Structure and regulation of rat long-chain acyl-CoA synthetase

Complementary DNAs encoding rat long-chain acyl-CoA synthetase have been isolated. The cDNAs were identified using synthetic oligonucleotide probes based on partial amino acid sequences of lysyl endopeptidase peptides of the purified enzyme. Rat long-chain acyl-CoA synthetase is predicted to contain 699 amino acid residues and to have a calculated molecular weight of 78,177. Significant sequence similarity was found between parts of long-chain acyl-CoA synthetase and firefly luciferase. Based on the similarity of the reaction mechanisms of the two enzymes, we propose a function for the similar region. The long-chain acyl-CoA synthetase mRNA is expressed in liver, heart, and epididymal adipose tissues and, to a much lesser extent, in brain, small intestine, and lung. The level of long-chain acyl-CoA synthetase mRNA is increased 7-8-fold in rat liver by feeding a diet high in carbohydrate or fat, consistent with the physiological significance of the enzyme in fatty acid metabolism.     

Sepharose-stearate as substrate for rat liver long-chain fatty acyl-CoA synthetase

Stearic acid coupled covalently to Sepharose 6B serves as substrate for thioesterification catalyzed by rat liver long-chain fatty acyl-CoA synthetase (ATP-forming) (EC 6.2.1.3). Availability as substrate is dependent upon the conservation of the free omega-terminal in addition to that of the free carboxyl function. The enzymatic overall formation of matrix-acyl-CoA in the presence of ATP and CoA as cosubstrates conforms to the stoichiometry reported for thioesterification of the free long-chain fatty acyl substrate. The preformed matrix-acyl-CoA serves as substrate for the backward synthetase reaction in the presence of AMP and PPi. The apparent Km values for ATP and CoA in the presence of the acyl matrix are similar to the respective Km values observed in the presence of the free acid substrate. The apparent Km for the acyl matrix is 10-fold higher (0.5 mM) than the apparent Km value for the free acid. The feasibility of enzymatic thioesterification of bound long-chain fatty acids implies that the exact nature of the bulky chain situated between the carboxy and omega-terminal plays a secondary role in defining the fatty acyl substrate specificity for long-chain fatty acyl-CoA synthetase. Also, dissociation of bound long-chain fatty acids does not constitute an obligatory preliminary step to fatty acid thioesterification.     

Human very-long-chain acyl-CoA synthetase: cloning, topography, and relevance to branched-chain fatty acid metabolism

Very-long-chain acyl-CoA synthetases (VLCS) activate very-long-chain fatty acids (VLCFA) containing 22 or more carbons to their CoA derivatives. We cloned the human ortholog (hVLCS) of the gene encoding the rat liver enzyme (rVLCS). Both hVLCS and rVLCS contain 620 amino acids, are expressed primarily in liver and kidney, and have a potential peroxisome targeting signal 1 (-LKL) at their carboxy termini. When expressed in COS-1 cells, hVLCS activated the VLCFA lignoceric acid (C24:0), a long-chain fatty acid (C16:0), and two branched-chain fatty acids, phytanic acid and pristanic acid. Immunofluorescence and immunoblot studies localized hVLCS to both peroxisomes and endoplasmic reticulum. In peroxisomes of HepG2 cells, hVLCS was topographically oriented facing the matrix and not the cytoplasm. This orientation, coupled with the observation that hVLCS activates branched-chain fatty acids, suggests that hVLCS could play a role in the intraperoxisomal reactivation of pristanic acid produced via alpha-oxidation of phytanic acid.     

Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin

The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.     

Palmitoyl-coenzyme A synthetase. Isolation of an enzyme-bound intermediate

An enzyme-bound intermediate of the overall reaction catalysed by rat liver microsomal long-chain fatty acyl-CoA synthetase is described. It was found to contain equimolar amounts of adenylate and fatty acid moieties bound to protein, and was stabilized by ATP. The intermediate reacted with CoA to give palmitoyl-CoA.     

Human long-chain acyl-CoA synthetase: structure and chromosomal location.

A complementary DNA clone encoding the entire human long-chain acyl-CoA synthetase was isolated and the total 698-amino acid sequence was deduced. The amino acid sequence of human long-chain acyl-CoA synthetase shows 84.9% identity to that of rat long-chain acyl-CoA synthetase. The nucleotide sequences of the protein coding regions between human and rat long-chain acyl-CoA synthetase mRNAs are highly conserved (85.6%), whereas those of the 3' untranslated regions are less conserved (72%). The location of the human long-chain acyl-CoA synthetase gene was identified on chromosome 4 by spot hybridization of flow-sorted chromosomes. Computer-assisted homology search revealed a significant similarity of the enzyme with the enzymes of the luciferase family. Based on this similarity, the structure of human long-chain acyl-CoA synthetase can be divided into five domains: the N-terminus, two domains similar to those in enzymes of the luciferase family, a long gap region between the similar domains and the C-terminus.     

