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.     

Convergent evolution of a 2-methylbutyryl-CoA dehydrogenase from isovaleryl-CoA dehydrogenase in Solanum tuberosum

The potato cDNAs Solanum tuberosum isovaleryl-CoA dehydrogenases 1 and 2 (St-IVD1 and St-IVD2) encode proteins that are 84% identical to each other and 65 and 64% identical to human IVD, respectively. St-IVD2 protein was previously partially purified from potato tubers and confirmed to be an IVD. The function of St-IVD1 is unknown. In these experiments, both proteins were expressed in Escherichia coli and purified as intact homotetramers. The substrate preference profile of the St-IVD2 protein was similar to that of human IVD. However, recombinant St-IVD1 had maximal activity with 2-methylbutyryl-CoA, which in humans is dehydrogenated by short/branched-chain acyl-CoA dehydrogenase (SBCAD). Whereas molecular modeling predicts that the 2-methylbutyryl-CoA dehydrogenase (2MBCD) and IVD substrate binding pockets are nearly identical, 2MBCD has amino acid substitutions at five residues that are invariant among all of the known and putative IVDs. Site-directed mutagenesis was used to match the human IVD active site with that of potato 2MBCD. The resulting mutant IVD had detectable activity with 2-methylbutyryl-CoA and no activity with isovaleryl-CoA. The 2MBCD active site was compared with that of human SBCAD using molecular modeling. Residues Met-361 and Ala-365 of 2MBCD appear to partially substitute for the function of Tyr-380 in human SBCAD, binding the methyl branch linked to C2 of 2-methylbutyryl-CoA, whereas residues Val-88, Val-92, and Val-96 appear to bind the distal C4 methyl group. The presence of a 2MBCD in potato that is highly homologous to IVD is an example of convergent evolution within the acyl-CoA dehydrogenase family, leading to the independent occurrence of two enzymes (SBCAD and 2MBCD) specific for 2-methylbutyryl-CoA.     

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.     

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.     

Two novel isovaleryl-CoA dehydrogenase gene mutations in a Chinese infant

Isovaleric acidemia (IVA) is a rare inherited metabolic disease caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD). Newborn screening with tandem mass spectrometry leads to early identification of individuals with risk of IVA. The family specific mutations are useful for prenatal diagnosis. Molecular genetic analysis helps to further confirm the clinical diagnosis of IVA. We describe here the clinical and metabolic features of a Chinese infant with early onset IVA. Sequence analysis of the IVD gene identifies compound heterozygous mutations in this patient, c.39G > A (p.W13X) nonsense mutation and c.597C > G (p.I199M) missense mutation, both of which are previously unreported. Structural analyses suggest that the p.I199M missense mutation may destabilize the IVD monomer structure and affect the interaction between IVD and flavin adenine dinucleotide. Both the clinical and genetic features of this patient help to further expand our knowledge of IVA.     

Molecular cloning and nucleotide sequence of cDNAs encoding the precursors of rat long chain acyl-coenzyme A, short chain acyl-coenzyme A, and isovaleryl-coenzyme A dehydrogenases. Sequence homology of four enzymes of the acyl-CoA dehydrogenase family

cDNAs encoding the entire coding regions of the precursors (p) of rat long chain acyl-CoA (LCAD), short chain acyl-CoA (SCAD) and isovaleryl-CoA dehydrogenase (IVD) have been cloned and sequenced. Three cDNAs for rat liver LCAD together cover a 1440-base pair region. These cDNAs encode the entire 430-amino acid sequence of pLCAD, including the 30-amino acid leader peptide and the 400-amino acid mature LCAD. A single 1773 base pair cDNA for rat SCAD covers the entire coding region (414 amino acids), including the 26-amino acid leader peptide and the 388-amino acid mature peptide. Four identified IVD cDNAs, when combined, encompass a 2104 base region, and encode 424 amino acids including a 30-amino acid leader peptide and the 394-amino acid mature peptide. The identities of all cDNA clones have been confirmed by matching the amino acid sequences predicted from the respective cDNAs to the amino-terminal and tryptic peptide sequences derived from the corresponding purified rat enzyme. Comparison of the sequences of four rat acyl-CoA dehydrogenases, including LCAD, MCAD, SCAD, and IVD, and two of their human counterparts (MCAD and SCAD) reveals a high degree of homology (57 invariant and 92 near invariant residues: 30.6-35.4% of identical residues in pairwise comparisons), suggesting that these enzymes belong to a gene family and have evolved from a common ancestral gene.     

