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:1.12.7.2 (hydrogenase)
3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A flavodoxin was purified to homogeneity from the nitrogen-fixing heterocystous cyanobacterium Anabaena sphaerica grown under iron-limited conditions. The protein has a molecular mass of 21 kDa, and its spectral properties and amino-acid composition are very close to that of flavodoxins from other cyanobacteria. A. sphaerica flavodoxin supported the activities of A. sphaerica NADP reductase and Clostridium butyricum hydrogenase in reconstituted systems with illuminated plant chloroplasts as reductant. With the use of polyclonal anti-flavodoxin antiserum it was found that nitrogen-fixing cultures of A. sphaerica grown under iron-sufficient conditions contain low but significant amounts of flavodoxin (0.2-0.6 micrograms/mg crude extract protein) which increased dramatically (to 8-15 micrograms/mg crude extract protein) after the iron concentration in the medium was decreased to below 1 microM Fe. The flavodoxin content of both iron-limited and iron-sufficient. A. sphaerica was also shown to depend upon the growth phase of the (batch) cultures with a maximum at early exponential phase, coinciding with maximal in-vivo nitrogenase activity. These results suggest that A. sphaerica flavodoxin not only substitutes for ferredoxin under iron-limiting conditions, but also fulfills some specific role under iron-sufficient conditions.
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PMID:Purification and properties of a flavodoxin from the heterocystous cyanobacterium Anabaena sphaerica. 834 30

Three different types of tungsten-containing enzyme have been previously purified from Pyrococcus furiosus (optimum growth temperature, 100 degrees C): aldehyde ferredoxin oxidoreductase (AOR), formaldehyde ferredoxin oxidoreductase (FOR), and glyceraldehyde-3-phosphate oxidoreductase (GAPOR). In this study, the organism was grown in media containing added molybdenum (but not tungsten or vanadium) or added vanadium (but not molybdenum or tungsten). In both cell types, there were no dramatic changes compared with cells grown with tungsten, in the specific activities of hydrogenase, ferredoxin:NADP oxidoreductase, or the 2-keto acid ferredoxin oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Compared with tungsten-grown cells, the specific activities of AOR, FOR, and GAPOR were 40, 74, and 1%, respectively, in molybdenum-grown cells, and 7, 0, and 0%, respectively, in vanadium-grown cells. AOR purified from vanadium-grown cells lacked detectable vanadium, and its tungsten content and specific activity were both ca. 10% of the values for AOR purified from tungsten-grown cells. AOR and FOR purified from molybdenum-grown cells contained no detectable molybdenum, and their tungsten contents and specific activities were > 70% of the values for the enzymes purified from tungsten-grown cells. These results indicate that P. furiosus uses exclusively tungsten to synthesize the catalytically active forms of AOR, FOR, and GAPOR, and active molybdenum- or vanadium-containing isoenzymes are not expressed when the cells are grown in the presence of these other metals.
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PMID:Molybdenum and vanadium do not replace tungsten in the catalytically active forms of the three tungstoenzymes in the hyperthermophilic archaeon Pyrococcus furiosus. 855 Apr 11

The present paper describes the sensitivity of the mitochondrial nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) to oxidative modification, and the effects of endogenous ubiquinol on this modification. A comparison is made between the effects of treatment with ADP-Fe3+ and ascorbate and with peroxynitrite, using kinetic, electrophoretic, and immunological analyses, together with lipid peroxidation measurements. The transhydrogenase was inactivated by both types of oxidative modification, but apparently through different mechanisms. Ubiquinol protected the enzyme against inactivation only when the modification was caused by ADP-Fe3+ and ascorbate treatment. Kinetic measurements revealed a threefold increase of the Km value of the enzyme for NADPH after exposure to ADP-Fe3+ and ascorbate, and a twofold increase of the Km values for both NADH and NADPH after exposure to peroxynitrite. NAD(H) exerted a protection against trans-hydrogenase inactivation when added to the preincubation in the case of peroxynitrite, but neither NAD(H) or NADP(H) protected in the case of ADP-Fe3+ and ascorbate. Using immunoblotting it was shown that the enzyme became both aggregated and fragmented, although to different extents, depending on the oxidative system used. Again, ubiquinol prevented these effects only in the case of ADP-Fe3+ and ascorbate treatment. Furthermore, there occurred a striking decrease in the 66-kDa trypsin fragment after exposure of the enzyme to ADP-Fe3+ and ascorbate, and of the 48-kDa trypsin fragment after exposure to peroxynitrite. It is concluded that the mitochondrial nicotinamide nucleotide transhydrogenase is sensitive to oxidative stress and that the mechanism underlying this can vary according to the challenge to which the enzyme is exposed. Endogenous ubiquinol may play a role in protecting the enzyme against agents perturbing the lipid phase of the membrane.
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PMID:Oxidative modification of nicotinamide nucleotide transhydrogenase in submitochondrial particles: effect of endogenous ubiquinol. 895 Oct 41

