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: EC:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be -420 mV and -285 mV, respectively. The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr, of 64000 and 31000, contained 9.9 +/- 0.4 S2- and 9.3 +/- 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity). The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400-nm region and a characteristic ESR signal at g = 2.01. The reduced form of the dimer was ESR-silent. The small dimer alone was diaphorase-inactive and did not reduce NAD with H2, but it displayed high H2-uptake activities with viologen dyes, methylene blue and FMN, and H2-evolving activity with reduced methyl viologen. Hydrogen-dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the ESR spectrum in the oxidized state.
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PMID:Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b. 608 43

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.
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PMID:Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties. 640 48

A bioluminescent assay for 12-alpha-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-alpha-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPH:FMN oxidoreductase or bacterial diaphorase. The assay was specific for 12-alpha-hydroxy bile acids and the lower limit of detection was 4 pmol/0.5 ml assay volume with a linear range of 4 to 2000 pmol. Intraassay precision was from 7.8 to 8.2%. Values obtained with this assay showed good agreement with those obtained by gas-liquid chromatography. The system using diaphorase was not stable at 4 degrees C in the absence of added thiol compounds, but could be stabilized by the addition of glutathione (0.5 mM). The assay is a convenient, a rapid, and an extremely sensitive method for the measurement of 12-alpha-hydroxy bile acid concentrations in the serum of patients or experimental animals.
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PMID:A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes. 657 65

A Japanese family with congenital methaemoglobinaemia is described. The family pedigree was compatible with autosomal recessive type of inheritance. The increased methaemoglobin concentration was ascribed to the red cell NADH diaphorase deficiency associated with the almost complete lack of one of the two peaks of the diaphorase activity as separated by DEAE Sephadex column chromatography. The NADH diaphorase and NADH methaemoglobin reductase deficiency was limited to the red cells. The methaemoglobin content in the blood of the propositus was 17.8% and isoelectric focusing analysis on a polyacrylamide gel plate showed that the haemoglobin consisted of 65.2% oxyhaemoglobin (alpha 2+ beta 2+)2, 29.6% half-oxidized forms, 20.9% (alpha 3+ beta 2+)2 and 8.7% (alpha 2+ beta 3+)2, and 3% full-oxidized methaemoglobin (alpha 3+ beta 3+)2. Oral administration of riboflavin 120 mg/d resulted in a gradual but significant decrease in the level of the met-form haemoglobins in parallel with a gradual increase in the red cell flavin content. Riboflavin is considered to be effective by activating the NADPH diaphorase (NADPH flavin reductase) system and appears to be useful for the treatment of congenital methaemoglobinaemia.
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PMID:Congenital methaemoglobinaemia due to NADH methaemoglobin reductase deficiency: successful treatment with oral riboflavin. 689 37

D-Lactate dehydrogenase, the starting enzyme for carbon and energy metabolism in dissimilatory sulfate-reducing bacteria, has been purified 36-fold from the soluble fraction of the sonicate of Desulfovibrio vulgaris, Miyazaki. The enzyme is specific for D-lactate (Km = 0.8 mM) and DL-2-hydroxybutyrate (probably its D-isomer) as the electron donor substrate. It reduces, in the presence of lactate, various artificial electron acceptors such as 1-methoxyphenazinium methyl sulfate, ferricyanide, tetrazolium dyes, methylene blue, and 2,6-dichlorophenol-indophenol. When 2 mol of ferricyanide was reduced, 1 mol of pyruvate was produced during the reaction. Among natural electron carriers, only cytochrome c-553 isolated from the same organism can be reduced by the enzyme. The ferric complex of pyridine-2,6-dicarboxylate can act as an electron acceptor if cytochrome c-553 is present in the reaction system. NAD+, NADP+, FAD, FMN, cytochrome c3, high-molecular-weight cytochrome, eucaryotic cytochromes c (yeast and horse) and O2 could not be reduced. The enzyme does not have any diaphorase activity. The D-lactate dehydrogenase of D. vulgaris must therefore be named D-lactate:ferricytochrome c-553 oxidoreductase [EC subclass 1.1.2]. A similar enzyme exists in the formate dehydrogenase-less mutant of D. vulgaris, Miyazaki, and in D. vulgaris, Hildenborough.
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PMID:D-lactate dehydrogenase of Desulfovibrio vulgaris. 727 46

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.
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PMID:The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction. 756 29

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.
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PMID:Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH. 1152 20

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.
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PMID:Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects. 1160 35

The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing [NiFe] hydrogenase (SH). This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities. The hydrogenase moiety mediates H(2):benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity. The SH of Rh. opacus resembles [NiFe] hydrogenases present in strains of the proteobacterium Ralstonia eutropha and in species of cyanobacteria. Heterologous expression of active [NiFe] hydrogenases failed in most cases due to protein-assisted maturation processes implicated in the assembly of the NiFe bimetallic site. This study reports on the construction of a recombinant plasmid harboring the four SH subunit genes hoxFUYH and the associated endopeptidase gene hoxW from Rh. opacus under the regime of the SH promoter from R. eutropha H16. The resulting recombinant plasmid restored lithoautotrophic growth in a R. eutropha mutant impaired in H(2)-oxidizing ability. The SH of Rh. opacus was functionally active in R. eutropha and displayed the typical features described for its natural host. It readily dissociated in vitro into two active subforms. Dissociation was accompanied by the loss of the H(2)-dependent NAD-reducing activity, which was partially reconstituted by addition of 5 mM MgSO(4) and 0.5 mM NiCl(2). Activity and stability of the SH from Rh. opacus were enhanced almost three-fold by co-overexpression of the SH-associated metal insertion genes hypA2B2F2 of R. eutropha. Under optimal conditions the heterologously expressed Rh. opacus SH catalyzed NAD-reduction at a specific activity of 1.7 units per mg protein, which is approximately 30% of the yield obtained for the R. eutropha SH. The results indicate that, despite an enormous phylogenetic distance of the two bacterial species, their SH proteins are highly related.
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PMID:Expression of a functional NAD-reducing [NiFe] hydrogenase from the gram-positive Rhodococcus opacus in the gram-negative Ralstonia eutropha. 1180 65

Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow-greenish enzyme complex [(alpha2betagamma)4; molecular mass 600 +/- 50 kDa] composed of a propionyl-CoA dehydrogenase (alpha2, 2 x 40 kDa) and an electron-transferring flavoprotein (ETF; beta, 38 kDa; gamma, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per alpha2betagamma subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 +/- 1 microm; kcat = 4.5 s-1 at 100 microm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 microm; kcat = 29 s-1 at 300 microm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 microm; kcat = 2.0 s-1) or butyryl-CoA (Km = 100 microm; kcat = 3.5 s-1) as electron donor and 200 microm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the beta and gamma subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.
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PMID:Acryloyl-CoA reductase from Clostridium propionicum. An enzyme complex of propionyl-CoA dehydrogenase and electron-transferring flavoprotein. 1260 23


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