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)

The NADP-reducing hydrogenase complex from Desulfovibrio fructosovorans is a heterotetramer encoded by the hndABCD operon. Sequence analysis indicates that the HndC subunit (52 kDa) corresponds to the NADP-reducing unit, and the HndD subunit (63.5 kDa) is homologous to Clostridium pasteurianum hydrogenase. The role of HndA and HndB subunits (18.8 kDa and 13.8 kDa, respectively) in the complex remains unknown. The HndA subunit belongs to the [2Fe-2S] ferredoxin family typified by C. pasteurianum ferredoxin. HndA is organized into two independent structural domains, and we report in the present work the NMR structure of its C-terminal domain, HndAc. HndAc has a thioredoxin-like fold consisting in four beta-strands and two relatively long helices. The [2Fe-2S] cluster is located near the surface of the protein and bound to four cysteine residues particularly well conserved in this class of proteins. Electron exchange between the HndD N-terminal [2Fe-2S] domain (HndDN) and HndAc has been previously evidenced, and in the present studies we have mapped the binding site of the HndDN domain on HndAc. A structural analysis of HndB indicates that it is a FeS subunit with 41% similarity with HndAc and it contains a possible thioredoxin-like fold. Our data let us propose that HndAc and HndB can form a heterodimeric intermediate in the electron transfer between the hydrogenase (HndD) active site and the NADP reduction site in HndC.
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PMID:Solution structure of HndAc: a thioredoxin-like domain involved in the NADP-reducing hydrogenase complex. 1673 71

The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 pmol Hz/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.
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PMID:Photoinduced hydrogen production by direct electron transfer from photosystem I cross-linked with cytochrome c3 to [NiFe]-hydrogenase. 1683 69

The main catalytic properties of the Hox type hydrogenase isolated from the Gloeocapsa alpicola cells have been studied. The enzyme effectively catalyzes reactions of oxidation and evolution of H2 in the presence of methyl viologen (MV) and benzyl viologen (BV). The rates of these reactions in the interaction with the physiological electron donor/acceptor NADH/NAD+ are only 3-8% of the MV(BV)-dependent values. The enzyme interacts with NADP+ and NADPH, but is more specific to NAD+ and NADH. Purification of the hydrogenase was accompanied by destruction of its multimeric structure and the loss of ability to interact with pyridine nucleotides with retained activity of the hydrogenase component (HoxYH). To show the catalytic activity, the enzyme requires reductive activation, which occurs in the presence of H2, and NADH accelerates this process. The final hydrogenase activity depends on the redox potential of the activation medium (E(h)). At pH 7.0, the enzyme activity in the MV-dependent oxidation of H2 increased with a decrease in E(h) from -350 mV and reached the maximum at E(h) of about -390 mV. However, the rate of H2 oxidation in the presence of NAD+ in the E(h) range under study was virtually constant and equal to 7-8% of the maximal rate of H2 oxidation in the presence of MV.
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PMID:Characterization of catalytic properties of hydrogenase isolated from the unicellular cyanobacterium Gloeocapsa alpicola CALU 743. 1722 91

A hydrogen-oxidizing bacterium strain AH-24 was isolated, which was classified in the genus Hydrogenophaga, based on the 16S rRNA gene sequence. The isolate possessed a typical yellow pigment of Hydrogenophaga species. Its closest relative was Hydrogenophaga pseudoflava, but the assimilation profile of sugar compounds resembled that of no species of Hydrogenophaga. The optimum temperature and pH for autotrophic growth were, respectively, 33-35 degrees C and 7.0. Most hydrogenase activity (benzyl viologen reducing activity) was localized in the membrane fraction (MF), but NAD(P)-reducing hydrogenase activity was detected in neither the membrane nor the soluble fractions. Cytochromes b561 and c551 were present in MF; both were reduced when hydrogen was supplied to the oxidized MF, suggesting involvement in respiratory H2 oxidation as electron carriers. Cytochrome b561 was inferred to function as the redox partner of the membrane-bound hydrogenase.
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PMID:Isolation and characterization of a new facultatively autotrophic hydrogen-oxidizing Betaproteobacterium, Hydrogenophaga sp. AH-24. 1803 33

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H2 evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80 degrees C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H2 production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low Km for H2 (approximately 20 microM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 microM dissolved CO). Since the Km for NADP+ is approximately 37 microM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.
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PMID:Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus. 1815 74

We have found the transcript of one of at least six ferredoxin encoding genes of the green alga Chlamydomonas reinhardtii, FDX5, strongly accumulating in anaerobiosis, indicating a vital role of the encoded protein in the anaerobic metabolism of the cells. According to absorption and electron paramagnetic resonance spectroscopy, Fdx5 is a plant-type [2Fe2S]-ferredoxin with a redox potential similar to that of the ferredoxin PetF. However, although Fdx5 seems to be located in the chloroplast, it is not able to photoreduce nicotinamide adenine dinucleotide phosphate (NADP(+)) via ferredoxin-NADP-reductase, nor to be an electron donor to the plastidic [FeFe]-hydrogenase HydA1. Thus, Fdx5 seems to have a special role in a yet to be identified anaerobic pathway.
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PMID:A novel, anaerobically induced ferredoxin in Chlamydomonas reinhardtii. 1910 55

