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)

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

The hndABCD operon from Desulfovibrio fructosovorans encodes an uncommon heterotetrameric NADP-reducing iron hydrogenase. The presence of a [2Fe-2S] cluster likely located in the C-terminal region of the HndA subunit has already been revealed. We have cloned and expressed the truncated hndA gene in Escherichia coli to isolate the structural [2Fe-2S] module. Optical and EPR spectra are found identical to that of the native HndA subunit and the midpoint redox potential (-385 mV) is similar to that of the native protein (-395 mV). These results clearly demonstrate that the C-terminal region of HndA is a structurally independent [2Fe2S] ferredoxin-like domain. In the same way, the N-terminal domain of the HndD subunit was overproduced in E. coli and characterized. The presence of a [2Fe-2S] cluster was evidenced by optical spectroscopy. The midpoint redox potential (-380 mV) of this domain was found very close to that of the truncated HndA subunit but the EPR properties were significantly different. The various EPR properties allowed us to observe an electron exchange between the two [2Fe-2S] ferredoxin-like domains of the HndA and HndD subunits. Moreover, domain-domain interactions, observed by far-western experiments, indicate that these subunits are direct partners in the native complex.
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PMID:The NADP-reducing hydrogenase from Desulfovibrio fructosovorans: functional interaction between the C-terminal region of HndA and the N-terminal region of HndD subunits. 1246 Jun 79

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.
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PMID:Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase. 1256 Sep 90

We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (T. kodakaraensis hydrogenase). A gene cluster encoding T. kodakaraensis hydrogenase was composed of four open reading frames (hyhBGSL(Tk)), where the hyhS(Tk) and hyhL(Tk) gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (hybD(Tk)) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL(Tk) were used for immunoaffinity purification of T. kodakaraensis hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified T. kodakaraensis hydrogenase was composed of four subunits (beta, gamma, delta, and alpha), corresponding to the products of hyhBGSL(Tk), respectively. Each alphabetagammadelta unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the T. kodakaraensis hydrogenase was 95 degrees C for H(2) uptake and 90 degrees C for H(2) production with methyl viologen as the electron carrier. We found that NADP(+) and NADPH promoted high levels of uptake and evolution of H(2), respectively, suggesting that the molecule is the electron carrier for the T. kodakaraensis hydrogenase.
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PMID:Characterization of a cytosolic NiFe-hydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. 1259 89

The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H2, CO2, and O2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D2. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O2, and could be quickly reactivated by NADH or NADPH (+H2). H2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H2 output was observed, followed by rapid H2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H2 and CO2 was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O2 in the light, H2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H2 production. A sustained rate of photoevolution of H2 corresponding to 6 micro mol of H2 mg of chlorophyll(-1) h(-1) or 2 ml of H2 liter(-1) h(-1) was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H2 production operate in M55, namely photolysis of water at the level of photosystem II and carbohydrate-mediated reduction of the plastoquinone pool.
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PMID:Sustained photoevolution of molecular hydrogen in a mutant of Synechocystis sp. strain PCC 6803 deficient in the type I NADPH-dehydrogenase complex. 1499 5

Factors that may initiate the metabolic transition for butanol production were investigated in batch cultures of Clostridium beijerinckii (synonym, Clostridium butylicum) VPI 13436. Cultures maintained at pH 6.8 produced nearly as much butanol as those incubated without pH control, indicating that neither a change in the culture pH nor acid conditions per se are always required to initiate solvent formation. Acetate and butyrate levels at the onset of butanol production were dependent on the pH at which the cultures were maintained. Cultures maintained at pH 6.8 could be accelerated into solvent production by artificially lowering the pH to 5.0 or by the addition of acetate plus butyrate without a pH change (but neither acid alone was effective). Solvent production was associated with slower rates of growth and general metabolism, and it did not show a requirement for mature spore formation. We speculate that a slowdown in metabolism, which may be brought about by several conditions, is mechanistically related to the onset of butanol production. Extracts of solvent-producing cells contained acetoacetate decarboxylase activity as well as higher NADP-linked butanol dehydrogenase and lower hydrogenase activities than extracts of acid-producing cells. Solvent production did not appear to involve an enhanced ability to catalyze H(2) oxidation.
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PMID:Acidic Conditions Are Not Obligatory for Onset of Butanol Formation by Clostridium beijerinckii (Synonym, C. butylicum). 1634 58

