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:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A clone that transforms the RKa mutant of Synechocystis PCC6803 defective in inorganic carbon (Ci) transport to the wild-type phenotype was isolated from a cyanobacterial genomic library. The clone contained an 11.8-kilobase-pair DNA insert. Sequencing of the insert DNA in the region of the mutation in RKa revealed an open reading frame (designated as ndhB), which showed extensive amino acid sequence homology to the subunit-2 genes of NADH dehydrogenase (EC 1.6.99.3) (ndhB) of chloroplasts and mitochondria. The homology was much stronger with the chloroplast genes. Sequence analysis of the ndhB gene of RKa mutant revealed a G----A substitution that results in a Gly----Asp substitution in the deduced amino acid. A defined mutant (M55), constructed by inactivating the ndhB gene in wild-type Synechocystis, required high CO2 conditions for growth and was unable to transport CO2 and HCO3- into the intracellular Ci pool. The results indicate that the ndhB gene is required for Ci transport. Dark respiration was also depressed by the inactivation of the ndhB gene. A possible role of the ndhB gene product in the energization of Ci transport is discussed.
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PMID:A gene homologous to the subunit-2 gene of NADH dehydrogenase is essential to inorganic carbon transport of Synechocystis PCC6803. 190 37

Two cDNA clones encoding bovine heart mitochondrial Rieske iron-sulfur protein were obtained by immunological screening of a bovine heart cDNA expression library in lambda gt11 with antiserum directed against Rieske iron-sulfur protein isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase. The cDNA inserts were 1005 and 1100 base pairs with an open reading frame of 807 base pairs which encoded a 196-amino acid mature Rieske iron-sulfur protein and a 73-amino acid presequence. The amino acid sequence of Rieske iron-sulfur protein deduced from nucleotide sequencing is the same as that obtained from protein sequencing except at residues #73 and #191 which are Ser and Asp instead of Ala and Gly, respectively.
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PMID:Cloning and sequencing of a cDNA encoding the Rieske iron-sulfur protein of bovine heart mitochondrial ubiquinol-cytochrome c reductase. 215 9

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) was purified to homogeneity from 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinol+ ++- labeled bovine heart mitochondrial ubiquinol-cytochrome c reductase. The N-terminal amino acid sequence of the isolated protein is Gly-Arg-Gln-Phe-Gly-His-Leu-Thr-Arg-Val-Arg-His-, which is identical with that of a Mr = 9500 protein in the reductase [Borchart et al. (1986) FEBS Lett. 200, 81-86]. A ubiquinone-binding peptide was prepared from [3H]azidoubiquinol-labeled QPc-9.5 kDa protein by trypsin digestion followed by HPLC separation. The partial N-terminal amino acid sequence of this peptide, Val-Ala-Pro-Pro-Phe-Val-Ala-Phe-Tyr-Leu-, corresponds to amino acid residues 48-57 in the reported Mr = 9500 protein. According to the proposed structural model for the Mr = 9500 protein, the azido-Q-labeled peptide is located in the membrane on the matrix side. These results confirm our previous assessment that the Mr = 13,400 subunit is not the small molecular weight Q-binding protein. Purified antibodies against QPc-9.5 kDa have a high titer with isolated QPc-9.5 kDa protein and complexes that contain it. Although antibodies against QPc-9.5 kDa do not inhibit intact succinate- and ubiquinol-cytochrome c reductases, a decrease of 85% and 20% in restoration of succinate- and ubiquinol-cytochrome c reductases, respectively, is observed when delipidated succinate- or ubiquinol-cytochrome reductases are incubated with antibodies prior to reconstitution with ubiquinone and phospholipid, indicating that epitopes at the catalytic site of QPc-9.5 kDa are buried in the phospholipid environment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) in mitochondrial ubiquinol-cytochrome c reductase: isolation, ubiquinone-binding domain, and immunoinhibition. 216 42

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

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.
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PMID:Identification of the domains of neuronal nitric oxide synthase by limited proteolysis. 866 Mar 10

Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.
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PMID:Thiol modification and site directed mutagenesis of the flavin domain of spinach NADH:nitrate reductase. 866 Jun 90

