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)

Activation of the NADPH oxidase of phagocytes in the cell-free system requires the association of several cytosolic components with membrane-bound cytochrome b. In this study we were able to fully reconstitute NADPH oxidase activity in the cell-free system with three recombinant proteins: p67-phox, p47-phox, p21rac1, and pure cytochrome b-245. Activity was dependent upon the concentration of the proteins, with maximal activity observed with roughly equimolar ratios of the cytochrome b and p67-phox (133 and 163 mol/s/mol, respectively) and concentrations of the other two proteins approximately 1 order of magnitude greater. No activity was observed in the absence of any one of these components. In addition, activation was dependent upon p21rac1 being preloaded with GTP, the cytochrome b being reconstituted with lipid, and the presence of FAD during activation. Half-maximal activity was observed at a concentration of NADPH of approximately 50 microM. These findings confirm our recent description of the membrane-bound cytochrome b as a FAD-containing flavocytochrome b containing the NADPH binding site, and implicate the three cytosolic proteins in its activation.
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PMID:Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245. 151 17

Although dihydropyrimidine dehydrogenase has been purified to varying degrees from several species, very little is known about the human enzyme. The importance of this enzyme has recently been shown with cancer chemotherapy, particularly in patients with genetic deficiency of this enzyme. In the present study, this enzyme was purified 7800-fold to homogeneity from human liver by introducing several novel methods including chromatofocusing, HPLC gel filtration, reversed-phase HPLC for the enzyme assay. Purified human enzyme has a molecular mass of 210 +/- 5 kDa and appears to be composed of two subunits. The apparent pI is pH 4.6 (+/- 0.2). The human enzyme contains approximately four flavin nucleotide molecules (two each of FAD and FMN) and 33 iron atoms per molecule of enzyme. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out. Amino acid composition and the N-terminal amino acid sequence of this enzyme were reported. A rabbit polyclonal antibody was raised and shown to be specific for the human liver enzyme. In conclusion, in the present manuscript, we report not only a novel procedure for purification of dihydropyrimidine dehydrogenase from human liver but also new data on its properties compared to other species, which will provide a basis for further biochemical and molecular studies of this enzyme.
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PMID:Purification and characterization of dihydropyrimidine dehydrogenase from human liver. 151 48

We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.
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PMID:Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179. 156 99

Microsomal P450s catalyze the monooxygenation of a large variety of hydrophobic compounds, including drugs, steroids, carcinogens, and fatty acids. The interaction of microsomal P450s with their electron transfer partner, NADPH-P450 reductase, during the transfer of electrons from NADPH to P450, for oxygen activation, may be important in regulating this enzyme system. Highly purified Bacillus megaterium P450BM-3 is catalytically self-sufficient and contains both the reductase and P450 domains on a single polypeptide chain of approximately 120,000 Da. The two domains of P450BM-3 appear to be analogous in their function and homologous in their sequence to the microsomal P450 system components. FAD, FMN, and heme residues are present in equimolar amounts in purified P450BM-3 and, therefore, this protein could potentially accept five electron equivalents per mole of enzyme during a reductive titration. The titration of P450BM-3 with sodium dithionite under a carbon monoxide atmosphere was complete with the addition of the expected five electron equivalents. The intermediate spectra indicate that the heme iron is reduced first, followed by the flavin residues. Titration of the protein with the physiological reductant, NADPH, also required approximately five electron equivalents when the reaction was performed under an atmosphere of carbon monoxide. Under an atmosphere of argon and in the absence of carbon monoxide, one of the flavin groups was reduced prior to the reduction of the heme group. The titration behavior of P450BM-3 with NADPH was surprising because no spectral changes characteristic of flavin semiquinone intermediates were observed. The results of the titration with NADPH can only be explained if (a) there was "rapid" intermolecular electron transfer between P450BM-3 molecules, (b) there is no kinetic barrier to the reduction of P450 by the one-electron-reduced form of the reductase, and (c) the "air-stable semiquinone" form of the reductase does not accumulate in this complex multidomain enzyme.
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PMID:P450BM-3: reduction by NADPH and sodium dithionite. 156 20

Quinoids undergo metabolism by a number of flavoenzymes. Reactive species formed during the metabolism of some quinoids might be anticipated to inhibit flavoenzyme activity. Several quinoids have been tested for their ability to inhibit rat liver thioredoxin reductase (TR). The antitumor quinones diaziquone and doxorubicin, and the quinoneimine 2,6-dichloroindophenol, were found to be inhibitors of the reduction of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) by TR. The inhibition was most marked after incubation of the quinoid with NADPH and the enzyme for 60 min before adding DTNB, with Ki values of 0.5 microM for diaziquone, 0.5 microM for doxorubicin, and 0.07 microM for 2,6-dichloroindophenol. The three quinoids all produced a time-dependent and first order loss of TR activity. There was formation of electron spin resonance-detectable semiquinoid free radicals upon incubation of diaziquone, doxorubicin and 2,6-dichloroindophenol with TR and NADPH under anaerobic conditions. Oxygen radicals formed by redox cycling of the quinoids did not make a major contribution to the inhibition of TR by the quinoids, as shown by the absence of significant reversal of the inhibition by anaerobic incubation conditions and the lack of effect of the oxygen radical scavengers dimethyl sulfoxide, superoxide dismutase and catalase. It was not possible to demonstrate NADPH-dependent covalent binding of radiolabeled diaziquone or doxorubicin to the TR apoprotein. It is possible that the quinoids bind noncovalently to the enzyme apoprotein, or bind to the FAD prosthetic group. The results of the study suggest that some antitumor quinoids are mechanism-based inhibitors of TR showing metabolism- and time-dependent irreversible inhibition of enzyme activity.
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PMID:Mechanism-based inhibition of thioredoxin reductase by antitumor quinoid compounds. 156 82

