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Tairo Oshima - One of the best experts on this subject based on the ideXlab platform.
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n1 aminopropylagmatine a new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme Thermophile thermus thermophilus
Journal of Biological Chemistry, 2005Co-Authors: Mio Ohnuma, Etsuko Kawashima, Masaru Niitsu, Yusuke Terui, Keijiro Samejima, Masatada Tamakoshi, Hidemichi Mitome, Tairo OshimaAbstract:Abstract In the extreme Thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the Thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.
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stabilization of nucleic acids by unusual polyamines produced by an extreme Thermophile thermus thermophilus
Biochemical Journal, 2005Co-Authors: Yusuke Terui, Kaori Hiraga, Etsuko Kawashima, Mio Ohnuma, Tairo OshimaAbstract:Extreme Thermophiles produce two types of unusual polyamine: long linear polyamines such as caldopentamine and caldohexamine, and branched polyamines such as quaternary ammonium compounds [e.g. tetrakis(3-aminopropyl)ammonium]. To clarify the physiological roles of long linear and branched polyamines in Thermophiles, we synthesized them chemically and tested their effects on the stability of ds (double-stranded) and ss (single-stranded) DNAs and tRNA in response to thermal denaturation, as measured by differential scanning calorimetry. Linear polyamines stabilized dsDNA in proportion to the number of amino nitrogen atoms within their molecular structure. We used the empirical results to derive formulae that estimate the melting temperature of dsDNA in the presence of polyamines of a particular molecular composition. ssDNA and tRNA were stabilized more effectively by tetrakis(3-aminopropyl)ammonium than any of the other polyamines tested. We propose that long linear polyamines are effective to stabilize DNA, and tetrakis(3-aminopropyl)ammonium plays important roles in stabilizing RNAs in Thermophile cells.
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studies on interdomain interaction of 3 isopropylmalate dehydrogenase from an extreme Thermophile thermus thermophilus by constructing chimeric enzymes
Extremophiles, 1999Co-Authors: Koichi Numata, Yoko Hayashiiwasaki, Katsuhide Yutani, Tairo OshimaAbstract:In our previous study, we showed that a chimeric isopropylmalate dehydrogenase, 2T2M6T, between an extreme Thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, isopropylmalate dehydrogenases (the name roughly denotes the primary structure; the first 20% from the N-terminal is coded by the Thermophile leuB gene, next 20% by mesophile, and the rest by the Thermophile gene) denatured in two steps with a stable intermediate, suggesting that in the chimera some of the interdomain interaction was lost by amino acid substitutions in the "2M" part. To identify the residues involved in the interdomain interactions, the first and the second halves of the 2M part of the chimera were substituted with the corresponding sequence of the Thermophile enzyme. Both chimeras, 3T1M6T and 2T1M7T, apparently showed one transition in the thermal denaturation without any stable intermediate state, suggesting that the cooperativity of the conformational stability was at least partly restored by the substitutions. The present study also suggested involvement of one or more basic residues in the unusual stability of the Thermophile enzyme.
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purification catalytic properties and thermostability of 3 isopropylmalate dehydrogenase from escherichia coli
Biochimica et Biophysica Acta, 1997Co-Authors: Gerlind Wallon, Susan T. Lovett, Hiromi Kirino, Gregory A Petsko, Kazutaka Yamamoto, Akihiko Yamagishi, Tairo OshimaAbstract:Abstract 3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the Thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg 2+ or Mn 2+ , with Mn 2+ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40°C the K m of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k cat was 69 s −1 . The half denaturationn temperature was 64°C, which was 20°C lower than that of the Thermophile enzyme.
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further stabilization of 3 isopropylmalate dehydrogenase of an extreme Thermophile thermus thermophilus by a suppressor mutation method
Journal of Bacteriology, 1996Co-Authors: Takashi Kotsuka, Masaaki Tomuro, Satoshi Akanuma, Akihiko Yamagishi, Tairo OshimaAbstract:We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme Thermophile, Thermus thermophilus, by a suppressor mutation method. We previously constructed a chimeric IPMDH consisting of portions of Thermophile and mesophile enzymes. The chimeric enzyme is less thermostable than the Thermophile enzyme. The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T. thermophilus. The transformants were screened at 76 degrees C in minimum medium, and three independent stabilized mutants were obtained. The leuB genes from these three mutants were cloned and analyzed. The sequence analyses revealed Ala-172-->Val substitution in all of the mutants. The thermal stability of the Thermophile IPMDH was improved by introducing the amino acid substitution.
