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)

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes 'malic enzyme,' NAD(P)H:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase, hydrogenase, acetate:succinate CoA transferase and succinate thiokinase leading to the formation of H2,CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role--especially in zoospores--as H2-evolving, ATP-generating and O2-scavenging organelles.
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PMID:Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2. 825 82

Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function. The first class contains nine H2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria. The second comprises four periplasmic and two membrane-bound H2-uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria. The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria. The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophusH16. The fifth is the H2-producing labile hydrogenase isoenzyme 3 of Escherichia coli. The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria. The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors. While those of class I, II, IV and V hydrogenases are homologous, i.e. sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes. Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively. These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases. Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected. Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains. The regions of hydrogen activation domains are highly conserved and can be divided into two categories. One corresponds to the 'nickel active center' of NiFe(Se)-hydrogenases. It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively. The other is the H-cluster of the Fe-hydrogenases. It might comprise three motifs on the C-terminal half of the large subunits. However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved. They are present essentially on the small subunits in NiFe-hydrogenases. Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Microbial hydrogenases: primary structure, classification, signatures and phylogeny. 831 59

The process of NAD+ photoreduction under the coupled action of CdS semiconductor and NAD-dependent hydrogenase from hydrogen-oxidizing bacterium Alcaligenes eutrophus may be divided into light and dark stages. At the first stage, illumination of the system leads to the photooxidation of the sacrificial electron donor and results in the reduction of the semiconductor surface. At the second dark stage NAD+ is reduced to NADH in the presence of hydrogenase. Atoms of metallic Cd(0) are shown to be the true substrate of the enzymatic reaction. The prerequisite for the electron transfer from Cd(0) to hydrogenase is enzyme adsorption on the semiconductor surface. The redox center of the hydrogenase reacting with Cd(0) atoms resides on the flavin-containing heterodimer of the protein. The activity of the hydrogenase immobilized on CdS in the reaction of NAD+ reduction by metallic Cd is close to the enzyme activity with the physiological substrates in solution. Thus, the first example of a metal being the substrate of the enzymatic process is presented.
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PMID:Metal as a novel type of the enzyme substrate. Metallic cadmium photogenerated in the system CdS-formate as a substrate of the NAD-dependent hydrogenase. 834 24

In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545-553]. Re-examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.
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PMID:hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system. 863 53

Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase was located in the 140,000 x g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in a 85% pure preparation with a specific activity of 6.0 U (mg protein)-1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The optimum of enzyme activity was at pH 9 and at 49 degrees C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme. The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50-100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase.
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PMID:Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans. 866 19

The specific activity of purified soluble hydrogenase of Alcaligenes eutrophus H16 was found to vary with enzyme concentration. Specific activity as a function of concentration of purified enzyme could be fit to an equation describing the dissociation of a compound into two components. An association constant, kappa(a), was determined in this way to be 39.4 +/- 8.7 micrograms protein/ml. The concentration of the enzyme affected its kinetic parameters: a tenfold decrease in enzyme concentration caused by a reduction of the V(max) and Kappa(m) (NAD) values to 45% and 58%, respectively, of the values for undiluted (0.64 mg/ml) enzyme. Diaphorase (NAD-dependent reduction of benzyl viologen) specific activity of the hydrogenase was unaffected by dilution. The extent of dilution-induced activity loss was dependent on pH, with greater activity loss observed at higher pH values. The substrate NAD prevented loss of specific activity due to dilution, while the product NADH did not. Specific activity loss due to dilution as reversed with the addition of the cofactor FMN. Dilution of the hydrogenase caused an increase in the enzyme's specific flavin fluorescence. These results suggest that dilution of the soluble hydrogenase of Alcaligenes eutrophus causes dissociation of the cofactor FMN, and this activity loss should be taken into account as an important factor governing hydrogenase activity and kinetic properties.
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PMID:FMN cofactor dissociation from the soluble hydrogenase of Alcaligenes eutrophus H16. 872 22

Biochemical decompression has been proposed as a method for reducing the amount of time required for deep-sea divers to return to the surface. Divers breathing H2/O2 mixtures would be presented with hydrogenase enzyme, and decompression would be accelerated by means of the enzymic removal of excess H2 from the tissues. We have studied FAD as a hydrogenase electron acceptor that is capable of transferring electrons derived from H2 oxidation directly to O2. Kinetic activity constants for the soluble hydrogenase from the bacterium Alcaligenes eutrophus H16 were determined with FAD, FMN and riboflavin as electron acceptors, and these values were compared with those obtained with the physiological electron acceptor NAD+. The Michaelis constants (K(m)) were similar for FAD, FMN and NAD. However, the maximal catalytic-centre activity (Kcat) was much lower for the flavins, and the catalytic efficiency (Kcat/K(m)) with FAD was 1/20th the value for NAD+. After enzyme-catalysed FAD reduction to FADH2, the FAD could be regenerated by addition of O2 and reduced again by the enzyme in the presence of H2. Thus FAD served as a regenerable electron shuttle between H2 and O2. H2O2, a by-product of FADH2 oxidation by O2, inhibited the enzyme. Much greater inhibition was observed with the reduced form of the enzyme. Active hydrogenase was efficiently encapsulated into human and pig red blood cells. Hydrogen consumption was seen with lysed carrier cells, but was demonstrated with unlysed carrier cells only when FAD was co-encapsulated along with enzyme. These results demonstrate that red blood cells encapsulating hydrogenase and FAD act as a system for continuous H2 consumption in a mammalian tissue without addition of exogenous factors, and such cells may provide a biotherapeutic method for reducing the risk and treatment of decompression sickness.
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PMID:Hydrogenase encapsulation into red blood cells and regeneration of electron acceptor. 886 3

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

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (betadelta) and an NADH:acceptor oxidoreductase (alphagamma)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed.
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PMID:Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22. 913 25

The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus MR11 into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7.4 kbp Asnl fragment of the linear plasmid pHG201 via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hoxF, -U, -Y, -H, -W and ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hoxF, -U, -Y and -H encode the subunits alpha, gamma, delta and beta, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the beta subunit. Finally, ORF7 encodes a protein which has similarities to cAMP- and cGMP-binding protein kinases, but its function is not known. ORF1, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus MR11 indicated that the hydrogenase genes are under control of a delta 70-like promoter located at the right end of ORF1 and are even transcribed under heterotrophic conditions at a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHY1 harbouring the 7.4 kbp Asnl fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.
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PMID:Genes encoding the NAD-reducing hydrogenase of Rhodococcus opacus MR11. 914 90


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