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)

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.
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PMID:Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme. 862 4

In order to test the proposal [Stehle, T., Claiborne, A., & Schulz, G. E. (1993) Eur. J. Biochem. 211, 221-226] that the active-site His10 of NADH peroxidase functions as an essential acid-base catalyst, we have analyzed mutants in which this residue has been replaced by Gln or Ala. The k(cat) values for both H10Q and H10A peroxidases, and the pH profile for k(cat) with H10Q, are very similar to those observed with wild-type peroxidase. Both mutants, however, exhibit K(m)(H2O2) values much higher (50-70-fold) than that for wild-type enzyme, and stopped-flow analysis of the H2O2 reactivity of two-electron reduced H10Q demonstrates that this difference is due to a 150-fold decrease in the second-order rate constant for this reaction with the mutant. Stopped-flow analyses also confirm that reduction of the enzyme by NADH is essentially unaffected by His10 replacement and remains largely rate-limiting in turnover; the formation of an E.NADH intermediate in the conversion of E-->EH2 is confirmed by diode-array spectral analyses with H10A. Both H10Q and H10A mutants, in their oxidized E(FAD, Cys42-sulfenic acid) forms, exhibit enhanced long-wavelength absorbance bands (lambda(max) = 650 nm and 550 nm, respectively), which most likely reflect perturbations in a charge-transfer interaction between the Cys42-sulfenic acid and FAD. Combined with the 50-fold increase in the second-order rate constant for H2O2 inactivation (via Cys42-sulfenic acid oxidation) of the H10Q mutant, these observations support the proposal that His10 functions in part to stabilize the unusual Cys42-sulfenic acid redox center within the active-site environment.
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PMID:The active-site histidine-10 of enterococcal NADH peroxidase is not essential for catalytic activity. 865 80

Inactivation of D-amino acid oxidase occurred by different mechanisms. The enzyme showed a rapid loss of activity in the presence of micromolar amounts of Cu2+ and Hg2+. It was also sensitive to oxidative inactivation by Fe2+ and H2O2 when both reagents were added in millimolar amounts. When oxidatively inactivated D-amino acid oxidase and a corresponding non-treated control were modified with the sulfhydryl-modifying, fluorescent reagent monobromobimane and subsequently digested with endoproteinase Glu-C, Cys-298 was identified to be a target for oxidative modification according to differences in the known peptide profile of fluorescence intensity. Another reason for the observed loss of enzyme activity in crude extracts was the specific proteolytic digestion of D-amino acid oxidase, which was dependent on the growth phase of the cells used. This cleavage was catalyzed by a serine-type proteinase and was the introductory step for the further complete degradation of the enzyme. In addition, a coenriched 50-kDa protein, identified as NADPH-specific glutamate dehydrogenase, significantly decreased the stability of the D-amino acid oxidase activity. Treatment of apo-D-amino acid oxidase from T. variabilis with monobromobimane resulted in a significantly increased fluorescence of two peptides, neither of which contained any cysteine residue. Thus, an involvement of cysteine residues in binding the FAD coenzyme should be excluded.
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PMID:Studies on the inactivation of the flavoprotein D-amino acid oxidase from Trigonopsis variabilis. 873 70

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H2O2 in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H2O2-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCl. This NADPH-ferricyanide reductase activity was shown to belong to the H2O2-generating system, although it did not produce H2O2. It was still Ca2+ dependent and H2O2 production was restored by decreasing the ionic strength by overnight dialysis. No H2O2 production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a well-defined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K3Fe(CN)6. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H2O2 production in porcine thyrocytes in primary culture, also induced the NADPH-K3Fe(CN)6 reductase activity associated with the H2O2-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.
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PMID:Solubilization and characterization of a thyroid Ca(2+)-dependent and NADPH-dependent K3Fe(CN)6 reductase. Relationship with the NADPH-dependent H2O2-generating system. 885 87

Biochemical decompression has been proposed as a method for reducing the amount of time required for deep-sea divers to return to the surface. Divers breathing H2/O2 mixtures would be presented with hydrogenase enzyme, and decompression would be accelerated by means of the enzymic removal of excess H2 from the tissues. We have studied FAD as a hydrogenase electron acceptor that is capable of transferring electrons derived from H2 oxidation directly to O2. Kinetic activity constants for the soluble hydrogenase from the bacterium Alcaligenes eutrophus H16 were determined with FAD, FMN and riboflavin as electron acceptors, and these values were compared with those obtained with the physiological electron acceptor NAD+. The Michaelis constants (K(m)) were similar for FAD, FMN and NAD. However, the maximal catalytic-centre activity (Kcat) was much lower for the flavins, and the catalytic efficiency (Kcat/K(m)) with FAD was 1/20th the value for NAD+. After enzyme-catalysed FAD reduction to FADH2, the FAD could be regenerated by addition of O2 and reduced again by the enzyme in the presence of H2. Thus FAD served as a regenerable electron shuttle between H2 and O2. H2O2, a by-product of FADH2 oxidation by O2, inhibited the enzyme. Much greater inhibition was observed with the reduced form of the enzyme. Active hydrogenase was efficiently encapsulated into human and pig red blood cells. Hydrogen consumption was seen with lysed carrier cells, but was demonstrated with unlysed carrier cells only when FAD was co-encapsulated along with enzyme. These results demonstrate that red blood cells encapsulating hydrogenase and FAD act as a system for continuous H2 consumption in a mammalian tissue without addition of exogenous factors, and such cells may provide a biotherapeutic method for reducing the risk and treatment of decompression sickness.
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PMID:Hydrogenase encapsulation into red blood cells and regeneration of electron acceptor. 886 3

