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Yasuo Igarashi - One of the best experts on this subject based on the ideXlab platform.
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nitrate reductases in Hydrogenobacter thermophilus with evolutionarily ancient features distinctive localization and electron transfer
Molecular Microbiology, 2017Co-Authors: Masafumi Kameya, Yasuo Igarashi, Hiroyuki Arai, Haruna Kanbe, Masaharu IshiiAbstract:Summary Dissimilatory nitrate reductase (NAR) and assimilatory nitrate reductase (NAS) serve as key enzymes for nitrogen catabolism and anabolism in many organisms. We purified NAR and NAS from H. thermophilus, a hydrogen-oxidizing chemolithoautotroph belonging to the phylogenetically deepest branch in the Bacteria domain. Physiological contribution of these enzymes to nitrate respiration and assimilation was clarified by transcriptomic analysis and gene disruption experiments. These enzymes showed several features unreported in bacteria, such as the periplasmic orientation of NAR anchored with a putative transmembrane subunit and the specific electron transfer from a [4Fe-4S]-type ferredoxin to NAS. While some of their enzymatic properties are shared with NARs from archaea and with NASs from phototrophs, phylogenetic analysis indicated that H. thermophilus NAR and NAS have deep evolutionary origins that cannot be explained by a recent horizontal gene transfer event from archaea and phototrophs. These findings revealed the diversity of NAR and NAS in nonphotosynthetic bacteria, and they also implied that the outward orientation of NAR and the ferredoxin-dependent electron transfer of NAS are evolutionarily ancient features preserved in H. thermophilus. This article is protected by copyright. All rights reserved.
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adaptation of Hydrogenobacter thermophilus toward oxidative stress triggered by high expression of alkyl hydroperoxide reductase
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Yuya Sato, Yasuo Igarashi, Hiroyuki Arai, Masaharu IshiiAbstract:Ferriperoxin is a novel peroxidase essential for aerobiosis of Hydrogenobacter thermophilus. Although the ferriperoxin-deficient mutant (Δfpx) was unable to grow aerobically, a suppressor mutant capable of aerobic growth was obtained after long aerobic cultivation. The alkyl hydroperoxide reductase gene was significantly upregulated in the suppressor mutant, indicating that the enzyme counteracts oxidative stress in the absence of ferriperoxin.
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transcriptome analyses of metabolic enzymes in thiosulfate and hydrogen grown Hydrogenobacter thermophilus cells
Bioscience Biotechnology and Biochemistry, 2012Co-Authors: Yuya Sato, Masaharu Ishii, Hiroyuki Arai, Yoshihiro Sambongi, Haruna Kanbe, Hirosuke Miyano, Yasuo IgarashiAbstract:Hydrogenobacter thermophilus is a chemolithoautotroph that utilizes not only hydrogen (H2) but also thiosulfate as sole source of energy and assimilates carbon dioxide via the reductive tricarboxylic acid (RTCA) cycle. We systematically carried out transcriptome analysis of metabolic enzymes in both H2- and thiosulfate-grown H. thermophilus cells. The analysis indicated that the expression of hydrogenase genes is repressed under thiosulfate oxidation conditions as compared with H2 oxidation conditions. This was confirmed by enzyme assay. In contrast, some genes for sulfur metabolism, including sox genes, showed almost the same expression levels under both conditions. In addition, the genes for the RTCA cycle showed high expression levels under both conditions. It was suggested that sulfur metabolism and the RTCA cycle function as forms of basal metabolism, and H2 oxidation is inducible. Switching of H2 oxidation can be advantageous for the lifestyle of this bacterium in nature.
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crystallization and preliminary x ray diffraction analysis of a novel type of phosphoserine phosphatase from Hydrogenobacter thermophilus tk 6
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2012Co-Authors: Yoko Chiba, Hiroyuki Arai, Yasuo Igarashi, Shoichiro Horita, Jun Ohtsuka, Koji Nagata, Masaru Tanokura, Masaharu IshiiAbstract:Two novel-type phosphoserine phosphatases (PSPs) with unique substrate specificity from the thermophilic and hydrogen-oxidizing bacterium Hydrogenobacter thermophilus TK-6 have previously been identified. Here, one of the PSPs (iPSP1) was heterologously expressed in Escherichia coli, purified and crystallized. Diffraction-quality crystals were obtained by the sitting-drop vapour-diffusion method using PEG 4000 as the precipitant. Two diffraction data sets with resolution ranges of 45.0–2.50 and 45.0–1.50 A were collected from a single crystal and were merged to give a highly complete data set. The space group of the crystal was identified as primitive orthorhombic P212121, with unit-cell parameters a = 49.8, b = 73.6, c = 124.3 A. The calculated Matthews coefficient (VM = 2.32 A3 Da−1) indicated that the crystal contained one iPSP1 complex per asymmetric unit.