Transfer of reducing equivalents from NADPH to the cytochromes P450 is mediated by NADPH-cytochrome P450 oxidoreductase, which contains stoichiometric amounts of tightly bound FMN and FAD. Hydrogen bonding and van der Waals interactions between FAD and amino acid residues in the FAD binding site of the reductase serve to regulate both flavin binding and reactivity. The precise orientation of key residues (Arg(454), Tyr(456), Cys(472), Gly(488), Thr(491), and Trp(677)) has been defined by x-ray crystallography (Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S., Kim, J.-J. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8411-8416). The current study examines the relative contributions of these residues to FAD binding and catalysis by site-directed mutagenesis and kinetic analysis. Mutation of either Tyr(456), which makes van der Waals contact with the FAD isoalloxazine ring and also hydrogen-bonds to the ribityl 4'-hydroxyl, or Arg(454), which bonds to the FAD pyrophosphate, decreases the affinity for FAD 8000- and 25,000-fold, respectively, with corresponding decreases in cytochrome c reductase activity. In contrast, substitution of Thr(491), which also interacts with the pyrophosphate grouping, had a relatively modest effect on both FAD binding (100-fold decrease) and catalytic activity (2-fold decrease), while the G488L mutant exhibited, respectively, 800- and 50-fold decreases in FAD binding and catalytic activity. Enzymic activity of each of these mutants could be restored by addition of FAD. Kinetic properties and the FMN content of these mutants were not affected by these substitutions, with the exception of a 3-fold increase in Y456S K(m)(cyt )(c) and a 70% decrease in R454E FMN content, suggesting that the FMN- and FAD-binding domains are largely, but not completely, independent. Even though Trp(677) is stacked against the re-face of FAD, suggesting an important role in FAD binding, deletion of both Trp(677) and the carboxyl-terminal Ser(678) decreased catalytic activity 50-fold without affecting FAD content.
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PMID:Differential contributions of NADPH-cytochrome P450 oxidoreductase FAD binding site residues to flavin binding and catalysis. 1102 49

There is a region exhibiting a similarity of amino acid sequence near the carboxyl-terminal segment of each FAD-containing oxidoreductase. In this region, four amino acid residues-Thr, Ala, Gly, and Asp-are highly conserved. To determine the involvement of the four amino acid residues (Thr-469, Ala-476, Gly-478, and Asp-479) in the activity of NADH dehydrogenase of an alkaliphilic Bacillus, mutations of these amino acid residues were conducted. In spite of high conservation, mutations of Thr-469 and Ala-476 to Ala and Ser, respectively, did not lead to a critical loss of enzyme activity. However, mutations of Gly-478 and Asp-479 to Ala caused a complete loss of the activity, which appears to result from the loss of binding capacity of FAD.
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PMID:Involvement of glycine and aspartate residues in the binding capacity of FAD in the NADH dehydrogenase from an alkaliphilic Bacillus. 1273 50

Mitochondrial superoxide (O(2)(.)) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O(2)(.) production; its mechanism of O(2)(.) generation is not known. Therefore, the catalytic function of NDH in the mediation of O(2)(.) generation was investigated by EPR spin-trapping. In the presence of NADH, O(2)(.) generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O(2)(.) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O(2)(.) generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O(2)(.) generation. O(2)(.) production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O(2)(.)-dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200)-Ala-Gly-Ala-Tyr-Ile-Cys(206)-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)-Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.
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PMID:Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation. 1615 Jul 35

The influence of 6-benzylaminopurine (BAP) on the respiration by mitochondria from bush bean (Phaseolus vulgaris L.), mung bean (P. aureus Roxburgh), soybean [Glycine max (L.) Merrill], maize (Zea mays L.), pea (Pisum sativum L.), and wheat (Triticum aestivum L.) was examined. BAP, a synthetic cytokinin, consistently inhibited oxygen uptake by mitochondria from all species when malate was used as the substrate. The decrease in respiration was especially evident in the presence of ADP or an uncoupler of oxidative phosphorylation. 6-Isopentenylaminopurine and 6-furfurylaminopurine also inhibited malate oxidation, but zeatin and adenine did not. In certain instances, BAP reduced succinate and NADH oxidation. With succinate as the substrate and with antimycin A present, inhibition by BAP paralleled that caused by salicylhydroxamic acid, an inhibitor of alternative respiration. A suggested scheme features a cytokinin-inhibited point located between NADH dehydrogenase and cytochrome b of the electron transport system. Electrons from the NADH generated by malate oxidation are assumed to flow through this point, with electrons from externally supplied or cytosolic NADH and succinate doing so only under certain conditions such as when alternative respiration is occurring. Cytokinin effects on respiration and perhaps on other phenomena may be mediated by this mechanism.
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PMID:Cytokinin inhibition of respiration in mitochondria from six plant species. 1659 62


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