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 bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli. hox DNA was identified by screening the E. coli gene bank for restoration of hydrogenase activity in A. eutrophus Hox mutants. Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants. An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment. A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins. hox DNA was subcloned into the vector pVK101. The resulting recombinant plasmids were used in complementation studies. The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A. eutrophus H16.
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PMID:Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16. 353 56

In Alcaligenes eutrophus, the formation of the hydrogenases and of five new peptides is subject to the hydrogenase control system. Of these, the B peptide was purified to homogeneity. This protein (Mr, 37,500) was composed of two identical subunits (Mr, 18,800). Antibodies against the B protein were used for its quantification by rocket immunoelectrophoresis. About 4% of the total protein consisted of the B protein; its molar ratio to the NAD-linked hydrogenase was about 4:1. The B protein appeared to be associated with the NAD-linked hydrogenase, as shown by gel filtration analysis with Sephadex G-200. The B protein was not detected in cells that had not expressed the hydrogenase proteins or that lacked the genetic information of the hydrogen-oxidizing character; it was also not detected in Tn5 insertional mutants that were unable to form soluble hydrogenase antigens. Immunochemical analysis of other species and genera than A. eutrophus revealed that only strains able to form a NAD-linked hydrogenase also formed B-protein antigens. The B protein is not required for the catalytic activity of soluble hydrogenase in vitro; its function is at present unknown.
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PMID:Purification and properties of a protein linked to the soluble hydrogenase of hydrogen-oxidizing bacteria. 355 56

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.
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PMID:Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain. 368 87

Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).
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PMID:Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. 388 93

The activity of hydrogenase was assayed in the intact cells and subcellular fractions of Brevibacterium flavum. The organism was shown to have the membrane-bound form of hydrogenase. The soluble NAD+-reducing hydrogenase was not found. Oxygen inhibited the hydrogenase activity, and its action was reversible. Molecular hydrogen activated the hydrogenase of B. flavum, which was shown to be a constitutive enzyme.
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PMID:[Hydrogenase activity in Brevibacterium flavum]. 390 43

Clostridium thermosulfurogenes displayed faster growth on either glucose, maltose, or starch than Clostridium thermohydrosulfuricum. Both species grew faster on glucose than on starch or maltose. The fermentation end product ratios were altered based on higher ethanol and lactate yields on starch than on glucose. In C. thermohydrosulfuricum, glucoamylase, pullulanase, and maltase were mainly responsible for conversion of starch and maltose into glucose, which was accumulated by a putative glucose permease. In C. thermosulfurogenes, beta-amylase was primarily responsible for degradation of starch to maltose, which was accumulated by a putative maltose permease and then hydrolyzed by glucoamylase. Regardless of the growth substrate, the rates of glucose, maltose, and starch transformation were higher in C. thermosulfurogenes than in C. thermohydrosulfuricum. Both species had a functional Embden-Meyerhof glycolytic pathway and displayed the following catabolic activities: ferredoxin-linked pyruvate dehydrogenase, acetate kinase, NAD(P)-ethanol dehydrogenase, NAD(P)-ferredoxin oxidoreductase, hydrogenase, and fructose-1,6-diphosphate-activated lactate dehydrogenase. Ferredoxin-NAD reductase activity was higher in C. thermohydrosulfuricum than NADH-ferredoxin oxidase activity, but the former activity was not detectable in C. thermosulfurogenes. Both NAD- and NADP-linked ethanol dehydrogenases were unidirectional in C. thermosulfurogenes but reversible in C. thermohydrosulfuricum. The ratio of hydrogen-producing hydrogenase to hydrogen-consuming hydrogenase was higher in C. thermosulfurogenes. Two biochemical models are proposed to explain the differential saccharide metabolism on the basis of species enzyme differences in relation to specific growth substrates.
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PMID:Differential amylosaccharide metabolism of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum. 393 39

When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.
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PMID:Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts. 409 65

1. The kinetic and metabolic properties of lactate dehydrogenase isoenzyme LDH(x) from human sperm cells and rat testes were studied. 2. LDH(x) shows a sensitivity to inhibition by stilboestrol diphosphate, urea and guanidinium chloride different from that of the LDH-H(4) and LDH-M(4) isoenzymes. 3. About 10 and 20% of the total lactate dehydrogenase activity of testes and sperm cells respectively were associated with particulate fractions. In sperm cells 11% was localized in the middle piece and 18.8% in the head fraction. LDH(x) was found in all particulate fractions of sperm cells. The middle piece contained 41.0% of total LDH(x) activity and showed high succinate dehydrogenase activity. 5. The pH-dependence of lactate/pyruvate and NAD(+)/NADH concentration ratios were estimated. Lactate dehydrogenase in sperm cells has maximal activity with NADH as coenzyme at pH7.5 and with NADPH as coenzyme at pH6.0. At pH6.0 a 10% greater oxidation of NADPH than of NADH was found. At acid pH lactate hydrogenase may function as an enzyme bringing about transhydrogenation from NADPH to NAD(+). 6. In agreement with the stoicheiometry of the lactate de- hydrogenase reaction, the lactate/pyruvate concentration ratio decreased with increasing pH. 7. The lactate/pyruvate and NAD(+)/NADH concentration ratios were estimated with glucose, fructose and sorbitol as substrates and as a function of time after addition of these substrates. During a 20min. period after the addition of the substrates, changes in lactate/pyruvate and NAD(+)/NADH concentration ratios were noticed. Increasing concentration of the substrates mentioned gave rise to asymptotic increases in lactate and pyruvate. 8. Sorbitol did not act as a substrate for LDH(x). 9. The findings described are consistent with the idea that LDH(x) is different from other known lactate dehydrogenase isoenzymes, but that it has a metabolic function similar to that of the isoenzymes of other tissues.
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PMID:Lactate dehydrogenase isoenzymes of sperm cells and tests. 430 63

The pathways of glucose and pyruvate metabolism in Spirochaeta litoralis, a free-living, strictly anaerobic marine spirochete, were studied. Addition of 0.2 to 0.4 M NaCl (final concentration) to suspending buffers prevented cell lysis and was necessary for gas evolution from various substrates by cell suspensions. The organism fermented glucose mainly to ethanol, acetate, CO(2), and H(2). Determination of radioactivity in products formed from (14)C-labeled glucose and assays of enzymatic activities in cell extracts indicated that S. litoralis catabolized glucose via the Embden-Meyerhof pathway. A clostridial-type clastic reaction was utilized by the spirochete to degrade pyruvate to acetyl-coenzyme A, CO(2), and H(2). Formation of acetate from acetyl-coenzyme A was catalyzed by phosphotransacetylase and acetate kinase. Nicotinamide adenine dinucleotide-dependent acetaldehyde and alcohol dehydrogenases converted acetyl-coenzyme A to ethanol. A reversible hydrogenase activity was detected in cell extracts. S. litoralis cell extracts contained a rubredoxin similar in spectral properties to other bacterial rubredoxins.
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PMID:Glucose and pyruvate metabolism of Spirochaeta litoralis, an anaerobic marine spirochete. 474 35

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be -420 mV and -285 mV, respectively. The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr, of 64000 and 31000, contained 9.9 +/- 0.4 S2- and 9.3 +/- 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity). The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400-nm region and a characteristic ESR signal at g = 2.01. The reduced form of the dimer was ESR-silent. The small dimer alone was diaphorase-inactive and did not reduce NAD with H2, but it displayed high H2-uptake activities with viologen dyes, methylene blue and FMN, and H2-evolving activity with reduced methyl viologen. Hydrogen-dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the ESR spectrum in the oxidized state.
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PMID:Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b. 608 43


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