Mitochondrial import and processing of wild type and type III mutant isovaleryl-CoA dehydrogenase

Isovaleric acidemia is a rare inborn error of metabolism caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD), a nucleus-encoded, homotetrameric, mitochondrial flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. We have previously identified a nucleotide deletion in the gene for IVD in fibroblasts from a patient with isovaleric acidemia leading to a shift in reading frame and premature termination of translation. The mutant IVD precursor is imported and processed to mature size, but no active enzyme is detected in mutant fibroblasts or expressed in Escherichia coli. Examination of the crystal structure of human IVD reveals that the C terminus is involved in tetramer stability. In vitro mitochondrial import experiments show that wild type IVD protein rapidly and stably forms mature homotetramer following import, whereas Type III mutant protein never forms stable oligomers. An additional series of mutant proteins with truncations and/or alterations in the C-terminal sequence implicates the C terminus of IVD in both enzyme activity and tetramer stability. Importantly, a dimeric intermediate in the folding pathway for wild type IVD has been identified in the in vitro mitochondrial import experiments, the first report of such an intermediate in the biogenesis of an acyl-CoA dehydrogenase.     

Inhibition in vitro of acyl-CoA dehydrogenases by 2-mercaptoacetate in rat liver mitochondria.

In rat liver hypo-osmotically treated mitochondria, 2-mercaptoacetate inhibits respiration induced by palmitoyl-CoA, octanoate or butyryl-CoA only when the reaction medium is supplemented with ATP. Under this condition, NADH-stimulated respiration is not affected. In liver mitochondrial matrix, the presence of ATP is also required to observe a 2-mercaptoacetate-induced inhibition of acyl-CoA dehydrogenases tested with palmitoyl-CoA, butyryl-CoA or isovaleryl-CoA as substrate. As the oxidation of these substrates is also inhibited by the incubation medium resulting from the reaction of 2-mercaptoacetate with acetyl-CoA synthase, with conditions under which 2-mercaptoacetate has no effect, 2-mercaptoacetyl-CoA seems to be the likely inhibitory metabolite responsible for the effects of 2-mercaptoacetate. Kinetic experiments show that the main effect of the 2-mercaptoacetate-active metabolite is to decrease the affinities of fatty acyl-CoA dehydrogenases towards palmitoyl-CoA or butyryl-CoA and of isovaleryl-CoA dehydrogenase towards isovaleryl-CoA. Addition of N-ethylmaleimide to mitochondrial matrix pre-exposed to 2-mercaptoacetate results in the immediate reversion of the inhibitions of palmitoyl-CoA and isovaleryl-CoA dehydrogenations and in a delayed reversion of butyryl-CoA dehydrogenation. These results led us to conclude that (i) the ATP-dependent conversion of 2-mercaptoacetate into an inhibitory metabolite takes place in the liver mitochondrial matrix and (ii) the three fatty acyl-CoA dehydrogenases and isovaleryl-CoA dehydrogenase are mainly competitively inhibited by this compound. Finally, the present study also suggests that the inhibitory metabolite of 2-mercaptoacetate may bind non-specifically to, or induce conformational changes at, the acyl-CoA binding sites of these dehydrogenases.     