Ken-ichi Uchida - One of the best experts on this subject based on the ideXlab platform.
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thermopile based on anisotropic magneto peltier effect
Applied Physics Letters, 2019Co-Authors: Raja Das, Ken-ichi UchidaAbstract:We propose thermopile structures for the anisotropic magneto-Peltier effect (AMPE) to enhance its heating/cooling power. The cross-shaped thermopile, one of the representative AMPE-based thermopile structures, consists of four L-shaped ferromagnetic metals arranged in a cross-shaped configuration, which allows the concentration of the AMPE-induced temperature modulation at the center of the cross structure. The AMPE-based thermopile does not require the use of any complicated junctions comprising different materials, enabling the design of compact and versatile temperature controllers for nanoscale devices.We propose thermopile structures for the anisotropic magneto-Peltier effect (AMPE) to enhance its heating/cooling power. The cross-shaped thermopile, one of the representative AMPE-based thermopile structures, consists of four L-shaped ferromagnetic metals arranged in a cross-shaped configuration, which allows the concentration of the AMPE-induced temperature modulation at the center of the cross structure. The AMPE-based thermopile does not require the use of any complicated junctions comprising different materials, enabling the design of compact and versatile temperature controllers for nanoscale devices.
Akihiko Yamagishi - One of the best experts on this subject based on the ideXlab platform.
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purification catalytic properties and thermostability of 3 isopropylmalate dehydrogenase from escherichia coli
Biochimica et Biophysica Acta, 1997Co-Authors: Gerlind Wallon, Susan T. Lovett, Hiromi Kirino, Gregory A Petsko, Kazutaka Yamamoto, Akihiko Yamagishi, Tairo OshimaAbstract:Abstract 3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the Thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg 2+ or Mn 2+ , with Mn 2+ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40°C the K m of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k cat was 69 s −1 . The half denaturationn temperature was 64°C, which was 20°C lower than that of the Thermophile enzyme.
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pyrimidine biosynthesis genes pyre and pyrf of an extreme Thermophile thermus thermophilus
Applied and Environmental Microbiology, 1996Co-Authors: Akihiko Yamagishi, T Tanimoto, Toshiharu Suzuki, T. OshimaAbstract:We have isolated uracil auxotrophic mutants of an extreme Thermophile, Thermus thermophilus. A part of the pyrimidine biosynthetic operon including genes for orotate phosphoribosyltransferase (pyrE) and for orotidine-5'-monophosphate decarboxylase (pyrF) was cloned and sequenced. The pyrE gene can be a bidirectional marker for the gene manipulation system of the Thermophile.
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further stabilization of 3 isopropylmalate dehydrogenase of an extreme Thermophile thermus thermophilus by a suppressor mutation method
Journal of Bacteriology, 1996Co-Authors: Takashi Kotsuka, Masaaki Tomuro, Satoshi Akanuma, Akihiko Yamagishi, Tairo OshimaAbstract:We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme Thermophile, Thermus thermophilus, by a suppressor mutation method. We previously constructed a chimeric IPMDH consisting of portions of Thermophile and mesophile enzymes. The chimeric enzyme is less thermostable than the Thermophile enzyme. The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T. thermophilus. The transformants were screened at 76 degrees C in minimum medium, and three independent stabilized mutants were obtained. The leuB genes from these three mutants were cloned and analyzed. The sequence analyses revealed Ala-172-->Val substitution in all of the mutants. The thermal stability of the Thermophile IPMDH was improved by introducing the amino acid substitution.
