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)

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.
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PMID:Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography. 82 51

The reaction mechanism of adenosine 5'-phosphosulfate (APS) reductase (EC 1.8.99.2) from Thiobacillus thioparus was studied using difference spectrum and stopped-flow techniques. The enzyme-bound FAD was rapidly reduced by sulfite with a first order rate constant of 97.1 s-1. The addition of AMP induced further spectral changes in the reduced enzyme which were consistent with the oxidation of FADH2 to the red (anionic) semiquinone FADH-) and the concomitant reduction of nonheme iron to the ferrous state. Superoxide dismutase (EC 1.15.1.1) or anaerobiosis inhibited the reduction of cytochrome c by the enzyme only to the extent of 25-35%, indicating the existence of a direct reduction of cytochrome c by the enzyme without involving O2-. the activity of enzyme with cytochrome c was inhibited by increasing the potassium phosphate concentration, the inhibition being more pronounced with horse heart cytochrome c than with Candida krusei cytochrome c.
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PMID:A study on the reaction mechanism of adenosine 5'-phosphosulfate reductase from Thiobacillus thioparus, an iron-sulfur flavoprotein. 83 49

NADPH-adrenodoxin reductase from steer adrenal cortex mitochrondria has been purified to homogeneity (on sodium dodecyl sulfate polyacrylamide gel electrophoresis) by chromatography on DEAE-cellulose, Sephadex, and hydroxylapatite. A molecular weight of 51,500 was determined from sodium dodecyl sulfate polyacrylamide gel electrophoresis, while sedimentation equilibrium ultracentrifugation gave a value of 49,500. All of the flavine present was identified as FAD; 1 mol/52,000 g of protein. The reductase contained 1.7% carbohydrate (using glucose as standard) by weight. Homogeneous adrenodoxin reductase exhibited a typical oxidized flavoprotein absorbance spectrum, with maxima at 270, 376, and 450 nm, and gave an absorbance ratio A450/A270 of 0.122-0.128 (depending on the preparation). Reduction of the flavoprotein with NADPH or dithionite gave progressive bleaching of the 450-nm peak. The reductase was absolutely required, in the presence of adrenodoxin, for electron transfer from NADPH to cytochrome c or to particulate cytochrome P450. Adrenodoxin refuctase is obligatory for reconstitution of 11beta-hydroxylation activity using deoxycorticosterone as substrate, and for the side-chain cleavage of 20alpha-hydroxycholesterol or cholesterol. The specific activity of the homogeneous preparation in cytochrome c reduction is at least 17,000 nmol min-1 mg of protein-1, corresponding to a turnover number of 850 min-1. No evidence for the existence of multiple forms or subunits was obtained.
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PMID:Purification and characterization of adrenodoxin reductase from bovine adrenal cortex. 112 83

A 37-yr-old woman with nontoxic goiter is presented. The thyroid 131I uptake at 3 and 24 hr were, respectively, 77.1% and 81.4% dose. Thiocyanate discharged 65.5% of the accumulated 131I in 30 min. In vitro organification of iodine in the thyroid homogenate from the patient was impaired and it was restored to normal by the addition of H2O2, glucose, and glucose oxidase system, FAD, or reduced cytochrome b5. Riboflavin, FMN, oxidized cytochrome b5, oxidized or reduced cytochrome c, NAD(H), and NADP(H) were ineffective in the reaction. The microsomal NADH-cytochrome b5 reductase activity was definitely low in the patient's thyroid. It was augmented to a normal level by incubation of the microsomes with FAD for 30 min or more. The activities of thyroid peroxidase, G6-PD, 6-PGD, catalase, protease, and NADPH-cytochrome c reductase were within normal limits. The major thyroid protein was normal thyroglobulin which could be readily iodinated in the presence of H2O2 and horse radish peroxidase. These findings suggest the correlation of an iodide organification defect with a cytochrome b5 reductase deficiency. Administration of high doses of FAD led to the restoration of thyroidal iodide organification mechanism associated with an increased thyroid hormone production and to a marked decrease of the goiter. Riboflavin was given without effect even at a high dosage level. Consequently, it seems likely that the deficient cytochrome b5 reductase activity in this patient is due to a defect in the biosynthesis of FAD, the coenzyme of the reductase, from riboflavin.
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PMID:Deficient cytochrome b5 reductase activity in nontoxic goiter with iodide organification defect. 116 26

Electron-transferring flavoprotein (ETF) and acyl dehydrogenases of pig liver mitochondria have been isolated in good yield by a new procedure. ETF and general acyl dehydrogenase appear homogenous, are free of reciprocal contamination, react with neither pyridine nucleotides not cytochrome c, and are completely dependent upon each other for reduction of dichlorophenol indophenol by acyl-CaA substrates. The properties of the present preparation (some of which differ significantly from those previously described) are presented. Sedimentation of ETF in 0.02 M KP-i yields a M-r for the native ETF of 58,00 plus or minus 3,000, whereas sedimentation of reduced and alkylated ETF in guanidine HCl yields a M-r of 26,000. Electrophoresis on sodium dodecyl sulfate gels in the presence or absence of mercaptoethanol gives a M-r of about 27,000 and flavin analysis gives a minimum molecular weight of about the same figure. Thus, ETF appears to contain one flavin (at least 90% FAD, by chromatographic and fluorescence characteristics) per 26,000 M-r, and therefore may be composed of two subunits with one flavin each. Sodium dodecyl sulfate gel electrophoresis of general acyl dehydrogenase in the absence of mercaptoethanol gives a band corresponding to a M-r of 84,000; in the presence of mercaptoethanol a band corresponding to a M-r of 42,000 is found. The minimum molecular weight based on flavin content is 40,500. These data considered in conjunction with previous reports from other laboratories, suggest a structure of four subunits per mol with one flavin per subunit..
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PMID:The purification and some properties of electron transfer flavoprotein and general fatty acyl coenzyme A dehydrogenase from pig liver mitochondria. 116 97

