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

---

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

Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.
...
PMID:Physicochemical properties of flavodoxin from Desulfovibrio vulgaris. 23 84

The hyp operon of Escherichia coli comprises several genes which are required for the synthesis of all three hydrogenase isoenzymes. Deletions were introduced into each of the hypA-E genes, transferred to the chromosome and the resulting mutants were analysed for hydrogenase 1, 2 and 3 activity. The products of three of the genes, hypB, hypD and hypE were found to be essential for the synthesis of all three hydrogenase isoenzymes. A defect in hypB, as previously observed, could be complemented by high nickel concentrations in the medium, whereas the effects of mutants in the other genes could not. Lesions in hypA prevented development of hydrogenase 3 activity, did not influence the level of hydrogenase 1 but led to a considerable increase in hydrogenase 2 activity although the amount of hydrogenase 2 protein was not drastically altered. Lesions in hypC, on the other hand, led to a reduction of hydrogenase 1 activity and abolished hydrogenase 3 activity. HYPA and HYPC, besides being required for hydrogenase 3 formation, therefore may have a function in modulating the activities of the three isoenzymes with respect to each other and adjusting their levels to the requirement imposed by the physiological situation. Mutations in all five hyp genes prevented the apparent processing of the large subunits of all three hydrogenase isoenzymes. It is concluded that the products of the hypA-E genes play a role in nickel incorporation into hydrogenase apoprotein and/or processing of the constituent subunits of this enzyme. The importance of their roles is also reflected in their phylogenetic conservation in distantly related organisms.
...
PMID:The hyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes in Escherichia coli. 148 71

A double mutant (JH103K10) was created from hydrogenase constitutive mutant (JH103) by replacement of a chromosomal 0.60 kb nickel metabolism related locus with a kanamycin resistance gene. The double mutant required 10 to 20 times more nickel (Ni) to achieve near parental strain levels of hydrogenase activity. In the absence of nickel, both JH103K10 and JH103 synthesized high levels of (inactive) hydrogenase apoprotein (large subunit, 65 kDa). With nickel, the double mutant JH103K10 synthesized the same level of hydrogenase apoenzyme (65-kDa subunit) as the JH103 parent strain; however, whole cell hydrogenase activity in JH103K10 was less than half of that in JH103, and the CPM (due to 63Ni in hydrogenase) of membranes and the calculated ratio of nickel per unit of hydrogenase enzyme of the double mutant were 40% of that in JH103. Therefore, the difference in hydrogenase activities between the double mutant and the Hupc strain can be accounted for by different abilities of the strains to incorporate nickel into the hydrogenase apoenzyme. The addition of nickel ions to previously Ni-starved and then chloramphenicol-treated Bradyrhizobium japonicum whole cells (JH103 and JH103K10) resulted in (an in vivo) restoration of hydrogenase activity, suggesting that the apoprotein synthesized in the Ni-free cultures could be activated by addition of nickel even in the absence of protein synthesis. The extent of reconstitution of active hydrogenase by nickel was greater in the absence of chloramphenicol. Hydrogenase apoprotein could not be activated by nickel in vitro even with the addition of ATP. The successful in vivo but not in vitro results suggest that enzymatic but cell-disruption labile factors are required for Ni incorporation into hydrogenase.
...
PMID:Nickel-dependent reconstitution of hydrogenase apoprotein in Bradyrhizobium japonicum Hupc mutants and direct evidence for a nickel metabolism locus involved in nickel incorporation into the enzyme. 150 31

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.
...
PMID:Redox properties and active center of phototrophic bacteria hydrogenases. 301 53

We found that Salmonella typhimurium strain LT2 (Z) possessed two immunologically distinct, membrane-bound hydrogenase isoenzymes, which were similar in electrophoretic mobilities and apoprotein contents to hydrogenase isoenzymes 1 and 2 of Escherichia coli. The S. typhimurium enzymes cross-reacted with antibodies raised to the respective hydrogenase isoenzymes of E. coli. As for E. coli, an additional membrane-bound hydrogenase activity (termed hydrogenase 3), which did not cross-react with antibodies raised against either hydrogenase 1 or 2, was also present in detergent-dispersed membrane preparations. The physiological role of each of the three isoenzymes in E. coli has remained unclear owing to the lack of mutants specifically defective for individual isoenzymes. However, analysis of two additional wild-type isolates of S. typhimurium revealed specific defects in their hydrogenase isoenzyme contents. S. typhimurium LT2 (A) lacked isoenzyme 2 but possessed normal levels of hydrogenases 1 and 3. S. typhimurium LT7 lacked both isoenzymes 1 and 2 but retained normal hydrogenase 3 activity. Characterization of hydrogen metabolism by these hydrogenase-defective isolates allowed us to identify the physiological role of each of the three isoenzymes. Hydrogenase 3 activity correlated closely with formate hydrogenlyase-dependent hydrogen evolution, whereas isoenzyme 2 catalyzed hydrogen uptake (oxidation) during anaerobic, respiration-dependent growth. Isoenzyme 1 also functioned as an uptake hydrogenase but only during fermentative growth. We postulate that this enzyme functions in a hydrogen-recycling reaction which operates during fermentative growth.
...
PMID:Characterization and physiological roles of membrane-bound hydrogenase isoenzymes from Salmonella typhimurium. 353 Nov 77

