Long-chain acyl-CoA oxidases of Arabidopsis

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium-and long-chain saturated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity with mono-unsaturated acyl-CoAs was slightly higher than with the corresponding saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showed maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl-CoAs was approximately twice as high as with the corresponding saturated acyl-CoA. Both enzymes have an apparent Km of approximately 5 microM with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady-state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA oxidases specific for shorter-chain acyl-CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long-chain oxidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.     

Substrate inhibition and affinities of the glyoxysomal β-oxidation of sunflower cotyledons

During the glyoxysomal β-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125–130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal β-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the β-oxidation of acyl-CoAs of various chain lengths showed that the β-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 μM. At substrate concentrations >20 μM, long-chain acyl-CoAs were β-oxidized more slowly than short-chain acyl-CoAs because the β-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5–10 μM substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the β-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the β-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (≤5 μM) of a long-chain acyl-CoA exerted an inhibitory effect on the β-oxidation rate of butyryl-CoA. Reversibility of the inhibition was observed as well as metabolization of the inhibiting long-chain acyl-CoA. Regarding the activities of the individual β-oxidation enzymes towards their C₄ substrates in the presence of a long-chain acyl-CoA, the MFP activity exhibited strong inhibition. This inhibition appears not to be due to the detergent-like physical properties of long-chain acyl-CoAs. The results of the studies, which are consistent with the observation that short-chain intermediates accumulate transiently during complete degradation of a long-chain acyl-CoA, suggest that the substrate concentration-dependent chain-length specificity of the β-oxidation and a metabolic regulation at the level of MFP are factors determining this transient accumulation. Received: 2 February 1999 / Accepted: 14 April 1999     

Effects of clofibric acid and tiadenol on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation in liver and extrahepatic tissues of rats

The effects of two peroxisome proliferators, p-chlorophenoxyisobutyric acid (clofibric acid) and 2,2'-(decamethylenedithio)diethanol (tiadenol), on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation were studied in several organs of rat. Among organs of control rats, the brain had the highest activity of long-chain acyl-CoA hydrolase, followed by testis, and a low activity was found in other tissues. Administration of the peroxisome proliferators caused a marked increase in activity of long-chain acyl-CoA hydrolase in both liver and intestinal mucosa and a slight increase in the activity in kidney, but little affected acyl-CoA hydrolase activity in either brain, testis, heart, spleen and skeletal muscle. In accordance with the change in the activity of acyl-CoA hydrolase, the activity of peroxisomal beta-oxidation was markedly increased in liver, intestinal mucosa and kidney, and a slight increase was found in brain and testis, whereas peroxisome proliferators little affected the activity in other organs tested. Gel filtration of cytosol from intestinal mucosa showed that clofibric acid caused an appearance of a new peak in intestinal mucosa. Although cytosol of liver, intestinal mucosa, brain and testis contained two 4-nitrophenyl acetate esterases with different molecular weights (about 105,000 and about 55,000), these esterases are different from cytosolic long-chain acyl-CoA hydrolases of these four organs in respect of molecular weight. The administration of clofibric acid little affected cytosolic 4-nitrophenyl acetate esterases. Comparative studies on cytosolic long-chain acyl-CoA hydrolases from these four organs showed that liver hydrolase I (molecular weight of about 80,000) had properties similar to those of brain and testis enzymes. On the other hand, intestinal mucosa enzyme was different from either hepatic hydrolase I or II (molecular weight of about 40,000). The results from the present study suggest that inductions of peroxisomal beta-oxidation and cytosolic long-chain acyl-CoA hydrolases are essential responses of rats to peroxisome proliferators not only in liver but also in intestinal mucosa and that induced hydrolases are not attributable to non-specific esterases.     

Human brain acyl-CoA hydrolase isoforms encoded by a single gene

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a-1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3(')-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.     

