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Tadayuki Imanaka - One of the best experts on this subject based on the ideXlab platform.
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random mutagenesis of a Hyperthermophilic Archaeon identified trna modifications associated with cellular hyperthermotolerance
Nucleic Acids Research, 2019Co-Authors: Izumi Orita, Satoshi Nakamura, Ryohei Futatsuishi, Kyoko Adachi, Takayuki Ohira, Akira Kaneko, Keiichi Minowa, Miho Suzuki, Takeshi Tamura, Tadayuki ImanakaAbstract:Random mutagenesis for the Hyperthermophilic Archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.
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engineering of the Hyperthermophilic Archaeon thermococcus kodakarensis for chitin dependent hydrogen production
Applied and Environmental Microbiology, 2017Co-Authors: Mehwish Aslam, Tadayuki Imanaka, Janrobert Simons, Ayumi Horiuchi, Toru Odani, Rikako Fujimoto, Ryoma Gunji, Yasuyuki Yamamoto, Masahiro Yamada, Tamotsu KanaiAbstract:Thermococcus kodakarensis is a Hyperthermophilic Archaeon that harbors a complete set of genes for chitin degradation to fructose 6-phosphate. However, wild-type T. kodakarensis KOD1 does not display growth on chitin. In this study, we developed a T. kodakarensis strain that can grow on chitin via genetic and adaptive engineering. First, a chitinase overproduction strain (KC01) was constructed by replacing the chitinase gene promoter with a strong promoter from the cell-surface glycoprotein gene, resulting in increased degradation of swollen chitin and accumulation of N -, N 9-diacetylchitobiose in the medium. To enhance N -, N 9-diacetylchitobiose assimilation in KC01, genes encoding diacetylchitobiose deacetylase, exo-β-D-glucosaminidase and glucosamine-6-phosphate deaminase were also overexpressed to obtain strain KC04. To strengthen the glycolytic flux of KC04, the gene encoding Tgr (transcriptional repressor of glycolytic genes) was disrupted to obtain strain KC04Δt. In both KC04 and KC04Δt strains, degradation of swollen chitin was further enhanced. In the culture broth of these strains, the accumulation of glucosamine was observed. KC04Δt was repeatedly inoculated in a swollen chitin-containing medium for 13 cultures. This adaptive engineering strategy resulted in the isolation of a strain (KC04ΔtM1) that showed almost complete degradation of 0.4% (w/v) swollen chitin after 90 h. The strain produced high levels of acetate and ammonium in the culture medium, and moreover, molecular hydrogen was generated. This strongly suggests that strain KC04ΔtM1 has obtained the ability to convert chitin to fructose 6-phosphate via deacetylation and deamination, and further convert fructose 6-phosphate to acetate via glycolysis coupled to hydrogen generation. IMPORTANCE Chitin is a linear homopolymer of β-1, 4-linked N -acetylglucosamine and is the second most abundant biomass next to cellulose. Compared to the wealth of research focused on the microbial degradation and conversion of cellulose, studies addressing microbial chitin utilization are still limited. In this study, using the Hyperthermophilic Archaeon Thermococcus kodakarensis as a host, we have constructed a strain that displays chitin-dependent hydrogen generation. The apparent hydrogen yield per unit sugar consumed was slightly higher with swollen chitin compared to starch. As gene manipulation in T. kodakarensis is relatively simple, the strain constructed in this study can also be used as a parent strain for the development and expansion of chitin-dependent biorefinery, in addition to its capacity to produce hydrogen.
