ACAD9

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Jerry Vockley - One of the best experts on this subject based on the ideXlab platform.

  • An acyl-CoA dehydrogenase microplate activity assay using recombinant porcine electron transfer flavoprotein.
    Analytical Biochemistry, 2019
    Co-Authors: Yuxun Zhang, Alwalid Mohsen, Jerry Vockley, Catherine Kochersperger, Keaton Solo, Alexandra V. Schmidt, Eric S. Goetzman
    Abstract:

    Abstract Acyl-CoA dehydrogenases (ACADs) play key roles in the mitochondrial catabolism of fatty acids and branched-chain amino acids. All nine characterized ACAD enzymes use electron transfer flavoprotein (ETF) as their redox partner. The gold standard for measuring ACAD activity is the anaerobic ETF fluorescence reduction assay, which follows the decrease of pig ETF fluorescence as it accepts electrons from an ACAD in vitro . Although first described 35 years ago, the assay has not been widely used due to the need to maintain an anaerobic assay environment and to purify ETF from pig liver mitochondria. Here, we present a method for expressing recombinant pig ETF in E coli and purifying it to homogeneity . The recombinant protein is virtually pure after one chromatography step, bears higher intrinsic fluorescence than the native enzyme, and provides enhanced activity in the ETF fluorescence reduction assay. Finally, we present a simplified protocol for removing molecular oxygen that allows adaption of the assay to a 96-well plate format. The availability of recombinant pig ETF and the microplate version of the ACAD activity assay will allow wide application of the assay for both basic research and clinical diagnostics.

  • Strategy for Disruption of the Mouse Acadm Gene
    2013
    Co-Authors: Ravi J Tolwani, Dietrich Matern, Piero Rinaldo, Jerry Vockley, Doug A Hamm, Liqun Tian, Daniel J. Sharer, Trenton R Schoeb, Philip A Wood
    Abstract:

    (A) The MCAD IV2 insertion targeting vector with a deleted 1.3-kb region encompassing exon 10 and flanking sequences. MCAD IV2 undergoes gap repair upon homologous recombination at the endogenous Acadm locus resulting in a duplication of exons 8, 9, and 10 at the disrupted allele.(B) Southern blot analysis of EcoRI-digested genomic DNA from ES cells screened by PCR. Probe A, a DNA fragment consisting of a portion of exon 10 that is not present in the targeting vector, hybridizes to an endogenous 3.1-kb fragment and, upon homologous recombination, to a 13.2-kb fragment. Lane 1 represents a wild-type ES cell line, and Lane 2 and 3 represent targeted ES cell lines.

  • a new genetic disorder in mitochondrial fatty acid β oxidation ACAD9 deficiency
    American Journal of Human Genetics, 2007
    Co-Authors: S L Rutledge, David R Kelly, C A Palmer, Geoffrey Murdoch, Nilanjana Majumder, Robert D Nicholls, Zhengtong Pei, Paul A Watkins, Jerry Vockley
    Abstract:

    The acyl-CoA dehydrogenases are a family of multimeric flavoenzymes that catalyze the α,β-dehydrogenation of acyl-CoA esters in fatty acid β-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.

  • human acyl coa dehydrogenase 9 plays a novel role in the mitochondrial β oxidation of unsaturated fatty acids
    Journal of Biological Chemistry, 2005
    Co-Authors: Regina Ensenauer, Jan Willard, Brian Berg Vandahl, Alwalid Mohsen, Grazia Isaya, Miao He, Thomas J Corydon, Eric S. Goetzman, Jerry Vockley
    Abstract:

    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.

Sander M Houten - One of the best experts on this subject based on the ideXlab platform.

  • ACAD9 a complex i assembly factor with a moonlighting function in fatty acid oxidation deficiencies
    Human Molecular Genetics, 2014
    Co-Authors: Jessica Nouws, Heleen Te Brinke, Leo G J Nijtmans, Sander M Houten
    Abstract:

    Oxidative phosphorylation and fatty acid oxidation are two major metabolic pathways in mitochondria. Acyl-CoA dehydrogenase 9 (ACAD9), an enzyme assumed to play a role in fatty acid oxidation, was recently identified as a factor involved in complex I biogenesis. Here we further investigated the role of ACAD9's enzymatic activity in fatty acid oxidation and complex I biogenesis. We provide evidence indicating that ACAD9 displays enzyme activity in vivo. Knockdown experiments in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts revealed that ACAD9 is responsible for the production of C14:1-carnitine from oleate and C12-carnitine from palmitate. These results explain the origin of these obscure acylcarnitines that are used to diagnose VLCAD deficiency in humans. Knockdown of ACAD9 in control fibroblasts did not reveal changes in the acylcarnitine profiles upon fatty acid loading. Next, we investigated whether catalytic activity of ACAD9 was necessary for complex I biogenesis. Catalytically inactive ACAD9 gave partial-to-complete rescue of complex I biogenesis in ACAD9-deficient cells and was incorporated in high-molecular-weight assembly intermediates. Our results underscore the importance of the ACAD9 protein in complex I assembly and suggest that the enzymatic activity is a rudiment of the duplication event.