Cytosolic modulators of activities of microsomal enzymes of cholesterol biosynthesis. Effects of Acyl-CoA inhibition and cytosolic Z-protein

Physiological concentrations of long-chain fatty acyl-CoAs have now been shown to inhibit microsomal methyl sterol oxidase. Acyl-CoA inhibition of hydroxymethylglutaryl-CoA reductase as well as methyl sterol oxidase can be either prevented or reversed by the addition of purified Z-protein (fatty acid-binding protein). Concomitantly, Z-protein addition decreases the extent of binding of radioactively labeled oleoyl-CoA to microsomal membranes. Free heme also inhibits hydroxymethylglutaryl-CoA reductase, and Z-protein reverses the extent of observed inhibition by binding heme analogous to the effect observed with acyl-CoAs. Similarly, Z-protein reverses substrate inhibition of acyl-CoA:cholesterol acyltransferase at high concentrations of acyl-CoA substrate. All these observations are consistent with the suggestion that, by binding acyl-CoAs and other enzyme effectors such as free heme, Z-protein modulates the effects of fluctuations of concentrations of major cellular metabolites. Furthermore, because the concentration of Z-protein is very low in rapidly growing hepatomas, such tumors may be very poorly buffered against the effects of acyl-CoAs, free fatty acids, heme and other effectors that may vary markedly by either altered metabolism or release of metabolites from necrotic tumor tissue.     

Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor alpha (PPARalpha)-regulated peroxisomal acyl-CoA thioesterases

Peroxisomes are organelles that function in the beta-oxidation of long- and very long-chain acyl-CoAs, bile acid-CoA intermediates, prostaglandins, leukotrienes, thromboxanes, dicarboxylic fatty acids, pristanic acid, and xenobiotic carboxylic acids. The very long- and long-chain acyl-CoAs are mainly chain-shortened and then transported to mitochondria for further metabolism. We have now identified and characterized two peroxisomal acyl-CoA thioesterases, named PTE-Ia and PTE-Ic, that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A. PTE-Ia and PTE-Ic show 82% sequence identity at the amino acid level, and a putative peroxisomal type 1 targeting signal of -AKL was identified at the carboxyl-terminal end of both proteins. Localization experiments using green fluorescent fusion protein showed PTE-Ia and PTE-Ic to be localized in peroxisomes. Despite their high level of sequence identity, we show that PTE-Ia is mainly active on long-chain acyl-CoAs, whereas PTE-Ic is mainly active on medium-chain acyl-CoAs. Lack of regulation of enzyme activity by free CoASH suggests that PTE-Ia and PTE-Ic regulate intraperoxisomal levels of acyl-CoA, and they may have a function in termination of beta-oxidation of fatty acids of different chain lengths. Tissue expression studies revealed that PTE-Ia is highly expressed in kidney, whereas PTE-Ic is most highly expressed in spleen, brain, testis, and proximal and distal intestine. Both PTE-Ia and PTE-Ic were highly up-regulated in mouse liver by treatment with the peroxisome proliferator WY-14,643 and by fasting in a peroxisome proliferator-activated receptor alpha-dependent manner. These data show that PTE-Ia and PTE-Ic have different functions based on different substrate specificities and tissue expression.     

NADH-sensitive propionyl-CoA hydrolase in brown-adipose-tissue mitochondria of the rat

Acyl-CoA hydrolase activity was studied in brown adipose tissue (BAT) mitochondria of rats. The substrate specificity was investigated: total hydrolase activity showed two activity peaks, one sharp peak for propionyl-CoA and a broad peak at medium- to long-chain acyl-CoAs. The propionyl-CoA activity fully comigrated with a mitochondrial matrix marker enzyme in fractionation studies of tissue and mitochondria. The hydrolytic activity against short-chain acyl-CoAs was inhibited by NADH, and analyses of the substrate specificity of the hydrolases in the presence and absence of NADH allowed for the delineation of two distinct acyl-CoA hydrolases. These hydrolases could also be separated by gel filtration. It was concluded that rat BAT mitochondria possess at least two matrix acyl-CoA hydrolases: one broad-spectrum acyl-CoA hydrolase with an apparent native molecular weight of less than 100,000, and a specific propionyl-CoA hydrolase with an apparent native molecular weight at least 240,000; this hydrolase is regulated by NADH. It is suggested that the function of the propionyl-CoA hydrolase is to ensure that the level of propionyl-CoA in the mitochondria is not detrimentally increased.     

