Human acyl-CoA dehydrogenase-9 plays a novel role in the mitochondrial beta-oxidation of unsaturated fatty acids

Unsaturated fatty acids play an important role in the prevention of human diseases such as diabetes, obesity, cancer, and neurodegeneration. However, their oxidation in vivo by acyl-CoA dehydrogenases (ACADs) that catalyze the first step of each cycle of mitochondrial fatty acid beta-oxidation is not entirely understood. Recently, a novel ACAD (ACAD-9) of unknown function that is highly homologous to human very-long-chain acyl-CoA dehydrogenase was identified by large-scale random sequencing. To characterize its enzymatic role, we have expressed ACAD-9 in Escherichia coli, purified it, and determined its pattern of substrate utilization. The N terminus of the mature form of the enzyme was identified by in vitro mitochondrial import studies of precursor protein. A 37-amino acid leader peptide was cleaved sequentially by two mitochondrial peptidases to yield a predicted molecular mass of 65 kDa for the mature subunit. Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane. Gel filtration analysis indicated that, like very-long-chain acyl-CoA dehydrogenase, ACAD-9 is a dimer, in contrast to the other known ACADs, which are tetramers. Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA). These results suggest a previously unrecognized role for ACAD-9 in the mitochondrial beta-oxidation of long-chain unsaturated fatty acids. Because of the substrate specificity and abundance of ACAD-9 in brain, we speculate that it may play a role in the turnover of lipid membrane unsaturated fatty acids that are essential for membrane integrity and structure.     

Crystal structure of rat short chain acyl-CoA dehydrogenase complexed with acetoacetyl-CoA: comparison with other acyl-CoA dehydrogenases.

The acyl-CoA dehydrogenases are a family of flavin adenine dinucleotide-containing enzymes that catalyze the first step in the beta-oxidation of fatty acids and catabolism of some amino acids. They exhibit high sequence identity and yet are quite specific in their substrate binding. Short chain acyl-CoA dehydrogenase has maximal activity toward butyryl-CoA and negligible activity toward substrates longer than octanoyl-CoA. The crystal structure of rat short chain acyl-CoA dehydrogenase complexed with the inhibitor acetoacetyl-CoA has been determined at 2.25 A resolution. Short chain acyl-CoA dehydrogenase is a homotetramer with a subunit mass of 43 kDa and crystallizes in the space group P321 with a = 143.61 A and c = 77.46 A. There are two monomers in the asymmetric unit. The overall structure of short chain acyl-CoA dehydrogenase is very similar to those of medium chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and bacterial short chain acyl-CoA dehydrogenase with a three-domain structure composed of N- and C-terminal alpha-helical domains separated by a beta-sheet domain. Comparison to other acyl-CoA dehydrogenases has provided additional insight into the basis of substrate specificity and the nature of the oxidase activity in this enzyme family. Ten reported pathogenic human mutations and two polymorphisms have been mapped onto the structure of short chain acyl-CoA dehydrogenase. None of the mutations directly affect the binding cavity or intersubunit interactions.     

Immunoprecipitation and electrophoretic analysis of four human acyl-CoA dehydrogenases and electron transfer flavoprotein using antibodies raised against the corresponding rat enzymes

We prepared monospecific antisera in rabbits against purified rat short-, medium-, and long-chain acyl-CoA dehydrogenases, isovaleryl-CoA dehydrogenase, and ETF and tested the immunocross-reactivity to the corresponding human enzymes. Each antiserum specifically reacted with the corresponding human enzyme. When immunoprecipitates were analyzed by SDS-PAGE, the mobilities of all the human acyl-CoA dehydrogenases and ETF subunits were identical to those of the rat counterparts with a single exception. Human medium-chain acyl-CoA dehydrogenase had a mobility on SDS-PAGE slightly slower than that of rat medium-chain acyl-CoA dehydrogenase, suggesting that human medium-chain acyl-CoA dehydrogenase was 1 kDa larger than the rat counterpart. The immunocross-reactivity of the antisera, raised against the rat acyl-CoA dehydrogenases and ETF to the human counterpart, provide useful tools for the study of mutant enzymes in cells from patients with a genetic defect of acyl-CoA dehydrogenases of ETF.     