  • acyl coa dehydrogenase 9 is required for the biogenesis of oxidative phosphorylation complex i
    Cell Metabolism, 2010
    Co-Authors: Jessica Nouws, Leo G J Nijtmans, Sander M Houten, Mariel Van Den Brand, Martijn A Huynen, Hanka Venselaar, Saskia J G Hoefs, Jolein Gloerich, Jonathan B Kronick, Timothy Hutchin
    Abstract:

    Summary Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified member of the acyl-CoA dehydrogenase family. It closely resembles very long-chain acyl-CoA dehydrogenase (VLCAD), involved in mitochondrial β oxidation of long-chain fatty acids. Contrary to its previously proposed involvement in fatty acid oxidation, we describe a role for ACAD9 in oxidative phosphorylation. ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I. Furthermore, ACAD9 mutations result in complex I deficiency and not in disturbed long-chain fatty acid oxidation. This strongly contrasts with its evolutionary ancestor VLCAD, which we show is not required for complex I assembly and clearly plays a role in fatty acid oxidation. Our results demonstrate that two closely related metabolic enzymes have diverged at the root of the vertebrate lineage to function in two separate mitochondrial metabolic pathways and have clinical implications for the diagnosis of complex I deficiency.

Salvatore Dimauro - One of the best experts on this subject based on the ideXlab platform.

  • Mitochondrial Encephalomyopathy Due to a Novel Mutation in ACAD9
    JAMA neurology, 2013
    Co-Authors: Caterina Garone, Maria Alice Donati, Michele Sacchini, Beatriz Garcia-diaz, Claudio Bruno, Sarah E. Calvo, Vamsi K. Mootha, Salvatore Dimauro
    Abstract:

    IMPORTANCE Mendelian forms of complex I deficiency are usually associated with fatal infantile encephalomyopathy. Application of “MitoExome” sequencing (deep sequencing of the entire mitochondrial genome and the coding exons of >1000 nuclear genes encoding the mitochondrial proteome) allowed us to reveal an unusual clinical variant of complex I deficiency due to a novel homozygous mutation in ACAD9. The patient had an infantile-onset but slowly progressive encephalomyopathy and responded favorably to riboflavin therapy.

  • infantile mitochondrial encephalomyopathy due to a novel mutation in ACAD9 p03 017
    Neurology, 2013
    Co-Authors: Caterina Garone, Maria Alice Donati, Michele Sacchini, Sarah E. Calvo, Vamsi K. Mootha, Beatriz Garciadiaz, Salvatore Dimauro
    Abstract:

    OBJECTIVE: To identify causative gene variants of early-onset complex I deficiency. BACKGROUND: Complex I deficiency is the most common respiratory chain defect with early-onset fatal encephalomyopathy. Although many molecular defects have been described both in structural subunits and in assembly factors, the genetic diagnosis remains unknown in a large cohort of patients. DESIGN/METHODS: We carried out biochemical and molecular studies in muscle and cultured fibroblasts from a patient with infantile mitochondrial encephalomyopathy. Next generation exome sequencing with a mitochondrial gene library (MitoExome) was applied to identify the molecular defect. RESULTS: A 9-year-old Italian boy had severe infantile-onset myopathy with exercise intolerance, weakness, muscle wasting. He also had mental retardation and severe complex I deficiency. Metabolic workup showed increased levels of plasma lactic acid, acylcarnitine C0, and alanine, and thyroid dysfunction. EMG was compatible with a myopathic process and muscle biopsy revealed mitochondrial proliferation. Cardiac function was normal and there were no abnormalities of brain MRI. Biochemical studies showed severe complex I and moderate complex III deficiencies both in muscle and in fibroblasts. Western blot analysis of mtDNA-encoded respiratory chain components showed reduced protein level of complex I. MitoExome sequencing revealed a new homozygous mutation in ACAD9 gene (p.R414C) that was confirmed by Sanger sequence and found in heterozygosity in both parents. Improvement of muscle strength was reported after treatment with high-dose riboflavin. CONCLUSIONS: ACAD9 is a complex I assembly factor whose defects have been associated with a protean clinical spectrum spanning from pure myopathy with exercise intolerance and lactic acidosis to rapidly progressive encephalomyopathy and hypertrophic cardiomyopathy. Our case contributes adds to the clinical heterogeneity of ACAD9 deficiency and confirms the importance of assembly factors in causing complex I deficiency. Disclosure: Dr. Garone has nothing to disclose. Dr. Donati has nothing to disclose. Dr. Sacchini has nothing to disclose. Dr. Calvo has nothing to disclose. Dr. Garcia-Diaz has nothing to disclose. Dr. Mootha has nothing to disclose. Dr. DiMauro has received personal compensation in an editorial capacity for MedLink Neurology.

Eric S. Goetzman - One of the best experts on this subject based on the ideXlab platform.