A specific and inexpensive assay of radiolabeled long-chain acyl-coenzyme A in isolated hepatocytes.

A simple procedure for determining radiolabeled long-chain acyl-CoA levels in isolated rat hepatocytes has been developed. It consists of a classical extraction of long-chain acyl-CoAs after preliminary removal of the excess labeled free fatty acid. A two-step TLC purification ensures elimination of long-chain acylcarnitine, the main interferent in most methods, and other common lipids. The purity of the acyl-CoA was confirmed by a second TLC system and by spectral analysis. Overall recovery was approximately 70%.     

Acyl-CoA synthesis,lipid metabolism and lipotoxicity

Although the underlying causes of insulin resistance have not been completely delineated, in most analyses, a recurring theme is dysfunctional metabolism of fatty acids. Because the conversion of fatty acids to activated acyl-CoAs is the first and essential step in the metabolism of long-chain fatty acid metabolism, interest has grown in the synthesis of acyl-CoAs, their contribution to the formation of signaling molecules like ceramide and diacylglycerol, and their direct effects on cell function. In this review, we cover the evidence for the involvement of acyl-CoAs in what has been termed lipotoxicity, the regulation of the acyl-CoA synthetases, and the emerging functional roles of acyl-CoAs in the major tissues that contribute to insulin resistance and lipotoxicity, adipose, liver, heart and pancreas.     

Access and utilization of long chain fatty acyl-CoA by zDHHC protein acyltransferases

S-acylation is a reversible posttranslational modification, where a long-chain fatty acid is attached to a protein through a thioester linkage. Being the most abundant form of lipidation in humans, a family of twenty-three human zDHHC integral membrane enzymes catalyze this reaction. Previous structures of the apo and lipid bound zDHHCs shed light into the molecular details of the active site and binding pocket. Here, we delve further into the details of fatty acyl-CoA recognition by zDHHC acyltransferases using insights from the recent structure. We additionally review indirect evidence that suggests acyl-CoAs do not diffuse freely in the cytosol, but are channeled into specific pathways, and comment on the suggested mechanisms for fatty acyl-CoA compartmentalization and intracellular transport, to finally speculate about the potential mechanisms that underlie fatty acyl-CoA delivery to zDHHC enzymes.     

Quantification of the concentration and 13C tracer enrichment of long-chain fatty acyl-coenzyme A in muscle by liquid chromatography/mass spectrometry

Recent diabetes and obesity research has been focused on the role of intracellular lipids in insulin resistance. Fatty acyl-coenzyme A (CoA) esters play a central role in the trafficking of intracellular lipids, but there has not previously been a method with which to quantify their kinetics using tracer methodology. We have therefore developed a high-performance liquid chromatography (HPLC)-mass spectrometry method to simultaneously measure the (13)C stable isotopic enrichment of palmitoyl-acyl-CoA ester and the concentrations of five individual long-chain fatty acyl-CoA esters extracted from muscle tissue samples. The long-chain fatty acyl-CoA can be effectively extracted from frozen muscle tissue samples and baseline separated by a reverse-phase HPLC with the presence of a volatile reagent-triethylamine. Negative ion electrospray mass spectrometry with selected ion monitoring was used to analyze the fatty acyl-CoAs to achieve reliable quantification of their concentrations and (13)C isotopic enrichment. Applying this protocol to rabbit muscle samples demonstrates that it is a sensitive, accurate, and precise method for the quantification of long-chain fatty acyl-CoA concentrations and enrichment.     