A NADH oxidase has been purified from the extreme thermophile Thermus thermophilus HB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N-terminal amino acids sequence. It is a monomeric flavin-adenine-dinucleotide-containing flavoprotein with an apparent molecular mass of 25 kDa and an 1:1 ratio of FAD to the polypeptide chain. The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H2O2. The apparent Km values for NADH and NADPH are 4.14 microM and 14.0 microM (pH 7.2 at room temperature), respectively, with a sixfold greater kcat/Km values for NADH compared to NADPH. The enzyme uses O2 as an electron acceptor in the presence of either FAD, riboflavin 5'-phosphate or riboflavin as cofactor. In addition, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (methylene blue, cytochrome c, p-nitroblue tetrazolium, 2,6-dichloroindophenol and potassium ferricyanide), even in the absence of flavin shuttles. No significant inhibition of the NADH oxidoreductase activity by superoxide dismutase was observed with these artificial electron acceptors, indicating that electron transfer occurs mainly from NADH directly to the electron acceptors, not via O2- as an intermediate. The purified NADH oxidase exhibits highest activity at pH 5.0 and is stable at elevated temperatures of up to 80 degrees C.
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PMID:Purification and characterization of a NADH oxidase from the thermophile Thermus thermophilus HB8. 157 5

Purification of aldehyde oxidoreductase from C. thermoaceticum, the first detected enzyme able to reduce reversibly non-activated carboxylic acids to the corresponding aldehydes (White, H., Strobl, G., Feicht, R. & Simon, H. (1989) Eur. J. Biochem. 184, 89-96), results in the generation of multiple forms of the enzyme. The specific activities for the viologen-mediated dehydrogenation of butyraldehyde for the two main forms of the purification procedure are 530 and 450 U/mg. Two forms of the enzyme composed of alpha,beta- and alpha,beta,gamma-subunits, can be differentiated. The latter binds to red-Sepharose and can be eluted very specifically with NADPH. In contrast to the alpha,beta-types the trimeric forms also catalyse the reversible reduction of oxidised viologen with NADPH (VAPOR activity). The dimer alpha,beta can oligomerize and the alpha,beta,gamma-trimer can easily form various oligomers or split off the gamma-subunit. The apparent molecular masses of the subunits alpha,beta and gamma are 64, 14 and 43 kDa. The alpha,beta-form reveals an apparent molecular mass of 86 kDa containing about 29 iron, 25 acid-labile sulphur, 0.8 tungsten and forms about 1 mol pterine-6-carboxylic acid by permanganate oxidation. The corresponding values of the trimer showing a mass of 300 kDa, are about 82 Fe, 54 S, 3.4 W and 2.5 pterine-6-carboxylic acid. In addition, 1.7 mol of FAD could be found which seems to be a component of the gamma-subunit. The aldehyde oxidoreductase from C. thermoaceticum and that from C. formicoaceticum (White, H., Feicht, R., Huber, C., Lottspeich, F. & Simon, H. (1991) Biol. Chem. Hoppe-Seyler 372, 999-1005) show qualitative similarities as far as the Fe, S, W and pterin content and the broad substrate specificity are concerned. However, there are also surprisingly marked differences with respect to composition and amino-acid sequence.
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PMID:The tungsten-containing aldehyde oxidoreductase from Clostridium thermoaceticum and its complex with a viologen-accepting NADPH oxidoreductase. 158 52

A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109. The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG). The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol. In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system. Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s. These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity. Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation.
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PMID:Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177. 162 1

Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils.
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PMID:Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system. 165 5

Following partial purification of macrophage nitric oxide (NO) synthase, enzyme activity requires L-arginine, NADPH, and constitutive cytosolic factors, one of which is tetrahydrobiopterin (BH4) (Kwon, N.S., Nathan, C.F. and Stuehr, D.J. [1989] J. Biol. Chem. 264, 20496). Here we identify FAD and GSH as two additional cofactors needed for full enzyme activity. With all defined cytosolic cofactors in excess, NO synthesis was linear over 3 h and was approximately 50% dependent on exogenous FAD, approximately 50% on glutathione (GSH), 84% on tetrahydrobiopterin (BH4), 95% on NADPH, and 98% on L-arginine. The concentrations of added FAD, GSH, and BH4 required for optimal activity were consistent with their levels in macrophage cytosol. Kinetic studies showed that GSH (or DTT) had little or no effect on the rate of NO generation over the first 20-30 min of the reaction, but prevented a subsequent dropoff in rate. This effect was distinct from thiol participation in BH4 regeneration. In contrast, exogenous FAD doubled the rate of NO synthesis throughout the assay period, consistent with a cofactor role. The role of NADPH was not to regenerate BH4, furnish NADP+, nor form reactive oxygen intermediates. These findings demonstrate NO synthesis by a partially purified enzyme in an otherwise defined system, and suggest that an NADPH-utilizing FAD flavoprotein may participate in the reaction.
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PMID:FAD and GSH participate in macrophage synthesis of nitric oxide. 169 11


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