The regulation of synthesis of the hydrogenase which is a component of the formate hydrogen-lyase complex was studied by means of a strain of Escherichia coli possessing a transcriptional fusion of the hydrogenase gene (hyd) with the lacZ gene (hyd::lac fusion). Formation of active hydrogenase in the wild strain requires the presence of nickel in the medium; transcription of the hyd gene, however, is independent from the presence of Ni2+. Ni2+ addition to Ni2+-prestarved cells did not lead to any activation of presumptive hydrogenase apoprotein. Regulatory mutants were isolated in which nitrate repression of hyd::lac expression was relieved. Two main classes of regulatory mutants were identified: (i) Mutants with a defect in nitrate reductase; (ii) mutants with a cis-dominant regulatory mutation closely linked to the hyd::lac fusion. In the presence of formate which acts as an inducer, the hyd::lac fusion was also expressed under aerobic conditions. The results infer that nitrate repression of transcription of the hydrogenase structural gene is not effected by nitrate itself but requires the function of the electron transport chain leading to nitrate and that mutations in the promoter/operator region of the hyd cistron may confer insensitivity to redox control both by oxygen and nitrate.
...
PMID:Regulation of the synthesis of hydrogenase (formate hydrogen-lyase linked) of E. coli. 644 May 7

Four microbial enzymes are known to require nickel: hydrogenase, methyl coenzyme M reductase, carbon monoxide dehydrogenase, and urease. Recent biochemical and molecular biological experiments have provided clear evidence for the existence of multiple auxiliary genes that facilitate nickel incorporation into urease and hydrogenase. Similarly, accessory factors are also likely to be required for the other two enzymes. One of the urease-related genes (ureE) encodes a cytoplasmic protein that has been purified and shown to bind nickel reversibly. We propose that the UreE protein serves as a nickel donor to urease apoprotein. A second urease-related auxiliary gene (ureG) possesses a sequence motif that is found in ATP- and GTP-binding proteins. We have shown that nickel incorporation into urease requires energy and speculate that the UreG protein may serve as an energy transducer, coupling the energy of NTP hydrolysis to metallocenter incorporation. The UreG protein is related in sequence to HypB, a protein that has been proposed to function in nickel processing in hydrogenases. Hence, the mechanisms for metallocenter biosynthesis in these two dissimilar enzymes may have evolved from a common nickel incorporation system.
...
PMID:Nickel enzymes in microbes. 802 91

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product.
...
PMID:Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme. 819 92

The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 microM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-1. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 microM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.
...
PMID:The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein. 842 37

Complex metalloenzymes (e.g., nitrogenase, hydrogenase, urease) are synthesized starting from the apoprotein via several intermediates by the action of accessory proteins. The isolation and biochemical characterization of such intermediates is hampered by their low abundance and their lability. Here we describe a technique for efficient single-step purification of a hydrogenase precursor under mild conditions using a N-terminal Strep-tag II affinity peptide and a novel StrepTactin Sepharose matrix. The tag was fused to the large subunit of [NiFe] hydrogenase 3 (HycE) of Escherichia coli. No significant influence of the affinity peptide on maturation or activity of the protein was observed when the modified gene was integrated into the chromosome by homologous recombination. A tagged nickel-free precursor form of HycE bound quantitatively to a recombinant StrepTactin Sepharose column. More than 90% pure subunit could be obtained after elution with desthiobiotin. The procedure was shown to be more efficient than purification by immobilized metal affinity chromatography using a N-terminal His-tag. General advantages of the novel Strep-tag II affinity purification especially for applications with metalloenzymes are discussed.
...
PMID:Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis. 960 45


1 2 Next >>

---