Human very long-chain acyl-CoA synthetase and two human homologs: initial characterization and relationship to fatty acid transport protein

Several human genes with a high degree of homology to rat very long-chain acyl-CoA synthetase (rVLCS) and mouse fatty acid transport protein (mFATP) were identified. Full-length cDNA clones were obtained for three genes, and predicted amino acid sequences were generated. Initial characterization indicated that one gene was most likely hVLCS, the human ortholog of rVLCS. The other two (hVLCS-H1 and hVLCS-H2) were more closely related to rVLCS than to mFATP. Phylogenetic analysis of amino acid sequences confirmed that hVLCS-H1 and hVLCS-H2 were evolutionarily closer to VLCSs than FATPs. Alignment of predicted amino acid sequences of human, rat and mouse VLCSs and FATPs revealed the existence of two highly conserved motifs. While one motif is also present in long-chain acyl-CoA synthetases, the other serves to distinguish the VLCS/FATP family from the long-chain synthetase family. Elucidation of the biochemical functions of all VLCS/FATP family members should provide new insights into cellular fatty acid metabolism.     

Inhibition of acyl-CoA synthetase by triacsins

Triacsin A, 1-hydroxy-3-(E,E-2',4'-undecadienylidine) triazene and triacsin C, 1-hydroxy-3-(E,E,E-2',4',7'-undecatrienylidine) triazene are potent inhibitors of acyl-CoA synthetase (EC 6.2.1.3). The concentrations of triacsin A required for 50% inhibition of acyl-CoA synthetase from Pseudomonas aeruginosa and from rat liver are 17 and 18 microM, and those of triacsin C are 3.6 and 8.7 microM, respectively. Kinetic analysis indicates that inhibition of triacsin A is non-competitive with respect to the two substrates ATP and coenzyme A, but is competitive with respect to long-chain fatty acids. The apparent Ki value is 8.97 microM when oleic acid is used as substrate. Acid hydrolysis of triacsins results in corresponding polyenic aldehydes with no activity. This suggests that the N-hydroxytriazene moiety is essential for inhibitory activity against acyl-CoA synthetase.     

Enzymatic and genetic characterization of firefly luciferase and Drosophila CG6178 as a fatty acyl-CoA synthetase

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.     

Binding of acyl-CoA to liver fatty acid binding protein: effect on acyl-CoA synthesis

The ability of purified rat liver and heart fatty acid binding proteins to bind oleoyl-CoA and modulate acyl-CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart fatty acid binding protein was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver fatty acid binding protein has a single binding site acyl-CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl-CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver fatty acid binding protein stimulated acyl-CoA production, whereas that from heart did not stimulate production over control values. 14C-labeled fatty acid-fatty acid binding protein complexes were prepared, incubated with membranes, and acyl-CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl-CoA in the presence of liver fatty acid binding protein but in the presence of heart fatty acid binding protein, only 45% of the fatty acid was converted. Liver but not heart fatty acid binding protein bound the acyl-CoA formed and removed it from the membranes. The amount of product formed was not changed by additional membrane, enzyme cofactors, or incubation time. Additional liver fatty acid binding protein was the only factor found that stimulated product formation. Acyl-CoA hydrolase activity was also shown in the absence of ATP and CoA. These studies suggest that liver fatty acid binding protein can increase the amount of acyl-CoA by binding this ligand, thereby removing it from the membrane and possibly aiding transport within the cell.     

Effects of experimental hypo- and hyperthyroidism on hepatic long-chain fatty acyl-CoA synthetase and hydrolase

The effects of T3 treatment and thyroidectomy on rat liver microsomal long-chain fatty acyl-CoA (LCFA-CoA) synthetase and LCFA-CoA hydrolase activities were determined. Hyperthyroid rats had a 36-42% decrease in LCFA-CoA synthetase with no change in hydrolase activity. This may contribute to the redirection of fatty acids from esterification to oxidation reactions in hyperthyroidism. Thyroidectomized rats had a 40-44% decrease in synthetase and a 27-42% decrease in LCFA-CoA hydrolase activity. The decrease in both LCFA-CoA synthetase and hydrolase activities in hypothyroidism may indicate that the LCFA-CoA turnover in this futile cycle is decreased in the liver.     

A fluorimetric assay for acyl-CoA synthetase activities.

In this paper we describe a fluorimetric assay for the measurement of long-chain acyl-CoA synthetase activity in rat liver postnuclear supernatants. The method is based upon the use of acyl-CoA oxidase which catalyzes the dehydrogenation of acyl-CoA esters to yield enoyl-CoA esters and H2O2. H2O2 subsequently reacts with homovanillic acid in a horseradish peroxidase-catalyzed reaction to form a highly fluorescent dimer (see G. G. Guilbault, P. J. Brignac, and M. Zimmer (1968) Anal. Chem. 40, 190-196). The increase in fluorescence can be followed either continuously or discontinuously. The method described is able to detect acyl-CoA synthetase activities as low as 20 microU/ml which is almost as sensitive as the standard isotopic assay used in most laboratories. The method is applicable to measure the activation of a variety of fatty acids. Finally, the method provides a simple means of carrying out kinetic studies.     

Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells

Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors α and β (LXRα and LXRβ), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.     