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engineering of the Hyperthermophilic Archaeon thermococcus kodakarensis for chitin dependent hydrogen production
Applied and Environmental Microbiology, 2017Co-Authors: Mehwish Aslam, Janrobert Simons, Ayumi Horiuchi, Toru Odani, Rikako Fujimoto, Ryoma Gunji, Yasuyuki Yamamoto, Masahiro Yamada, Savyasachee Jha, Tadayuki ImanakaAbstract:Thermococcus kodakarensis is a Hyperthermophilic Archaeon that harbors a complete set of genes for chitin degradation to fructose 6-phosphate. However, wild-type T. kodakarensis KOD1 does not display growth on chitin. In this study, we developed a T. kodakarensis strain that can grow on chitin via genetic and adaptive engineering. First, a chitinase overproduction strain (KC01) was constructed by replacing the chitinase gene promoter with a strong promoter from the cell surface glycoprotein gene, resulting in increased degradation of swollen chitin and accumulation of N-,N'-diacetylchitobiose in the medium. To enhance N-,N'-diacetylchitobiose assimilation in KC01, genes encoding diacetylchitobiose deacetylase, exo-β-d-glucosaminidase, and glucosamine-6-phosphate deaminase were also overexpressed to obtain strain KC04. To strengthen the glycolytic flux of KC04, the gene encoding Tgr (transcriptional repressor of glycolytic genes) was disrupted to obtain strain KC04Δt. In both KC04 and KC04Δt strains, degradation of swollen chitin was further enhanced. In the culture broth of these strains, the accumulation of glucosamine was observed. KC04Δt was repeatedly inoculated in a swollen-chitin-containing medium for 13 cultures. This adaptive engineering strategy resulted in the isolation of a strain (KC04ΔtM1) that showed almost complete degradation of 0.4% (wt/vol) swollen chitin after 90 h. The strain produced high levels of acetate and ammonium in the culture medium, and, moreover, molecular hydrogen was generated. This strongly suggests that strain KC04ΔtM1 has acquired the ability to convert chitin to fructose 6-phosphate via deacetylation and deamination and further convert fructose 6-phosphate to acetate via glycolysis coupled to hydrogen generation.IMPORTANCE Chitin is a linear homopolymer of β-1,4-linked N-acetylglucosamine and is the second most abundant biomass next to cellulose. Compared to the wealth of research focused on the microbial degradation and conversion of cellulose, studies addressing microbial chitin utilization are still limited. In this study, using the Hyperthermophilic Archaeon Thermococcus kodakarensis as a host, we have constructed a strain that displays chitin-dependent hydrogen generation. The apparent hydrogen yield per unit of sugar consumed was slightly higher with swollen chitin than with starch. As gene manipulation in T. kodakarensis is relatively simple, the strain constructed in this study can also be used as a parent strain for the development and expansion of chitin-dependent biorefinery, in addition to its capacity to produce hydrogen.
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characterization of a thermostable glucose dehydrogenase with strict substrate specificity from a Hyperthermophilic Archaeon thermoproteus sp gdh 1
Bioscience Biotechnology and Biochemistry, 2015Co-Authors: Hiroshi Aiba, Haruyuki Atomi, Yoshiaki Nishiya, Masayuki Azuma, Yuusuke Yokooji, Tadayuki ImanakaAbstract:A Hyperthermophilic Archaeon was isolated from a terrestrial hot spring on Kodakara Island, Japan and designated as Thermoproteus sp. glucose dehydrogenase (GDH-1). Cell extracts from cells grown in medium supplemented with glucose exhibited NAD(P)-dependent glucose dehydrogenase activity. The enzyme (TgGDH) was purified and found to display a strict preference for d-glucose. The gene was cloned and expressed in Escherichia coli, resulting in the production of a soluble and active protein. Recombinant TgGDH displayed extremely high thermostability and an optimal temperature higher than 85 °C, in addition to its strict specificity for d-glucose. Despite its thermophilic nature, TgGDH still exhibited activity at 25 °C. We confirmed that the enzyme could be applied for glucose measurements at ambient temperatures, suggesting a potential of the enzyme for use in measurements in blood samples.
