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)

Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of two-component regulatory systems present in a number of H2-oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine protein kinases. Introduction of the complete set of genes on a broad-host-range plasmid into A. eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H2. This repression was released by truncation of hoxJ. H2-dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H2-independent mode of control characteristic of A. eutrophus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H2 in response to the availability of the carbon and energy source. RNA dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.
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PMID:A hydrogen-sensing system in transcriptional regulation of hydrogenase gene expression in Alcaligenes species. 904 26

Oxidation of molecular hydrogen catalyzed by [NiFe] hydrogenases is a widespread mechanism of energy generation among prokaryotes. Biosynthesis of the H2-oxidizing enzymes is a complex process subject to positive control by H2 and negative control by organic energy sources. In this report we describe a novel signal transduction system regulating hydrogenase gene (hox) expression in the proteobacterium Alcaligenes eutrophus. This multicomponent system consists of the proteins HoxB, HoxC, HoxJ*, and HoxA. HoxB and HoxC share characteristic features of dimeric [NiFe] hydrogenases and form the putative H2 receptor that interacts directly or indirectly with the histidine protein kinase HoxJ*. A single amino acid substitution (HoxJ*G422S) in a conserved C-terminal glycine-rich motif of HoxJ* resulted in a loss of H2-dependent signal transduction and a concomitant block in autophosphorylating activity, suggesting that autokinase activity is essential for the response to H2. Whereas deletions in hoxB or hoxC abolished hydrogenase synthesis almost completely, the autokinase-deficient strain maintained high-level hox gene expression, indicating that the active sensor kinase exerts a negative effect on hox gene expression in the absence of H2. Substitutions of the conserved phosphoryl acceptor residue Asp55 in the response regulator HoxA (HoxAD55E and HoxAD55N) disrupted the H2 signal-transduction chain. Unlike other NtrC-like regulators, the altered HoxA proteins still allowed high-level transcriptional activation. The data presented here suggest a model in which the nonphosphorylated form of HoxA stimulates transcription in concert with a yet unknown global energy-responsive factor.
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PMID:A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus. 977 May 10

Previous genetic studies have revealed a multicomponent signal transduction chain, consisting of an H(2) sensor, a histidine protein kinase, and a response regulator, which controls hydrogenase gene transcription in the proteobacterium Ralstonia eutropha. In this study, we isolated the H(2) sensor and demonstrated that the purified protein forms a complex with the histidine protein kinase. Biochemical and spectroscopic analysis revealed that the H(2) sensor is a cytoplasmic [NiFe]-hydrogenase with unique features. The H(2)-oxidizing activity was 2 orders of magnitude lower than that of standard hydrogenases and insensitive to oxygen, carbon monoxide, and acetylene. Interestingly, only H(2) production but no HD formation was detected in the D(2)/H(+) exchange assay. Fourier transform infrared data showed an active site similar to that of standard [NiFe]-hydrogenases. It is suggested that the protein environment accounts for a restricted gas diffusion and for the typical kinetic parameters of the H(2) sensor. EPR analysis demonstrated that the [4Fe-4S] clusters within the small subunit were not reduced under hydrogen even in the presence of dithionite. Optical spectra revealed the presence of a novel, redox-active, n = 2 chromophore that is reduced by H(2). The possible involvement of this chromophore in signal transduction is discussed.
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PMID:The H2 sensor of Ralstonia eutropha. Biochemical characteristics, spectroscopic properties, and its interaction with a histidine protein kinase. 1127 70

Molecular hydrogen is widely used by microorganisms as a source of energy. One of the best studied aerobic hydrogen oxidizers, the beta-proteobacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), harbors two distinct [NiFe]-hydrogenases which catalyze the heterolytic cleavage of H2 into 2H+ and 2e-. The genes encoding the hydrogenase subunits are arranged in two large operons together with accessory and regulatory genes involved in hydrogenase biosynthesis. Both operons are transcribed from strong sigma54-dependent promoters. Transcription requires the activation by the HoxA protein, a member of the NtrC family of response regulators. HoxA is only active when H2 is present in the environment. H2 recognition is mediated by a signal transduction complex consisting of the soluble histidine protein kinase HoxJ and a regulatory [NiFe]-hydrogenase which acts as an H2 receptor. Biochemical and genetic data suggest that signal transduction between the RH and HoxJ involves an electron transport process. According to our current model the histidine protein kinase HoxJ inactivates HoxA by phosphorylation in the absence H2. This property of the HoxJ-HoxA regulator pair is quite different from the behaviour of common two-component regulatory systems. Phosphorylation of HoxA is blocked in the presence of H2 provided the RH can contact HoxJ and transmit the signal to the kinase. Furthermore, hydrogenase gene expression is subject to a global regulatory network in response to the carbon and energy source. HoxA is a major component of this epistatic control the molecular mechanism of which is not yet understood.
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PMID:The hydrogen-sensing apparatus in Ralstonia eutropha. 1193 56