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dissimilatory fe iii reduction by cytochrome c 552 in a thermophilic obligately chemolithoautotrophic bacterium Hydrogenobacter thermophilus tk 6
Journal of Japanese Society for Extremophiles, 2010Co-Authors: Naoki Yoshida, Masaharu Ishii, Hiroyuki Arai, Hiroshi Saiki, Naoya Ohmura, Norio Matsumoto, Kazuhiro Sasaki, Yasuo IgarashiAbstract:A thermophilic, obligately chemolithoauto- trophic H2-oxidizing bacterium, Hydrogenobacter thermophilus TK-6 can utilize O2 or NO3- as an electron acceptor. Here we show that this bacterium can grow autotrophically by the reduction of Fe(III)-EDTA or Fe(III)-DTPA (diethylene-triamine-pentaacetic acid) as an electron acceptor, through oxidation of H2 as an electron donor. A 7.6 kDa c-type cytochrome was purified from soluble fraction as the iron reducing protein and the protein was identified as cytochrome c-552. A rate constant for the reduction of Fe(III)-EDTA by the cytochrome was kinetically determined to be 1.73 x 107 s-1 by stopped-flow spectrophotometry, which is more than 20-fold higher than the constants of other iron reductases. The redox potential was measured as E = +0.27 V (vs NHE [Normal Hydrogen Electrode]), which is high enough to reduce Fe(III)-EDTA. These results suggest that cytochrome c-552 works as a terminal electron donor for Fe(III) in the periplasmic space.
Masaharu Ishii - One of the best experts on this subject based on the ideXlab platform.
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phosphoserine phosphatase is required for serine and one carbon unit synthesis in Hydrogenobacter thermophilus
Journal of Bacteriology, 2017Co-Authors: Keug Tae Kim, Yoko Chiba, Hiroyuki Arai, Azusa Kobayashi, Masaharu IshiiAbstract:Hydrogenobacter thermophilus is an obligate chemolithoautotrophic bacterium of the phylum Aquificae and is capable of fixing carbon dioxide through the reductive tricarboxylic acid (TCA) cycle. The recent discovery of two novel-type phosphoserine phosphatases (PSPs) in H. thermophilus suggests the presence of a phosphorylated serine biosynthesis pathway; however, the physiological role of these novel-type metal-independent PSPs (iPSPs) in H. thermophilus has not been confirmed. In the present study, a mutant strain with a deletion of pspA, the catalytic subunit of iPSPs, was constructed and characterized. The generated mutant was a serine auxotroph, suggesting that the novel-type PSPs and phosphorylated serine synthesis pathway are essential for serine anabolism in H. thermophilus. As an autotrophic medium supplemented with glycine did not support the growth of the mutant, the reversible enzyme serine hydroxymethyltransferase does not appear to synthesize serine from glycine and may therefore generate glycine and 5,10-CH2-tetrahydrofolate (5,10-CH2-THF) from serine. This speculation is supported by the lack of glycine cleavage activity, which is needed to generate 5,10-CH2-THF, in H. thermophilus Determining the mechanism of 5,10-CH2-THF synthesis is important for understanding the fundamental anabolic pathways of organisms, because 5,10-CH2-THF is a major one-carbon donor that is used for the synthesis of various essential compounds, including nucleic and amino acids. The findings from the present experiments using a pspA deletion mutant have confirmed the physiological role of iPSPs as serine producers and show that serine is a major donor of one-carbon units in H. thermophilusIMPORTANCE Serine biosynthesis and catabolism pathways are intimately related to the metabolism of 5,10-CH2-THF, a one-carbon donor that is utilized for the biosynthesis of various essential compounds. For this reason, determining the mechanism of serine synthesis is important for understanding the fundamental anabolic pathways of microorganisms. In the present study, we experimentally confirmed that a novel phosphoserine phosphatase in the obligate chemolithoautotrophic bacterium Hydrogenobacter thermophilus is essential for serine biosynthesis. This finding indicates that serine is synthesized from an intermediate of gluconeogenesis in H. thermophilus In addition, because glycine cleavage system activity and genes encoding an enzyme capable of producing 5,10-CH2-THF were not detected, serine appears to be the major one-carbon donor to tetrahydrofolate (THF) in H. thermophilus.