Protists that live in low oxygen conditions often oxidize pyruvate to acetate via anaerobic ATP-generating pathways. Key enzymes that commonly occur in these pathways are pyruvate:ferredoxin oxidoreductase (PFO) and [FeFe]-hydrogenase (H(2)ase) as well as the associated [FeFe]-H(2)ase maturase proteins HydE, HydF, and HydG. Determining the origins of these proteins in eukaryotes is of key importance to understanding the origins of anaerobic energy metabolism in microbial eukaryotes. We conducted a comprehensive search for genes encoding these proteins in available whole genomes and expressed sequence tag data from diverse eukaryotes. Our analyses of the presence/absence of eukaryotic PFO, [FeFe]-H(2)ase, and H(2)ase maturase sequences across eukaryotic diversity reveal orthologs of these proteins encoded in the genomes of a variety of protists previously not known to contain them. Our phylogenetic analyses revealed: 1) extensive lateral gene transfers of both PFO and [FeFe]-H(2)ase in eubacteria, 2) decreased support for the monophyly of eukaryote PFO domains, and 3) that eukaryotic [FeFe]-H(2)ases are not monophyletic. Although there are few eukaryote [FeFe]-H(2)ase maturase orthologs characterized, phylogenies of these proteins do recover eukaryote monophyly, although a consistent eubacterial sister group for eukaryotic homologs could not be determined. An exhaustive search for these five genes in diverse genomes from two representative eubacterial groups, the Clostridiales and the alpha-proteobacteria, shows that although these enzymes are nearly universally present within the former group, they are very rare in the latter. No alpha-proteobacterial genome sequenced to date encodes all five proteins. Molecular phylogenies and the extremely restricted distribution of PFO, [FeFe]-H(2)ases, and their associated maturases within the alpha-proteobacteria do not support a mitochondrial origin for these enzymes in eukaryotes. However, the unexpected prevalence of PFO, pyruvate:NADP oxidoreductase, [FeFe]-H(2)ase, and the maturase proteins encoded in genomes of diverse eukaryotes indicates that these enzymes have an important role in the evolution of microbial eukaryote energy metabolism.
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PMID:Phylogenetic distributions and histories of proteins involved in anaerobic pyruvate metabolism in eukaryotes. 1980 39

Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.
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PMID:Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production. 2046 92

Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H(2)), a carbon-neutral, renewable product derived from water, in low yields during autofermentation (anaerobic metabolism) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have constructed a mutant (the ldhA mutant) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to D-lactate, the major fermentative reductant sink in this AMOP. Both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolomic methods have shown that autofermentation by the ldhA mutant resulted in no D-lactate production and higher concentrations of excreted acetate, alanine, succinate, and hydrogen (up to 5-fold) compared to that by the wild type. The measured intracellular NAD(P)(H) concentrations demonstrated that the NAD(P)H/NAD(P)(+) ratio increased appreciably during autofermentation in the ldhA strain; we propose this to be the principal source of the observed increase in H(2) production via an NADH-dependent, bidirectional [NiFe] hydrogenase. Despite the elevated NAD(P)H/NAD(P)(+) ratio, no decrease was found in the rate of anaerobic conversion of stored carbohydrates. The measured energy conversion efficiency (ECE) from biomass (as glucose equivalents) converted to hydrogen in the ldhA mutant is 12%. Together with the unimpaired photoautotrophic growth of the ldhA mutant, these attributes reveal that metabolic engineering is an effective strategy to enhance H(2) production in AMOPs without compromising viability.
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PMID:Redirecting reductant flux into hydrogen production via metabolic engineering of fermentative carbon metabolism in a cyanobacterium. 2054 51

Photosynthetic water splitting, coupled to hydrogenase-catalyzed hydrogen production, is considered a promising clean, renewable source of energy. It is widely accepted that the oxygen sensitivity of hydrogen production, combined with competition between hydrogenases and NADPH-dependent carbon dioxide fixation are the main limitations for its commercialization. Here we provide evidence that, under the anaerobic conditions that support hydrogen production, there is a significant loss of photosynthetic electrons toward NADPH production in vitro. To elucidate the basis for competition, we bioengineered a ferredoxin-hydrogenase fusion and characterized hydrogen production kinetics in the presence of Fd, ferredoxin:NADP(+)-oxidoreductase (FNR), and NADP(+). Replacing the hydrogenase with a ferredoxin-hydrogenase fusion switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction. These results suggest a new direction for improvement of biohydrogen production and a means to further resolve the mechanisms that control partitioning of photosynthetic electron transport.
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PMID:Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro. 2160 30


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