Two [NiFe] hydrogenases enable the proteobacterium Ralstonia eutropha H16 to grow on molecular hydrogen as the sole energy source. A third [NiFe] hydrogenase (RH) acts as an H2 sensor in a multiple component signal transduction chain that controls hydrogenase gene transcription. The RH forms a dimeric heterodimer (HoxBC)2 in which HoxC contains the H2-sensing active site and HoxB the electron-transferring components including an organic, not yet identified redox cofactor. This oligomer forms a tight complex with the histidine protein kinase HoxJ. Both the sensor and the kinase were analysed by mutagenesis for functional domains that are instrumental in H2 signal transmission. A mutant deleted for a C-terminal peptide of 55 amino acids in HoxB lost its H2-sensing ability but still catalysed H2 oxidation. The mutant protein failed to form the dimeric heterodimer and a complex with HoxJ. The organic redox cofactor was no longer detectable in the truncated sensor. H2 sensing was also abolished by deletion of the PAS domain of HoxJ, indicating that this domain is involved in signal transduction. A truncated version of HoxJ consisting of only the input domain of the kinase was still capable of forming a complex with the RH. Mass determination of the purified HoxJ protein revealed that the kinase forms a homotetramer. The unique oligomeric structure of the H2-sensing complex with respect to its regulatory function is discussed.
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PMID:The H2-sensing complex of Ralstonia eutropha: interaction between a regulatory [NiFe] hydrogenase and a histidine protein kinase. 1500 94

H(2) is an attractive energy source for many microorganisms and is mostly consumed before it enters oxic habitats. Thus aerobic H(2)-oxidizing organisms receive H(2) only occasionally and in limited amounts. Metabolic adaptation requires a robust oxygen-tolerant hydrogenase enzyme system and special regulatory devices that enable the organism to respond rapidly to a changing supply of H(2). The proteobacterium Ralstonia eutropha strain H16 that harbours three [NiFe] hydrogenases perfectly meets these demands. The unusual biochemical and structural properties of the hydrogenases are described, including the strategies that confer O(2) tolerance to the NAD-reducing soluble hydrogenase and the H(2)-sensing regulatory hydrogenase. The regulatory hydrogenase that forms a complex with a histidine protein kinase recognizes H(2) in the environment and transmits the signal to a response regulator, which in turn controls transcription of the hydrogenase genes.
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PMID:A hydrogen-sensing multiprotein complex controls aerobic hydrogen metabolism in Ralstonia eutropha. 1566 76

Clostridium thermocellum is a thermophilic, anaerobic, cellulolytic bacterium that produces ethanol and acetic acid as major fermentation end products. The effect of growth conditions on gene expression in C. thermocellum ATCC 27405 was studied using cells grown in continuous culture under cellobiose or cellulose limitation over a approximately 10-fold range of dilution rates (0.013 to 0.16 h(-1)). Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of 17 genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes (recA and the 16S rRNA gene) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cyclic AMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed >10-fold-reduced levels of expression when grown on cellobiose at dilution rates of >0.05 h(-1): cipA (cellulosomal scaffolding protein), celS (exoglucanase), manA (mannanase), and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution.
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PMID:Expression of 17 genes in Clostridium thermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture. 1608 62

In proteobacteria capable of H(2) oxidation under (micro)aerobic conditions, hydrogenase gene expression is often controlled in response to the availability of H(2). The H(2)-sensing signal transduction pathway consists of a heterodimeric regulatory [NiFe]-hydrogenase (RH), a histidine protein kinase and a response regulator. To gain insights into the signal transmission from the Ni-Fe active site in the RH to the histidine protein kinase, conserved amino acid residues in the L0 motif near the active site of the RH large subunit of Ralstonia eutropha H16 were exchanged. Replacement of the strictly conserved Glu13 (E13N, E13L) resulted in loss of the regulatory, H(2)-oxidizing and D(2)/H(+) exchange activities of the RH. According to EPR and FTIR analysis, these RH derivatives contained fully assembled [NiFe] active sites, and para-/ortho-H(2) conversion activity showed that these centres were still able to bind H(2). This indicates that H(2) binding at the active site is not sufficient for the regulatory function of H(2) sensors. Replacement of His15, a residue unique in RHs, by Asp restored the consensus of energy-linked [NiFe]-hydrogenases. The respective RH mutant protein showed only traces of H(2)-oxidizing activity, whereas its D(2)/H(+)-exchange activity and H(2)-sensing function were almost unaffected. H(2)-dependent signal transduction in this mutant was less sensitive to oxygen than in the wild-type strain. These results suggest that H(2) turnover is not crucial for H(2) sensing. It may even be detrimental for the function of the H(2) sensor under high O(2) concentrations.
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PMID:Impact of alterations near the [NiFe] active site on the function of the H(2) sensor from Ralstonia eutropha. 1722 78

