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Haruyuki Atomi - One of the best experts on this subject based on the ideXlab platform.
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effects of high level expression of a1 atpase on h2 production in Thermococcus Kodakarensis
Journal of Bioscience and Bioengineering, 2020Co-Authors: Janrobert Simons, Tadayuki Imanaka, Tamotsu Kanai, Haruki Beppu, Haruyuki AtomiAbstract:The hyperthermophilic archaeon Thermococcus Kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. Kodakarensis. Additional A1-type ATPase genes were introduced into T. Kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. Kodakarensis.
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Integration of large heterologous DNA fragments into the genome of Thermococcus Kodakarensis
Extremophiles, 2020Co-Authors: Takaaki Sato, Daisuke Takada, Takashi Itoh, Moriya Ohkuma, Haruyuki AtomiAbstract:In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus Kodakarensis . Using the uracil auxotroph T. Kodakarensis KU216 (∆ pyrF ) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii . The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus , and only when the distances between the two regions were relatively short (2–5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. Kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. Kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.
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Crystal structure of pantoate kinase from Thermococcus Kodakarensis.
Proteins, 2019Co-Authors: Akiko Kita, Yuusuke Yokooji, Hiroya Tomita, Haruyuki Atomi, Tadayuki Imanaka, A. Kishimoto, Takahiro Shimosaka, Kunio MikiAbstract:: The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus Kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested.
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microbe profile Thermococcus Kodakarensis the model hyperthermophilic archaeon
Microbiology, 2019Co-Authors: Haruyuki Atomi, John N ReeveAbstract:: Thermococcus Kodakarensis is a hyperthermophilic Euryarchaeon that grows well under laboratory conditions and, being naturally competent for genetic transformation, it has become a widely studied experimental model species. With the genome sequence available since 2004, combining genetic, enzymological and structural biochemical approaches has revealed previously unknown and unanticipated features of archaeal molecular biology and metabolism. T. Kodakarensis DNA polymerase is already commercialized and with the details of metabolism and hydrogenase available, generating H2 from biopolymers solubilized at high temperatures, most notably chitin, now seems a very attractive possibility as a renewable energy bioprocess.
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the tk0271 protein activates transcription of aromatic amino acid biosynthesis genes in the hyperthermophilic archaeon Thermococcus Kodakarensis
Mbio, 2019Co-Authors: Yasuyuki Yamamoto, Tamotsu Kanai, Tsuyoshi Kaneseki, Haruyuki AtomiAbstract:ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus Kodakarensis is not involved in such regulation. There are 87 genes on the T. Kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. Kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcalesaromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. Kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. Kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus Kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.
Tadayuki Imanaka - One of the best experts on this subject based on the ideXlab platform.
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effects of high level expression of a1 atpase on h2 production in Thermococcus Kodakarensis
Journal of Bioscience and Bioengineering, 2020Co-Authors: Janrobert Simons, Tadayuki Imanaka, Tamotsu Kanai, Haruki Beppu, Haruyuki AtomiAbstract:The hyperthermophilic archaeon Thermococcus Kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. Kodakarensis. Additional A1-type ATPase genes were introduced into T. Kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. Kodakarensis.
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Crystal structure of pantoate kinase from Thermococcus Kodakarensis.
Proteins, 2019Co-Authors: Akiko Kita, Yuusuke Yokooji, Hiroya Tomita, Haruyuki Atomi, Tadayuki Imanaka, A. Kishimoto, Takahiro Shimosaka, Kunio MikiAbstract:: The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus Kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested.
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pol b a family b dna polymerase in Thermococcus Kodakarensis is important for dna repair but not dna replication
Microbes and Environments, 2019Co-Authors: Takashi Kushida, Tadayuki Imanaka, Shinsuke Fujiwara, Sonoko Ishino, Yoshizumi Ishino, Issay Narumi, Hiroki HigashibataAbstract:: Thermococcus Kodakarensis possesses two DNA polymerases, Pol B and Pol D. We generated a T. Kodakarensis strain (DPB1) in which polB was completely deleted and a derivative of DPB1 in which polB was overexpressed; neither of the generated strains exhibited any growth delay, indicating that the lack or overexpression of Pol B in T. Kodakarensis did not affect cell growth. We also found that DPB1 showed higher sensitivity to four DNA-damaging agents (ultraviolet C irradiation, γ-ray irradiation, methyl methanesulfonate, and mitomycin C) than the parental strain. The sensitivity of DPB1 was restored to the level of the parent strain by the introduction of a plasmid harboring polB, suggesting that the DNA damage-sensitive phenotype of DPB1 was due to the loss of polB. Collectively, these results indicate that Pol B is involved in DNA repair, but not DNA replication, which, in turn, implies that Pol D is the sole replicative DNA polymerase in Thermococcus species.
