Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purification and partial characterisation of an NADP(H) dependent artificial mediator accepting pyridine nucleotide oxidoreductase (AMAPOR) from the anaerobic Clostridium thermoaceticum is described. Depending on the redox potential of the artificial mediators the AMAPOR is able to regenerate NADP+ or NADPH rendering the enzyme useful for preparative work applying NADP(H) dependent oxidoreductases. At 37 degrees C crude extracts of C. thermoaceticum have an AMAPOR activity of 5-7 U mg(-1). This is 28 degrees under the optimal growth temperature of this microrganism. Out of apparently more than 10 AMAPOR active proteins in the crude cell extracts visible after electrophoresis and activity staining on the gel, two of these proteins were isolated. They seem to be two different oligomers. According to gel electrophoresis they show apparent molecular masses of about 200 and 400 kDa. These two forms showed after SDS gel electrophoresis two monomers with apparent molecular masses of 42 and 56 kDa which we call alpha and beta. The two oligomers may have the compositions alpha2beta2 and alpha4beta4. They contain Fe/S cluster and FAD. Various amounts of the FAD were lost during the purification procedure. This loss is partially reversible after addition of FAD. The AMAPOR reacts with rather different artificial mediators such as viologens, quinones e.g. 1,4-benzoquinone or anthraquinone-2,6-disulphonate, 2,6-dichloro-indophenol and clostridial rubredoxin. Two different ferredoxins from C. thermoaceticum, oxygen or lipoamide are no substrates indicating the here described AMAPOR is not a diaphorase in the usual sense.
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PMID:On a new artificial mediator accepting NADP(H) oxidoreductase from Clostridium thermoaceticum. 1105 22

Cytochrome b(5) reductase (cb5r) catalyzes the transfer of reducing equivalents from NADH to cytochrome b(5). Utilizing an efficient heterologous expression system that produces a histidine-tagged form of the hydrophilic, diaphorase domain of the enzyme, site-directed mutagenesis has been used to generate cb5r mutants with substitutions at position 91 in the primary sequence. Arginine 91 is an important residue in binding the FAD prosthetic group and part of a conserved "RxY(T)(S)xx(S)(N)" sequence motif that is omnipresent in the "ferredoxin:NADP(+) reductase" family of flavoproteins. Arginine 91 was replaced with K, L, A, P, D, Q, and H residues, respectively, and all the mutant proteins purified to homogeneity. Individual mutants were expressed with variable efficiency and all exhibited molecular masses of approximately 32 kDa. With the exception of R91H, all the mutants retained visible absorption spectra typical of a flavoprotein, the former being produced as an apoprotein. Visible absorption spectra of R91A, L, and P were red shifted with maxima at 458 nm, while CD spectra indicated an altered FAD environment for all the mutants except R91K. Fluorescence spectra showed a reduced degree of intrinsic flavin fluorescence quenching for the R91K, A, and P, mutants, while thermal stability studies suggested all the mutants, except R91K, were somewhat less stable than the wild-type domain. Initial-rate kinetic measurements demonstrated that the mutants exhibited decreased NADH:ferricyanide reductase activity with the R91P mutant retaining the lowest activity, corresponding to a k(cat) of 283 s(-1) and a K(NADH)(m) of 105 microM, when compared to the wild-type domain (k(cat) = 800 s(-1) K(NADH)(m) = 6 microM). These results demonstrate that R91 is not essential for FAD binding in cb5r; however, mutation of R91 perturbs the flavin environment and alters both diaphorase substrate recognition and utilization.
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PMID:Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b(5) reductase. 1133 12

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.
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PMID:Succinate dehydrogenase and other respiratory pathways in thylakoid membranes of Synechocystis sp. strain PCC 6803: capacity comparisons and physiological function. 1141 66

A calcium and NAD(P)H-dependent H(2)O(2)-generating activity has been studied in paranodular thyroid tissues from four patients with cold thyroid nodules and from nine diffuse toxic goiters. H(2)O(2) generation was detected both in the particulate (P 3,000 g) and in the microsomal (P 100,000 g) fractions of paranodular tissue surrounding cold thyroid nodules (PN), with the same biochemical properties described for NADPH oxidase found in porcine and human thyroids. In PN tissues, the particulate NADPH oxidase activity (224 +/- 38 nmol H(2)O(2) x h(-1) x mg(-1) protein) was similar to that described for the porcine thyroid enzyme. However, no NADPH oxidase activity was detectable in the particulate fractions from eight diffuse toxic goiter patients treated with iodine before surgery; all but one also received propylthiouracil or methimazole in the preoperative period. Thyroid cytochrome c reductase (diffuse toxic goiters = 438 +/- 104 nmol NADP(+) x h(-1) x mg(-1) protein; PN = 78 +/- 10 nmol NADP(+) x h(-1) x mg(-1) protein) and thyroperoxidase (diffuse toxic goiters = 621 +/- 179 U x g(-1) protein; PN = 232 +/- 121 U x g(-1) protein) activities were unaffected by iodide. Thus, the human NADPH oxidase seems to be inhibited by iodinated compounds in vivo and probably is an enzyme involved in the Wolff-Chaikoff effect. Our findings reinforce the hypothesis that thyroid NADPH oxidase is responsible for the production of H(2)O(2) necessary for thyroid hormone biosynthesis.
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PMID:Ca(2+)/nicotinamide adenine dinucleotide phosphate-dependent H(2)O(2) generation is inhibited by iodide in human thyroids. 1154 71