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genetic studies on the virus like regions in the genome of Hyperthermophilic Archaeon thermococcus kodakarensis
Extremophiles, 2013Co-Authors: Kenta Tagashira, Wakao Fukuda, Tamotsu Kanai, Haruyuki Atomi, Masaaki Matsubara, Tadayuki ImanakaAbstract:Four virus-like integrated elements (TKV1, TKV2, TKV3, and TKV4) have been found in the genome of Hyperthermophilic Archaeon, Thermococcus kodakarensis, but virus particle formation has not been observed in the culture of T. kodakarensis. As the result of growth property analyses, mutants lacking each of the four virus-like regions exhibited decrease in the cell concentration and/or less growth rates compared to growth of parental strain (KU216), when the T. kodakarensis strains were grown at 85 °C in nutrient-rich medium. These results indicated that the genes in virus-like regions stimulated the cell growth under the observed growth condition. As the result of transcriptome analyses, genes involved in amino acid, energy or nucleotide metabolisms, and transport systems were up- or down-regulated in the cells of mutant strains. Interestingly, a decrease in transcriptional levels of glutamine synthetase (TK1796) gene (Tk-glnA) was observed in the cells of four mutant strains. Growths of TKV1 disrupted strain and TKV4 disrupted strain have shown no difference compared with that of KU216 by the addition of glutamate or glutamine, and the result suggested that TKV1 and TKV4 contributed to supply of amino acids to the cell.
Toshihisa Ohshima - One of the best experts on this subject based on the ideXlab platform.
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Data_Sheet_1_A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3.docx
2018Co-Authors: Ryushi Kawakami, Haruhiko Sakuraba, Tatsuya Ohshida, Toshihisa OhshimaAbstract:We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea.
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A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3
Frontiers Media S.A., 2018Co-Authors: Ryushi Kawakami, Haruhiko Sakuraba, Tatsuya Ohshida, Toshihisa OhshimaAbstract:We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea
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gene expression and characterization of a third type of dye linked l proline dehydrogenase from the aerobic Hyperthermophilic Archaeon aeropyrum pernix
Bioscience Biotechnology and Biochemistry, 2012Co-Authors: Takenori Satomura, Haruhiko Sakuraba, Shinichiro Suye, Yusuke Hara, Toshihisa OhshimaAbstract:A third novel type of dye-linked L-proline dehydrogenase (LPDH) has recently been found in the Hyperthermophilic Archaeon, Pyrobaculum calidifontis, by Satomura et al. The gene encoding the enzyme homologue was identified in the aerobic Hyperthermophilic Archaeon, Aeropyrum pernix. The gene was successfully expressed in Escherichia coli, and the product was purified to homogeneity and characterized. The expressed enzyme was highly thermostable LPDH having a molecular mass of about 88 kDa and a homodimeric structure. The preferred substrate for the enzyme was L-proline with 2,6-dichloroindophenol (DCIP) as the electron acceptor. However, the enzyme did not utilize ferricyanide as the electron acceptor, in contrast to all other known LPDHs. The electrochemical determination of L-proline at concentrations from 0 to 0.7 mM was achieved by using A. pernix LPDH. A phylogenetic analysis revealed A. pernix LPDH to be clustered with the third type of LPDHs, and to be clearly separated from the clusters of previous...
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expression purification crystallization and preliminary x ray diffraction analysis of a galactose 1 phosphate uridylyltransferase from the Hyperthermophilic Archaeon pyrobaculum aerophilum
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2012Co-Authors: Takenori Satomura, Akihiro Hiraki, Tomoyuki Kawai, Ryushi Kawakami, Toshihisa Ohshima, Haruhiko SakurabaAbstract:A galactose 1-phosphate uridylyltransferase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the tetragonal space group P41, with unit-cell parameters a = b = 73.3, c = 126.1 A, and diffracted to 2.73 A resolution on beamline BL5A at the Photon Factory. The overall R merge was 7.3% and the data completeness was 99.8%.
