Molecular characterization of an Escherichia coli mutant with a temperature-sensitive malonyl coenzyme A-acyl carrier protein transacylase.
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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. (+info)
Expression in Escherichia coli and refolding of the malonyl-/acetyltransferase domain of the multifunctional animal fatty acid synthase.
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A cDNA encoding residues 429-815 of the multifunctional rat fatty acid synthase has been expressed in Escherichia coli and the recombinant protein refolded in vitro as a catalytically active malonyl-/acetyltransferase. Kinetic properties of the refolded recombinant enzyme were indistinguishable from those of a transferase preparation derived from the natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independent protein. Replacement of the active site Ser-581 (full-length fatty acid synthase numbering) with alanine completely eliminated catalytic activity, whereas replacement with cysteine resulted in retention of about 1% activity. The wild type transferase was extremely susceptible to inhibition by diethyl pyrocarbonate, and protection against inhibition was afforded by both malonyl- and acetyl-CoA. Replacement of the highly conserved residue His-683 with Ala reduced activity by 99.95%, and the residual activity was relatively unaffected by diethyl pyrocarbonate. The rate of acylation of the active site serine residue was also reduced by several orders of magnitude in the His-683 --> Ala mutant. These results indicate that His-683 plays an essential role in catalysis, likely by accepting a proton from the active site serine, thus increasing its nucleophilicity. (+info)
Characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in Escherichia coli and refolding in vitro.
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cDNAs of various lengths encoding the second domain of the multifunctional fatty acid synthase (FAS) have been expressed in Escherichia coli and the recombinant proteins refolded in vitro to catalytically active monomeric malonyl-/acetyltransacylases. FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, can be omitted from the recombinant enzyme with no loss in catalytic activity. This shortened transacylase, consisting of FAS residues 488-809, can be repeatedly denatured and renatured in vitro with reproducibly high recovery and no loss in specific activity. When expressed as a soluble enzyme in Spodoptera frugiperda cells, this transacylase has the same specific activity as the enzyme that has been refolded in vitro. The refolded transacylase consisting of FAS residues 488-809, but not the longer enzyme consisting of residues 428-815, can be crystallized readily. These results suggest that FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, are not required for catalysis of the transacylase reaction. This region of the FAS is less well conserved than the transacylase catalytic domain and may constitute an extended structural linker that facilitates the functional interaction between the transacylase and acyl carrier protein domains. (+info)
Antibodies to long-chain acyl-CoAs. A new tool for lipid biochemistry.
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Antibodies directed against long chain acyl-CoAs (having 16 and 18 carbon atoms) have been prepared and are reported for the first time. A modified ELISA procedure adapted to these amphiphilic molecules has been developed: it is a rapid, simple and sensitive test permitting to detect as little as 3 pmol of acyl-CoA. These antibodies represent a new tool for studying long-chain acyl-CoAs. Their use in an immunochemical approach for the study of protein-acyl-CoA interactions is presented. (+info)
Fatty acid elongation in yeast--biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.