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.     

Beta-oxidation of long-chain fatty acids by human fibroblasts: evidence for a novel long-chain acyl-coenzyme A dehydrogenase.

Fibroblasts from patients with long-chain acyl-CoA dehydrogenase deficiency were found to oxidize [1-14C]linoleate at an average rate of 60% of normal but [9,10(n)-3H]myristate at an average rate of only 37% of normal, a relationship reverse from that predicted by the chain-length specificities of the three known straight-chain mitochondrial acyl-CoA dehydrogenases. The residual long-chain beta-oxidative activity was found to be mitochondrial and associated with the accumulation of tetradecadienoate (C14:2w6) when the mutant fibroblasts were incubated with 100 mumol/L linoleate (C18:2w6) or eicosadienoate (C20:2w6). The results suggest the presence in human fibroblasts of a novel acyl-CoA dehydrogenase with activity toward 15 to 20 carbon-length fatty acids.     

Mouse models for disorders of mitochondrial fatty acid beta-oxidation

Mitochondrial beta-oxidation of fatty acids is vital for energy production in periods of fasting and other metabolic stress. Human patients have been identified with inherited disorders of mitochondrial beta-oxidation of fatty acids with enzyme deficiencies identified at many of the steps in this pathway. Although these patients exhibit a range of disease processes, Reye-like illness (hypoketotic-hypoglycemia, hyperammonemia and fatty liver) and cardiomyopathy are common findings. There have been several mouse models developed to aid in the study of these disease conditions. The characterized mouse models include inherited deficiencies of very long-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein-alpha, and medium-/short-chain hydroxyacyl-CoA dehydrogenase. Mouse mutants developed, but presently incompletely characterized as models, include carnitine palmitoyltransferase-1a and medium-chain acyl-CoA dehydrogenase deficiencies. In general, the mouse models of disorders of mitochondrial fatty acid beta-oxidation have shown clinical signs that include Reye-like syndrome and cardiomyopathy, and many are cold intolerant. It is expected that these mouse models will provide vital contributions in understanding the mechanisms of disease pathogenesis of fatty acid oxidation disorders and the development of appropriate treatments and supportive care.     

Purification and properties of acyl coenzyme A dehydrogenases from bovine liver. Formation of 2-trans,4-cis-decadienoyl coenzyme A

Three straight chain acyl-CoA dehydrogenases were purified to apparent homogeneity from bovine liver using 40-70% (NH4)2SO4 precipitation, gel filtration, DEAE-cellulose column chromatography, and preparative electrophoresis. Separation of the acyl-CoA dehydrogenases by these procedures has been efficiently monitored by two newly developed analytical methods: (i) native staining of acyl-CoA dehydrogenases following separation by electrophoresis in polyacrylamide gels and (ii) determination of general acyl-CoA dehydrogenase by means of a specific substrate, 4-cis-decenoyl-CoA. The three acyl-CoA dehydrogenases were classified into short chain, general, and long chain acyl-CoA dehydrogenases on the basis of their chain length specificities according to the nomenclature proposed by Hall and Kamin (Hall, C. L., and Kamin, H. (1975) J. Biol. Chem. 250, 3470-3486). The enzymes gave single protein bands in polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions, and their subunit and native molecular weights were estimated to be 40,300 and 188,000 for short chain acyl-CoA dehydrogenase, 43,300 and 205,000 for general acyl-CoA dehydrogenase, and 45,200 and 172,000 for long chain acyl-CoA dehydrogenase. Long chain and general acyl-CoA dehydrogenases markedly differed in their substrate specificities toward unsaturated acyl-CoA esters with a double bond at position 4. The former oxidized 4-cis-decenoyl-CoA at a rate of only 2.7% of that obtained with decanoyl-CoA as substrate, while for the latter enzyme 4-cis-decenoyl-CoA was even a slightly better substrate than decanoyl-CoA. 2-trans,4-cis-Decenoyl-CoA was identified as the product of this reaction.     