Dark H(2) metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas, and mosses. A time variable and temperature-sensitive anaerobic incubation was required prior to H(2) evolution. H(2) evolution was sensitive to disalicylidenepropanediamine. An immediate H(2) uptake was observed in these algae. Immediate dark H(2) uptake but no evolution was observed in the mosses.A cell-free hydrogenase preparation was obtained from anaerobically adapted Chlamydomonas reinhardii by means of sonic oscillation. The hydrogenase was not sedimented at 100,000g. It catalyzed the reduction of methylene blue, p-benzoquinone, NAD, NADP, but not spinach ferredoxin. H(2) evolution was noted with dithionite and with reduced methyl viologen as donors but not with reduced spinach ferredoxin. Similarly, hydrogenase activities were not affected by disalicylidenepropanediamine. The pH optima for H(2) evolution and for H(2) uptake were 7.2 and 7.5 to 9.5, respectively. Extracts prepared from the anaerobically adapted red alga, Chondrus crispus, and the moss, Leptobryum pyriforme, consumed but did not evolve H(2). Uptake was slightly stimulated by methylene blue. It is proposed that red algae and mosses appear to metabolize H(2) by a different pathway than Chlamydomonas.
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PMID:H(2) metabolism in photosynthetic organisms: I. Dark h(2) evolution and uptake by algae and mosses. 1665 60

Chloroplast and photosystem I particles were encapsulated in small spheres (about 20 mum diameter) with an artificial membrane built up by cross-linking amino groups of protamine with toluenediisocyanate. The artificial membrane was permeable to small substrate and product molecules but not to soluble proteins. Photosystem I activity was retained by the encapsulated chloroplast particles. Washed photosystem I particles were encapsulated with the soluble proteins, ferredoxin, and ferredoxin-NADP oxidoreductase, and the microcapsules photoreduced NADP using ascorbate plus dichlorophenolindophenol as the electron donor. The photosystem I particles were also encapsulated with hydrogenase from Chromatium and a very low rate of photoevolution of hydrogen was obtained. The results show that chloroplast membrane fragments can be encapsulated with soluble proteins that couple transfer reactions to the primary photochemical apparatus.
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PMID:Microencapsulation of chloroplast particles. 1665 64

A spinach (Spinacia oleracia var. America) chloroplast particle fortified with ferredoxin, fructose-1,6-bisphosphate, or ribose-5-phosphate and NADP has been shown to generate NADPH by the oxidation of glyceraldehyde-3 phosphate to glycerate-3-phosphate (PGA) and to reduce ferredoxin with the NADPH. The resulting reduced ferredoxin can reduce O(2) to H(2)O(2), nitrite to ammonia, or protons to H(2). Hydrogen production was the result of adding hydrogenase from Chlamydomonas reinhardii to the chloroplast preparation. The predicted stoichiometry of 1 PGA:1 O(2) in the absence of and 2 PGA:1 O(2) in the presence of catalase was observed indicating H(2)O(2) as the end product of O(2) reduction. The predicted stoichiometry of 3 PGA:1 nitrite:1 ammonia was also observed. A scheme is presented to account for a sustained generation of NADP and ATP necessary for the dissimilation of starch in the darkened chloroplast. The unifying term chloroplast respiration is introduced to account for those reactions in which reduced ferredoxin interacts with physiological acceptors other than NADP or nitrite, hydrogen, or O(2) respiration when nitrite, protons, or O(2) is the ultimate electron acceptor.
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PMID:Chloroplast Respiration : A MEANS OF SUPPLYING OXIDIZED PYRIDINE NUCLEOTIDE FOR DARK CHLOROPLASTIC METABOLISM. 1666 26

Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of a constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogense in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process.Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg(2+), Ca(2+), and iron does not lead to active hydrogenase formation. Futhermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place.The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase.
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PMID:Activation and de novo synthesis of hydrogenase in chlamydomonas. 1666 54


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