We report herein the study of two siblings (DESM and DSM) with hypothyroidism, goiter, and positive perchlorate discharge tests (50% and 70%) in a family (M) with no history of consanguinity. Thyroid gland histology showed a predominance of hyperactive follicles, with high epithelial cells and variable colloid content. Thyroid peroxidase iodide oxidation (DESM, 1034; DSM, 1064 U/g protein) and albumin iodination (DESM, 16; DSM, 8 nmol I/mg protein) activities were within the normal range. Tg content was normal in both glands compared with that in diffuse toxic goiter (DESM, 28; DSM, 17; diffuse toxic goiter, 19 mg/g tissue), and Tg could be normally iodinated by thyroid peroxidase in vitro (DESM, 3.4; DSM, 4.3; diffuse toxic goiter, 6.3 nmol I/mg Tg). Thyroid cytochrome c reductase activities in these goiters were higher than that in paranodular tissues (DESM, 473; DSM, 567; paranodular tissues, 78 nmol NADP(+)/h/mg protein). However, thyroid NADPH oxidase activities were very low both in the particulate 3,000 x g (DESM, 4.8; DSM, 44; paranodular tissues, 224 nmol H(2)O(2)/h/mg protein) and in the particulate 100,000 x g fractions (DESM, 40; DSM, 47; paranodular tissues, 200 nmol H(2)O(2)/h/mg protein). Thus, a decreased Ca(2+)/NAD(P)H-dependent H(2)O(2) generation is the probable cause of the organification defect in these goiters.
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PMID:Goiter and hypothyroidism in two siblings due to impaired Ca(+2)/NAD(P)H-dependent H(2)O(2)-generating activity. 1160 May 51

Ferredoxin-NAD(P)(+) reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP(+) and NAD(+). When concentrations of NADP(+) exceeded 10 microM, NADP(+) photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD(+). It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.
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PMID:Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum. 1200 11

Glucose metabolism of bifidobacteria in the presence of 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), a specific growth stimulator for bifidobacteria, and ferricyanide (Fe(CN)(6)(3-)) as an extracellular electron acceptor was examined using resting cells of Bifidobacterium longum and Bifidobacterium breve. NAD(P)H in the cells is oxidized by ACNQ with the aid of diaphorase activity, and reduced ACNQ donates the electron to Fe(CN)(6)(3-). Exogenous oxidation of NADH by the ACNQ/Fe(CN)(6)(3-) system suppresses the endogenous lactate dehydrogenase reaction competitively, which results in the remarkable generation of pyruvate and a decrease in lactate production. In addition, a decrease in acetate generation is also observed in the presence of ACNQ and Fe(CN)(6)(3-). This phenomenon could not be explained in terms of the fructose-6-phosphate phosphoketolase pathway, but suggests rather that glucose is partially metabolized via the hexose monophosphate pathway. This was verified by NADP(+)-induced reduction of Fe(CN)(6)(3-) in cell-free extracts in the presence of ACNQ. Effects of the ACNQ/Fe(CN)(6)(3-) system on anaerobically harvested cells were also examined. Stoichiometric analysis of the metabolites from the pyruvate-formate lyase pathway suggests that exogenous oxidation of NADH is an efficient method to produce ATP in this pathway.
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PMID:2-Amino-3-carboxy-1,4-naphthoquinone affects the end-product profile of bifidobacteria through the mediated oxidation of NAD(P)H. 1207 35