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an ornithine ω aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus Kodakarensis
Journal of Biological Chemistry, 2018Co-Authors: Shinichi Hachisuka, Hiroya Tomita, Makoto Nishiyama, Tadayuki Imanaka, Renchao Zheng, Yuguo Zheng, Haruyuki AtomiAbstract:: Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus Kodakarensis We overexpressed the TK2101 gene in T. Kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene-disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.
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Identification of the glucosamine kinase in the chitinolytic pathway of Thermococcus Kodakarensis.
Journal of Bioscience and Bioengineering, 2017Co-Authors: Mehwish Aslam, Tadayuki Imanaka, Tamotsu Kanai, Naoya Takahashi, Kohei Matsubara, Haruyuki AtomiAbstract:Although the chitinolytic pathway of the hyperthermophilic archaeon Thermococcus Kodakarensis is well-studied, the genome does not contain genes homologous to previously identified glucosamine kinase genes. As some ADP-dependent glucokinases in the order Thermococcales exhibit phosphorylation activities for both glucose and glucosamine in vitro, the homolog in T. Kodakarensis, encoded by TK1110, was selected as a candidate for the missing glucosamine kinase gene. The purified, recombinant TK1110 enzyme exhibited phosphorylation activities for not only glucose but also glucosamine and N-acetylglucosamine. Kinetic analysis indicated that activity towards glucosamine was as significant as that towards glucose. In order to determine the physiological role of TK1110 in the chitinolytic pathway of T. Kodakarensis, a gene disruption strain of TK1110 was constructed. When grown in chitin-containing medium, the TK1110 disruption resulted in almost complete impairment in chitin degradation, and a complete loss of chitin-dependent H2 production. As H2 production is tightly linked to cell growth in T. Kodakarensis, the present results strongly suggest that TK1110 functions as the glucosamine kinase responsible for the chitin degradation in T. Kodakarensis.
Naeem Rashid - One of the best experts on this subject based on the ideXlab platform.
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heterologous gene expression and characterization of tk2246 a highly active and thermostable plant type l asparaginase from Thermococcus Kodakarensis
International Journal of Biological Macromolecules, 2020Co-Authors: Shahid Mahmood Chohan, Muhammad Sajed, Sabeel Un Naeem, Naeem RashidAbstract:Abstract The genome sequence of the hyperthermophilic archaeon Thermococcus Kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l -asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l -asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 μmol min−1 mg−1 towards l -asparagine. The enzyme exhibited a 10% activity towards d -asparagine as compared to 100% against l -asparagine. No detectable activity was observed towards l - or d -glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent Km and Vmax values of 3.1 mM and 833 μmol min−1 mg−1, respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol−1. To the best of our knowledge, this is the first characterization of a plant-type l -asparaginase from class Thermococci of phylum Euryarchaeota.
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An overview of 25 years of research on Thermococcus Kodakarensis, a genetically versatile model organism for archaeal research
Folia Microbiologica, 2020Co-Authors: Naeem Rashid, Mehwish AslamAbstract:Almost 25 years have passed since the discovery of a planktonic, heterotrophic, hyperthermophilic archaeon named Thermococcus Kodakarensis KOD1, previously known as Pyrococcus sp. KOD1, by Imanaka and coworkers. T. Kodakarensis is one of the most studied archaeon in terms of metabolic pathways, available genomic resources, established genetic engineering techniques, reporter constructs, in vitro transcription/translation machinery, and gene expression/gene knockout systems. In addition to all these, ease of growth using various carbon sources makes it a facile archaeal model organism. Here, in this review, an attempt is made to reflect what we have learnt from this hyperthermophilic archaeon.