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structure of a multicopper oxidase from the Hyperthermophilic Archaeon pyrobaculum aerophilum
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2011Co-Authors: Haruhiko Sakuraba, Kazunari Yoneda, Kohtaroh Koga, Yasuhiro Kashima, Toshihisa OhshimaAbstract:The crystal structure of an extremely thermostable multicopper oxidase (McoP) from the Hyperthermophilic Archaeon Pyrobaculum aerophilum was determined at a resolution of 2.0 A. The overall fold was comprised of three cupredoxin-like domains and the main-chain coordinates of the enzyme were similar to those of multicopper oxidases from Escherichia coli (CueO) and Bacillus subtilis (CotA). However, there were clear topological differences around domain 3 between McoP and the other two enzymes: a methionine-rich helix in CueO and a protruding helix in CotA were not present in McoP. Instead, a large loop (PL-1) covered the T1 copper centre of McoP and a short α-helix in domain 3 extended near the N-terminal end of PL-1. In addition, the sizes of several surface loops in McoP were markedly smaller than the corresponding loops in CueO and CotA. Structural comparison revealed that the presence of extensive hydrophobic interactions and a smaller cavity volume are likely to be the main factors contributing to the hyperthermostability of McoP.
V. Popov - One of the best experts on this subject based on the ideXlab platform.
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structural characterization of geranylgeranyl pyrophosphate synthase gace1337 from the Hyperthermophilic Archaeon geoglobus acetivorans
Extremophiles, 2018Co-Authors: T. Petrova, A Y Nikolaeva, Eugeny V Gruzdev, Viktor S Stroilov, Konstantin M. Boyko, T N Stekhanova, Jennifer A. Littlechild, Andrey V Mardanov, V. PopovAbstract:A novel type 1 geranylgeranyl pyrophosphate synthase GACE1337 has been identified within the genome of a newly identified Hyperthermophilic Archaeon Geoglobus acetivorans. The enzyme has been cloned and over-expressed in Escherichia coli. The recombinant enzyme has been biochemically and structurally characterized. It is able to catalyze the synthesis of geranylgeranyl pyrophosphate as a major product and of farnesyl pyrophosphate in smaller amounts, as measured by gas chromatography–mass spectrometry at an elevated temperature of 60 °C. Its ability to produce two products is consistent with the fact that GACE1337 is the only short-chain isoprenyl diphosphate synthase in G. acetivorans. Attempts to crystallize the enzyme were successful only at 37 °C. The three-dimensional structure of GACE1337 was determined by X-ray diffraction to 2.5 A resolution. A comparison of its structure with those of related enzymes revealed that the Geoglobus enzyme has the features of both type I and type III geranylgeranyl pyrophosphate synthases, which allow it to regulate the product length. The active enzyme is a dimer and has three aromatic amino acids, two Phe, and a Tyr, located in the hydrophobic cleft between the two subunits. It is proposed that these bulky residues play a major role in the synthetic reaction by controlling the product elongation.
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characterization of a thermostable short chain alcohol dehydrogenase from the Hyperthermophilic Archaeon thermococcus sibiricus
Applied and Environmental Microbiology, 2010Co-Authors: T N Stekhanova, E Y Bezsudnova, V M Gumerov, Andrey V Mardanov, Nikolay V. Ravin, Konstantin G. Skryabin, V. PopovAbstract:Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the Hyperthermophilic Archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.
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characterization of a thermostable short chain alcohol dehydrogenase from the Hyperthermophilic Archaeon thermococcus sibiricus
Applied and Environmental Microbiology, 2010Co-Authors: T N Stekhanova, E Y Bezsudnova, V M Gumerov, Andrey V Mardanov, Nikolay V. Ravin, Konstantin G. Skryabin, V. PopovAbstract:Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the Hyperthermophilic Archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.
Haruyuki Atomi - One of the best experts on this subject based on the ideXlab platform.
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Hyperthermophilic Archaeon thermococcus kodakarensis utilizes a four step pathway for nad salvage through nicotinamide deamination
Journal of Bacteriology, 2018Co-Authors: Shinichi Hachisuka, Takaaki Sato, Haruyuki AtomiAbstract:ABSTRACT Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the Hyperthermophilic Archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway. IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD+ from nicotinamide (Nm) in the Hyperthermophilic Archaeon Thermococcus kodakarensis. The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD+ itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.