The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism

The importance of peroxisomes in lipid metabolism is now well established and peroxisomes contain approximately 60 enzymes involved in these lipid metabolic pathways. Several acyl-CoA thioesterase enzymes (ACOTs) have been identified in peroxisomes that catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. A number of acyltransferase enzymes, which are structurally and functionally related to ACOTs, have also been identified in peroxisomes, which conjugate (or amidate) bile acid-CoAs and acyl-CoAs to amino acids, resulting in the production of amidated bile acids and fatty acids. The function of ACOTs is to act as auxiliary enzymes in the α- and β-oxidation of various lipids in peroxisomes. Human peroxisomes contain at least two ACOTs (ACOT4 and ACOT8) whereas mouse peroxisomes contain six ACOTs (ACOT3, 4, 5, 6, 8 and 12). Similarly, human peroxisomes contain one bile acid-CoA:amino acid N-acyltransferase (BAAT), whereas mouse peroxisomes contain three acyltransferases (BAAT and acyl-CoA:amino acid N-acyltransferases 1 and 2: ACNAT1 and ACNAT2). This review will focus on the human and mouse peroxisomal ACOT and acyltransferase enzymes identified to date and discuss their cellular localizations, emerging structural information and functions as auxiliary enzymes in peroxisomal metabolic pathways. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Nature of the reaction product of [1-14C]stearoyl-CoA elongation by etiolated leek seeding microsomes

The elongation of [1-14C]stearoyl-CoA by microsomes from etiolated leek seedlings, in the presence of malonyl-CoA and NADPH, has been studied at different substrate and enzyme concentrations. The HPTLC analysis of the whole reaction mixture, followed by the analysis of the label in the fatty acid methyl esters of long-chain acyl-CoAs, phosphatidylcholine (PC), and neutral lipids, showed that the acyl-CoA fraction contained most of the labeled very-long-chain fatty acids. The very-long-chain fatty acids were rapidly formed and released from the elongase(s) as acyl-CoAs. The label of long-chain acyl-CoAs increased for 20 min and then decreased, whereas it increased in PC. Labeled very-long-chain fatty acids appeared in the neutral lipid + free fatty acid fraction after a 20-min lag.     

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.     

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.     

A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency

The acyl-CoA dehydrogenases are a family of multimeric flavoenzymes that catalyze the alpha,beta -dehydrogenation of acyl-CoA esters in fatty acid beta -oxidation and amino acid catabolism. Genetic defects have been identified in most of the acyl-CoA dehydrogenases in humans. Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified acyl-CoA dehydrogenase that demonstrates maximum activity with unsaturated long-chain acyl-CoAs. We now report three cases of ACAD9 deficiency. Patient 1 was a 14-year-old, previously healthy boy who died of a Reye-like episode and cerebellar stroke triggered by a mild viral illness and ingestion of aspirin. Patient 2 was a 10-year-old girl who first presented at age 4 mo with recurrent episodes of acute liver dysfunction and hypoglycemia, with otherwise minor illnesses. Patient 3 was a 4.5-year-old girl who died of cardiomyopathy and whose sibling also died of cardiomyopathy at age 21 mo. Mild chronic neurologic dysfunction was reported in all three patients. Defects in ACAD9 mRNA were identified in the first two patients, and all patients manifested marked defects in ACAD9 protein. Despite a significant overlap of substrate specificity, it appears that ACAD9 and very-long-chain acyl-CoA dehydrogenase are unable to compensate for each other in patients with either deficiency. Studies of the tissue distribution and gene regulation of ACAD9 and very-long-chain acyl-CoA dehydrogenase identify the presence of two independently regulated functional pathways for long-chain fat metabolism, indicating that these two enzymes are likely to be involved in different physiological functions.     

Acyl coenzyme A thioesterase Them5/Acot15 is involved in cardiolipin remodeling and fatty liver development

Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/β-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5(-/-) mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and β-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions.