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

The respiratory chain of a marine Vibrio alginolyticus contains two types of NADH-quinone reductase (NQR): one is an Na(+)-dependent NQR functioning as an Na+ pump (NQR-1) and the other is an Na(+)-independent NQR (NQR-2). NQR-2 was purified about 55-fold from the membrane of mutant Nap-1 which is devoid of NQR-1, and its properties were compared with those of NQR-1. In contrast to NQR-1, the purified NQR-2 does not require any salts for activity and is not inhibited by up to 0.4 M salts. The optimum pH of NQR-2 is between 6.8 and 7.8, which is about 0.7 ph units lower than that of NQR-1. NQR-2 is insensitive to strong inhibitors of NQR-1 such as p-chloromercuribenzoate, Ag+ and 2-heptyl-4-hydroxyquinoline N-oxide. Using inverted membrane vesicles, it was confirmed that NQR-2 has no capacity to generate a membrane potential. NQR-2 reduces menadione and ubiquinone-1 by a two-electron reduction pathway. Since the NADH-reacting FAD-containing beta-subunit of NQR-1 reduces quinones by a one-electron reduction pathway, the mode of quinone reduction is closely related to energy coupling; the formation of semiquinone radicals as an intermediate is likely to be essential to functioning as an ion pump.
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PMID:Properties of respiratory chain-linked Na(+)-independent NADH-quinone reductase in a marine Vibrio alginolyticus. 154 99

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

It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction. The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM. The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.
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PMID:Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli. 211 86

NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed enzyme which promotes two-electron reductions of quinones and thereby protects cells against damage by reactive oxygen species generated during oxidative cycling of quinones and semiquinone radicals. Quinone reductase activity represents a minor component (about 0.006%) of mouse liver cytosolic proteins under basal (uninduced) conditions. Two isofunctional forms of this quinone reductase have been purified to homogeneity (1700-fold) in 30% yield from the liver cytosols of female CD-1 mice in which the enzymes were induced by administration of 2(3)-tert-butyl-4-hydroxyanisole. The purification involved ion exchange, hydrophobic, and affinity chromatographies. The two enzyme forms have been designated "hydrophilic" and "hydrophobic" based on the order of elution from phenyl-Sepharose. The more abundant hydrophilic form has been crystallized in the presence of FAD in the form of macroscopic tetragonal crystals. The two forms have similar isoelectric points (pI 9.2) and subunit molecular weights (Mr = 30,000) and probably exist as dimers in the native state. Purified preparations of the enzymes are equiactive with NADH and NADPH and show almost complete dependence on added FAD for catalytic activity. The Km values for FAD of the hydrophilic and hydrophobic forms are 2.72 and 1.72 nM, respectively. Their catalytic activities are the same and are remarkably high for nicotinamide nucleotide-linked dehydrogenases; maximum velocities (expressed per mg of pure enzyme) approach 4000 units/mg of protein under appropriate assay conditions. When menadione is the electron acceptor, the Km value for this quinone is very low (Km congruent to 2 microM). Both enzyme forms are potently inhibited by dicoumarol. Rabbit antisera against the hydrophilic quinone reductase precipitate quantitatively the entire quinone reductase activity of mouse liver cytosols obtained from animals maintained on a standard diet or those induced with 3-tert-butyl-4-hydroxyanisole. The quinone reductase activity of rat liver cytosols is also quantitatively precipitated by this antiserum.
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PMID:Purification and characterization of two isofunctional forms of NAD(P)H: quinone reductase from mouse liver. 241 14

NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed dicoumarol-inhibitable FAD-containing protein that catalyzes the obligatory two-electron reduction of quinones. The enzyme plays an important role in protecting animal cells against quinone toxicity and may be involved in the vitamin K-dependent blood coagulation cascade. Cocrystallization of rat liver quinone reductase with Cibacron blue, a potent inhibitor with respect to NAD(P)H, was achieved by the method of vapor diffusion in the presence of ammonium sulfate and low concentrations of polyethylene glycol. X-ray diffraction analysis showed these blue crystalline platelets to be monoclinic and to belong to the space group P2(1) (a = 71.6 A, b = 107.1 A, c = 87.8 A and beta = 92.60 degrees) with two dimers in the asymmetric unit. The crystals diffract to a resolution of at least 2.8 A.
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PMID:X-ray diffraction analyses of crystals of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase containing cibacron blue. 249 4

The respiratory chain-linked NADH-quinone reductase (NQR) and NADH-ferricyanide dehydrogenase (NFD) were extracted from membranes of Escherichia coli by n-dodecyl octaethyleneglycol monoether detergent and purified by DEAE-Sephacel, DEAE-5PW and Bio-Gel HTP column chromatography. The purified NQR contained FAD as a cofactor, catalyzed the reduction of ubiquinone-1 (Q1) and reacted with NADH, but not with deamino-NADH (d-NADH), with an apparent Km of 48 microM. On the other hand, the purified NFD contained FMN as a cofactor, reacted with both NADH and d-NADH, and catalyzed the reduction of ferricyanide but not Q1. NFD showed a high affinity for both NADH and d-NADH with a Km of 7-9 microM. NFD was inactivated, whereas NQR was rather activated, by preincubation with an electron donor in the absence of electron acceptor. These properties were compared with those of activities observed with inverted membrane vesicles with special reference to the generation of inside-positive membrane potential (delta psi). It was found that d-NADH-reactive FMN-containing NFD is a dehydrogenase part of energy-generating NADH-quinone reductase complex. The FAD-containing NQR was very similar to that purified by Jaworowski et al. (Biochemistry (1981) 20, 2041-2047), and reduced Q1 without generating delta psi.
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PMID:Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain. 267 83

The respiratory chain of a marine bacterium, Vibrio alginolyticus, required Na+ for maximum activity, and the site of Na+ -dependent activation was localized on the NADH-quinone reductase segment. The Na+ -dependent NADH-quinone reductase extruded Na+ as a direct result of redox reaction. It was composed of three subunits, alpha, beta, and gamma, with apparent Mr of 52, 46, and 32 KDa, respectively. The reduction of ubiquinone-1 to ubiquinol proceeded via ubisemiquinone radicals. The former reaction was catalyzed by the FAD-containing beta subunit. This reaction showed no specific requirement for Na+. For the formation of ubiquinol, the presence of the gamma subunit and the FMN-containing alpha subunit was essential. The latter reaction specifically required Na+ for activity and was strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide. It was assigned to the coupling site for Na+ transport. The mode of energy coupling of redox-driven Na+ pump was compared with those of decarboxylase- and ATP-driven Na+ pumps found in other bacteria.
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PMID:Sodium-transport NADH-quinone reductase of a marine Vibrio alginolyticus. 268 59

Cytosolic NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed, FAD-containing enzyme that catalyzes the obligatory two-electron reduction of quinones. Cibacron Blue is an inhibitor of this enzyme comparable in potency to dicoumarol. Pure quinone reductase was obtained from the livers of Sudan II (1-[2,4-dimethylphenylazo]-2-naphthol)-treated rats in a single step by Cibacron Blue-agarose chromatography. Cibacron Blue is a competitive inhibitor with respect to NADH (Ki = 170 nM) and is a noncompetitive inhibitor with respect to menadione (Ki = 540 nM). Addition of Cibacron Blue to quinone reductase resulted in a decrease and red shift of the enzyme-bound FAD peak at 450 nm. The titration of the absorbance changes for both FAD and Cibacron Blue could be fitted to curves describing an equilibrium binding equation with a KD of 300 nM and one binding site per enzyme subunit. Furthermore, the Cibacron Blue difference spectrum that resulted from binding to quinone reductase was abolished by dicoumarol. Significant amino acid homology between quinone reductase and the nucleotide binding regions of enzymes that bind to Cibacron Blue was found. These data indicate that Cibacron Blue is a useful ligand for the purification of quinone reductase and a new probe for its NAD(P)H binding site. Conditions for crystallizing rat liver quinone reductase are also described.
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PMID:Purification and crystallization of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue affinity chromatography: identification of a new and potent inhibitor. 321 67

Two types of the NADH-quinone reductase were isolated from Thermus thermophilus HB-8 membranes, by use of the nonionic detergent, dodecyl beta-maltoside, and NAD-agarose affinity, DEAE-cellulose, hydroxyapatite, and Superose 6 column chromatography. One of these (NADH dehydrogenase 1) is a complex composed of 10 unlike polypeptides, and the other (NADH dehydrogenase 2) exhibits a single band (Mr 53,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase 1 was about 14 times higher than that of the dodecyl beta-maltoside extract and partially rotenone sensitive. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase 2 was about 30-fold as high as that of the dodecyl beta-maltoside extract and rotenone insensitive. The purified NADH dehydrogenase 1 contained noncovalently bound FMN, non-heme iron, and acid-labile sulfide. The ratio of FMN to non-heme iron to acid-labile sulfide was 1:11-12:7-9. The high content of iron and labile sulfide is suggestive of the presence of several iron-sulfur clusters. The purified NADH dehydrogenase 2 contained noncovalently bound FAD and no non-heme iron or acid-labile sulfide. The activities of both NADH dehydrogenases were stable at temperatures of greater than or equal to 80 degrees C. The occurrence of two distinct types of NADH dehydrogenase as a common feature in the membranes of various aerobic bacteria is discussed.
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PMID:Purification and characterization of two types of NADH-quinone reductase from Thermus thermophilus HB-8. 337 42

NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.
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PMID:A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase. 751 54


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