Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.3.1.8 (acyl-CoA dehydrogenase)
 785 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.
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PMID:Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. 146 45

The most prominent biochemical consequence of riboflavin deficiency in rats is a drastic decrease in various acyl-CoA dehydrogenase activities, especially that of short chain and isovaleryl-CoA dehydrogenase (IVD). As a result, oxidation of fatty acids and leucine is severely inhibited. We studied the effects of FAD at various stages of acyl-CoA dehydrogenase biogenesis. Immunoblot revealed severe losses of various acyl-CoA dehydrogenases and electron transfer flavoprotein in riboflavin-deficient rat liver mitochondria. The decreases in IVD and short chain acyl-CoA dehydrogenase were particularly severe, reaching values of 17 and 34% of controls, respectively. With the exception of IVD, the rate of in vitro transcription of the respective genes and the amounts of mRNAs of these flavoproteins in tissues increased 3-8.5-fold over controls. The amount of IVD mRNA and its transcription rate remained unchanged, suggesting that IVD expression is regulated separately from other acyl-CoA dehydrogenases. When riboflavin was depleted, in vitro translation of acyl-CoA dehydrogenase and electron transfer flavoprotein alpha-subunit mRNAs was moderately inhibited. Translation of non-flavoproteins was also inhibited. The stability of precursor acyl-CoA dehydrogenases and their mitochondrial import/processing were unaffected. However, mature acyl-CoA dehydrogenases degraded markedly faster in deficient mitochondria than in controls. Regardless of whether precursors were translated under riboflavin-depleted or riboflavin replete conditions, mature acyl-CoA dehydrogenases survived well when imported into normal mitochondria but degraded faster when imported into deficient mitochondria. These findings indicate that FAD ligand binds to mature acyl-CoA dehydrogenase inside the mitochondria.
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PMID:FAD-dependent regulation of transcription, translation, post-translational processing, and post-processing stability of various mitochondrial acyl-CoA dehydrogenases and of electron transfer flavoprotein and the site of holoenzyme formation. 151 28

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.
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PMID:Beta-oxidation of long-chain fatty acids by human fibroblasts: evidence for a novel long-chain acyl-coenzyme A dehydrogenase. 154 Jan 49

beta-Oxidation of palmitate and tetradecanedioic acid was studied in cell-free extracts of the Gram-positive bacterium Corynebacterium sp. strain 7E1C, and the acyl-CoA ester intermediates formed were analysed by h.p.l.c. beta-Oxidation assays displayed a lag phase before a constant rate of NAD+ reduction was obtained. The length of the lag phase was inversely proportional to the number of units of activity added to assays. This is a characteristic feature of a system of consecutive reactions proceeding via free intermediates. During beta-oxidation of palmitate all the saturated acyl-CoAs from C16 to C8 were detected together with trace amounts of unsaturated and 3-hydroxy-intermediates. The time-course of intermediate formation again indicated a precursor-product relationship indicative of free intermediates being formed. When 3-hydroxyacyl-CoA dehydrogenase was inhibited by completely removing NAD+ from assays, the major acyl-CoAs, detected during palmitate beta-oxidation were palmitoyl-CoA, hexadeca-2-enoyl-CoA and 3-hydroxypalmitoyl-CoA. These compounds also displayed a precursor-product relationship. Under normal assay conditions the acyl-CoA dehydrogenase(s) are the probable rate-limiting enzyme(s) of the beta-oxidation spiral. These results indicate that in cell-free extracts of Corynebacterium sp. strain 7E1C, beta-oxidation proceeds via free acyl-CoA intermediates and is at variance with the concept of substrate channelling or of a 'leaky hose pipe' model as proposed for mitochondrial beta-oxidation in eukaryotic cells. The significant accumulation of chain-shortened acyl-CoA esters is similar to the situation observed for mammalian peroxisomal beta-oxidation.
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PMID:Long-chain acyl-CoA ester intermediates of beta-oxidation of mono- and di-carboxylic fatty acids by extracts of Corynebacterium sp. strain 7E1C. 163 89

The theory of steady-state flux control was applied to characterize the regulation of beta-oxidation flux in uncoupled rat liver mitochondria oxidizing palmitoylcarnitine in the presence of rotenone, malonate and the beta-hydroxybutyrate/acetoacetate redox buffer. By titrations with inhibitors such as antimycin, myxothiazol, azide and 4-pentenoic acid, the flux control coefficients of the b-c1 complex, cytochrome c oxidase and thiolase, were determined experimentally. The flux control coefficients of carnitine palmitoyltransferase II, ETF:CoQ oxidoreductase and beta-hydroxybutyrate dehydrogenase were determined from elasticity coefficients obtained by measuring the flux dependencies of acyl-CoA and acetyl-CoA+CoASH concentrations, the electron transfer flavoprotein redox state, the CoQ redox state and the NAD redox state. It was found that at low flux rates the flux control was distributed mainly between acyl-CoA dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase (Ci = 0.89). At maximum flux rates, carnitine palmitoyltransferase II (Ci = 0.35) and thiolase (Ci = 0.13) contribute additionally to the flux control. Thus, the phenomena of regulation of mitochondrial beta-oxidation can be described as multistep control.
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PMID:Application of the theory of steady-state flux control to mitochondrial beta-oxidation. 166 35

