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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

H2 oxidation in Azotobacter vinelandii is catalyzed by a membrane-bound, alpha beta dimeric [NiFe] hydrogenase. Maturation of the enzyme involves cleavage of a putative N-terminal signal sequence in the beta subunit and removal of 15 amino acids from the C terminus of the alpha subunit. Cells limited for nickel exhibited low hydrogenase activities and contained an apparently large form of the alpha subunit. Addition of nickel to such cells increased hydrogenase activities fivefold over 2 h. The increase in the first hour did not require transcription and translation and correlated with processing of the large form of the alpha subunit (pre-alpha) to the small form (alpha) resembling the alpha subunit from the purified enzyme. In vivo, pre-alpha appeared soluble whereas the majority of alpha was membrane bound. Processing of pre-alpha to alpha was reproduced in vitro in membrane-depleted extracts of nickel-limited cells. Processing specifically required the addition of Ni2+, whereas Co2+, Cu2+, Ca2+, Fe2+, Mn2+, and Zn2+ were ineffective. However, Zn2+, Co2+, and Cu2+ inhibited nickel-dependent processing. Mg-ATP and Mg-GTP stimulated processing, whereas anaerobic conditions and/or the addition of dithiothreitol and sodium dithionite was unnecessary. Processing was not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, E64, and pepstatin.
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PMID:In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii. 828 21

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.
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PMID:Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri. 832 4

Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes required for transfer of the methyl group of methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 binds the methyl group of methanol to its corrinoid prosthetic group only when the central cobalt atom of the corrinoid is present in the highly reduced Co(I) state. However, upon manipulation of MT1 and even during catalysis, the enzyme becomes inactivated as the result of Co(I) oxidation. Reactivation requires H2, hydrogenase, and ATP. Ferredoxin stimulated the apparent reaction rate of methyl group transfer. Here we report that one more protein fraction was found essential for the overall reaction and, more specifically, for formation of the methylated MT1 intermediate. The more of the protein that was present, the shorter the delay of the start of methyl group transfer. The maximum velocity of methyl transfer was not substantially affected by these varying amounts of protein. This demonstrated that the protein was involved in the activation of MT1. Therefore, it was called methyltransferase activation protein.
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PMID:Involvement of an activation protein in the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri. 844 90

The bioenergetic role of the reduction of elemental sulfur (S0) in the hyperthermophilic archaeon (formerly archaebacterium) Pyrococcus furiosus was investigated with chemostat cultures with maltose as the limiting carbon source. The maximal yield coefficient was 99.8 g (dry weight) of cells (cdw) per mol of maltose in the presence of S0 but only 51.3 g (cdw) per mol of maltose if S0 was omitted. However, the corresponding maintenance coefficients were not found to be significantly different. The primary fermentation products detected were H2, CO2, and acetate, together with H2S, when S0 was also added to the growth medium. If H2S was summed with H2 to represent total reducing equivalents released during fermentation, the presence of S0 had no significant effect on the pattern of fermentation products. In addition, the presence of S0 did not significantly affect the specific activities in cell extracts of hydrogenase, sulfur reductase, alpha-glucosidase, or protease. These results suggest either that S0 reduction is an energy-conserving reaction, i.e., S0 respiration, or that S0 has a stimulatory effect on or helps overcome a process that is yield limiting. A modification of the Entner-Doudoroff glycolytic pathway has been proposed as the primary route of glucose catabolism in P. furiosus (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208-14216, 1991). Operation of this pathway should yield 4 mol of ATP per mol of maltose oxidized, from which one can calculate a value of 12.9 g (cdw) per mol of ATP for non-S0 growth. Comparison of this value to the yield data for growth in the presence of S0 reduction is equivalent to an ATP yield of 0.5 mol of ATP per mol of S0 reduced. Possible mechanism to account for this apparent energy conservation are discussed.
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PMID:Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus. 844 88