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nitrate reductases in Hydrogenobacter thermophilus with evolutionarily ancient features distinctive localization and electron transfer
Molecular Microbiology, 2017Co-Authors: Masafumi Kameya, Yasuo Igarashi, Hiroyuki Arai, Haruna Kanbe, Masaharu IshiiAbstract:Summary Dissimilatory nitrate reductase (NAR) and assimilatory nitrate reductase (NAS) serve as key enzymes for nitrogen catabolism and anabolism in many organisms. We purified NAR and NAS from H. thermophilus, a hydrogen-oxidizing chemolithoautotroph belonging to the phylogenetically deepest branch in the Bacteria domain. Physiological contribution of these enzymes to nitrate respiration and assimilation was clarified by transcriptomic analysis and gene disruption experiments. These enzymes showed several features unreported in bacteria, such as the periplasmic orientation of NAR anchored with a putative transmembrane subunit and the specific electron transfer from a [4Fe-4S]-type ferredoxin to NAS. While some of their enzymatic properties are shared with NARs from archaea and with NASs from phototrophs, phylogenetic analysis indicated that H. thermophilus NAR and NAS have deep evolutionary origins that cannot be explained by a recent horizontal gene transfer event from archaea and phototrophs. These findings revealed the diversity of NAR and NAS in nonphotosynthetic bacteria, and they also implied that the outward orientation of NAR and the ferredoxin-dependent electron transfer of NAS are evolutionarily ancient features preserved in H. thermophilus. This article is protected by copyright. All rights reserved.
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discovery of an intermolecular disulfide bond required for the thermostability of a heterodimeric protein from the thermophile Hydrogenobacter thermophilus
Bioscience Biotechnology and Biochemistry, 2016Co-Authors: Keug Tae Kim, Yoko Chiba, Hiroyuki Arai, Masaharu IshiiAbstract:Factors that increase protein thermostability are of considerable interest in both scientific and industrial fields. Disulfide bonds are one of such factors that increase thermostability, but are rarely found in intracellular proteins because of the reducing environment of the cytosol. Here, we report the first example of an intermolecular disulfide bond between heteromeric subunits of a novel-type phosphoserine phosphatase from a thermophilic bacterium Hydrogenobacter thermophilus, which contributes to the protein thermostability at the physiological temperature. Comparison of remaining soluble proteins between wild-type and cysteine-deleted mutant using SDS-PAGE revealed that the disulfide bond increases the thermostability of the whole protein by tightly connecting a subunit with low solubility to the partner with higher solubility. Furthermore, it was strongly suggested that the disulfide bond is formed and contributes to the stability in vivo. This finding will open new avenues for the design of proteins with increased thermostability.
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adaptation of Hydrogenobacter thermophilus toward oxidative stress triggered by high expression of alkyl hydroperoxide reductase
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Yuya Sato, Yasuo Igarashi, Hiroyuki Arai, Masaharu IshiiAbstract:Ferriperoxin is a novel peroxidase essential for aerobiosis of Hydrogenobacter thermophilus. Although the ferriperoxin-deficient mutant (Δfpx) was unable to grow aerobically, a suppressor mutant capable of aerobic growth was obtained after long aerobic cultivation. The alkyl hydroperoxide reductase gene was significantly upregulated in the suppressor mutant, indicating that the enzyme counteracts oxidative stress in the absence of ferriperoxin.
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tk 6 from Hydrogenobacter thermophilus purification and characterization of atp citrate
2013Co-Authors: Masaharu Ishii, Y Igarashi, T KodamaAbstract:drase (31,000), soybdan trypsin inhibitor (21,500), and lyso-zyme (14,400).Gel filtration analysis. Purified enzyme solution was con-densed by the procddure described above. The solution,together with molecular weight standards, was put on acolumn (3.2 by 100 cm) of Toyopearl 650S preequilibratedwith 100 mMTris hydrochloride (pH 8.0) containing 1 mMMgCl2, 100 puM ATP, and 10 mM2-mercaptoethanol. Pro-teins were eluted with the same buffer, and the-eluate wasfractionated (5 gperfraction). ATP:citrate lyase activity andA280 ofthe eluate were mneasured, andthe molecular weightof native enzyme was determined. The molecular weightstandards were thyroglobulin (670,000), gamma globulin(158,000), ovalbumin (44,000), myoglobin (17,000), and vita-min B12 (1,350).Determination of Km values for each component. The
Hiroyuki Arai - One of the best experts on this subject based on the ideXlab platform.
