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 gene capable of encoding a protein sharing 45% identical amino acids with the alpha subunit of the integration host factor (IHF) of Escherichia coli was isolated from the photosynthetic bacterium Rhodobacter capsulatus strain B10 by complementation of a hydrogenase-deficient (Hup-) mutant, IR4. A DNA fragment of 274 base pairs containing an IHF binding consensus sequence, isolated from the promoter region of the hydrogenase structural genes (hupSL), was shown by gel retardation assays to bind the IHF protein from E. coli. The product of the R. capsulatus gene was shown to bind specifically to the 274-base-pair DNA fragment from the hupSL promoter. By analogy to the E. coli himA gene, which encodes the alpha subunit of IHF, the gene complementing the IR4 mutant was named himA of R. capsulatus. The wild-type himA gene, cloned in plasmid pBO2, was introduced into the IR4 strain and shown to restore, in trans, hydrogenase activity and autotrophic growth in the mutant. In IR4, a C----T transition mutation had replaced Arg-8 by Cys-8. Gel mobility shifts of the 274-base-pair DNA fragment, not observed with the himA gene product of IR4, were restored with extracts from IR4(pBO2) cells, containing the himA gene on the recombinant plasmid pBO2.
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PMID:A mutation in a Rhodobacter capsulatus gene encoding an integration host factor-like protein impairs in vivo hydrogenase expression. 196 42

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.
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PMID:Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata. 301 42

This review surveys recent work done in the laboratory of the author and related laboratories on the properties and possible practical applications of hydrogenases of phototrophic microorganisms. Homogeneous hydrogenase preparations were obtained from purple non-sulfur (Rhodospirillum rubrum S1, Rhodobacter capsulatus B10) and purple sulfur (Chromatium vinosum D, Thiocapsa roseopersicina BBS) bacteria, and from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum L; highly purified hydrogenase samples were prepared from the cyanobacterium Anabaena cylindrica and from the green alga Chlamydomonas reinhardii. It was shown that hydrogenases of R. capsulatus and T. roseopersicina contain Ni and Fe-S cluster. The cytochromes of the c or b type serve as native electron acceptors for the hydrogenases of the purple bacteria and cyanobacteria; rubredoxin or cytochrome c for the hydrogenase of the green sulfur bacterium; and ferredoxin for Ch. reinhardii hydrogenase. The hydrogenase of T. roseopersicina BBS reversibly activates H2 at Eh less than -290 mV (pH 7), whereas those from R. capsulatus and from C. limicola f. thiosulfatophilum exhibit their maximum activity at Eh greater than -300 mV and are thus favourable only for the H2 uptake. Hydrogenase synthesis in different phototrophs depends on pO2, H2 concentrations and organic substrates. Organic compounds, which serve as electron donors and carbon sources, repress hydrogenase synthesis in R. rubrum, R. capsulatus and in Ectothiorhodospira shaposhnikovii when present at high concentrations. The synthesis of T. roseopersicina hydrogenase is constitutive. H2 notably stimulates hydrogenase activity in R. capsulatus. The synthesis of hydrogenase in R. sphaeroides 2R occurs only in the presence of H2 and does not depend on the presence of organic compounds in the medium.
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PMID:Hydrogenases of phototrophic microorganisms. 301 44

It is shown that the activity of phototrophic bacteria hydrogenases depends on the redox potential (Eh) of the medium. Hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina strain BBS reversibly activates H2 at Eh less than -290 mV (pH 7.0). When Eh is increased from -290 to -170 mV, the enzyme is converted into an inactive form which is accompanied by one-electron oxidation of its Fe-S cluster. In contrast, the hydrogenases of the purple nonsulfur bacterium Rhodobacter capsulatus B10 and the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum exhibit maximum activity at Eh greater than -300 mV, favourable only for H2 uptake. When Eh decreases the activities of these enzymes drop dramatically; this accounts for their unidirectional effect directed mainly towards H2 uptake. Such dependence on Eh of activity of hydrogenases from these bacteria correlates with their physiological function in the metabolism of phototrophic bacteria, i.e. with the catalysis of the H2 uptake reaction. Hydrogenases from purple bacteria contain nickel and a single Fe-S cluster. Metal chelators do not affect the activity of these enzymes, which indicates that iron and nickel are tightly bound to the apoprotein. Sulfhydryl compounds irreversibly inactivate T. roseopersicina hydrogenase by 30-40% in the presence of sulfide. Acetylene and carbon monoxide are reversible inhibitors of the enzyme. EPR and inhibitory analysis indicate a direct interaction of H2 with the nickel ion in the active center of the T. roseopersicina hydrogenase.
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PMID:Redox properties and active center of phototrophic bacteria hydrogenases. 301 53

Non-autotrophic ( Aut -) mutants of Rhodopseudomonas capsulata B10 were tested for their efficiency of nitrogenase-mediated H2 production. Three of these mutants ( IR3 , IR4 and IR5 ) showed an increase stoichiometry of H2 production, mediated by nitrogenase, from certain organic substrates. For example, in a medium containing 7 mM-L-glutamate as nitrogen source, strain IR4 produced 10-20% more H2 than did the wild type with DL-lactate or L-malate as major carbon source, 20-50% more H2 with DL-malate, and up to 70% more with D-malate. Strain IR4 was deficient in 'uptake' hydrogenase activity as measured by H2-dependent reduction of Methylene Blue or Benzyl Viologen. However, this observation did not explain the increased efficiency of H2 production, since H2 uptake (H2 recycling) was undetectable in cells of the wild type. Instead, increased H2 production by the mutant appeared to be due to an improved conversion of organic substrates to H2 and CO2, presumably due to an altered carbon metabolism. The metabolism of D-malate by different strains was studied. An NAD+-dependent D-malic enzyme was synthesized constitutively by the wild type, and showed a Km for D-malate of 3 mM. The activity of this enzyme was approx. 50% higher in strain IR4 than in the wild type, and the mutant also grew twice as fast as the wild type with D-malate as sole carbon source.
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PMID:Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata. 614 10