Palmitoyl-CoA synthetase on the external surface of isolated rat hepatocytes

After incubating isolated rat hepatocytes with [1-¹⁴C]palmitic acid, CoA and ATP (+MgCl₂), a significant amount of [1-¹⁴C]palmitoyl-CoA was found in the incubation medium. There was no correlation between its rate of synthesis and the degree of intactness of the cells. The results indicate that there is a long-chain fatty acyl-CoA synthetase active on the external surface of the hepatocyte plasma membrane. The activity of this enzyme was negligible in primary cultures of rat hepatocytes, suggesting that the exofacial long-chain acyl-CoA synthetase is an artifact of the collagenase perfusion technique used to prepare the hepatocytes.     

Characterization of an acyl-coA thioesterase that functions as a major regulator of peroxisomal lipid metabolism.

Peroxisomes function in beta-oxidation of very long and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. Here we have cloned and characterized a peroxisomal acyl-CoA thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA thioesterase 2). PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor alpha. Recombinant PTE-2 showed a broad chain length specificity with acyl-CoAs from short- and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA, and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acid-CoA:amino acid N-acyltransferase activity, suggesting that this thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.     

Correlation between the cellular level of long-chain acyl-CoA, peroxisomal beta-oxidation, and palmitoyl-CoA hydrolase activity in rat liver. Are the two enzyme systems regulated by a substrate-induced mechanism?

Data obtained in earlier studies with rats fed diets containing high doses of peroxisome proliferators (niadenate, tiadenol, clofibrate, or nitotinic acid) are used to look for a quantitative relationship between peroxisomal beta-oxidation, palmitoyl-CoA hydrolase, palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities, and the cellular concentration of their substrate and reaction products. The order of the hyperlipidemic drugs with regard to their effect on CoA derivatives and enzyme activities was niadenate greater than tiadenol greater than clofibrate greater than nicotinic acid. Linear regression analysis of long-chain acyl-CoA content versus palmitoyl-CoA hydrolase and peroxisomal beta-oxidation activity showed highly significant linear correlations both in the total liver homogenate and in the peroxisome-enriched fractions. A dose-response curve of tiadenol showed that carnitine palmitoyltransferase and palmitoyl-CoA synthetase activities and the ratio of long-chain acyl-CoA to free CoASH in total homogenate rose at low doses before detectable changes occurred in the peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. A plot of this ratio parallelled the palmitoyl-CoA synthetase activity. The specific activity of microsomally localized carnitine palmitoyl-transferase was low and unchanged up to a dose where no enhanced peroxisomal beta-oxidation was observed, but over this dose the activity increased considerably so that the specific of the enzyme in the mitochondrial and microsomal fractions became comparable. The mitochondrial palmitoyl-CoA synthetase activity decreased gradually. The correlations may be interpreted as reflecting a common regulation mechanism for palmitoyl-CoA hydrolase and peroxisomal beta-oxidation enzymes, i.e., the cellular level of long-chain acyl-CoA acting as the metabolic message for peroxisomal proliferation resulting in induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. The findings are discussed with regard to their possible consequences for mitochondrial fatty acid oxidation and the conversion of long-chain acyl-L-carnitine to acyl-CoA derivatives.     

Regulation of mitochondrial carbamoyl-phosphate synthetase 1 activity by active site fatty acylation

In addition to its role in reversible membrane localization of signal-transducing proteins, protein fatty acylation could play a role in the regulation of mitochondrial metabolism. Previous studies have shown that several acylated proteins exist in mitochondria isolated from COS-7 cells and rat liver. Here, a prominent fatty-acylated 165-kDa protein from rat liver mitochondria was identified as carbamoyl-phosphate synthetase 1 (CPS 1). Covalently attached palmitate was linked to CPS 1 via a thioester bond resulting in an inhibition of CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to irreversible inactivation of CPS 1 and occurred in a time- and concentration-dependent manner. Fatty acylation of CPS 1 was prevented by preincubation with N-ethylmaleimide and 5'-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues. Our results suggest that fatty acylation of CPS 1 is specific for long-chain fatty acyl-CoA and very likely occurs on at least one of the essential cysteine residues inhibiting the catalytic activity of CPS 1. Inhibition of CPS 1 by long-chain fatty acyl-CoAs could reduce amino acid degradation and urea secretion, thereby contributing to nitrogen sparing during starvation.     

Identification of a fatty acyl-CoA synthetase gene, lcf2+, which affects viability after entry into the stationary phase in Schizosaccharomyces pombe

The lcf1(+) gene, which encodes a long chain fatty acyl-CoA synthetase, is necessary for the maintenance of viability after entry into the stationary phase in Schizosaccharomyces pombe. In this study, we analyzed a paralogous gene, SPBP4H10.11c (named lcf2(+)), and we present evidence that the gene encodes a new fatty acyl-CoA synthetase. The enzyme preferentially recognized myristic acid as a substrate. A Deltalcf2 mutant showed increased viability after entry into the stationary phase in SD medium. A Deltalcf1Deltalcf2 double mutant showed a severe decrease in long-chain fatty acyl-CoA synthetase activity and a rapid loss of viability after entry into the stationary phase. These results suggest that fatty acid utilization and/or metabolism is important to determine viability in the stationary phase.