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)

The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source. The hydrogenase is membrane-bound and relatively oxygen-sensitive. The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase. The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction). SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000. The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors. The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.
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PMID:Purification and properties of the membrane-bound hydrogenase from N2-fixing Alcaligenes latus. 630 22

A highly specific inducible membrane-bound 4-pyridoxic acid dehydrogenase has been solubilized and purified to apparent homogeneity from Pseudomonas MA-1 grown with pyridoxine as a sole source of carbon and nitrogen. The undenatured enzyme migrates as a single band on gel electrophoresis; denatured preparations show two barely resolved bands (Mr = 63,000 and 61,000). Undenatured preparations aggregate readily, as evidenced by Mr values of 148,000, 470,000, and greater than 670,000 obtained by density gradient centrifugation or by gel filtration under various conditions. The enzyme contains FAD but no Fe or acid-labile S; an average minimum molecular weight of 131,000 was calculated from the FAD content. In the presence of 2,6-dichloroindophenol, the enzyme dehydrogenates 4-pyridoxic acid to the corresponding aldehyde; this reaction is not inhibited by CN-. At the pH optimum of 8.0, a Vm of approximately 7.0 mumol min-1 mg-1 and a Km of 9 microM were obtained. 2,6-Dichloroindophenol, phenazine methosulfate, and menadione are effective electron acceptors; ubiquinones are less active, while NAD, FAD, and O2 are inactive. However, in membrane fractions, oxygen supports 4-pyridoxic acid oxidation via a CN--sensitive electron transport chain, indicating that the dehydrogenase probably is coupled to ATP generation in such preparations.
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PMID:The bacterial oxidation of vitamin B6. 4-Pyridoxic acid dehydrogenase: a membrane-bound enzyme from Pseudomonas MA-1. 634 42

The flavin prosthetic group (FAD) of thioredoxin reductase has been replaced by 1-deazaFAD (carbon substituted for nitrogen at position 1). Reduction of 1-deazaFAD-thioredoxin reductase by four electrons proceeds in two stages having midpoint potentials that are separated by 0.063 V. Two-electron reduced 1-deazaFAD-thioredoxin reductase (EH2) has spectral characteristics that are different from both the fully oxidized and fully reduced enzyme. The fluorescence of the 2-electron reduced enzyme shows a mixture of two EH2 species. The spectrum of one EH2 species has a single absorption peak (lambda max, 414 nm; epsilon 414, 8750 M-1 cm-1) which is similar to the spectrum of 1-deazaFAD-C-4a adducts (referred to as the 414-nm absorbing species). In the other EH2 species the electrons are in the dithiol, and it has an oxidized 1-deazaFAD spectrum (referred to as the 550-nm EH2 species). The equilibrium between the two EH2 species of 1-deazaFAD-thioredoxin reductase is pH dependent, forming more of the 414-nm absorbing species as the pH is lowered. The pH dependence suggests the presence of an active center base having a pK of 7.41 on the 414-nm EH2 species and a thiol of pK 6.73 on the 550-nm EH2 species. These pK values are similar to the pK values determined for native enzyme having a disulfide or a dithiol (7.59 and 6.98, respectively). Thus, the pH dependence of the equilibrium between the two EH2 species of 1-deazaFAD-thioredoxin reductase is further evidence for an active site base with an ionization behavior that is linked to the chemical state of the active site disulfide moiety. The nature of the linked ionization is consistent with a thiol base ion pair formed upon disulfide reduction.
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PMID:Reconstitution of Escherichia coli thioredoxin reductase with 1-deazaFAD. Evidence for 1-deazaFAD C-4a adduct formation linked to the ionization of an active site base. 636 6

Antibodies to NADPH-cytochrome P-450 reductase have been used to essentially abolish the contribution of cytochrome P-450 to xenobiotic metabolism by mammalian microsomes. This permits the determination of the activity of the FAD-containing mono-oxygenase and the stoichiometry between substrate, O2 and NADPH, in the microsomal membrane, and in the absence of cytochrome P-450-dependent activity. FAD-containing mono-oxygenase oxidation rates were determined for sulphur- and nitrogen-containing substrates, including: thiols; sulphides; thioamides; primary, secondary and tertiary amines; hydrazines. Although the enzyme in mouse, rabbit, rat and pig microsomes displays similar substrate specificity, some catalytic characteristics are different between species and tissues.
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PMID:The measurement of FAD-containing mono-oxygenase activity in microsomes containing cytochrome P-450. 650 63

By techniques involving differential centrifugation and specific precipitation with CaCl2, it was shown that dimethylamine and trimethylamine mono-oxygenase activities co-sediment with NADPH-cytochrome c reductase activity in sphaeroplast lysates of Candida utilis grown on trimethylamine as sole nitrogen source. Since the active fraction also contained low levels of cytochromes P-450 and P-420, it was concluded that the two amine mono-oxygenases are located in the smooth endoplasmic reticulum and thus end up in the microsomal fraction on cell fractionation. Ten to twenty-fold enrichment of mono-oxygenase specific activity could be achieved by separation of activity from soluble protein by centrifugation or gel filtration. Cell-free extracts prepared in the absence of FAD showed only very low mono-oxygenase activity for either substrate. Some activity could be restored by addition of flavin nucleotides: there was a fivefold stimulation by FAD and a fourfold stimulation by FMN. All trimethylamine mono-oxygenase activity was lost when a partially purified preparation containing both activities was incubated for more than 24 h at 0 degrees C, suggesting that separate enzymes are responsible for the oxidation of secondary and tertiary amines. The enzyme preparation oxidized a wide range of secondary alkylamines up to dibutylamine and tertiary alkylamines up to tributylamine. Primary amines, choline, di- and triethanolamine, spermine, spermidine and substituted anilines were not oxidized. NADH had a lower apparent Km value and higher Vmax value than NADPH. Secondary and tertiary alkylamines containing more than one kind of alkyl group gave more than one kind of aldehyde on oxidation. Stoicheiometry determinations showed a consumption of 1 mol NAD(P)H and 1 mol O2 per mol aldehyde formed. Carbon monoxide, cyanide, proadifen hydrochloride (SKF 525-A), mercurials and mercaptoethanol all inhibited both activities.
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PMID:Subcellular localization and properties of partially purified dimethylamine and trimethylamine mono-oxygenase activities in Candida utilis. 654 83

