Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.3.99.3 (acyl-CoA dehydrogenase)
 1,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ninety percent of variant medium-chain acyl-CoA dehydrogenase (MCAD) alleles in patients with MCAD deficiency carry a 985 A-->G transition which causes glutamate substitution for lysine 329 in precursor (p) MCAD (K-304 in mature MCAD). We have used site-directed mutagenesis to produce three variant cDNAs encoding variant pMCAD with glutamate (Kp329E2), aspartate (Kp329D), or arginine (Kp329R) substitution for Kp329. We carried out in vitro expression of cDNAs, and incubated the translation products with isolated rat liver mitochondria. Kp329E was imported into mitochondria and processed into the mature subunit as efficiently as wild-type. Gel filtration analysis of the mitochondria revealed that at 10 min after import, markedly more K304E eluted as a monomer than did wild-type, and the amount of K304E tetramer formed was distinctly less than wild-type at any point up to 60 min after import, indicating that the assembly of K304E is defective. After further incubation, K304E decayed more rapidly than did wild-type, indicating a reduced stability. In similar studies, K304R behaved like the wild-type, while K304D closely resembled K304E, indicating that the presence of a basic residue at 304 is essential for tetramer formation and intramitochondrial stability of mature MCAD.
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PMID:Impaired tetramer assembly of variant medium-chain acyl-coenzyme A dehydrogenase with a glutamate or aspartate substitution for lysine 304 causing instability of the protein. 136 Nov 90

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

Pig kidney medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) is irreversibly and stoichiometrically inactivated by [1-14C]-2-octynoyl coenzyme A. The linkage is stable at pH 2-6, but labile under basic conditions. The inhibitor labels a unique tryptic peptide, Ile-Tyr-Gln-Ile-Tyr-Glu-Gly-Thr-Ala-Gln-Ile-Gln-Arg, close to the C-terminus of the protein. The peptide is labeled at Glu-401 with the acyl moiety of the inhibitor but does not contain detectable coenzyme A. Both the inactivation of the dehydrogenase and the appearance of an absorption band at 800 nm show large primary deuterium isotope effects using 4,4'-dideuterio-2-octynoyl-CoA (7.3 and 6.3, respectively). Thus, 2-octynoyl-CoA is a mechanism-based inactivator of the dehydrogenase and is activated by rate-limiting gamma-proton abstraction. Glutamate-401 may be the base that abstracts the pro-R alpha-proton during the dehydrogenation of normal substrates.
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PMID:2-octynoyl coenzyme A is a mechanism-based inhibitor of pig kidney medium-chain acyl coenzyme A dehydrogenase: isolation of the target peptide. 323 92

The flavoenzyme pig kidney general acyl-CoA dehydrogenase (EC 1.3.99.3) is inactivated by cyclohexane-1,2-dione in borate buffer in a reaction that exhibits pseudo-first-order kinetics. Strong protection is afforded by the substrate octanoyl-CoA, as well as by heptadecyl-CoA, a potent competitive inhibitor of the dehydrogenase that does not reduce enzyme flavin. Enzyme exhibiting 10% residual activity in borate buffer contains about 1.3 modified arginine residues per flavin molecule. Very little reduction of the modified enzyme in borate buffer occurs at high concentrations of octanoyl-CoA, in marked contrast with the stoicheiometric reduction of the native enzyme. However, in phosphate buffer alone, the modified enzyme exhibits 55% residual activity and, although binding of substrate is still seriously impaired (apparent Kd=14 microM), excess substrate effects the formation of the characteristic reduced flavin X enoyl-CoA charge-transfer complex. These results suggest that the susceptible arginine residue, though not catalytically essential, is probably within the acyl-CoA-binding site of general acyl-CoA dehydrogenase.
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PMID:Modification of an arginine residue in pig kidney general acyl-coenzyme A dehydrogenase by cyclohexane-1,2-dione. 716 2

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.
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PMID:Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood. 747 27

The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids. The degree of overlap in substrate specificity is narrow among these enzymes. The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity. In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD). Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified. The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates. None of the other mutants exhibited enzyme activity. A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified. It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively. These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity.
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PMID:Functional role of the active site glutamate-368 in rat short chain acyl-CoA dehydrogenase. 895 87