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Elongation of long-chain fatty acids was investigated in yeast mutants lacking endogenous de novo fatty acid synthesis. In this background, in vitro fatty acid elongation was dependent strictly on the substrates malonyl-CoA, NADPH and a medium-chain or long-chain acyl-CoA primer of 10 or more carbon atoms. Maximal activity was observed with primers containing 12-14 carbon atoms, while shorter-chain-length acyl-CoA were almost (octanoyl-CoA) or completely (hexanoyl-CoA, acetyl-CoA) inactive. In particular, acetyl-CoA was inactive as a primer and as extender unit. The Michaelis constants for octanoyl-CoA (0.33 mM), decanoyl-CoA (0.83 mM) lauroyl-CoA (0.05 mM), myristoyl-CoA (0.4 mM) and palmitoyl-CoA (0.13 mM) were determined and were comparable for fatty acid synthesis and elongation. In contrast, the affinity of malonyl-CoA was 17-fold lower for elongation (Km = 0.13 mM) than for the fatty acid synthase (FAS) system. With increasing chain length of the primer (> or = 12:0), fatty acid elongation becomes increasingly sensitive to substrate inhibition. Due to the activation of endogenous fatty acids, ATP exhibits a stimulatory effect at suboptimal but not at saturating substrate concentrations. In the yeast cell homogenate, the specific activity of fatty acid elongation is about 10-20-fold lower than that of de novo fatty acid synthesis. The same elongation activity is observed in respiratory competent and in mitochondrially defective cells. The products of in vitro fatty acid elongation are fatty acids of 15-17 or 22-26 carbon atoms, depending on whether tridecanoyl-CoA or stearoyl-CoA is used as a primer. In vitro, the elongation products are converted in part, by alpha-oxidation, to their odd-chain-length lower homologues or are hydrolyzed to fatty acids. In contrast, no odd-chain-length elongation products or very-long-chain fatty acids (VLCFA) shorter than 26:0 are observed in vivo. Hence, VLCFA synthesis exhibits a higher processivity in vivo than in the cell homogenate. In addition, the in vivo process appears to be protected against side reactions such as hydrolysis or alpha-oxidation. Yeast mutants defective in 12:0 or 13:0 elongation were derived from fas-mutant strains according to their failure to grow on 13:0-supplemented media. In vivo, 12:0 elongation was reduced to 0-10% of the normal level, while 16:0 elongation and VLCFA synthesis were unimpaired. It is concluded that yeast contains either two different elongation systems, or that the respective mutation interferes differentially with medium-chain and long-chain fatty acid elongation. The yeast gene affected in the elongation-defective mutants was isolated and, upon sequencing, identified as the known ELO1 sequence. It encodes a putative membrane protein of 32-kDa molecular mass with no obvious similarity to any of the known FAS component enzymes. (+info)
Arginyl residues are involved in acyl-CoA binding to the elongase from etiolated leek seedlings.
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The C18:0-CoA elongase from etiolated leek seedling microsomes was inactivated by treatment with phenylglyoxal, a reagent which specifically modifies arginyl residues. In the presence of 20 mM phenylglyoxal, 95% of the C18:0-CoA elongation was inhibited. The condensation and dehydration reactions of the overall elongation were totally inhibited, whereas enoyl-CoA reductase activity was diminished by 75%, but the nature of the final elongation product was unchanged. Phenylglyoxal did not modify the C18:0-CoA partition between membrane and aqueous compartments; moreover, [1-14C]phenylglyoxal labeling experiments showed a covalent binding of the inhibitor to membrane proteins. The ability of several substrates to prevent the inactivation by phenylglyoxal was investigated. NADH and NADPH had no effect. CoA led to a 75% protection, and the incorporation of [14C]phenylglyoxal was strongly affected by 10 mM CoA. The acyl chain length of the acyl-CoAs played also a crucial role in preventing the binding of phenylglyoxal. The maximal prevention of phenylglyoxal inhibition was obtained with C18:0-CoA. This suggests that arginyl residues could be present in the vicinity of the acyl-CoA binding site of the subunits of C18:0-CoA elongase. (+info)
Overproduction of a functional fatty acid biosynthetic enzyme blocks fatty acid synthesis in Escherichia coli.
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beta-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase. (+info)
cDNA cloning of Brassica napus malonyl-CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant.
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The GenBank database was searched using the E. coli malonyl CoA:ACP transacylase (MCAT) sequence, for plant protein/cDNA sequences corresponding to MCAT, a component of plant fatty acid synthetase (FAS), for which the plant cDNA has not been isolated. A 272-bp Zea mays EST sequence (GenBank accession number: AA030706) was identified which has strong homology to the E. coli MCAT. A PCR derived cDNA probe from Zea mays was used to screen a Brassica napus (rape) cDNA library. This resulted in the isolation of a 1200-bp cDNA clone which encodes an open reading frame corresponding to a protein of 351 amino acids. The protein shows 47% homology to the E. coli MCAT amino acid sequence in the coding region for the mature protein. Expression of a plasmid (pMCATrap2) containing the plant cDNA sequence in Fab D89, an E. coli mutant, in MCAT activity restores growth demonstrating functional complementation and direct function of the cloned cDNA. This is the first functional evidence supporting the identification of a plant cDNA for MCAT. (+info)