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.     

Long-chain acyl-CoA dehydrogenase is a key enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids

The first reaction of mitochondrial beta-oxidation, which is catalyzed by acyl-CoA dehydrogenases, was studied with unsaturated fatty acids that have a double bond either at the 4,5 or 5,6 position. The CoA thioesters of docosahexaenoic acid, arachidonic acid, 4,7,10-cis-hexadecatrienoic acid, 5-cis-tetradecenoic acid, and 4-cis-decenoic acid were effectively dehydrogenated by both rat and human long-chain acyl-CoA dehydrogenases (LCAD), whereas they were poor substrates of very long-chain acyl-CoA dehydrogenases (VLCAD). VLCAD, however, was active with CoA derivatives of long-chain saturated fatty acids or unsaturated fatty acids that have double bonds further removed from the thioester function. Although bovine LCAD effectively dehydrogenated 5-cis-tetradecenoyl-CoA (14:1) and 4,7,10-cis-hexadecatrienoyl-CoA, it was nearly inactive toward the other unsaturated substrates. The catalytic efficiency of rat VLCAD with 14:1 as substrate was only 4% of the efficiency determined with tetradecanoyl-CoA, whereas LCAD acted equally well on both substrates. The conclusion of this study is that LCAD serves an important, if not essential function in the beta-oxidation of unsaturated fatty acids.     

Identification and properties of an inducible phenylacyl-CoA dehydrogenase in Pseudomonas putida KT2440

A novel acyl-CoA dehydrogenase that initiates beta-oxidation of the side chains of phenylacyl-CoA compounds by Pseudomonas putida was induced by growth with phenylhexanoate as carbon source. It was identified as the product of gene PP_0368, which was cloned and overexpressed in Escherichia coli. This phenylacyl-CoA dehydrogenase was found to be dimeric with a subunit molecular mass of 66 kDa, to contain FAD and to be active with phenylacyl-CoA substrates having side chains from four to at least 11 carbon atoms. The same enzyme was induced by the aliphatic alkanoate octanoate. The optimal aliphatic substrates for the enzyme were palmitoyl-CoA and stearoyl-CoA, a property shared with mammalian very-long-chain acyl-CoA dehydrogenases. The FAD in the enzyme was reduced by aromatic and aliphatic substrates, with changes to the oxidation-reduction potential. Chemical reduction by dithionite ion and oxidation by ferricyanide ion showed that the enzyme can accept four electrons: two to reduce the flavin and two to slowly reduce an unknown acceptor, which in its reduced form interacts with the oxidized flavin in a charge-transfer complex. The experiments identify for the first time an acyl-CoA dehydrogenase that oxidizes the activated forms of aromatic acids similar to those used to first demonstrate the biological beta-oxidation of fatty acids.     

Effect of Randomly Interesterified Triacylglycerol Containing Medium- and Long-Chain Fatty Acids on Hepatic Fatty Acid Oxidation after a Single Administration to Rats

MLCTs, which are randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule, showed significantly higher acyl-CoA dehydrogenase activity when measured by using butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA as substrates than long-chain triacylglycerol one hour after a single administration to rats. These results suggest that not only medium-chain fatty acid oxidation, but also long-chain fatty acid oxidation were increased in the liver of rats administered with MLCT.     

Effect of randomly interesterified triacylglycerol containing medium- and long-chain fatty acids on hepatic fatty acid oxidation after a single administration to rats

MLCTs, which are randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule, showed significantly higher acyl-CoA dehydrogenase activity when measured by using butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA as substrates than long-chain triacylglycerol one hour after a single administration to rats. These results suggest that not only medium-chain fatty acid oxidation, but also long-chain fatty acid oxidation were increased in the liver of rats administered with MLCT.     

