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:1.6.99.1 (NADPH-diaphorase)
3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Old yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88,000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.
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PMID:Occurrence of old yellow enzyme in Gluconobacter suboxydans, and the cyclic regeneration of NADP. 4 38

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.
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PMID:Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16. 18 26

A dihydrodipicolinate reductase containing flavin was purified from sporulating Bacillus subtilis PCI 219. The purified enzyme appeared homogeneous by dise gel electrophoresis. Its molecular weight was estimated as 74,000 by gel filtration on Sephadex G-200, and as 18,500 by electrophoresis on sodium dodecylsulfate polyacrylamid gel. These results suggest that the enzyme is composed of four subunits. The prosthetic group was identified as FMN, and one mole of the enzyme contained two moles of FMN. Both NADPH and NADH acted as coenzyme, though NADH was less effective. The enzyme also exhibited diaphorase activity. The pH optimum was 6.1. The enzyme was inhibited by dipicolinate but not by lysine or alpha, epsilon-diaminopimelate.
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PMID:A new flavin enzyme catalyzing the reduction of dihydrodipicolinate in sporulating Bacillus subtilis I. Purification and properties. 23 91

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities. 137 27

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).
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PMID:The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT). 147 94

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.
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PMID:NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst. 184 86

The brewer's yeast old yellow enzyme (OYE) was reconstituted with 8-fluoro-8-demethyl FMN (8F-FMN). The reconstituted enzyme exhibited absorption maxima at 355 and 450 nm in the visible region. This reconstituted enzyme underwent no further spectral changes, showing no evidence of modification in the flavin moiety. However, when the reconstituted enzyme was subjected to specific limited proteolysis with bovine alpha-chymotrypsin, gradual spectral changes were observed with disappearance of the 355- and 450-nm bands accompanied by the appearance of a new band at 496 nm. Identical spectral changes were observed when the proteolytically cleaved OYE (nicked OYE) was reconstituted with 8F-FMN. The process associated with these spectral changes was found to be unimolecular by kinetic analysis. Reverse-phase HPLC analysis revealed that these spectral changes resulted from covalent bond formation between 8F-FMN and the protein moiety after the proteolytic cleavage of the protein into 14K and 34K fragments. The reverse-phase HPLC monitored at 490 nm showed that the chromophore with 496 nm absorption maximum was covalently attached to the 14K fragment. The amino acid sequence analysis of the flavinylated 14K fragment together with that of the 14K fragment of native OYE indicated that the N-terminal leucine of the 14K fragment is the site of flavinylation. These findings imply that the amino group of the N-terminal leucine of the 14K fragment became available as the result of proteolysis and that this amino group nucleophilically attacked the 8-position of 8F-FMN, forming a covalent bond between the flavin moiety and the 14K fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies on the flavin-binding region of old yellow enzyme with an active site probe, 8-fluoro-8-demethyl FMN. 201 74

The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH-ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH-cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin-NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.
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PMID:Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin. 250 11

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.
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PMID:Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. 255 Apr 23

The 31P- and 13C-NMR spectra of old yellow enzyme (OYE) were measured. The 31P-NMR signal of FMN bound to apo OYE-I, one of the pure forms of OYE, was observed at a substantially lower field compared to that of free FMN. While the 31P-signal of free FMN is pH-titratable with a pK value of about 6.5, which corresponds to the monoanion-dianion transition of the phosphate group, the 31P-signal of FMN bound to OYE-I shows no pH-dependence at pH 5-9, indicating that the phosphate group of FMN bound to OYE-I is fixed in the dianionic form in the pH region of 5-9. Apo OYE(0), i.e., the OYE preparation obtained by the conventional method, was reconstituted with [2-13C]FMN or [4,10a-13C2]FMN, while apo OYE-I was reconstituted with [4a-13C]FMN. The 13C-NMR spectra of these reconstituted OYE species were measured in the absence and presence of phenolic compounds which form complexes with OYE. Each 13C-signal of the 13C-labeled FMN became broader in the bound state compared to the free state, indicating restriction of flavin mobility in the bound form. Complex formation of the reconstituted OYE species with p-bromophenol did not shift the 10a-13C signal but shifted the 2- and 4-13C signals slightly upfield, whereas the 4a-13C signal was shifted significantly upfield in the complexed form. This complex-induced upfield shift of the 4a-13C signal was measured with various p-substituted phenols.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:31P- and 13C-NMR studies on the flavin-protein and flavin-ligand interactions in brewer's yeast old yellow enzyme. 287 11


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