Purification and properties of short chain acyl-CoA, medium chain acyl-CoA, and isovaleryl-CoA dehydrogenases from human liver

Short chain acyl-CoA (SCA), medium chain acyl-CoA (MCA), and isovaleryl-CoA (IV) dehydrogenases were purified to homogeneity from human liver using ammonium sulfate fractionation followed by DEAE-Sephadex A-50, hydroxyapatite, Matrex Gel Blue A, agarose-hexane-CoA, and Bio-Gel A-0.5 column chromatographies. The specific activities of the final preparations were enriched 507-, 750-, and 588-fold over those from the second ammonium sulfate fractionation step. The native molecular weights were estimated to be 168,000, 178,000, and 172,000, respectively, by gel filtration. Each of them exhibited, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single protein band with molecular weights of 41,000, 44,000, and 42,000, respectively, indicating a homotetrameric structure. UV/visual spectra, fluorescence spectra, and other evidence indicated that each contains 1 mol of FAD per subunit. They all utilized electron transfer flavoprotein (ETF) or phenazine methosulfate (PMS) as an electron acceptor. The products of SCA dehydrogenase/butyryl-CoA, MCA dehydrogenase/octanoyl-CoA, and IV dehydrogenase/isovaleryl-CoA reactions were identified as crotonyl-CoA, 2-octenoyl-CoA, and 3-methylcrotonyl-CoA, respectively, using gas chromatography. Kinetic parameters Vappmax and Kappm) of these enzymes for various acyl-CoA substrates, as well as Kappm values for ETF and PMS are presented. In general, the substrate specificities of human SCA, MCA, and IV dehydrogenases are slightly less stringent than those of their rat counterparts and resemble those of their bovine and porcine counterparts. The pattern of substrate specificity for these enzymes determined using ETF as electron acceptor significantly differed from that determined using PMS. All of them were severely inhibited by (methylenecyclopropyl)acetyl-CoA.     

Acyl-CoA Dehydrogenases: Dynamic History of Protein Family Evolution

The acyl-CoA dehydrogenases (ACADs) are enzymes that catalyze the α,β-dehydrogenation of acyl-CoA esters in fatty acid and amino acid catabolism. Eleven ACADs are now recognized in the sequenced human genome, and several homologs have been reported from bacteria, fungi, plants, and nematodes. We performed a systematic comparative genomic study, integrating homology searches with methods of phylogenetic reconstruction, to investigate the evolutionary history of this family. Sequence analyses indicate origin of the family in the common ancestor of Archaea, Bacteria, and Eukaryota, illustrating its essential role in the metabolism of early life. At least three ACADs were already present at that time: ancestral glutaryl-CoA dehydrogenase (GCD), isovaleryl-CoA dehydrogenase (IVD), and ACAD10/11. Two gene duplications were unique to the eukaryotic domain: one resulted in the VLCAD and ACAD9 paralogs and another in the ACAD10 and ACAD11 paralogs. The overall patchy distribution of specific ACADs across the tree of life is the result of dynamic evolution that includes numerous rounds of gene duplication and secondary losses, interdomain lateral gene transfer events, alteration of cellular localization, and evolution of novel proteins by domain acquisition. Our finding that eukaryotic ACAD species are more closely related to bacterial ACADs is consistent with endosymbiotic origin of ACADs in eukaryotes and further supported by the localization of all nine previously studied ACADs in mitochondria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Exon skipping in IVD RNA processing in isovaleric acidemia caused by point mutations in the coding region of the IVD gene

Isovaleric acidemia (IVA) is a recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). We have reported elsewhere nine point mutations in the IVD gene in fibroblasts of patients with IVA, which lead to abnormalities in IVD protein processing and activity. In this report, we describe eight IVD gene mutations identified in seven IVA patients that result in abnormal splicing of IVD RNA. Four mutations in the coding region lead to aberrantly spliced mRNA species in patient fibroblasts. Three of these are amino acid altering point mutations, whereas one is a single-base insertion that leads to a shift in the reading frame of the mRNA. Two of the coding mutations strengthen pre-existing cryptic splice acceptors adjacent to the natural splice junctions and apparently interfere with exon recognition, resulting in exon skipping. This mechanism for missplicing has not been reported elsewhere. Four other mutations alter either the conserved gt or ag dinucleotide splice sites in the IVD gene. Exon skipping and cryptic splicing were confirmed by transfection of these mutations into a Cos-7 cell line model splicing system. Several of the mutations were predicted by individual information analysis to inactivate or significantly weaken adjacent donor or acceptor sites. The high frequency of splicing mutations identified in these patients is unusual, as is the finding of missplicing associated with missense mutations in exons. These results may lead to a better understanding of the phenotypic complexity of IVA, as well as provide insight into those factors important in defining intron/exon boundaries in vivo.     