Purification, characterization and cloning of isovaleryl-CoA dehydrogenase from higher plant mitochondria.

Between the different types of Acyl-CoA dehydrogenases (ACADs), those specific for branched chain acyl-CoA derivatives are involved in the catabolism of amino acids. In mammals, isovaleryl-CoA dehydrogenase (IVD), an enzyme of the leucine catabolic pathway, is a mitochondrial protein, as other acyl-CoA dehydrogenases involved in fatty acid beta-oxidation. In plants, fatty acid beta-oxidation takes place mainly in peroxisomes, and the cellular location of the enzymes involved in the catabolism of branched-chain amino acids had not been definitely assigned. Here, we describe that highly purified potato mitochondria have important IVD activity. The enzyme was partially purified and cDNAs from two different genes were obtained. The partially purified enzyme has enzymatic constant values with respect to isovaleryl-CoA comparable to those of the mammalian enzyme. It is not active towards straight-chain acyl-CoA substrates tested, but significant activity was also found with isobutyryl-CoA, implying an additional role of the enzyme in the catabolism of valine. The present study confirms recent reports that in plants IVD activity resides in mitochondria and opens the way to a more detailed study of amino-acid catabolism in plant development.     

Mechanism of action of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases. Direct evidence for carbanion formation as an intermediate step using enzyme-catalyzed C-2 proton/deuteron exchange in the absence of C-3 exchange

The mechanisms of the initial interactions of three rat liver acyl-CoA dehydrogenases (short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases) and their fatty acyl-CoA substrate were studied using enzyme-catalyzed deuterium exchange. The reaction products were identified and quantitated using mass spectroscopy and 1H-NMR. When fatty acyl-CoA substrates were incubated with catalytic amounts of acyl-CoA dehydrogenase in D2O in the absence of an electron acceptor, a rapid monodeuteration of the substrate occurred to replace one of the prochiral C-2 hydrogens, while no C-3 hydrogens were exchanged with deuterium. The C-2 monodeuteration proceeded to the extent of 80% of the total amount of substrate added at 90 min and almost to completion at 120 min. The pKa values and optimum pD values for the C-2 proton/deuteron exchange reactions were 6.0 and 7.5, respectively, for each of the three acyl-CoA dehydrogenases. The apparent turnover numbers were 3.0, 3.3, and 0.5 s-1 for short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases, respectively. These results provide the first direct evidence for carbanion formation via abstraction of a C-2 hydrogen by a base in the enzyme, as the first step of the catalytic pathway of acyl-CoA dehydrogenation. When the acyl-CoA dehydrogenases were reacted with moderate excesses of acyl-CoA substrates in D2O in the absence of an electron acceptor, maximum bleaching of the FAD absorbance and the appearance of the long wavelength absorbance, attributed to a charge transfer complex, were observed. However, the dehydrogenation products, 2-enoyl-CoAs, were produced either not at all or in an amount which represented only a minor fraction of the amount of the enzyme added, while the substrates in the enzyme-substrate complexes rapidly turned over as indicated by the extensive monodeuteration which concomitantly occurred. Unlike previous hypothesis, these results indicate that the hydride ion transfer from C-3 of the substrate to the enzyme-FAD is not yet complete in the charge-transfer complex. The transfer of the hydride ion to alloxazine N-5 and the release of products are completed only in the presence of electron-transfer flavoprotein or another suitable electron acceptor.     

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.     

Chemical diagnosis of inherited defects of fatty acid metabolism and ketogenesis

Urinary organic acid profiles in patients with inherited defects of fatty acid metabolism and ketogenesis are described. Medium-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, multiple acyl-CoA dehydrogenase, and 3-hydroxy-3-methyl-glutaryl-CoA lyase deficiencies can be recognized at the metabolite level. Data on long-chain acyl-CoA dehydrogenase and systemic carnitine deficiencies are scarce. In the latter disorders, dicarboxylic aciduria is rather nonspecific and points to a modest omega-oxidation of long chain fatty acids.     

Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism

Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors.     

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.