We describe the purification of a H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K(m) value of 4.2 microM for NADH and 16 microM for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce O.-2 nor H2O2 as stoichiometric products of oxygen reduction, implicating H2O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (Cu2+ and Zn2+) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin:NAD(P)1 oxidoreductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.
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PMID:A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. 889 1

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

A novel activity producing gamma-aminobutyric acid (GABA) from L-ornithine in the presence of NAD(P)+ was found in the crude extract of L-ornithine-induced Hafnia alvei, in addition to L-ornithine decarboxylase (ODC) activity. The reaction system for the former activity consisted of two enzymes, L-ornithine oxidase (decarboxylating, OOD) and gamma-aminobutyraldehyde (GABL) dehydrogenase (GDH). OOD catalyzed the conversion of L-ornithine into GABL, CO2, NH3, and H2O2 in the presence of O2, and GDH dehydrogenated GABL to GABA in the presence of NAD(P)+. OOD, purified to homogeneity, had a high ODC activity and the activity ratio of ODC to OOD was almost constant throughout the purification (ODC/ OOD=160:1). The molecular mass of the OOD was about 230 kDa, probably consisting of three identical subunits of a 77 kDa peptide, and OOD had an absorption maximum at 420 nm as well as at 278 nm, the specific absorption for an enzyme containing pyridoxal phosphate (PLP). The content of PLP was estimated at about 1 mol per subunit. OOD was specific to L-ornithine, and other L-amino acids and polyamines including putrescine were inert. The enzyme was activated by PLP, but not by pyridoxamine 5'-phosphate, FAD, FMN, or pyrroloquinoline quinone, and it was inactivated by hydrazine, semicarbazide, and hydroxylamine. The holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine, and reconstituted with PLP. These properties of OOD were almost the same as those of ODC separately purified to homogeneity from H. alvei. Zn2+ and Cu2+, butanedione, and sodium borohydride inhibited both OOD and ODC in a similar manner. The OOD reaction required O2 and only the ODC reaction proceeded under anaerobic conditions. The substitution of air for oxygen in the reaction vessel and the addition of catalase-H2O, enhanced only the OOD reaction, resulting in an increase of the ratio of OOD/ODC to 1:30 and 1:4.1, respectively. These results suggested that OOD and ODC are identical and that the former is a side reaction of the latter in the presence of O2.
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PMID:L-ornithine decarboxylase from Hafnia alvei has a novel L-ornithine oxidase activity. 944 11

The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN.
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PMID:Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica. 949 88

The flavoprotein NADH oxidase (O2 --> 2H2O) from Enterococcus faecalis 10C1 contains a cysteinyl redox center, in addition to FAD. We have proposed a cysteine-sulfenic acid (Cys-SOH) structure for the oxidized form of Cys42; the presence of this redox center is consistent with the stoichiometries reported for earlier reductive titrations of wild-type oxidase, and we have proposed that Cys42-SH plays a key role in the overall four-electron reduction of O2 --> 2H2O. To test these proposals, we provide in this report an analysis of the oxidative half-reaction of an oxidase mutant in which Cys42 is replaced by Ser. NADH titrations lead to direct flavin reduction with 1.05 equiv of NADH/FAD and give rise to the formation of a very stable E-FADH2.NAD+ complex. Kinetic analyses indicate that this species is catalytically competent, and its reactivity with O2 has been analyzed in detail by stopped-flow spectrophotometry using both single-wavelength and diode-array modes of data acquisition. The combined results of this analysis demonstrate that replacement of Cys42 with Ser provides for an altered O2 reduction stoichiometry in which H2O2, not 2H2O, is the product. The two subunits of the reduced enzyme.NAD+ complex react with O2 in an asymmetric mechanism, consistent with an alternating sites cooperativity model such as that proposed [Miller, S. M., Massey, V., Williams, C. H., Jr., Ballou, D. P., and Walsh, C. T. (1991) Biochemistry 30, 2600-2612] for mercuric reductase. An FAD C(4a)-hydroperoxide is identified as the primary oxygenated intermediate in reoxidation of the complex, but the reaction of O2 with the complementary subunit does not proceed until full reoxidation has occurred at the primary subunit. To our knowledge, this is the first report of a C(4a)-peroxyflavin intermediate outside the flavoprotein monooxygenase class.
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PMID:Oxygen reactivity of an NADH oxidase C42S mutant: evidence for a C(4a)-peroxyflavin intermediate and a rate-limiting conformational change. 962 41


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