Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been postulated that the peroxisomal fatty acid-oxidizing system [Lazarow & de Duve (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2043--2046; Lazarow (1978) J. Biol. Chem. 253, 1522--1528] resembles that of mitochondria, except for the first oxidative reaction. In this step, O2 would be directly reduced to H2O2 by an oxidase. Two specific procedures developed to detect the activity of the characteristic enzyme fatty acyl-CoA oxidase are presented, namely polarographic detection of palmitoyl-CoA-dependent cyanide-insensitive O2 consumption and palmitoyl-CoA-dependent H2O2 generation coupled to the peroxidation of methanol in an antimycin A-insensitive reaction. Fatty acyl-CoA oxidase activity is stimulated by FAD, which supports the flavoprotein nature postulated for this enzyme. Its activity increases 7-fold per g wet wt. of liver in rats treated with nafenopin, a hypolipidaemic drug. Subcellular fractionation of livers from normal and nafenopin-treated animals provides evidence for its peroxisomal localization. The stoicheiometry for palmitoyl-CoA-dependent O2 consumption, H2O2 generation and NAD+ reduction is 1 : 1 : 1. This suggests that fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal fatty acid-oxidizing system.
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PMID:Detection of peroxisomal fatty acyl-coenzyme A oxidase activity. 51 63

The peroxisomal acyl-CoA oxidase has been purified from extracts of the yeast Candida tropicalis grown with alkanes as the principal energy source. The enzyme has a molecular weight of 552,000 and a subunit molecular weight of 72,100. Using an experimentally determined molar extinction coefficient for the enzyme-bound flavin, a minimum molecular weight of 146,700 was determined. Based on these data, the oxidase contains eight perhaps identical subunits and four equivalents of FAD. No other beta-oxidation enzyme activities are detected in purified preparations of the oxidase. The oxidase flavin does not react with sulfite to form an N(5) flavin-sulfite complex. Photochemical reduction of the oxidase flavin yields a red semiquinone; however, the yield of semiquinone is strongly pH dependent. The yield of semiquinone is significantly reduced below pH 7.5. The flavin semiquinone can be further reduced to the hydroquinone. The behavior of the oxidase flavin during photoreduction and its reactivity toward sulfite are interpreted to reflect the interaction in the N(1)-C(2)O region of the flavin with a group on the protein which acts as a hydrogen-bond acceptor. Like the acyl-CoA dehydrogenases which catalyze the same transformation of acyl-CoA substrates, the oxidase is inactivated by the acetylenic substrate analog, 3-octynoyl-CoA, which acts as an active site-directed inhibitor.
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PMID:Chemical and catalytic properties of the peroxisomal acyl-coenzyme A oxidase from Candida tropicalis. 663 56

The acyl-CoA oxidase, catalysing the peroxisomal desaturation of the CoA-ester of trihydroxycoprostanic acid, a bile acid intermediate, has been purified to homogeneity from rat liver. Its native molecular mass, as determined by gel filtration and native gel electrophoresis, was 120 and 175 kDa respectively, suggesting a homodimeric protein consisting of 68.6 kDa subunits. If isolated in the presence of FAD, the enzyme showed a typical flavoprotein spectrum and contained most likely 2 mol of FAD per mol of enzyme. The cofactor, however, was loosely bound. The enzyme acted exclusively on 2-methyl-branched compounds, including pristanoyl-CoA and 2-methylhexanoyl-CoA if albumin was present. Important parameters to obtain a pure and active enzyme were the following: (1) using chromatographic separations like hydrophobic interaction and metal affinity, which allow the presence of high salt concentrations, conditions which stabilize the oxidase; (2) avoiding dialysis and (NH4)2SO4 precipitation; (3) including, when appropriate, FAD, dithiothreitol and a diol-compound in the solvents; and (4) carefully monitoring the removal of other acyl-CoA oxidases which possess the same native molecular mass and subunit size.
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PMID:Purification and further characterization of peroxisomal trihydroxycoprostanoyl-CoA oxidase from rat liver. 799 33