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phosphoserine phosphatase is required for serine and one carbon unit synthesis in Hydrogenobacter thermophilus
Journal of Bacteriology, 2017Co-Authors: Keug Tae Kim, Yoko Chiba, Hiroyuki Arai, Azusa Kobayashi, Masaharu IshiiAbstract:Hydrogenobacter thermophilus is an obligate chemolithoautotrophic bacterium of the phylum Aquificae and is capable of fixing carbon dioxide through the reductive tricarboxylic acid (TCA) cycle. The recent discovery of two novel-type phosphoserine phosphatases (PSPs) in H. thermophilus suggests the presence of a phosphorylated serine biosynthesis pathway; however, the physiological role of these novel-type metal-independent PSPs (iPSPs) in H. thermophilus has not been confirmed. In the present study, a mutant strain with a deletion of pspA, the catalytic subunit of iPSPs, was constructed and characterized. The generated mutant was a serine auxotroph, suggesting that the novel-type PSPs and phosphorylated serine synthesis pathway are essential for serine anabolism in H. thermophilus. As an autotrophic medium supplemented with glycine did not support the growth of the mutant, the reversible enzyme serine hydroxymethyltransferase does not appear to synthesize serine from glycine and may therefore generate glycine and 5,10-CH2-tetrahydrofolate (5,10-CH2-THF) from serine. This speculation is supported by the lack of glycine cleavage activity, which is needed to generate 5,10-CH2-THF, in H. thermophilus Determining the mechanism of 5,10-CH2-THF synthesis is important for understanding the fundamental anabolic pathways of organisms, because 5,10-CH2-THF is a major one-carbon donor that is used for the synthesis of various essential compounds, including nucleic and amino acids. The findings from the present experiments using a pspA deletion mutant have confirmed the physiological role of iPSPs as serine producers and show that serine is a major donor of one-carbon units in H. thermophilusIMPORTANCE Serine biosynthesis and catabolism pathways are intimately related to the metabolism of 5,10-CH2-THF, a one-carbon donor that is utilized for the biosynthesis of various essential compounds. For this reason, determining the mechanism of serine synthesis is important for understanding the fundamental anabolic pathways of microorganisms. In the present study, we experimentally confirmed that a novel phosphoserine phosphatase in the obligate chemolithoautotrophic bacterium Hydrogenobacter thermophilus is essential for serine biosynthesis. This finding indicates that serine is synthesized from an intermediate of gluconeogenesis in H. thermophilus In addition, because glycine cleavage system activity and genes encoding an enzyme capable of producing 5,10-CH2-THF were not detected, serine appears to be the major one-carbon donor to tetrahydrofolate (THF) in H. thermophilus.
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nitrate reductases in Hydrogenobacter thermophilus with evolutionarily ancient features distinctive localization and electron transfer
Molecular Microbiology, 2017Co-Authors: Masafumi Kameya, Yasuo Igarashi, Hiroyuki Arai, Haruna Kanbe, Masaharu IshiiAbstract:Summary Dissimilatory nitrate reductase (NAR) and assimilatory nitrate reductase (NAS) serve as key enzymes for nitrogen catabolism and anabolism in many organisms. We purified NAR and NAS from H. thermophilus, a hydrogen-oxidizing chemolithoautotroph belonging to the phylogenetically deepest branch in the Bacteria domain. Physiological contribution of these enzymes to nitrate respiration and assimilation was clarified by transcriptomic analysis and gene disruption experiments. These enzymes showed several features unreported in bacteria, such as the periplasmic orientation of NAR anchored with a putative transmembrane subunit and the specific electron transfer from a [4Fe-4S]-type ferredoxin to NAS. While some of their enzymatic properties are shared with NARs from archaea and with NASs from phototrophs, phylogenetic analysis indicated that H. thermophilus NAR and NAS have deep evolutionary origins that cannot be explained by a recent horizontal gene transfer event from archaea and phototrophs. These findings revealed the diversity of NAR and NAS in nonphotosynthetic bacteria, and they also implied that the outward orientation of NAR and the ferredoxin-dependent electron transfer of NAS are evolutionarily ancient features preserved in H. thermophilus. This article is protected by copyright. All rights reserved.