The regulatory Ni-Fe hydrogenase (RH) from the H(2)-oxidizing bacterium Ralstonia eutropha functions as an oxygen-resistant hydrogen sensor, which is composed of the large, active-site-containing HoxC subunit and the small subunit HoxB carrying Fe-S clusters. In vivo, the HoxBC subunits form a dimer designated as RH(wt). The RH(wt) protein transmits its signals to the histidine protein kinase HoxJ, which itself forms a homotetramer and a stable complex with RH(wt) (RH(wt)-HoxJ(wt)), located in the cytoplasm. In this study, we used X-ray absorption (XAS), electron paramagnetic resonance (EPR), and Fourier transform infrared (FTIR) spectroscopy to investigate the impact of various complexes between RH and HoxJ on the structural and electronic properties of the Ni-Fe active site and the Fe-S clusters. Aside from the RH(wt) protein and the RH(wt)-HoxJ(wt) complex, we investigated the RH(stop) protein, which consists of only one HoxB and HoxC unit due to the missing C-terminus of HoxB, as well as RH(wt)-HoxJ(Deltakinase), in which the histidine protein kinase lacks the transmitter domain. All constructs reacted with H(2), leading to the formation of the EPR-detectable Ni(III)-C state of the active site and to the reduction of Fe-S clusters detectable by XAS, thus corroborating that H(2) cleavage is independent of the presence of the HoxJ protein. In RH(stop), presumably one Fe-S cluster was lost during the preparation procedure. The coordination of the active site Ni in RH(stop) differed from that in RH(wt) and the RH(wt)-HoxJ complexes, in which additional Ni--O bonds were detected by XAS. The Ni--O bonds caused only very minor changes of the EPR g-values of the Ni-C and Ni-L states and of the IR vibrational frequencies of the diatomic CN(-) and CO ligands at the active-site Fe ion. Both one Fe-S cluster in HoxB and an oxygen-rich Ni coordination seem to be stabilized by RH dimerization involving the C-terminus of HoxB and by complex formation with HoxJ.
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PMID:Protein-protein complex formation affects the Ni-Fe and Fe-S centers in the H2-sensing regulatory hydrogenase from Ralstonia eutropha H16. 2034 Jan 24

Ruminococcus albus 7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO2, and 2.6 H2 when growing in batch culture and to 2 acetate, 2 CO2, and 4 H2 when growing in continuous culture in syntrophic association with H2-consuming microorganisms that keep the H2 partial pressure low. The organism uses NAD(+) and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO2, NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H2. Of all the enzymes involved, only the enzyme catalyzing the formation of H2 from NADH remained unknown. Here, we report that R. albus 7 grown in batch culture on glucose contained, besides a ferredoxin-dependent [FeFe]-hydrogenase (HydA2), a ferredoxin- and NAD-dependent electron-bifurcating [FeFe]-hydrogenase (HydABC) that couples the endergonic formation of H2 from NADH to the exergonic formation of H2 from reduced ferredoxin. Interestingly, hydA2 is adjacent to the hydS gene, which is predicted to encode an [FeFe]-hydrogenase with a C-terminal PAS domain. We showed that hydS and hydA2 are part of a larger transcriptional unit also harboring putative genes for a bifunctional acetaldehyde/ethanol dehydrogenase (Aad), serine/threonine protein kinase, serine/threonine protein phosphatase, and a redox-sensing transcriptional repressor. Since HydA2 and Aad are required only when R. albus grows at high H2 partial pressures, HydS could be a H2-sensing [FeFe]-hydrogenase involved in the regulation of their biosynthesis.
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PMID:Hydrogen formation and its regulation in Ruminococcus albus: involvement of an electron-bifurcating [FeFe]-hydrogenase, of a non-electron-bifurcating [FeFe]-hydrogenase, and of a putative hydrogen-sensing [FeFe]-hydrogenase. 2515 86


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