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an overview of 25 years of research on Thermococcus Kodakarensis a genetically versatile model organism for archaeal research
Folia Microbiologica, 2020Co-Authors: Naeem Rashid, Mehwish AslamAbstract:: Almost 25 years have passed since the discovery of a planktonic, heterotrophic, hyperthermophilic archaeon named Thermococcus Kodakarensis KOD1, previously known as Pyrococcus sp. KOD1, by Imanaka and coworkers. T. Kodakarensis is one of the most studied archaeon in terms of metabolic pathways, available genomic resources, established genetic engineering techniques, reporter constructs, in vitro transcription/translation machinery, and gene expression/gene knockout systems. In addition to all these, ease of growth using various carbon sources makes it a facile archaeal model organism. Here, in this review, an attempt is made to reflect what we have learnt from this hyperthermophilic archaeon.
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biochemical characterization of a highly active adp dependent phosphofructokinase from Thermococcus Kodakarensis
Journal of Bioscience and Bioengineering, 2020Co-Authors: Nisar A Shakir, Mehwish Aslam, Tahira Bibi, Naeem RashidAbstract:The genome sequence of Thermococcus Kodakarensis contains an open reading frame, TK0376, annotated as ADP-dependent phosphofructokinase belonging to pfkC family. The encoding gene was expressed in Escherichia coli and the gene product was characterized. The recombinant protein was produced in soluble and active form. Phosphofructokinase activity of TK0376 was metal-ion dependent and the highest activity (5090 μmol min−1 mg−1) was found in the presence of Co2+ followed by Mg2+ (3280 μmol min−1 mg−1) at 90°C and pH 7.5. TK0376 preferred ADP as phosphoryl donor, however, it could be replaced by ATP but with a 5-fold lower activity. It catalyzed the phosphorylation of fructose 6-phosphate and dephosphorylation of fructose 1,6-bisphosphate. In addition, it was able to phosphorylate glucose and nucleosides but with a much lower rate compared to that of fructose 6-phosphate. The apparent kcat and Km values against fructose 6-phosphate were 4238 s−1 and 0.74 mM, respectively. The rate of dephosphorylation of fructose 1,6-bisphosphate was 3-times lower at 50°C than the phosphorylation of fructose 6-phosphate. Similarly, the rate of phosphorylation of glucose was 450-fold lower than that of fructose 6-phosphate. Phosphofructokinase activity was not allosterically regulated, but it was slightly enhanced by phosphoenol pyruvate, and inhibited by ATP and AMP in a competitive manner.
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characterization of tk1646 a highly thermostable 3 5 single strand specific exonuclease from Thermococcus Kodakarensis
International Journal of Biological Macromolecules, 2019Co-Authors: Muhammad Sulaiman Saeed, Naeem RashidAbstract:Abstract Exonucleases catalyze the hydrolysis of terminal phosphodiester bond in nucleic acid. They play important role in maintaining the integrity of DNA in eukaryotes, prokaryotes and archaea. Limited studies have been done on archaeal exonucleases. Here we report molecular cloning of TK1646, a putative exonuclease from the hyperthermophilic archaeon Thermococcus Kodakarensis, and expression of the gene in Escherichia coli. Recombinant TK1646, produced in soluble and active form, was purified to apparent homogeneity. Characterization of the recombinant enzyme indicated that it was single strand specific 3′–5′ exonuclease which cleaved the substrate DNA after every two nucleotides. It exhibited highest activity at 85–100 °C and pH 9.0. Unique property of TK1646 was its thermostability as it maintained its activity even at 100 °C with a half-life of 180 min. Recombinant TK1646 followed Michaelis-Menten kinetics and exhibited apparent Km and Vmax values of 33 ± 4 μM and 812 ± 48 nmol/min/mg, respectively. To the best of our knowledge this is the most thermostable single strand specific 3′–5′ exonuclease characterized to date.
Gang-won Cheong - One of the best experts on this subject based on the ideXlab platform.