It is shown that -2H+/K(+)-exchange through the H(+)-K(+)-pump, formed by the F0F1-ATPase and the Trk H system, H(+)-K(+)-exchange via H(+)-K(+)-antiporter, formed by the F0 and the Trk G (core) system [1-2], and production of H2 in anaerobically grown E.coli are changed in the mutants with defects in components of formate hydrogen lyase complex, oxidizing formate to CO2 and H2. 2H+/K(+)-exchange and H2 production are destroyed, but H(+)-K(+)-exchange with a variable stoichiometry for N,N'-dicyclohexyl-carbodiimide-sensitive ion fluxes is displayed in the fdhF mutant E.coli FM911, where formate dehydrogenase(H) is absent. 2H+/K(+)-exchange does not occur, but H(+)-K(+)-exchange with variable stoichiometry for N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes and H2 production are observed in the uncD mutant E.coli AN817 with defect in beta subunit of the F1. Deletion of the hyc-operon in mutant E.coli HD700, led to absence of hydrogenase 3, destroys H(+)-K(+)-exchange and H2 production. H2 evaluation is shown in the E.coli K12(lambda) protoplasts, treated with toluene, by adding of NADH into the medium, containing ATP and K+. It is inhibited by N,N'-dicyclohexylcarbodiimide. H2 production is increased by adding of dithiothreitol, when NADH is changed by formate. It is lost in the mutants with defects in the F0 (E.coli AN936) or in the Trk A protein (E.coli TK2242). Dehydrogenase(H) and hydrogenase 3 are assumed to link mutually with a H(+)-K(+)-pump operation, reducing equivalents, necessary for a dithiol-disulfide interconversion within a mechanism of pump, are transferred from formate by means of dehydrogenase(H) to hydrogenase 3 through the F0F1 and the Trk H system to produce H2. It is assumed that hydrogenase 3 can interact with a mechanism of H(+)-K(+)-antiporter, NADH could serve as a donor of reducing equivalents. A role of thiol-groups and dithiol-disulfide interconversion in a functions of both mechanism for H(+)-K(+)-exchange is confirmed.
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PMID:[Role of components of formate-hydrogen-lyase in forming molecular hydrogen and their connection with proton-potassium exchange in anaerobically grown Escherichia coli]. 872 54

In Methanosarcina barkeri the transfer of the methyl group from methanol to 2-mercaptoethanesulfonic acid is catalyzed by the concerted action of two methyltransferases. The first one is the corrinoid-containing methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1), which binds the methyl group of methanol to its corrinoid prosthetic group. MT1 is only catalytically active when the cobalt atom of the corrinoid is present in the highly reduced Co(I) state. In the course of its purification and even during catalysis, MT1 becomes oxidatively inactivated. The enzyme, however, may be reductively reactivated by a suitable reducing system (hydrogen and hydrogenase), ATP, and an enzyme called methyltransferase activation protein (MAP). In order to elucidate its role in the reactivation process, MAP was purified to apparent homogeneity. The protein had an Mr = 60,000. Preincubation of the enzymic components involved with 8-azido-ATP or with ATP demonstrated MAP to be the primary site of action of ATP. In agreement herewith, the protein was autophosphorylated by [gamma-32P]ATP in a 1:1 stoichiometry. Phosphorylated MAP substituted for ATP in the activation of MT1, and the addition of increasing amounts of MAP phosphate resulted in a corresponding increase of active MT1. However, in the presence of limiting amounts of MAP, maximal activation of MT1 could be achieved during a lag phase provided ATP was present, indicating that MAP acts as a catalyst. This paper is the first to report on the presence, isolation, and function of a phosphorylated protein in a methanogenic archaeon.
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PMID:Purification and properties of an enzyme involved in the ATP-dependent activation of the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri. 879 94

Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes involved in the transmethylation reaction from methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 only binds the methyl group of methanol when the cobalt atom of its corrinoid prosthetic groups is present in the highly reduced Co(I) state. Formation of this redox state requires H2, hydrogenase, methyltransferase activation protein, and ATP. Optical and electron paramagnetic resonance spectroscopy studies were employed to determine the oxidation states and coordinating ligands of the corrinoids of MT1 during the activation process. Purified MT1 contained 1.7 corrinoids per enzyme with cobalt in the fully oxidized Co(III) state. Water and N-3 of the 5-hydroxybenzimidazolyl base served as the upper and lower ligands, respectively. Reduction to the Co(II) level was accomplished by H2 and hydrogenase. The cob(II)amide of MT1 had the base coordinated at this stage. Subsequent addition of methyltransferase activation protein and ATP resulted in the formation of base-uncoordinated Co(II) MT1. The activation mechanism is discussed within the context of a proposed model and compared to those described for other corrinoid-containing methyl group transferring proteins.
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PMID:Activation mechanism of methanol:5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri. 879 95

The HupT protein of Rhodobacter capsulatus, involved in negative regulation of hydrogenase gene expression, is predicted to be a histidine kinase on the basis of sequence comparisons. The protein was overproduced in Escherichia coli, purified to homogeneity, and demonstrated to autophosphorylate in vitro in the presence of [gamma-32P]ATP. An H217N hupt mutant was constructed, and the mutant protein was shown to have lost kinase activity. This result, and the fact that the phosphoryl group in phosphorylated HupT appeared to be bound to an N atom, support the suggestion from sequence comparisons that HupT is a histidine kinase, which can autophosphorylate on the His217 residue.
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PMID:Purification and in vitro phosphorylation of HupT, a regulatory protein controlling hydrogenase gene expression in Rhodobacter capsulatus. 900 58

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H2 plus hydrogenase purified from strain MC.
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PMID:Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC. 923 5


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