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discovery of an intermolecular disulfide bond required for the thermostability of a heterodimeric protein from the thermophile Hydrogenobacter thermophilus
Bioscience Biotechnology and Biochemistry, 2016Co-Authors: Keug Tae Kim, Yoko Chiba, Hiroyuki Arai, Masaharu IshiiAbstract:Factors that increase protein thermostability are of considerable interest in both scientific and industrial fields. Disulfide bonds are one of such factors that increase thermostability, but are rarely found in intracellular proteins because of the reducing environment of the cytosol. Here, we report the first example of an intermolecular disulfide bond between heteromeric subunits of a novel-type phosphoserine phosphatase from a thermophilic bacterium Hydrogenobacter thermophilus, which contributes to the protein thermostability at the physiological temperature. Comparison of remaining soluble proteins between wild-type and cysteine-deleted mutant using SDS-PAGE revealed that the disulfide bond increases the thermostability of the whole protein by tightly connecting a subunit with low solubility to the partner with higher solubility. Furthermore, it was strongly suggested that the disulfide bond is formed and contributes to the stability in vivo. This finding will open new avenues for the design of proteins with increased thermostability.
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the crystal structure of siroheme decarboxylase in complex with iron uroporphyrin iii reveals two essential histidine residues
Journal of Molecular Biology, 2014Co-Authors: Kristin Haufschildt, Stefan Schmelz, Theresa M Kriegler, Dirk W Heinz, Judith Streif, Alexander Neumann, Hiroyuki Arai, Gunhild LayerAbstract:Abstract The isobacteriochlorin heme d 1 serves as an essential cofactor in the cytochrome cd 1 nitrite reductase NirS that plays an important role for denitrification. During the biosynthesis of heme d 1 , the enzyme siroheme decarboxylase catalyzes the conversion of siroheme to 12,18-didecarboxysiroheme. This enzyme was discovered recently (Bali S, Lawrence AD, Lobo SA, Saraiva LM, Golding BT, Palmer DJ et al. Molecular hijacking of siroheme for the synthesis of heme and d 1 heme. Proc Natl Acad Sci USA 2011;108:18260–5) and is only scarcely characterized. Here, we present the crystal structure of the siroheme decarboxylase from Hydrogenobacter thermophilus representing the first three-dimensional structure for this type of enzyme. The overall structure strikingly resembles those of transcriptional regulators of the Lrp/AsnC family. Moreover, the structure of the enzyme in complex with a substrate analog reveals first insights into its active-site architecture. Through site-directed mutagenesis and subsequent biochemical characterization of the enzyme variants, two conserved histidine residues within the active site are identified to be involved in substrate binding and catalysis. Based on our results, we propose a potential catalytic mechanism for the enzymatic reaction catalyzed by the siroheme decarboxylase.
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adaptation of Hydrogenobacter thermophilus toward oxidative stress triggered by high expression of alkyl hydroperoxide reductase
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Yuya Sato, Yasuo Igarashi, Hiroyuki Arai, Masaharu IshiiAbstract:Ferriperoxin is a novel peroxidase essential for aerobiosis of Hydrogenobacter thermophilus. Although the ferriperoxin-deficient mutant (Δfpx) was unable to grow aerobically, a suppressor mutant capable of aerobic growth was obtained after long aerobic cultivation. The alkyl hydroperoxide reductase gene was significantly upregulated in the suppressor mutant, indicating that the enzyme counteracts oxidative stress in the absence of ferriperoxin.
Tohru Kodama - One of the best experts on this subject based on the ideXlab platform.
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Chemical structure of a novel aminophospholipid from Hydrogenobacter thermophilus strain TK-6.