Medium- and short-chain acyl-CoA oxidases were identified in and subsequently purified from dark-grown maize plantlets. The oxidase showing preference for medium-chain fatty acyl-CoAs (C10-C14) was purified to homogeneity. The oxidase showing preference for short-chain fatty acyl-CoAs (C4-C8) was purified over 150-fold. Various catalytic properties confirmed these enzymes to be true acyl-CoA oxidases. They produced trans-2-enoyl-CoA and H2O2 from the saturated acyl-CoA, as verified by various independent assay techniques. They also exhibited FAD-dependent activity; i.e. removal of loosely bound FAD by gel filtration markedly reduced activity, which could be restored upon re-addition of FAD. They showed apparent Km values between 2 and 10 microM for the acyl-CoA substrate giving maximal activity, no activity with the corresponding free fatty acid, high pH optima (8.3-8.6) and a peroxisomal subcellular location. The medium-chain acyl-CoA oxidase was determined to be a monomeric protein with a molecular mass of 62 kDa. The short-chain acyl-CoA oxidase was shown to have a native molecular mass of 60 kDa, but exhibited a labile multimeric structure, as indicated by the elution of multiple peaks of activity during several chromatographic steps, and ultimately by the purification of a subunit of molecular mass 15 kDa. The medium- and short-chain acyl-CoA oxidases were demonstrated to be distinct from the maize equivalent of the cucumber glyoxysomal long-chain acyl-CoA oxidase previously purified and characterized [Kirsch, Loffler and Kindl (1986) J. Biol. Chem. 261, 8570-8575]. The maize long-chain acyl-CoA oxidase was partially purified to permit determination of its substrate specificity; it showed activity with a broad range of acyl-CoAs of chain length greater than C8, and maximal activity with C16. The implications of the existence of multiple acyl-CoA oxidases in the regulation of plant peroxisomal beta-oxidation are discussed.
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PMID:Higher-plant medium- and short-chain acyl-CoA oxidases: identification, purification and characterization of two novel enzymes of eukaryotic peroxisomal beta-oxidation. 897 74

Two forms of rat peroxisomal acyl-CoA oxidase (ACO-I and -II) interact with the substrate analogs, 3-ketoacyl-CoAs, forming a complex characterized by the so-called charge-transfer (CT) band around 575 nm in the absorption spectra. The CT band of ACO-I exhibited a broad dependency on the acyl chain-length from C4 to C16, whereas that of ACO-II showed increased intensity with a longer acyl chain to reach a maximum with a chain-length of C12. These chain-length dependencies of the CT bands were compared with those of the enzymatic activities reported previously [Setoyama et al. (1995) Biochem. Biophys. Res. Commun. 217, 482-487]. The differences in spectroscopic and enzymatic properties between ACO-I and -II suggest that the amino acid stretch corresponding to the third exon in the ACO sequence affects the binding of the ligand and substrate, since the difference in the primary structure between ACO-I and -II lies in the short amino acid stretch corresponding to the third of the total of 14 exons. On the other hand, resonance Raman spectra of the complexes of ACO-I and -II with 3-ketoacyl-CoAs excited in the CT band showed similar features. The two prominent FAD bands II and III, associated with the C(4a)=N(5) moiety of FAD, were observed at 1,577 and 1,545 cm(-1), respectively. In contrast, the bands at 1,615 and 1,493 cm(-1) in the ACO-I x 3-keto-C8-CoA complex were assigned to the stretching modes of C=O at positions 3 and 1 of the ligand, respectively, by using the isotopically labeled ligands. Both C=O stretching bands were shifted to lower wave numbers upon complex formation with ACO-I, implying that the C=O bond involves the single bond (C-O-) character in the active site cavity. The downshift of the C(1)=O stretching band was larger than that of the C(3)=O stretching band. Therefore, the ligand lies in the active site as the anionic form with a major contribution from C(1)-O-. These observations demonstrate that the CT band around 575 nm arises from the charge-transfer interaction between the oxidized FAD and the enolate transformed after the elimination of the a-proton. The band II of FAD in the complexes reveals a significant decrease in the frequency in comparison with the complexes of medium-chain acyl-CoA dehydrogenase (MCAD) with 3-ketoacyl-CoA. This observation suggests a difference between ACO and MCAD in the hydrogen-bonding network associated with enzyme-bound FAD.
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PMID:Spectroscopic studies of rat liver acyl-CoA oxidase with reference to recognition and activation of substrate. 935 89

A range of 4-thiaacyl-CoA derivatives has been synthesized to study the bioactivation of cytotoxic fatty acids by the mitochondrial medium-chain acyl-CoA dehydrogenase and the peroxisomal acyl-CoA oxidase. Both enzymes catalyze alpha-proton abstraction from normal acyl-CoA substrates with elimination of a beta-hydride equivalent to the FAD prosthetic group. In competition with this oxidation reaction, 4-thiaacyl-CoA thioesters undergo dehydrogenase-catalyzed beta-elimination, providing that the corresponding thiolates are sufficiently good leaving groups and can be accommodated by the active site of the enzyme. Thus, the dehydrogenase catalyzes the elimination of 2-mercaptobenzothiazole and 4-nitrothiophenolate from 4-(2-benzothiazole)-4-thiabutanoyl-CoA and 4-(4-nitrophenyl)-4-thiabutanoyl-CoA, respectively. However, the 2,4-dinitrophenyl-analogue appears too bulky and the unsubstituted thiophenyl-derivative is insufficiently activated for significant elimination. Molecular modeling shows that steric interference from the flavin ring dictates a syn rather than an anti elimination. Acryloyl-CoA, the other product of 4-thiaacyl-CoA elimination reactions, is not a significant inactivator of the medium-chain dehydrogenase. In contrast, the irreversible inactivation observed during beta-elimination using 5,6-dichloro-4-thia-5-hexenoyl-CoA (DCTH-CoA), 5,6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA), and 6-chloro-5,5,6-trifluoro-4-thiahexanoyl-CoA (CTFTH-CoA) is caused by release of cytotoxic thiolate products within the active site of the dehydrogenase. The double bond between C5 and C6 found in the vinylic analogues DCTH- and DCTFTH-CoA is not essential for enzyme inactivation, although CTFTH-CoA is a weaker inhibitor of the dehydrogenase. Mechanism-based inactivation with CTFTH-CoA requires elimination, is unaffected by exogenous nucleophiles, and is strongly protected by octanoyl-CoA. The peroxisomal acyl-CoA oxidase efficiently oxidizes 4-thiaacyl-CoA analogues, but is only rapidly inactivated by DCTFTH-CoA. The variable ratio of elimination to oxidation observed for DCTH-, DCTFTH-, and CTFTH-CoA may influence the metabolism of the corresponding cytotoxic alkanoic acids in vivo.
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PMID:Elimination reactions in the medium-chain acyl-CoA dehydrogenase: bioactivation of cytotoxic 4-thiaalkanoic acids. 947 67