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hydrophobic interaction at the subunit interface contributes to the thermostability of 3 isopropylmalate dehydrogenase from an extreme Thermophile thermus thermophilus
FEBS Journal, 1994Co-Authors: Hiromi Kirino, Yumiko Hayashi, Masayuki Ohba, Miho Aoshima, Takayoshi Wakagi, Akihiko Yamagishi, Makoto Aoki, Tairo OshimaAbstract:We cloned and sequenced the leuB gene encoding 3-isopropylmalate dehydrogenase from Escherichia coli K-12 (JM103). Errors (33 residues) were found and corrected in the sequence previously reported for the leuB gene of Thermus thermophilus. The three-dimensional structure of the Thermophile enzyme and the amino acid sequence comparison suggested that a part of the high stability of the T. thermophilus enzyme is conferred by increased hydrophobic interaction at the subunit-subunit interface. Two residues at the interface of the T. thermophilus enzyme, Leu246 and Val249, are substituted with less hydrophobic residues, Glu and Met, respectively, in the E. coli enzyme, whereas other residues in this region are highly conserved. The mutated T. thermophilus enzyme [L246E, V249M]IPMDH had reduced stability to heat. Two residues of the E. coli dehydrogenase, Glu256 and Met259, were replaced with the corresponding residues from the Thermophile sequence. The resulted mutant enzyme was more resistant to heat than the wild-type enzyme.
Hiromi Kirino - One of the best experts on this subject based on the ideXlab platform.
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purification catalytic properties and thermostability of 3 isopropylmalate dehydrogenase from escherichia coli
Biochimica et Biophysica Acta, 1997Co-Authors: Gerlind Wallon, Susan T. Lovett, Hiromi Kirino, Gregory A Petsko, Kazutaka Yamamoto, Akihiko Yamagishi, Tairo OshimaAbstract:Abstract 3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the Thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg 2+ or Mn 2+ , with Mn 2+ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40°C the K m of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k cat was 69 s −1 . The half denaturationn temperature was 64°C, which was 20°C lower than that of the Thermophile enzyme.
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hydrophobic interaction at the subunit interface contributes to the thermostability of 3 isopropylmalate dehydrogenase from an extreme Thermophile thermus thermophilus
FEBS Journal, 1994Co-Authors: Hiromi Kirino, Yumiko Hayashi, Masayuki Ohba, Miho Aoshima, Takayoshi Wakagi, Akihiko Yamagishi, Makoto Aoki, Tairo OshimaAbstract:We cloned and sequenced the leuB gene encoding 3-isopropylmalate dehydrogenase from Escherichia coli K-12 (JM103). Errors (33 residues) were found and corrected in the sequence previously reported for the leuB gene of Thermus thermophilus. The three-dimensional structure of the Thermophile enzyme and the amino acid sequence comparison suggested that a part of the high stability of the T. thermophilus enzyme is conferred by increased hydrophobic interaction at the subunit-subunit interface. Two residues at the interface of the T. thermophilus enzyme, Leu246 and Val249, are substituted with less hydrophobic residues, Glu and Met, respectively, in the E. coli enzyme, whereas other residues in this region are highly conserved. The mutated T. thermophilus enzyme [L246E, V249M]IPMDH had reduced stability to heat. Two residues of the E. coli dehydrogenase, Glu256 and Met259, were replaced with the corresponding residues from the Thermophile sequence. The resulted mutant enzyme was more resistant to heat than the wild-type enzyme.
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molecular cloning and nucleotide sequence of 3 isopropylmalate dehydrogenase gene leub from an extreme Thermophile thermus aquaticus yt 1
Journal of Biochemistry, 1991Co-Authors: Hiromi Kirino, Tairo OshimaAbstract:: A gene (leuB) coding for 3-isopropylmalate dehydrogenase [EC 1.1.1.85] from an extreme Thermophile, Thermus aquaticus YT-1 was cloned in Escherichia coli and the nucleotide sequence was determined. It contains an open reading frame of 1,035 bp encoding 344 amino acid residues. The homology with that from T. thermophilus HB8 is 87.0% in nucleotide and 91.3% in amino acid sequences. No overlapped gene was found in the present leuB gene, in contrast to the previous prediction that Thermus leuD gene is overlapped with leuB [Croft et al. (1987) Mol. Gen. Genet. 210, 490-497]. Substitutions in the primary structure which are unique for the Thermophile sequences are discussed in relation to the unusual stability of the Thermophile dehydrogenase based on amino acid sequence comparison of 9 microorganisms including Thermophiles and mesophiles.
Joseph Groom - One of the best experts on this subject based on the ideXlab platform.
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deletion of the clostridium thermocellum reca gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous dna sequences
Journal of Industrial Microbiology & Biotechnology, 2018Co-Authors: Joseph Groom, Janet Westpheling, Daehwan Chung, Adam M. GussAbstract:A limitation to the engineering of cellulolytic Thermophiles is the availability of functional, thermostable (≥ 60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic Thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic Thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ∆recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic Thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.