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metabolism dealing with thermal degradation of nad in the Hyperthermophilic Archaeon thermococcus kodakarensis
Journal of Bacteriology, 2017Co-Authors: Shinichi Hachisuka, Takaaki Sato, Haruyuki AtomiAbstract:: NAD+ is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD+ is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD+ concentrations and possibly remove and/or reuse undesirable degradation products of NAD+ Here we confirmed that at 85°C, thermal degradation of NAD+ results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The Hyperthermophilic Archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD+ in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD+ breakdown.IMPORTANCE Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD+ is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal degradation to ADP-ribose and nicotinamide. Here we show that an ADP-ribose pyrophosphatase homolog from the Hyperthermophilic Archaeon Thermococcus kodakarensis converts the detrimental ADP-ribose to ribose 5-phosphate and AMP, compounds that can be directed to central carbon metabolism. This physiological role for ADP-ribose pyrophosphatases might be universal in hyperthermophiles, as their homologs are widely distributed among both Hyperthermophilic bacteria and archaea.
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characterization of a thermostable glucose dehydrogenase with strict substrate specificity from a Hyperthermophilic Archaeon thermoproteus sp gdh 1
Bioscience Biotechnology and Biochemistry, 2015Co-Authors: Hiroshi Aiba, Haruyuki Atomi, Yoshiaki Nishiya, Masayuki Azuma, Yuusuke Yokooji, Tadayuki ImanakaAbstract:A Hyperthermophilic Archaeon was isolated from a terrestrial hot spring on Kodakara Island, Japan and designated as Thermoproteus sp. glucose dehydrogenase (GDH-1). Cell extracts from cells grown in medium supplemented with glucose exhibited NAD(P)-dependent glucose dehydrogenase activity. The enzyme (TgGDH) was purified and found to display a strict preference for d-glucose. The gene was cloned and expressed in Escherichia coli, resulting in the production of a soluble and active protein. Recombinant TgGDH displayed extremely high thermostability and an optimal temperature higher than 85 °C, in addition to its strict specificity for d-glucose. Despite its thermophilic nature, TgGDH still exhibited activity at 25 °C. We confirmed that the enzyme could be applied for glucose measurements at ambient temperatures, suggesting a potential of the enzyme for use in measurements in blood samples.
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genetic studies on the virus like regions in the genome of Hyperthermophilic Archaeon thermococcus kodakarensis
Extremophiles, 2013Co-Authors: Kenta Tagashira, Wakao Fukuda, Tamotsu Kanai, Haruyuki Atomi, Masaaki Matsubara, Tadayuki ImanakaAbstract:Four virus-like integrated elements (TKV1, TKV2, TKV3, and TKV4) have been found in the genome of Hyperthermophilic Archaeon, Thermococcus kodakarensis, but virus particle formation has not been observed in the culture of T. kodakarensis. As the result of growth property analyses, mutants lacking each of the four virus-like regions exhibited decrease in the cell concentration and/or less growth rates compared to growth of parental strain (KU216), when the T. kodakarensis strains were grown at 85 °C in nutrient-rich medium. These results indicated that the genes in virus-like regions stimulated the cell growth under the observed growth condition. As the result of transcriptome analyses, genes involved in amino acid, energy or nucleotide metabolisms, and transport systems were up- or down-regulated in the cells of mutant strains. Interestingly, a decrease in transcriptional levels of glutamine synthetase (TK1796) gene (Tk-glnA) was observed in the cells of four mutant strains. Growths of TKV1 disrupted strain and TKV4 disrupted strain have shown no difference compared with that of KU216 by the addition of glutamate or glutamine, and the result suggested that TKV1 and TKV4 contributed to supply of amino acids to the cell.
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histone and tk0471 trmbl2 form a novel heterogeneous genome architecture in the Hyperthermophilic Archaeon thermococcus kodakarensis
Molecular Biology of the Cell, 2011Co-Authors: Hugo Maruyama, Tadayuki Imanaka, Haruyuki Atomi, Minsang Shin, Toshiyuki Oda, Rie Matsumi, Ryosuke L Ohniwa, Takehiko Itoh, Katsuhiko Shirahige, Shige H YoshimuraAbstract:This study demonstrates that the chromosome of the Hyperthermophilic Archaeon Thermococcus kodakarensis is organized into a heterogeneous structure created with histone and a novel protein TK0471/T...