Selective inactivation of various acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA

Inactivation of five distinct acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA (MCPA-CoA), the toxic metabolite of hypoglycin from unripe ackee fruit, was investigated using purified enzyme preparations. Short-chain acyl-CoA (SCADH), medium-chain acyl-CoA (MCADH) and isovaleryl-CoA (IVDH) dehydrogenases were severely and irreversibly inactivated by MCPA-CoA, while 2-methyl-branched chain acyl-CoA dehydrogenase (2-meBCADH) was only slowly and mildly inactivated. Long-chain acyl-CoA dehydrogenase (LCADH) was not significantly inactivated, even after prolonged incubation with MCPA-CoA. Inactivation of SCADH, MCADH and IVDH was effectively prevented by the addition of substrate. This mode of inactivation by MCPA-CoA explains the urinary metabolite profile in hypoglycin treated-rats, which includes large amounts of metabolites from fatty acids and leucine, and relatively small amounts of those from valine and isoleucine. Spectrophotometric titration of SCADH and MCADH with MCPA-CoA, together with the protective effects of substrate, indicates that MCPA-CoA is acted upon by, and exerts in turn irreversible inactivation of, SCADH and MCADH, confirming that MCPA-CoA is a suicide inhibitor (Wenz et al. (1981) J. Biol. Chem. 256, 9809-9812). Spectrophotometric titration data of LCADH and MCPA-CoA is typical of non-reacting CoA ester.     

Cloning and characterization of a human cDNA ACAD10 mapped to chromosome 12q24.1

Mitochondrial fatty acid -oxidation is an important energy resource for many mammal tissues. Acyl-CoA dehydrogenases (ACADs) are a family of flavoproteins that are involved in the -oxidation of the fatty acyl-CoA derivatives. Deficiency of these ACADs can cause metabolic disorders including muscle fatigue, hypoglycaemia, hepatic lipidosis and so on. By large scale sequencing, we identified a cDNA sequence of 3960 base pairs with a typical acyl-CoA dehydrogenase function domain. RT-PCR result shows that it is widely expressed in human tissues, especially high in liver, kidney, pancreas and spleen. It is hypothesized that this is a novel member of ACADs family. Abbreviations: ACADs – acyl-CoA dehydrogenases, FAD – flavinadenine dinucleotide, SCAD – short-chain acyl-CoA dehydrogenase,MCAD – medium-chain acyl-CoA dehydrogenase, LCAD – long-chain acyl-CoAdehydrogenase, VLCAD – very long- chain acyl-CoA dehydrogenase, IVD –isocalery-CoA dehydrogenase, SBCAD – short/branched chain acyl-CoAdehydrogenase, GCD – glutaryl- CoA dehydrogenase, ETF – electron transferflavoprotein, ACAD8 – acyl-CoA dehydrogenase 8, ACAD9 – acyl-CoAdehydrogenase 9, ACAD10 – acyl-CoA dehydrogenase 10.     

Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: rationale for enhanced reactivity

2-Pentynoyl-CoA inactivates glutaryl-CoA dehydrogenase at a rate that considerably exceeds the rates of inactivation of short chain and medium chain acyl-CoA dehydrogenases by this inhibitor and related 2-alkynoyl-CoAs. To determine the rate of inactivation by 2-pentynoyl-CoA, we investigated the inactivation in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the binding site. The enhanced rate of inactivation does not reflect an alteration in specificity for the acyl group, nor does it reflect the covalent modification of a residue other than the active site glutamate. In addition to determining the inactivation of catalytic activity a spectral intermediate was detected by stopped-flow spectrophotometry, and the rate constants of formation and decay of this charge transfer complex (lambdamax approximately 790 nm) were determined by global analysis. Although the rate-limiting step in the inactivation of the other acyl-CoA dehydrogenases can involve the abstraction of a proton at C-4, this is not the case with glutaryl-CoA dehydrogenase. Glutaryl-CoA dehydrogenase is also differentiated from other acyl-CoA dehydrogenases in that the catalytic base must access both C-2 and C-4 in the normal catalytic pathway. Access to C-4 is not obligatory for the other dehydrogenases. Analysis of the distance from the closest carboxylate oxygen of the glutamate base catalyst to C-4 of a bound acyl-CoA ligand for medium chain, short chain, and isovaleryl-CoA dehydrogenases suggests that the increased rate of inactivation reflects the carboxylate oxygen to ligand C-4 distance in the binary complexes. This distance for wild type glutaryl-CoA dehydrogenase is not known. Comparison of the rate constants of inactivation and formation of a spectral species between wild type glutaryl-CoA dehydrogenase and a E370D mutant are consistent with the idea that this distance in glutaryl-CoA dehydrogenase contributes to the enhanced rate of inactivation and the 1,3-prototropic shift catalyzed by the enzyme.     