Significant dissociation of FMN from NADPH:cytochrome P-450 reductase resulted in loss of the activity for reduction of cytochrome b5 as well as cytochrome c and cytochrome P-450. However, the ability to reduce these electron acceptors was greatly restored upon incubation of FMN-depleted enzyme with added FMN. The reductions of cytochrome c and detergent-solubilized cytochrome b5 by NADPH:cytochrome P-450 reductase were greatly increased in the presence of high concentrations of KCl, although the stimulatory effect of the salt on cytochrome P-450 reduction was less significant. No apparent effect of superoxide dismutase could be seen on the rate or extent of cytochrome reduction in solutions containing high-salt concentrations. Complex formation of the flavoprotein with cytochrome c, which is known to be involved in the mechanism of non-physiological electron transfer, caused a perturbation in the absorption spectrum in the Soret-band region of cytochrome c, and its magnitude was enhanced by addition of KCl. Similarly, an appreciable increase in ellipticity in the Soret band of cytochrome c was observed upon binding with the flavoprotein. However, only small changes were found in absorption and circular dichroism spectra for the complex of NADPH:cytochrome P-450 reductase with either cytochrome b5 or cytochrome P-450. It is suggested that the high-salt concentration allows closer contact between the heme and flavin prosthetic groups through hydrophobic-hydrophobic interactions rather than electrostatic-charge pairing between the flavoprotein and the cytochrome which causes a faster rate of electron transfer. Neither alterations in the chemical shift nor in the line width of the bound FMN and FAD phosphate resonances were observed upon complex formation of NADPH:cytochrome P-450 reductase with the cytochrome.
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PMID:Effect of KCl on the interactions between NADPH:cytochrome P-450 reductase and either cytochrome c, cytochrome b5 or cytochrome P-450 in octyl glucoside micelles. 131 30

Several catalytic properties of the FAD enzyme cellobiose:quinone oxidoreductase (CBQ) and the heme/FAD enzyme, cellobiose oxidase (CBO) have been investigated and compared. Dichlorophenol-indophenol was found to be a very good electron acceptor for cellobiose oxidation by both enzymes. The optimal pH value for this oxidation with dichlorophenol-indophenol as a co-substrate was observed around pH 4 for both enzymes. The turnover numbers of this reaction were also very similar. The Km values for cellobiose oxidation were identical, whereas the Km for CBO with dichlorophenol-indophenol is lower than that of CBQ. Atmospheric oxygen is a very poor electron acceptor for both CBO and CBQ, however, CBO can utilize cytochrome c as an effective electron acceptor, while CBQ cannot. The specific activity of CBO for cytochrome c is thus about 200-times higher than for oxygen. Thus, one way to distinguish the two enzymes is by the cytochrome-c-reducing ability of CBO. Therefore, we propose that the nomenclature for CBO is tentatively changed to cellobiose:cytochrome c oxidoreductase until a rational name can be installed. Both enzymes have radical-reducing activities. The cation radical, derived from 1,2,4,5-tetramethoxybenzene, was reduced by both enzymes at almost the same reaction rate. The phenoxyradical produced by lignin peroxidase, catalyzing the oxidation of acetosyringon, was also reduced by both enzymes. The reduction of phenoxyradicals formed by phenoloxidases (lignin peroxidases, as well as laccases) may be important in preventing repolymerization reactions which we suggest would significantly facilitate lignin degradation.
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PMID:A comparison of the catalytic properties of cellobiose:quinone oxidoreductase and cellobiose oxidase from Phanerochaete chrysosporium. 132 Oct 38

Cellobiose oxidase (CBO) from Phanerochaete chrysosporium can utilize dichlorphenol-indophenol (Cl2Ind) and cytochrome c as effective electron acceptors for the oxidation of cellobiose. However, the pH dependencies of activity for these electron acceptors are significantly different. Both compounds act as effective electron acceptors at pH 4.2, whereas only dichlorophenol-indophenol is active at pH 5.9. To explain this discrepancy, the pH dependencies of the reduction rates of FAD and heme, respectively, in CBO by cellobiose have been investigated by stopped-flow spectrophotometry. Both FAD and heme are reduced with a high rate constant at pH 4.2. In contrast, at pH 5.9, only FAD reduction is fast, while the reduction of the heme is extremely slow. As a conclusion, the reduction of cytochrome c by CBO is dependent on heme, which functions at a lower pH range compared to reduction of FAD.
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PMID:Cellobiose oxidase from Phanerochaete chrysosporium. Stopped-flow spectrophotometric analysis of pH-dependent reduction. 132 33

The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> [2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.
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PMID:Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2. 132 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


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