Journal of bacteriology, 2001Co-Authors: Jun-ichiro Yoshino, Masaharu Ishii, Tohru Kodama, Yasumasa Sugiyama, Shohei Sakuda, Hiromichi Nagasawa, Yasuo IgarashiAbstract:The phospholipid composition of Hydrogenobacter thermophilus strain TK-6, an obligately chemolithoautotrophic, extremely thermophilic hydrogen bacterium, was analyzed. Two of four phospholipids detected from the strain were assumed to be phosphatidylinositol and phosphatidylglycerol. An aminophospholipid named PX, whose content among the phospholipids was 65%, was found to have a novel chemical structure by analysis of the dilyso form with nuclear magnetic resonance and fast atom bombardment-mass spectrometry (FAB-MS) and by analysis of the intact PX with FAB-MS as 1,2-diacyl-3-O-(phospho-2-O-(1-amino)-2,3,4,5-pentanetetrol)-sn-glycerol. Structurally similar phospholipids have been identified in Methanospirillum hungatei, Methanolacinia paynteri, and Methanogenium cariaci, which all belong to the Archaea.
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purification and characterization of membrane bound hydrogenase from Hydrogenobacter thermophilus strain tk 6 an obligately autotrophic thermophilic hydrogen oxidizing bacterium
Bioscience Biotechnology and Biochemistry, 2000Co-Authors: Masaharu Ishii, Tohru Kodama, Seiichi Takishita, Jun-ichiro Yoshino, Toshio Iwasaki, Yuwadee Peerapornpisal, Yasuo IgarashiAbstract:A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with alpha2beta2 structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent Km of around 300 microM and with a turnover number around 2900 (min(-1)). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilus strain TK-6.
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Reductive TCA cycle in an aerobic bacterium, Hydrogenobacter thermophilus strain TK-6
Studies in Surface Science and Catalysis, 1998Co-Authors: Masaharu Ishii, Ki Seok Yoon, Yasufumi Ueda, Tohru Kodama, Toshihiro Ochiai, Nare Yun, Seiichi Takishita, Yasuo IgarashiAbstract:Publisher Summary Hydrogenobacter thermophilus strain TK-6 is an aerobic thermophilic hydrogen-oxidizing bacterium isolated from hot spring in Izu, Japan. As for CO 2 fixation pathway of the strain, 14 CO 2 labeling experiment, in vitro enzyme assays, and purification of ATP:citrate lyase in the group supported the idea that the reductive TCA cycle is operative in strain TK-6. However, identification of a strong reductant that is needed for the pyruvate synthase and 2-oxoglutarate synthase and clarification of an electron transport system were definitely needed. H. thermophilus was shown to belong to a very early branching order, the Aquificales . Also, strain TK-6 was isolated from hot spring, which implies that the strain may have its original ecological niche underground where little or no oxygen exists. Taking these things into consideration, it is of great interest to examine if the strain has its ability to grow anaerobically.
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Heterologous expression of Hydrogenobacter thermophilus cytochrome c-552 in the periplasm of Pseudomonas aeruginosa
Journal of Fermentation and Bioengineering, 1998Co-Authors: Yan Zhang, Hiroyuki Arai, Yasuo Igarashi, Yoshihiro Sambongi, Tohru KodamaAbstract:Thermostable cytochrome c-552 of Hydrogenobacter thermophilus was heterologously expressed in the periplasm of Pseudomonas aeruginosa using a fusion gene coding for the P. aeruginosa cytochrome c-551 signal peptide and the H. thermophilus mature cytochrome c-552. The values of the thermal denaturation parameters of periplasmically expressed cytochrome c-552 were identical to those of the authentic protein purified from H. thermophilus.
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purification and characterization of pyruvate ferredoxin oxidoreductase from Hydrogenobacter thermophilus tk 6
Archives of Microbiology, 1997Co-Authors: Ki Seok Yoon, Masaharu Ishii, Tohru Kodama, Yasuo IgarashiAbstract:Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH optimum was 7.6–7.8. The apparent K m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t 50%) at 70° C was approximately 8 h.
Masahiro Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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carboxylation reaction catalyzed by 2 oxoglutarate ferredoxin oxidoreductases from Hydrogenobacter thermophilus
Extremophiles, 2010Co-Authors: Masahiro Yamamoto, Masaharu Ishii, Hiroyuki Arai, Takeshi Ikeda, Yasuo IgarashiAbstract:Hydrogenobacter thermophilus TK-6 is a thermophilic, chemolithoautotrophic, hydrogen-oxidizing bacterium that fixes carbon dioxide via the reductive tricarboxylic acid (rTCA) cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is the key enzyme in this cycle that fixes carbon dioxide. The genome of strain TK-6 encodes at least two distinct OGOR enzymes, termed For and Kor. We report here a method for measuring the carboxylation of succinyl-CoA catalyzed by OGORs. The method involves the in vitro coupling of OGOR with ferredoxin and pyruvate:ferredoxin oxidoreductase from strain TK-6, and glutamate dehydrogenase from Sulfolobus tokodaii. Using this method, we determined both the apparent maximum velocities and the Km values of For and Kor for the carboxylation of succinyl-CoA. This is the first reported kinetic analysis of carbon fixation catalyzed by OGOR enzymes from the rTCA cycle.