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel nucleotide derived from NADP that has now been shown to be active in releasing Ca(2+) from intracellular stores in a wide variety of cells ranging from plant to human. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, no assay has been reported for NAADP to date. In the present study, a widely applicable assay for NAADP with high sensitivity is described. NAADP was first dephosphorylated to nicotinic acid-adenine dinucleotide by treatment with alkaline phosphatase. The conversion was shown to be stoichiometric. NMN-adenylyltransferase was then used to convert nicotinic acid-adenine dinucleotide into NAD in the presence of high concentrations of NMN. The resultant NAD was amplified by a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD cycled through these coupled reactions, a molecule of highly fluorescent resorufin was generated. The reaction could be performed for hours, resulting in more than a 1000-fold amplification. Concentrations of NAADP over the 10-20 nM range could be routinely measured. This novel cycling assay was combined with an enzymic treatment to provide the necessary specificity for the assay. NAADP was found to be resistant to NADase and apyrase. Pretreatment of samples with a combination of the hydrolytic enzymes completely eliminated the interference from common nucleotides. The versatility of the cycling assay can also be extended to measure nicotinic acid, which is a substrate in the synthesis of NAADP catalysed by ADP-ribosyl cyclase, over the micromolar range. All the necessary reagents for the cycling assay are widely available and it can be performed using a multi-well fluorescence plate reader, providing a high-throughput method. This is the first assay reported for NAADP and nicotinic acid, which should be valuable in elucidating the messenger functions of NAADP.
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PMID:A novel cycling assay for nicotinic acid-adenine dinucleotide phosphate with nanomolar sensitivity. 1211 13

Ferredoxin NADP(H) oxidoreductases (FNR) are flavoenzymes that catalyze the electron transfer between NADP(H) and a wide range of compounds including ferredoxins and bacterial flavodoxins. FNRs are classified into two major groups: plant- and vertebrate-type. Plant-type FNRs are implicated in photosynthesis and nitrogen fixation in plastids and photosynthetic bacteria, and were recently implicated in cell protection against reactive oxygen species (ROS). Vertebrate-type FNRs are mitochondrial enzymes implicated in steroid hormone biosynthesis in mammals and in Fe(+) uptake and metabolism in yeasts. We have cloned and sequenced a cDNA coding for the vertebrate-type Schistosoma mansoni FNR. Gel diaphorase activity and western blot assays demonstrated that SmFNR represented the major diaphorase activity of adult worms. An active recombinant SmFNR was expressed in Escherichia coli that made the bacteria tolerant to oxygen peroxide, cumene hydroperoxide and the superoxide-generating herbicide, methyl viologen (MV).
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PMID:Schistosoma mansoni ferredoxin NADP(H) oxidoreductase and its role in detoxification. 1238 48

Microsomal cytochrome b(5) reductase (EC 1.6.2.2) catalyzes the reduction of ferricytochrome b(5) using NADH as the physiological electron donor. Site-directed mutagenesis has been used to engineer the soluble rat cytochrome b(5) reductase diaphorase domain to utilize NADPH as the preferred electron donor. Single and double mutations at residues D239 and F251 were made in a recombinant expression system that corresponded to D239E, S and T, F251R, and Y, D239S/F251R, D239S/F251Y, and D239T/F251R, respectively. Steady-state turnover measurements indicated that D239S/F251Y was bispecific while D239T, D239S/F251R, and D239T/F251R were each NADPH-specific. Wild-type (WT) cytochrome b(5) reductase showed a 3700-fold preference for NADH whereas the mutant with the highest NADPH efficiency, D239T, showed an 11-fold preference for NADPH, a 39200-fold increase. Wild-type cytochrome b(5) reductase only formed a stable charge-transfer complex with NADH while D239T formed complexes with both NADH and NADPH. The rates of hydride ion transfer, determined by stopped-flow kinetics, were k(NADH-WT) = 130 s(-1), k(NADPH-WT) = 5 s(-1), k(NADH-D239T) = 180 s(-1), and k(NADPH-D239T) = 73 s(-1). K(s) determinations by differential spectroscopy demonstrated that D239T could bind nonreducing pyridine nucleotides with a phosphate or a hydroxyl substituent at the 2' position, whereas wild-type cytochrome b(5) reductase would only bind 2' hydroxylated molecules. Oxidation-reduction potentials (E degrees ', n = 2) for the flavin cofactor were WT = -268 mV, D239T = -272 mV, WT+NAD(+) = -190 mV, D239T+NAD(+) = -206 mV, WT+NADP(+) = -253 mV, and D239T+NADP(+) = -215 mV, which demonstrated the thermodynamic contribution of NADP(+) binding to D239T. The crystal structures of D239T and D239T in complex with NAD(+) indicated that the loss of the negative electrostatic surface that precluded 2' phosphate binding in the wild-type enzyme was primarily responsible for the observed improvement in the use of NADPH by the D239T mutant.
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PMID:Engineering and characterization of a NADPH-utilizing cytochrome b5 reductase. 1450 67


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