When Rhodopseudomonas capsulata B10 grows in media with different organic compounds, the hydrogenase activity estimated both by the evolution and uptake of H2 is lowest in cells taken from the middle of the exponential growth phase, and highest in cells from the beginning of the stationary phase. Cells grown in a medium containing malate have a higher hydrogenase activity than those cultivated in a medium with lactate or other compounds (900 and 20 nmoles of H2 per 1 min per 1 mg of protein, respectively). In the experiments with chloramphenicol (10(-5) M), organic compounds (not CO2) were shown to repress hydrogenase synthesis. When the cells were incubated in a medium without an organic substrate or in its presence, the exogenous H2 or H2 evolved as the result of nitrogenase action causes an increase in the activity of hydrogenase.
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PMID:[Hydrogenase activity of Rhodopseudomonas capsulata growing on organic media]. 675 94

Hydrogenase activity was found in cells of Rhodopseudomonas capsulata strain B10 cultured under a variety of growth conditions either anaerobically in the light or aerobically in the dark. The highest activities were found routinely in cells grown in the presence of H2. The hydrogenase of R. capsulata was localized in the particulate fraction of the cells. High hydrogenase activities were usually observed in cells possessing an active nitrogenase. The hydrogen produced by the nitrogenase stimulated the activity of hydrogenase in growing cells. However, the synthesis of hydrogenase was not closely linked to the synthesis of nitrogenase. Hydrogenase was present in dark-grown cultures, whereas nitrogenase synthesis was not significant in the absence of light. Unlike nitrogenase, hydrogenase was present in cultures grown on NH4+. Conditions were established which allowed the synthesis of either nitrogenase or hydrogenase by resting cells. We concluded that hydrogenase can be synthesized independently of nitrogenase.
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PMID:Hydrogenase activity in Rhodopseudomonas capsulata: relationship with nitrogenase activity. 699 43

A 25 kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium leguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF-hupS-hupL-hupM-hu pD-hupF-hupG-hupH-hupJ-hupK-hypA- hypB-hupR1- hypC-hypD-hypE-ORF19-ORF20, all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup- mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD gene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant prevented the expression of a hupS::lacZ translational fusion from being stimulated by H2 as it is observed in the wild-type strain B10. It is inferred that the HypF protein is a factor involved in H2 stimulation of hydrogenase expression.
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PMID:Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants. 849 90

The hupT, hupU, and hupV genes, which are located upstream from the hupSLC and hypF genes in the chromosome of Rhodobacter capsulatus, form the hupTUV operon expressed from the hupT promoter. The hupU and hupV genes, previously thought to belong to a single open reading frame, encode HupU, of 34.5 kDa (332 amino acids), and HupV, of 50.4 kDa (476 amino acids), which are >/= 50% identical to the homologous Bradyrhizobium japonicum HupU and HupV proteins and Rhodobacter sphaeroides HupU1 and HupU2 proteins, respectively; they also have 20 and 29% similarity with the small subunit (HupS) and the large subunit (HupL), respectively, of R. capsulatus [NiFe]hydrogenase. HupU lacks the signal peptide of HupS and HupV lacks the C-terminal sequence of HupL, which are cleaved during hydrogenase processing. Inactivation of hupV by insertional mutagenesis or of hupUV by in-frame deletion led to HupV- and Hup(UV)- mutants derepressed for hydrogenase synthesis, particularly in the presence of oxygen. These mutants were complemented in trans by plasmid-borne hupTUV but not by hupT or by hupUV, except when expressed from the inducible fru promoter. Complementation of the HupV- and Hup(UV)- mutants brought about a decrease in hydrogenase activity up to 10-fold, to the level of the wild-type strain B10, indicating that HupU and HupV participate in negative regulation of hydrogenase expression in concert with HupT, a sensor histidine kinase involved in the repression process. Plasmid-borne gene fusions used to monitor hupTUV expression indicated that the operon is expressed at a low level (50- to 100-fold lower than hupS).
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PMID:The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus. 875 35

The photosynthetic bacterium Rhodobacter capsulatus contains a membrane-bound [NiFe]hydrogenase encoded by the hupSL genes. We show in this study that hypF mutants are devoid of hydrogenase activity and lack the HupL protein. We also observed that, in contrast to the wild-type strain B10, transcription of the hupSL genes was not stimulated by H2 in the hypF mutants RS13 and BSE19. Complementation of the hypF mutants with the plasmid borne hypF gene restored hydrogenase activity to wild-type levels and inducibility by H2. The R. capsulatus hupU and hupV gene products share significant similarities with the small (HupS) and the large (HupL) hydrogenase subunits, respectively. Active HupUV proteins can catalyze the hydrogen-deuterium exchange reaction. In whole cells, this H-D exchange is distinguishable from the H-D exchange catalyzed by the membrane-bound HupSL proteins by its insensitivity to O2 and to acetylene. By measuring the formation of H2 and HD in exchange with D2 uptake, we demonstrated that the hypF mutants have no active HupUV nor HupSL proteins. H-D exchange activity, of both HupUV and HupSL, was restored by hypF gene complementation. These data indicate that the HypF protein participates not only in the maturation of HupSL, but also in the maturation of the HupUV proteins and that the latter are involved in the cellular response to H2.
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PMID:Rhodobacter capsulatus HypF is involved in regulation of hydrogenase synthesis through the HupUV proteins. 949 69


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