Sponges (Porifera) are the phylogenetically oldest metazoan organisms. From one member of the siliceous sponges, Geodia cydonium, the cDNA encoding a putative SOS protein, the AidB-like protein of the Ada system from bacteria, was isolated and characterized. The cDNA, GCaidB, comprises an open reading frame of 446 amino acid (aa) residues encoding a polypeptide with a calculated Mr of 49,335. This molecule shows high similarity to the bacterial AidB proteins from Mycobacterium tuberculosis and Escherichia coli and somewhat lower similarities to acyl-CoA dehydrogenases (ADHs) and acyl-CoA oxidases (AOXs). Northern blot analysis confirmed the presence of the complete transcript. The deduced sponge aa sequence, GC_aidB, possesses the two characteristic acyl-CoA dehydrogenase signatures 1 and 2. Incubation of the sponge with N-methyl-N'-nitro-N-nitrosoguanidine causes a strong increase in the 2.1-kb large transcript of GCaidB; maximal expression is seen after 24 h of incubation with this DNA methylating agent. ADHs and AOXs can be grouped, depending on the position of the catalytically important Glu residue, into the Glu-Gly (Glu adjacent to Gly) class and the Glu-Arg (Glu adjacent to Arg) class. The phylogenetically oldest metazoan AidB-like molecule, GC_aidB of G. cydonium, belongs to the Glu-Gly class of ADHs. Phylogenetic analyses of the Glu-Gly class enzymes, with the described AidB-like protein from G. cydonium and the bacterial AidB polypeptides, together with metazoan ADHs and AOXs, revealed that the AidB(-like) proteins diverged first from a common ancestor, while the eukaryotic AOX and ADA polypeptides as well as the GHDs appeared later. According to the analyses, the very long-chain ADHs are older than the medium-chain, short-chain, and branched-chain ADHs. Inclusion of the phylogenetical oldest member of the Glu-Arg class of enzymes, the bacterial ADH-CaiA sequence in these analyses, revealed that this class of enzymes appeared later in evolution and arose from the Glu-Gly class perhaps after gene duplication.
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PMID:Identification and expression of the SOS response, aidB-like, gene in the marine sponge Geodia cydonium: implication for the phylogenetic relationships of metazoan acyl-CoA dehydrogenases and acyl-CoA oxidases. 973 61

A novel mutation was identified in two unrelated patients with medium-chain acyl-CoA dehydrogenase deficiency. First, a 19-year-old Caucasian female presented with a devastating illness, resulting in sudden death in adulthood which is unusual. The second patient, now a 3.5-year-old male, presented at 17 months of age with a hypoglycemic seizure and dehydration. Sequence analysis revealed a novel mutation G617T in exon 8 resulting in an arginine to leucine substitution at codon 206 (R206L). Both patients were compound heterozygous for this G617T and the common mutation A985G.
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PMID:Identification of a novel mutation in patients with medium-chain acyl-CoA dehydrogenase deficiency. 1076 81

Glutaryl-CoA dehydrogenase catalyzes the oxidation and decarboxylation of glutaryl-CoA to crotonyl-CoA and CO(2). Inherited defects in the protein cause glutaric acidemia type I, a fatal neurologic disease. Glutaryl-CoA dehydrogenase is the only member of the acyl-CoA dehydrogenase family with a cationic residue, Arg-94, situated in the binding site of the acyl moiety of the substrate. Crystallographic investigations suggest that Arg-94 is within hydrogen bonding distance of the gamma-carboxylate of glutaryl-CoA. Substitution of Arg-94 by glycine, a disease-causing mutation, and by glutamine, which is sterically more closely related to arginine, reduced k(cat) of the mutant dehydrogenases to 2-3% of k(cat) of the wild type enzyme. K(m) of these mutant dehydrogenases for glutaryl-CoA increases 10- to 16-fold. The steady-state kinetic constants of alternative substrates, hexanoyl-CoA and glutaramyl-CoA, which are not decarboxylated, are modestly affected by the mutations. The latter changes are probably due to steric and polar effects. The dissociation constants of the non-oxidizable substrate analogs, 3-thiaglutaryl-CoA and acetoacetyl-CoA, are not altered by the mutations. However, abstraction of a alpha-proton from 3-thiaglutaryl-CoA, to yield a charge transfer complex with the oxidized flavin, is severely limited. In contrast, abstraction of the alpha-proton of acetoacetyl-CoA by Arg-94 --> Gln mutant dehydrogenase is unaffected, and the resulting enolate forms a charge transfer complex with the oxidized flavin. These experiments indicate that Arg-94 does not make a major contribution to glutaryl-CoA binding. However, the electric field of Arg-94 may stabilize the dianions resulting from abstraction of the alpha-proton of glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain gamma-carboxylates. It is also possible that Arg-94 may orient glutaryl-CoA and 3-thiaglutaryl-CoA for abstraction of an alpha-proton.
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PMID:The function of Arg-94 in the oxidation and decarboxylation of glutaryl-CoA by human glutaryl-CoA dehydrogenase. 1102 31

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.
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PMID:Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue. 1469 13


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