiothreitol was shown to stimulate enzyme activity approximately 5-fold. These experiments suggest that a reduced sulfhydryl group(s) on the enzyme is required for maximal catalytic activity.

Characterization of a Malonyl-Enzyme Intermediate and Identification of the Malonyl Binding Site in Malonyl Coenzyme A-Acyl Carrier Protein Transacylase of Escherichia coli

Abstract A malonyl-enzyme intermediate, which functions in the transacylation of malonate from coenzyme A to acyl carrier protein (ACP) in Escherichia coli fatty acid synthesis, was isolated and characterized using purified and reduced malonyl-CoA-ACP transacylase. An enzymatically active malonyl-enzyme complex was isolated by Sephadex chromatography in 8 m urea, by trichloroacetic acid precipitation, and by Sephadex chromatography in aqueous buffer. The intermediate isolated by these procedures was shown to be free of CoA and capable of transferring the covalently bound malonyl group to CoA or ACP. Formation of malonyl-enzyme appeared to be a readily reversible reaction with a pH optimum between 6.0 and 7.5. Stability measurements of the malonyl-enzyme indicated a marked lability at pH values above 6.0, with a half-life of 30 to 40 min at pH 7.0 at 0°. The malonyl-enzyme linkage was shown to be stable to performic acid oxidation and labile to neutral hydroxylamine and alkaline pH. Denaturation of the malonyl-enzyme by urea at concentrations of 2.0 m or greater increased the stability of the malonyl-enzyme at various pH values and stabilized it to cleavage by hydroxylamine. Dilution of the urea concentration to 0.1 m permitted renaturation of the malonyl-enzyme, as indicated by its ability to transfer bound malonyl groups to CoA and its sensitivity to hydroxylamine and alkaline pH. A pH dependent inhibition of malonyl-enzyme formation was observed with the alkylating agents, N-ethylmaleimide and iodoacetamide. Treatment of the enzyme with phenylmethylsulfonylfluoride and p-chloromercuribenzoate also resulted in inhibition of malonyl-enzyme formation. p-Chloromercuribenzoate effects were shown to be reversed by 2-mercaptoethanol. Preincubation of the enzyme with malonyl-CoA prevented the inhibitory effects of all agents tested. Identification of the malonyl binding site on the enzyme was accomplished by proteolytic hydrolysis of [14C]malonyl-enzyme. Digestion of carboxymethylated [14C]malonyl-enzyme with thermolysin and purification of the labeled peptides by paper chromatography and Dowex 50 column chromatography resulted in the separation of five groups of labeled peptides, B through F. Treatment with performic acid indicated that the [14C]malonyl groups were not cleaved from these peptides by this treatment. Further purification of the major [14C]malonyl-peptide, D, by paper electrophoresis and by Dowex 50 chromatography after performic acid oxidation resulted in the isolation of a pure peptide with the amino acid composition alanine, glycine, histidine, and serine. Digestion of two of the [14C]malonyl-peptides, E and F, with aminopeptidase plus pronase resulted in the quantitative release of radioactive products having chromatographic properties identical with those of synthetic malonyl-O-serine. These experiments indicate that a serine residue of malonyl transacylase is the covalent binding site of malonyl groups in the malonyl-enzyme intermediate.

Studies on the Mechanism of Fatty Acid Synthesis: XVII. PREPARATION AND GENERAL PROPERTIES OF ACETYL COENZYME A AND MALONYL COENZYME A-ACYL CARRIER PROTEIN TRANSACYLASES

Acetyl coenzyme A-acyl carrier protein transacylase (acetyl transacylase) and malonyl coenzyme A-acyl carrier protein transacylase (malonyl transacylase) have been purified 90- and 255-fold, respectively, from Escherichia coli extracts and exhibited the following properties. Acetyl transacylase is heat-labile whereas malonyl transacylase is comparatively heat-stable, 80% of its activity surviving for 20 min at 80°. Both enzymes have pH optima in the region of 6.5. The reactions are readily reversible, and equilibrium constants are 2.09 for acetyl transacylase and 2.33 for malonyl transacylase. Inhibition of the enzymes by N-ethylmaleimide and iodoacetamide demonstrates that both are sulfhydryl enzymes. Acetyl transacylase is relatively specific for acetyl-CoA but can transacylate coenzyme A esters of propionic, butyric, and hexanoic acids at a rate of 23.4, 9.8, and 4.5% that of acetyl-CoA, respectively. Evidence is presented in support of a mechanism of action of acetyl transacylase involving the intermediary formation of an acetyl—S—enzyme which can then transfer the acetyl group to acyl carrier protein or coenzyme A.