The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the H2-dependent NADP reduction was completely abolished. The double-mutated DM4 (DeltahynABC, DeltahndD) strain was still able to grow on hydrogen plus sulfate as the sole energy source. The growth may have occurred under these culture conditions because of the presence of the remaining [Fe] hydrogenase. The cells grew differently on various substrates depending on whether fructose, lactate, or pyruvate was used in the presence of sulfate. The (hnd mutant growth rates were 25-70% lower than those of the wild-type strain, although the molar growth yield remained unchanged. By contrast, mutants devoid of both [NiFe] hydrogenase and NADP-reducing hydrogenase had 24-38% lower growth yields and showed a corresponding drop in the growth rates. We concluded that each of the three hydrogenases may contribute to the energy supply in D. fructosovorans and that the loss of one enzyme might be compensated for by another. However, the loss of two hydrogenases affected the phosphorylation accompanying the metabolism of fructose, lactate, and pyruvate.
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PMID:Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans. 900 Mar 40

Based on the DNA sequence of its structural genes, clustered in the hnd operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovorans is thought to be a heterotetrameric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit, respectively. The weak representativity of the enzyme among cell proteins has prevented its purification. This paper discusses the purification and characterization of the HndA subunit of this unique tetrameric iron hydrogenase overproduced in Escherichia coli. The purified subunit contains 1.7 mol of non-heme iron and 1.7 mol of acid-labile sulfide/mol. EPR analysis of the reduced form of HndA indicates that it contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with values of gx, gy, and gz equal to 1.915, 1.950, and 2. 000, respectively, and a midpoint redox potential of -395 mV. The UV-visible and EPR spectra of the [2Fe-2S] cluster indicate that HndA belongs to the [2Fe-2S] family typified by the Clostridium pasteurianum [2Fe-2S] ferredoxin. The C-terminal sequence of HndA shows 27% identity with the C-terminal sequence of the 25-kDa subunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from Bos taurus complex I, and 32% identity with the entire sequence of C. pasteurianum [2Fe-2S] ferredoxin. The four cysteine residues involved in HndA cluster binding have been tentatively identified on the basis of sequence identity considerations. Evidence of a HndA organization based on two independent structural domains is discussed.
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PMID:Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli. 948 16

The NADP-reducing hydrogenase of Desulfovibrio fructosovorans represents a novel class of [Fe] hydrogenases which is encoded by the well-characterized hndABCD operon containing the genes hndA, hndB, hndC, and hndD. Expression of this operon, monitored by measuring the NADP-reducing activity, was found to be maximum during the exponential phase of growth on fructose and then decreased when the concentration of the carbon and energy source became limiting. The optimum pH for the H2-driven NADP reduction was 8, and the apparent K(m) and Vmax were determined to be 0.09 mM and 13 x 10(-3) u/mg, respectively. Heterologous expression of the hnd genes in Escherichia coli was carried out to raise antisera against the different subunits of the NADP-reducing hydrogenase. The antisera were used to detect the four subunits in cell extract of D. fructosovorans after separation by SDS- and native PAGE. The four subunits of the NADP-reducing hydrogenase were demonstrated to be associated in a complex which exhibited H2-driven methyl viologen reduction. Furthermore, on native gel, a form lacking HndD, with no hydrogen-dependent methyl viologen reductase activity was also shown to be present in D. fructosovorans.
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PMID:The NADP-reducing hydrogenase of Desulfovibrio fructosovorans: evidence for a native complex with hydrogen-dependent methyl-viologen-reducing activity. 970 71