Haruhiko Sakuraba - One of the best experts on this subject based on the ideXlab platform.
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A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3
Frontiers Media S.A., 2018Co-Authors: Ryushi Kawakami, Haruhiko Sakuraba, Tatsuya Ohshida, Toshihisa OhshimaAbstract:We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea
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Data_Sheet_1_A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3.docx
2018Co-Authors: Ryushi Kawakami, Haruhiko Sakuraba, Tatsuya Ohshida, Toshihisa OhshimaAbstract:We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea.
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gene expression and characterization of a third type of dye linked l proline dehydrogenase from the aerobic Hyperthermophilic Archaeon aeropyrum pernix
Bioscience Biotechnology and Biochemistry, 2012Co-Authors: Takenori Satomura, Haruhiko Sakuraba, Shinichiro Suye, Yusuke Hara, Toshihisa OhshimaAbstract:A third novel type of dye-linked L-proline dehydrogenase (LPDH) has recently been found in the Hyperthermophilic Archaeon, Pyrobaculum calidifontis, by Satomura et al. The gene encoding the enzyme homologue was identified in the aerobic Hyperthermophilic Archaeon, Aeropyrum pernix. The gene was successfully expressed in Escherichia coli, and the product was purified to homogeneity and characterized. The expressed enzyme was highly thermostable LPDH having a molecular mass of about 88 kDa and a homodimeric structure. The preferred substrate for the enzyme was L-proline with 2,6-dichloroindophenol (DCIP) as the electron acceptor. However, the enzyme did not utilize ferricyanide as the electron acceptor, in contrast to all other known LPDHs. The electrochemical determination of L-proline at concentrations from 0 to 0.7 mM was achieved by using A. pernix LPDH. A phylogenetic analysis revealed A. pernix LPDH to be clustered with the third type of LPDHs, and to be clearly separated from the clusters of previous...
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expression purification crystallization and preliminary x ray diffraction analysis of a galactose 1 phosphate uridylyltransferase from the Hyperthermophilic Archaeon pyrobaculum aerophilum
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2012Co-Authors: Takenori Satomura, Akihiro Hiraki, Tomoyuki Kawai, Ryushi Kawakami, Toshihisa Ohshima, Haruhiko SakurabaAbstract:A galactose 1-phosphate uridylyltransferase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the tetragonal space group P41, with unit-cell parameters a = b = 73.3, c = 126.1 A, and diffracted to 2.73 A resolution on beamline BL5A at the Photon Factory. The overall R merge was 7.3% and the data completeness was 99.8%.
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structure of a multicopper oxidase from the Hyperthermophilic Archaeon pyrobaculum aerophilum
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2011Co-Authors: Haruhiko Sakuraba, Kazunari Yoneda, Kohtaroh Koga, Yasuhiro Kashima, Toshihisa OhshimaAbstract:The crystal structure of an extremely thermostable multicopper oxidase (McoP) from the Hyperthermophilic Archaeon Pyrobaculum aerophilum was determined at a resolution of 2.0 A. The overall fold was comprised of three cupredoxin-like domains and the main-chain coordinates of the enzyme were similar to those of multicopper oxidases from Escherichia coli (CueO) and Bacillus subtilis (CotA). However, there were clear topological differences around domain 3 between McoP and the other two enzymes: a methionine-rich helix in CueO and a protruding helix in CotA were not present in McoP. Instead, a large loop (PL-1) covered the T1 copper centre of McoP and a short α-helix in domain 3 extended near the N-terminal end of PL-1. In addition, the sizes of several surface loops in McoP were markedly smaller than the corresponding loops in CueO and CotA. Structural comparison revealed that the presence of extensive hydrophobic interactions and a smaller cavity volume are likely to be the main factors contributing to the hyperthermostability of McoP.