  • An acyl-CoA dehydrogenase microplate activity assay using recombinant porcine electron transfer flavoprotein.
    Analytical Biochemistry, 2019
    Co-Authors: Yuxun Zhang, Alwalid Mohsen, Jerry Vockley, Catherine Kochersperger, Keaton Solo, Alexandra V. Schmidt, Eric S. Goetzman
    Abstract:

    Abstract Acyl-CoA dehydrogenases (ACADs) play key roles in the mitochondrial catabolism of fatty acids and branched-chain amino acids. All nine characterized ACAD enzymes use electron transfer flavoprotein (ETF) as their redox partner. The gold standard for measuring ACAD activity is the anaerobic ETF fluorescence reduction assay, which follows the decrease of pig ETF fluorescence as it accepts electrons from an ACAD in vitro . Although first described 35 years ago, the assay has not been widely used due to the need to maintain an anaerobic assay environment and to purify ETF from pig liver mitochondria. Here, we present a method for expressing recombinant pig ETF in E coli and purifying it to homogeneity . The recombinant protein is virtually pure after one chromatography step, bears higher intrinsic fluorescence than the native enzyme, and provides enhanced activity in the ETF fluorescence reduction assay. Finally, we present a simplified protocol for removing molecular oxygen that allows adaption of the assay to a 96-well plate format. The availability of recombinant pig ETF and the microplate version of the ACAD activity assay will allow wide application of the assay for both basic research and clinical diagnostics.

  • complex i assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency
    Human Molecular Genetics, 2015
    Co-Authors: Manuel Schiff, Alwalid Mohsen, Eric S. Goetzman, Birgit Haberberger, Chuanwu Xia, Yudong Wang, Radha Uppala, Yuxun Zhang, Anuradha Karunanidhi, Dolly Prabhu
    Abstract:

    Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions.

  • human acyl coa dehydrogenase 9 plays a novel role in the mitochondrial β oxidation of unsaturated fatty acids
    Journal of Biological Chemistry, 2005
    Co-Authors: Regina Ensenauer, Jan Willard, Brian Berg Vandahl, Alwalid Mohsen, Grazia Isaya, Miao He, Thomas J Corydon, Eric S. Goetzman, Jerry Vockley
    Abstract:

    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.

Alwalid Mohsen - One of the best experts on this subject based on the ideXlab platform.

  • An acyl-CoA dehydrogenase microplate activity assay using recombinant porcine electron transfer flavoprotein.
    Analytical Biochemistry, 2019
    Co-Authors: Yuxun Zhang, Alwalid Mohsen, Jerry Vockley, Catherine Kochersperger, Keaton Solo, Alexandra V. Schmidt, Eric S. Goetzman
    Abstract:

    Abstract Acyl-CoA dehydrogenases (ACADs) play key roles in the mitochondrial catabolism of fatty acids and branched-chain amino acids. All nine characterized ACAD enzymes use electron transfer flavoprotein (ETF) as their redox partner. The gold standard for measuring ACAD activity is the anaerobic ETF fluorescence reduction assay, which follows the decrease of pig ETF fluorescence as it accepts electrons from an ACAD in vitro . Although first described 35 years ago, the assay has not been widely used due to the need to maintain an anaerobic assay environment and to purify ETF from pig liver mitochondria. Here, we present a method for expressing recombinant pig ETF in E coli and purifying it to homogeneity . The recombinant protein is virtually pure after one chromatography step, bears higher intrinsic fluorescence than the native enzyme, and provides enhanced activity in the ETF fluorescence reduction assay. Finally, we present a simplified protocol for removing molecular oxygen that allows adaption of the assay to a 96-well plate format. The availability of recombinant pig ETF and the microplate version of the ACAD activity assay will allow wide application of the assay for both basic research and clinical diagnostics.

  • evaluation of mitochondrial bioenergetics dynamics endoplasmic reticulum mitochondria crosstalk and reactive oxygen species in fibroblasts from patients with complex i deficiency
    Scientific Reports, 2018
    Co-Authors: Alwalid Mohsen, Anuradha Karunanidhi, Guilhian Leipnitz, Bianca Seminotti, Vera Roginskaya, Desiree M Markantone, Mateus Grings, Stephanie J Mihalik
    Abstract:

    Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.

  • complex i assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency
    Human Molecular Genetics, 2015
    Co-Authors: Manuel Schiff, Alwalid Mohsen, Eric S. Goetzman, Birgit Haberberger, Chuanwu Xia, Yudong Wang, Radha Uppala, Yuxun Zhang, Anuradha Karunanidhi, Dolly Prabhu
    Abstract:

    Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions.

  • human acyl coa dehydrogenase 9 plays a novel role in the mitochondrial β oxidation of unsaturated fatty acids
    Journal of Biological Chemistry, 2005
    Co-Authors: Regina Ensenauer, Jan Willard, Brian Berg Vandahl, Alwalid Mohsen, Grazia Isaya, Miao He, Thomas J Corydon, Eric S. Goetzman, Jerry Vockley
    Abstract:

    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.