Chlorophyllin a was conjugated with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene), PEG-NH(2), to form the PEG-chlorophyllin conjugate through acid-amide bonds. The PEG-chlorophyllin conjugate was stable toward light illumination under anaerobic condition in comparison with chlorophyllin a. The conjugate catalyzed the reduction of methyl viologen in the presence of 2-mercaptoethanol and the evolution of hydrogen gas in the presence of methyl viologen (an electron carrier), 2-mercaptoethanol (an electron donor) and hydrogenase (Scheme 1). Furthermore, the PEG-chlorophyllin conjugate catalyzed the photoreduction of NADP(+) or NAD(+) in the presence of ascorbate as an electron donor and ferredoxin-NADP(+) reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by the PEG-chlorophyllin conjugate under the illumination, CO(2) fixation was accomplished by the synthesis of malate (C(4)) from pyruvate (C(3)) and CO(2) in the presence of malic enzyme (Scheme 2). These reactions mentioned above did never proceed in dark or without each enzyme.
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PMID:Hydrogen gas evolution and carbon dioxide fixation with visible light by chlorophyllin coupled with polyethylene glycol. 1063 79

The green alga Scenedesmus obliquus is capable of both uptake and production of H(2) after anaerobic adaptation (photoreduction of CO(2) or photohydrogen production). The essential enzyme for H(2)-metabolism is a NiFe-hydrogenase with a [2Fe-2S]-ferredoxin as its natural redox partner. Western blot analysis showed that the hydrogenase is constitutively expressed. The K(m) values were 79.5 microM and 12.5 microM, determined with ferredoxin and H(2), respectively, as electron donor for the hydrogenase. In vitro, NADP(+) was reduced by H(2) in the presence of the hydrogenase, the ferredoxin and a ferredoxin-NADP reductase. From these results and considerations on the stoichiometry we propose that this light-independent electron transfer is part of the photoreduction of CO(2) in vivo. For ATP synthesis, necessary for the photoreduction of CO(2), light-dependent cyclic electron transfer around Photosystem (PS) I accompanies this 'dark reaction'. PS II fluorescence data suggest that (a) in S. obliquus H(2)-reduction might function as the anaerobic counterpart of the O(2)-dependent Mehler reaction, and (b) the presence of either a ferredoxin quinone-reductase or NAD(P)-dehydrogenase (complex I) in S. obliquus chloroplasts.
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PMID:Electron pathways involved in H(2)-metabolism in the green alga Scenedesmus obliquus. 1111 39

Cadmium and lead metals deposited on CdS particles are shown to act as substrates--electron donors for enzymes, hydrogenase from Thiocapsa roseopersicina (HG), NAD-dependent hydrogenase from Alcaligenes eutrophus (NLH), and ferredoxin:NADP oxidoreductase (FNR) from Chlorella in the formation of hydrogen, NADH and NADPH, respectively. Adsorption of the enzyme on the surface of the metallized CdS particle is required for enzymatic oxidation of metal. The maximum rates for the formation of hydrogen and NADH catalyzed by hydrogenase and NAD-dependent hydrogenase with metals as electron donors are comparable with the rates obtained for these enzymes using soluble substrates. Kinetic analysis of the enzymatic oxidation of cadmium metal has revealed that the rate decreases mainly due to the formation of a solid product, which is supposed to be Cd(OH)2. The deceleration of lead oxidation catalyzed by hydrogenase proceeds at the expense of the inhibitory effect of the formed Pb2+. The enzymatic oxidation of electrochemically prepared cadmium metal is also shown. Based on these results, a new mechanism of action of the enzymes involved in anaerobic biocorrosion is proposed. By this mechanism, the enzyme accelerates the process of metal dissolution through a mediatorless catalysis of the reduction of the enzyme substrate.
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PMID:Enzymatic oxidation of cadmium and lead metals photodeposited on cadmium sulfide. 1120 26

Desulfovibrio fructosovorans possesses two periplasmic hydrogenases (a nickel-iron and an iron hydrogenase) and a cytoplasmic NADP-dependent hydrogenase. The hydAB genes encoding the periplasmic iron hydrogenase were replaced, in the wild-type strain as well as in single mutants depleted of one of the other two hydrogenases, by the acc1 gene encoding resistance to gentamycin. Molecular characterization and remaining activity measurements of the resulting single and double mutants were performed. All mutated strains exhibited similar growth when H(2) was the electron donor but they grew differently on fructose, lactate or pyruvate as electron donors. Our results indicate that the loss of one enzyme might be compensated by another even though hydrogenases have different localization in the cells.
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PMID:Construction and physiological studies of hydrogenase depleted mutants of Desulfovibrio fructosovorans. 1220 80


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