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sequencing and reverse transcription polymerase chain reaction rt pcr analysis of four hydrogenase gene clusters from an obligately autotrophic hydrogen oxidizing bacterium Hydrogenobacter thermophilus tk 6
Journal of Bioscience and Bioengineering, 2007Co-Authors: Yasufumi Ueda, Masaharu Ishii, Hiroyuki Arai, Masahiro Yamamoto, Takashi Urasaki, Yasuo IgarashiAbstract:A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive tricarboxylic acid cycle and grows rapidly with a generation time of about 1 h. TK-6 is believed to have an efficient hydrogen-oxidizing ability to support such rapid growth. We cloned hydrogenase genes from TK-6 and found that this strain has at least four clusters of hydrogenase genes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that all four hydrogenase gene clusters were transcribed under aerobic condition at hydrogen concentrations of 45% and 60%. One of them was not transcribed at a hydrogen concentration of 20%. All the four hydrogenase gene clusters were expressed under anaerobic denitrifying condition at a hydrogen concentration of 75%.
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role of two 2 oxoglutarate ferredoxin oxidoreductases in Hydrogenobacter thermophilus under aerobic and anaerobic conditions
Fems Microbiology Letters, 2006Co-Authors: Masahiro Yamamoto, Masaharu Ishii, Hiroyuki Arai, Yasuo IgarashiAbstract:Hydrogenobacter thermophilus TK-6 is a thermophilic, hydrogen-oxidizing bacterium that fixes carbon dioxide as a sole carbon source via the reductive tricarboxylic acid cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes in the pathway. Strain TK-6 has at least two isozymes of OGOR, namely For and Kor. These OGORs showed different expression patterns under aerobic conditions than under anaerobic conditions. In this work, we developed a homologous recombination method for Hydrogenobacter, and constructed a For mutant and a Kor mutant. Observation of phenotypes of the mutant strains showed that Kor was essential for anaerobic growth and that For activity supported robust aerobic growth of the organism.
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two tandemly arranged ferredoxin genes in the Hydrogenobacter thermophilus genome comparative characterization of the recombinant 4fe 4s ferredoxins
Bioscience Biotechnology and Biochemistry, 2005Co-Authors: Takeshi Ikeda, Daijiro Ohmori, Masaharu Ishii, Hiroyuki Arai, Masahiro Yamamoto, Yasuo IgarashiAbstract:A thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene encoding a ferredoxin involved in this cycle as an electron donor (HtFd1) was cloned and sequenced. Interestingly, another ferredoxin gene (encoding HtFd2) was found in tandem with the HtFd1 gene. These two ferredoxin genes overlapped by four bp, and transcriptional analysis revealed that they are co-transcribed as an operon. The deduced amino acid sequences of HtFd1 and HtFd2 were 42.9% identical and each contained four cysteine residues that serve as probable ligands to an iron-sulfur cluster. Spectroscopic analyses of the purified recombinant ferredoxins heterologously expressed in Escherichia coli indicated that each ferredoxin contains a single [4Fe–4S]2+⁄1+ cluster.
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characterization of two different 2 oxoglutarate ferredoxin oxidoreductases from Hydrogenobacter thermophilus tk 6
Biochemical and Biophysical Research Communications, 2003Co-Authors: Masahiro Yamamoto, Masaharu Ishii, Hiroyuki Arai, Yasuo IgarashiAbstract:A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive TCA cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes of this cycle. Strain TK-6 has two distinct OGOR enzymes termed For and Kor. These enzymes were purified and characterized following heterologous expression in Escherichia coli. The specific activity of For was approximately one-tenth of that of Kor. Additionally, For showed higher thermo-stability than Kor under both aerobic and anaerobic conditions. Western blot analysis showed that both of For and Kor were expressed when strain TK-6 was grown under aerobic conditions. In contrast, only Kor was expressed when the strain was grown under anaerobic conditions using nitrate as a terminal electron acceptor. These results indicate that For supports the optimal growth of strain TK-6 in the presence of oxygen.