Structures of isobutyryl-CoA dehydrogenase and enzyme-product complex: comparison with isovaleryl- and short-chain acyl-CoA dehydrogenases

The acyl-CoA dehydrogenases are a family of mitochondrial flavoproteins involved in the catabolism of fatty and amino acids. Isobutyryl-CoA dehydrogenase (IBD) is involved in the catabolism of valine and catalyzes the conversion of isobutyryl-CoA to methacrylyl-CoA. The crystal structure of IBD with and without substrate has been determined to 1.76-A resolution. The asymmetric unit contains a homotetramer with substrate/product bound in two monomers. The overall structure of IBD is similar to those of previously determined acyl-CoA dehydrogenases and consists of an NH2-terminal alpha-helical domain, a medial beta-strand domain and a C-terminal alpha-helical domain. The enzyme-bound ligand has been modeled in as the reaction product, methacrylyl-CoA. The location of Glu-376 with respect to the C-2-C-3 of the bound product and FAD confirms Glu-376 to be the catalytic base. IBD has a shorter and wider substrate-binding cavity relative to short-chain acyl-CoA dehydrogenase, permitting the optimal binding of the isobutyryl-CoA substrate. The dramatic lateral expansion of the binding cavity seen in isovaleryl-CoA dehydrogenase is not observed in IBD. The conserved tyrosine or phenylalanine that defines a side of the binding cavity in other acyl-CoA dehydrogenases is replaced by a leucine (Leu-375) in the current structure. Substrate binding changes the position of some residues lining the binding pocket as well as the position of the loop containing the catalytic glutamate and subsequent helix. Three clinical mutations have been modeled to the structure. The mutations do not affect substrate binding but instead appear to disrupt protein folding and/or stability.     

Mammalian electron transferring flavoprotein.flavoprotein dehydrogenase complexes observed by microelectrospray ionization-mass spectrometry and surface plasmon resonance

Microelectrospray ionization-mass spectrometry was used to directly observe electron transferring flavoprotein.flavoprotein dehydrogenase interactions. When electron transferring flavoprotein and porcine dimethylglycine dehydrogenase or sarcosine dehydrogenase were incubated together in the absence of substrate, a relative molecular mass corresponding to the flavoprotein.electron transferring flavoprotein complex was observed, providing the first direct observation of these mammalian complexes. When an acyl-CoA dehydrogenase family member, human short chain acyl-CoA dehydrogenase, was incubated with dimethylglycine dehydrogenase and electron transferring flavoprotein, the microelectrospray ionization-mass spectrometry signal for the dimethylglycine dehydrogenase.electron transferring flavoprotein complex decreased, indicating that the acyl-CoA dehydrogenases have the ability to compete with the dimethylglycine dehydrogenase/sarcosine dehydrogenase family for access to electron transferring flavoprotein. Surface plasmon resonance solution competition experiments revealed affinity constants of 2.0 and 5.0 microm for the dimethylglycine dehydrogenase-electron transferring flavoprotein and short chain acyl-CoA dehydrogenase-electron transferring flavoprotein interactions, respectively, suggesting the same or closely overlapping binding motif(s) on electron transferring flavoprotein for dehydrogenase interaction.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.