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.
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PMID:Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase. 173 Jun 32

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.
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PMID:Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation. 174 86

A trifunctional beta-oxidation protein, designated TFP, was purified to apparent homogeneity from oleate-induced mycelia of Neurospora crassa. 2-Enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities copurified in constant ratios with this protein when crude extracts were subjected to cation-exchange, dye-ligand, and adsorption chromatography. Trifunctionality was substantiated by coinciding enzyme activity ratios during the last two purification steps and additional chromatographic steps. The enzyme was shown to be a 365-kDa tetramer of subunits with a molecular mass of 93 kDa. Several lines of evidence suggest that these subunits are identical. Monospecific antibodies raised against the homogenous protein specifically precipitated the three enzymatic activities of TFP. Immunoblotting of fractions obtained after sucrose density gradient centrifugation of a crude extract indicated that TFP was exclusively localized in glyoxysome-like microbodies. The beta-oxidation system of N. crassa is structurally related to those of peroxisomes despite the presence of an acyl-CoA dehydrogenase rather than an acyl-CoA oxidase. A mitochondrial 2-enoyl-CoA hydratase activity was separated from TFP and purified to apparent homogeneity. The absence of all other beta-oxidation activities from mitochondria suggests that this organelle and its 2-enoyl-CoA hydratase are not involved in fatty acid degradation in N. crassa.
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PMID:The beta-oxidation system in catalase-free microbodies of the filamentous fungus Neurospora crassa. Purification of a multifunctional protein possessing 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities. 183 48

The free two-electron-reduced form of medium-chain acyl-CoA dehydrogenase is reoxidized by 120 microM molecular oxygen (50 mM phosphate buffer, pH 7.6, 2 degrees C) with a half-time of approximately 7 s. Reoxidation yields hydrogen peroxide as a major product with only traces of the superoxide anion. In contrast, enzyme reduced with octanoyl-CoA is extremely slowly reoxidized oxygen, and so a series of 14 different substrate analogues have been tested to assess the structural factors responsible for this effect. Complexes with redox-inactive ligands such as 3-thia- and 2-azaoctanoyl-CoA lead to an approximately 3000-fold slowing of the rate of reoxidation of the free dihydroflavin form of the enzyme. Comparable ligands lacking the thioester carbonyl function are much less effective with rates some 1.3-4-fold slower than the free enzyme. The strong suppression of oxygen reactivity observed with certain ligands is probably not simply a steric effect but may reflect desolvation of the active site and consequent destabilization of the superoxide anion intermediate formed during reoxidation of the flavin. The profound differences in oxygen reactivity between acyl-CoA dehydrogenase and acyl-CoA oxidase and the unusual stability of certain flavoprotein semiquinones in air are discussed in terms of these thermodynamic and kinetic arguments.
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PMID:Reactivity of medium-chain acyl-CoA dehydrogenase toward molecular oxygen. 186 64

The cDNA of human medium chain acyl-CoA dehydrogenase (MCADH) was modified by in vitro mutagenesis, and the sequence encoding the mature form of MCADH was introduced into an inducible expression plasmid. We observed synthesis of the protein in Escherichia coli cells transformed with this plasmid with measurable MCADH enzyme activity in cell extracts. Glutamic acid 376, which has been proposed by Powell and Thorpe (Powell, P. J., and Thorpe, J. (1988) Biochemistry 27, 8022-8028) as an essential residue and the proton-abstracting base at the active site of the enzyme, was mutated to glutamine. After expression in bacteria of this plasmid, the corresponding extracts show no detectable MCADH activity, although mutant MCADH-protein production was detected by protein immunoblots. The mature enzyme and the Gln376 mutant were purified to apparent homogeneity. The wild-type enzyme is a yellow protein due to the content of stoichiometric FAD and had a specific activity which is 50% of MCADH purified from pig kidney. The Gln376 mutant is devoid of activity (less than 0.02% that of wild type, expressed enzyme) and is green because of bound CoA persulfide. Properties of the mutant enzyme suggest that the Glu376----Gln change specifically affects substrate binding. These results prove that Glu376 plays an important role in the initial step of dehydrogenation catalysis.
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PMID:Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli. 197 May 66


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