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proteome profiling of heat oxidative and salt stress responses in Thermococcus Kodakarensis kod1
Frontiers in Microbiology, 2015Co-Authors: Le Van Duyet, Yuan H Xuan, Gang-won CheongAbstract:The thermophilic species, Thermococcus Kodakarensis KOD1, a model microorganism for studying hyperthermophiles, has adapted to optimal growth under conditions of high temperature and salinity. However, the environmental conditions for the strain are not always stable, and this strain might face different stresses. In the present study, we compared the proteome response of T. Kodakarensis to heat, oxidative, and salt stresses using two-dimensional electrophoresis, and protein spots were identified through MALDI-TOF/MS. Fifty-nine, forty-two, and twenty-nine spots were induced under heat, oxidative, and salt stresses, respectively. Among the up-regulated proteins, four proteins (a hypothetical protein, pyridoxal biosynthesis lyase, peroxiredoxin, and protein disulphide oxidoreductase) were associated with all three stresses. Gene ontology analysis showed that these proteins were primarily involved metabolic and cellular processes. The KEGG pathway analysis suggested that the main metabolic pathways involving these enzymes were related to carbohydrate metabolism, secondary metabolite synthesis, and amino acid biosynthesis. These data might enhance our understanding of the functions and molecular mechanisms of thermophilic Archaea for survival and adaptation in extreme environments.
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architecture and characterization of a thermostable moxr family aaa atpase from Thermococcus Kodakarensis kod1
Extremophiles, 2014Co-Authors: Bang Phuong Pham, Jae Myeong Kwak, Gang-won CheongAbstract:AAA+ ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus Kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths. In addition, the two-stack hexameric protein catalyzed dsDNA decomposition to form and then release ssDNA, whereas the hexamer TkMoxR structure interacted with but did not release dsDNA. These results suggest that TkMoxR has DNA helicase activity involved in gene expression control.
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Architecture and characterization of a thermostable MoxR family AAA(+) ATPase from Thermococcus Kodakarensis KOD1.
Extremophiles, 2014Co-Authors: Bang Phuong Pham, Jae Myeong Kwak, Gang-won CheongAbstract:AAA+ ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus Kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths. In addition, the two-stack hexameric protein catalyzed dsDNA decomposition to form and then release ssDNA, whereas the hexamer TkMoxR structure interacted with but did not release dsDNA. These results suggest that TkMoxR has DNA helicase activity involved in gene expression control.
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cloning purification and biochemical characterisation of an organic solvent detergent and thermo stable amylopullulanase from Thermococcus Kodakarensis kod1
Process Biochemistry, 2013Co-Authors: Q. Guan, Gang-won Cheong, F. Zeng, Yuhan Wang, Zhe Li, Shihong ZhangAbstract:Abstract Thermostable amylopullulanases can catalyse the hydrolysis of both α-1,4 and α-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus Kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 °C. Apu-Tk was also active at a broad range of pH (4–7), with an optimum pH of ~5.0–5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% β-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications.
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high level expression and characterization of a thermostable lysophospholipase from Thermococcus Kodakarensis kod1
Extremophiles, 2012Co-Authors: Yuhan Wang, Bang Phuong Pham, Gang-won Cheong, Fangfang Ping, Shihong ZhangAbstract:Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus Kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783 bp encodes a 260-amino acid protein with a molecular mass of 29 kDa. LysoPL-tk has a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence. LysoPL-tk was expressed in Escherichia coli and purified to homogeneity. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent K m value for p-nitrophenyl butyrate was 607.1 μM with V max values of 95.5 U/mg. The enzyme was active at a broad range of pH (5–8) and temperatures (70–95 °C) with the optimum pH and temperature being 8.0 and 85 °C, respectively. The high yield, broad substrate range along with its thermo-stability indicates that LysoPL-tk is a potential enzyme in industrial application.
Tamotsu Kanai - One of the best experts on this subject based on the ideXlab platform.
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effects of high level expression of a1 atpase on h2 production in Thermococcus Kodakarensis
Journal of Bioscience and Bioengineering, 2020Co-Authors: Janrobert Simons, Tadayuki Imanaka, Tamotsu Kanai, Haruki Beppu, Haruyuki AtomiAbstract:The hyperthermophilic archaeon Thermococcus Kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. Kodakarensis. Additional A1-type ATPase genes were introduced into T. Kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. Kodakarensis.