Many preparations of flavin nucleotides contain nucleotide isomers of the natural compounds which are difficult to remove or separate. The method of dynamic complex-exchange (or paired-ion) chromatography has been used with high-performance liquid chromatography to achieve resolution and purification of isomers. A solution of nucleotide in water was chromatographed isocratically on a C18-substituted silica column with a mobile phase of methanol, water, and tetrabutylammonium phosphate at neutral pH. Commercial preparations of FMN and FAD contained multiple components. The purified isomers were subjected to ion-exchange chromatography directly on a quaternary nitrogen-substituted silica column to remove methanol and tetrabutylammonium cation, and thus obtain pure nucleotide in aqueous buffer suitable for use with proteins. With analytical equipment, a milligram of pure FMN or FAD was produced in 1 day. The same procedure was useful for the rapid identification and quantitation of flavin nucleotides in proteins. After exposure of a protein solution to heat treatment, the supernatant was subjected to dynamic complex-exchange chromatography, as described above.
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PMID:The purification and identification of flavin nucleotides by high-performance liquid chromatography. 663 2

The flavin prosthetic group (FAD) of the aromatic hydroxylases melilotate hydroxylase (EC 1.14.13.4) and phenol hydroxylase (EC 1.14.13.7) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). Neither modified enzyme could hydroxylate its substrate, both catalyzed the oxidation of NAD(P)H to NAD(P)+ and H2O2. The rate of the reduction of the enzymes by NAD(P)H was increased by the binding of substrate. Both enzymes formed a detectable flavin C(4a) hydroperoxide intermediate upon reaction of the reduced enzyme-substrate complex with oxygen. Reduced 1-deaza-FAD phenol hydroxylase also showed a detectable C(4a) hydroperoxide intermediate when reacted with oxygen in the absence of substrate. The C(4a) hydroperoxide of 1-deaza-FAD phenol hydroxylase, in the absence of phenol, decayed to an intermediate which showed a perturbed oxidized enzyme spectrum, Eox. This intermediate in turn decayed to give the original oxidized enzyme. In the presence of phenol, a second oxidized species with a perturbed spectrum, intermediate X, was apparent after formation of the flavin C(4a) hydroperoxide and before Eox formation. Steady state kinetic analysis of 1-deaza-FAD phenol hydroxylase demonstrated that the Eox to Eox conversion was not in the catalytic cycle. During turnover Eox was reduced by NADPH.
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PMID:Reactions of 1-deaza-FAD-substituted phenol hydroxylase and melilotate hydroxylase. 669 23

Human neutrophils were fractionated by nitrogen cavitation and Percoll density centrifugation, and the subcellular localization of FAD-flavoprotein, b-cytochrome, NADH-cytochrome b5 reductase, and NADPH-dependent cytochrome c reductase were determined in normal cells, cells from two patients with chronic granulomatous disease (CGD), and normal cells that had been stimulated with phorbol myristate acetate. In normal cells, a FAD-flavoprotein is found in a 1:2 molar ratio, with cytochrome b in the fractions containing the specific granules. Triton X-114 phase distribution indicates that the b-cytochrome but not the b-cytochrome-associated flavoprotein is an integral membrane protein. 80% of this flavoprotein, as well as all the b-cytochrome, was absent in these fractions from 2 CGD patients, although these patients had normal quantities of FAD in the fractions containing plasma membranes and cytosol. During stimulation the b-cytochrome-associated flavoprotein of the granules translocates with the b-cytochrome to the plasma membrane where NADPH oxidase is localized. Definition of the role of these NADPH oxidase constituents may provide a molecular description of the normal neutrophil respiratory burst and the molecular defect(s) in CGD.
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PMID:Subcellular localization of the human neutrophil NADPH oxidase. b-Cytochrome and associated flavoprotein. 670 48

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.
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PMID:Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD. 676 49

Whole cells of Candida boidinii grown on di- or tri-methylamine as sole nitrogen source readily oxidized both amines. The oxidation was potently inhibited by carbon monoxide. Cell-free extracts required the presence of 20 microM FAD before mono-oxygenase activity with both amines could be demonstrated. NADH was a better electron donor than NADPH. Activity was present in cells grown on secondary and tertiary amines but not on primary amines, and was detected in a number of different yeasts. Enzyme activity could be sedimented at 187 000 x g, and was associated with NADPH-cytochrome c reductase activity. It is thus probably microsomal. Activity was inhibited by cyanide, mercaptoethanol, carbon monoxide and proadifen hydrochloride (SKF 525-A).
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PMID:Oxidation of dimethylamine and trimethylamine in methazotrophic yeasts by microsomal mono-oxygenases sensitive to carbon monoxide. 687 Sep 1


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