Peroxisomal beta-oxidation, consisting of four steps catalysed by an acyl-CoA oxidase, a multifunctional protein and a thiolase, is responsible for the shortening of a variety of lipid compounds. The first reaction of this pathway is catalysed by a FAD-containing acyl-CoA oxidase, three isotypes of which have been so far recognised. Among these, straight-chain acyl-CoA oxidase (ACOX) acts on long and very long chain fatty acids, prostaglandins and some xenobiotics. We investigated ACOX localisation by means of a sensitive, tyramide based, immunocytochemical technique, thus obtaining a complete distribution atlas of the enzyme in adult rat CNS. Granular immunoreaction product was found in the cytoplasm of neuronal and glial cells, both in the perikarya and in the cell processes. ACOX immunoreactive neurons were present to variable extent, in either forebrain or hindbrain areas. Specifically, the strongest signal was detected in the pallidum, septum, red nucleus, reticular formation, nuclei of the cranial nerves, and motoneurons of the spinal cord. We then compared the ACOX immunoreactivity pattern with our previous distribution maps of other peroxisomal enzymes in the adult rat brain. While ACOX appeared to colocalise with catalase in the majority of cerebral regions, some differences with respect to d-amino acid oxidase were noted. These observations support the hypothesis of heterogeneous peroxisomal populations in the nervous tissue. The wide distribution of the enzyme in the brain is consistent with the severe and generalised neurological alterations characterising the peroxisomal disorder caused by ACOX deficiency (pseudo-neonatal adrenoleukodystrophy).
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PMID:Immunocytochemical localization of acyl-CoA oxidase in the rat central nervous system. 1157 43

Five isoforms of acyl-CoA oxidase (Aox), designated Aox1p to Aox5p, constitute a 443-kD heteropentameric complex containing one polypeptide chain of each isoform within the peroxisomal matrix of the yeast Yarrowia lipolytica. Assembly of the Aox complex occurs in the cytosol and precedes its import into peroxisomes. Peroxisomal targeting of the Aox complex is abolished in a mutant lacking the peroxin Pex5p, a component of the matrix protein targeting machinery. Import of the Aox complex into peroxisomes does not involve the cytosolic chaperone Pex20p, which mediates the oligomerization and import of peroxisomal thiolase. Aox2p and Aox3p play a pivotal role in the formation of the Aox complex in the cytosol and can substitute for one another in promoting assembly of the complex. In vitro, these subunits retard disassembly of the Aox complex and increase the efficiency of its reassembly. Neither Aox2p nor Aox3p is required for acquisition of the cofactor FAD by other components of the complex. We provide evidence that the Aox2p- and Aox3p-assisted assembly of the Aox complex in the cytosol is mandatory for its import into peroxisomes and that no component of the complex can penetrate the peroxisomal matrix as a monomer.
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PMID:Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica. 1181 35

One of the acyl-CoA oxidases from the yeast Yarrowia lipolytica, acyl-CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His)(6) fusion protein. The specific activity of the purified enzyme, containing FAD, was 19.7 micromolmin(-1)mg(-1) using myristoyl-CoA as substrate. Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed. Straight-chain acyl-CoAs, with a chain length of 10-14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies. Acyl-CoAs containing more than 14C were also desaturated, if their concentration was below 25 microM or if proteins capable of binding these CoA-esters, such as albumin or beta-casein, were added to the assay. These long-chain acyl-CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates. Compared to palmitoyl-CoA, activity toward hexadecadioyl-CoA, containing a omega-carboxy group, was similar. Taken together, these data suggest that micelles of long-chain acyl-CoAs are able to bind and inhibit Aox2p. The enzyme was also active toward acyl-CoA-esters containing a 2-methyl group, but only the 2S isomer was recognized.
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PMID:The acyl-CoA oxidases from the yeast Yarrowia lipolytica: characterization of Aox2p. 1239 12

An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro.
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PMID:Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays. 1263 Dec 94


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