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the tk0271 protein activates transcription of aromatic amino acid biosynthesis genes in the hyperthermophilic archaeon Thermococcus Kodakarensis
Mbio, 2019Co-Authors: Yasuyuki Yamamoto, Tamotsu Kanai, Tsuyoshi Kaneseki, Haruyuki AtomiAbstract:ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus Kodakarensis is not involved in such regulation. There are 87 genes on the T. Kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. Kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcalesaromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. Kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. Kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus Kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.
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Identification of the glucosamine kinase in the chitinolytic pathway of Thermococcus Kodakarensis.
Journal of Bioscience and Bioengineering, 2017Co-Authors: Mehwish Aslam, Tadayuki Imanaka, Tamotsu Kanai, Naoya Takahashi, Kohei Matsubara, Haruyuki AtomiAbstract:Although the chitinolytic pathway of the hyperthermophilic archaeon Thermococcus Kodakarensis is well-studied, the genome does not contain genes homologous to previously identified glucosamine kinase genes. As some ADP-dependent glucokinases in the order Thermococcales exhibit phosphorylation activities for both glucose and glucosamine in vitro, the homolog in T. Kodakarensis, encoded by TK1110, was selected as a candidate for the missing glucosamine kinase gene. The purified, recombinant TK1110 enzyme exhibited phosphorylation activities for not only glucose but also glucosamine and N-acetylglucosamine. Kinetic analysis indicated that activity towards glucosamine was as significant as that towards glucose. In order to determine the physiological role of TK1110 in the chitinolytic pathway of T. Kodakarensis, a gene disruption strain of TK1110 was constructed. When grown in chitin-containing medium, the TK1110 disruption resulted in almost complete impairment in chitin degradation, and a complete loss of chitin-dependent H2 production. As H2 production is tightly linked to cell growth in T. Kodakarensis, the present results strongly suggest that TK1110 functions as the glucosamine kinase responsible for the chitin degradation in T. Kodakarensis.
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the cdc45 recj like protein forms a complex with gins and mcm and is important for dna replication in Thermococcus Kodakarensis
Nucleic Acids Research, 2017Co-Authors: Mariko Nagata, Haruyuki Atomi, Janrobert Simons, Tamotsu Kanai, Sonoko Ishino, Hiromi Ogino, Takeshi Yamagami, Yoshizumi IshinoAbstract:: The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus Kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. Kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells.
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Gene regulation of two ferredoxin:NADP^+ oxidoreductases by the redox-responsive regulator SurR in Thermococcus Kodakarensis
Extremophiles, 2017Co-Authors: Ryota Hidese, Haruyuki Atomi, Tadayuki Imanaka, Tamotsu Kanai, Keita Yamashita, Kohei Kawazuma, Shinsuke FujiwaraAbstract:The redox-responsive regulator SurR in the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus Kodakarensis binds to the SurR-binding consensus sequence (SBS) by responding to the presence of elemental sulfur. Here we constructed a surR gene disruption strain (DTS) in T. Kodakarensis , and identified the genes that were under SurR control by comparing the transcriptomes of DTS and parent strains. Among these genes, transcript levels of ferredoxin:NADP^+ oxidoreductases 1 and 2 (FNOR1 and FNOR2) genes displayed opposite responses to surR deletion, indicating that SurR repressed FNOR1 transcription while enhancing FNOR2 transcription. Each promoter region contains an SBS upstream (uSBS) and downstream (dSBS) of TATA. In addition to in vitro binding assays, we examined the roles of each SBS in vivo. In FNOR1 , mutations in either one of the SBSs resulted in a complete loss of repression, indicating that the presence of both SBSs was essential for repression. In FNOR2 , uSBS indeed functioned to enhance gene expression, whereas dSBS functioned in gene repression. SurR bound to uSBS2 of FNOR2 more efficiently than to dSBS2 in vitro, which may explain why SurR overall enhances FNOR2 transcription. Further analyses indicated the importance in the distance between uSBS and TATA for transcriptional activation in FNOR2 .