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Tairo Oshima - One of the best experts on this subject based on the ideXlab platform.
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Enigmas of biosyntheses of unusual polyamines in an extreme thermophile, Thermus thermophilus.
Plant Physiology and Biochemistry, 2010Co-Authors: Tairo OshimaAbstract:Thermus thermophilus, an extreme thermophile belonging to Domain Bacteria, produces unusual polyamines in addition to standard polyamines. To understand mechanisms of changes of polyamine compositions of the thermophile upon change of growth conditions such as environmental temperature, metabolic pathways of polyamine biosyntheses of T. thermophilus have been studied and a new polyamine metabolic pathway was proposed. However, many enigmas remain to be solved in future studies. In this paper, biosyntheses of two non-standard polyamines, thermospermine and sym-homospermidine which are also produced and play important roles in plant cells, of the extreme thermophile are discussed in relation to the biosynthetic reactions in plants.
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stabilization of nucleic acids by unusual polyamines produced by an extreme thermophile thermus thermophilus
Biochemical Journal, 2005Co-Authors: Yusuke Terui, Mio Ohnuma, Kaori Hiraga, Etsuko Kawashima, 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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urea induced unfolding and conformational stability of 3 isopropylmalate dehydrogenase from the thermophile thermus thermophilus and its mesophilic counterpart from escherichia coli
Biochemistry, 1999Co-Authors: Chie Motono, And Akihiko Yamagishi, Tairo OshimaAbstract:To reveal the basis of the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli has been studied. The urea-induced equilibrium unfolding of T. thermophilus and E. coli IPMDHs at 27 °C was monitored by measuring the changes in far-UV CD, intrinsic fluorescence, anilinonaphthalenesulfonic acid (ANS) binding, and catalytic activity in the presence of nonionic detergent Tween 20. For both enzymes, the spectral methods revealed a biphasic unfolding transition. The first transition was protein concentration-independent, whereas the second was protein concentration-dependent for both enzymes. The observation suggested a three-state unfolding mechanism with a dimeric intermediate. However, the intermediates of the E. coli and the T. thermophilus IPMDHs seemed to be different from each other. The intermediate of the E. coli IPMDH lost its secondary and tertiary str...
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The organization of the leuC, leuD and leuB genes of the extreme thermophile Thermus thermophilus.
Gene, 1998Co-Authors: Masatada Tamakoshi, Akihiko Yamagishi, Tairo OshimaAbstract:Abstract 3-Isopropylmalate dehydrogenase is encoded by leuB gene while leuC and leuB genes encode the large and small subunits of isopropylmalate isomerase in leucine biosynthetic pathway, respectively. Organization of the leuB , leuC and leuD genes of an extreme thermophile, Thermus thermophilus , was investigated by sequence analysis. Location of the genes was also tested by complementation analysis of leu deficiency of the thermophile and Escherichia coli . The order was the leuC , leuD , and leuB genes and, in contrast to a previous report, they did not overlap with each other. Sequence analysis of the leuC and leuD genes suggested that cysteine residues for iron–sulfur binding and other amino acid residues involved in isomerase activity, which have been inferred from analysis of a related protein, aconitase, were highly conserved.
Natsuki Tomariguchi - One of the best experts on this subject based on the ideXlab platform.
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occurrence of randomly recombined functional 16s rrna genes in thermus thermophilus suggests genetic interoperability and promiscuity of bacterial 16s rrnas
Scientific Reports, 2019Co-Authors: Kentaro Miyazaki, Natsuki TomariguchiAbstract:Based on the structural complexity of ribosomes, 16S rRNA genes are considered species-specific and hence used for bacterial phylogenetic analysis. However, a growing number of reports suggest the occurrence of horizontal gene transfer, raising genealogical questions. Here we show the genetic interoperability and promiscuity of 16S rRNA in the ribosomes of an extremely thermophilic bacterium, Thermus thermophilus. The gene in this thermophile was systematically replaced with a diverse array of heterologous genes, resulting in the discovery of various genes that supported growth, some of which were from different phyla. Moreover, numerous functional chimeras were spontaneously generated. Remarkably, cold-adapted mutants were obtained carrying chimeric or full-length heterologous genes, indicating that horizontal gene transfer promoted adaptive evolution. The ribosome may well be understood as a patchworked supramolecule comprising patchworked components. We here propose the “random patch model” for ribosomal evolution.
Akihiko Yamagishi - One of the best experts on this subject based on the ideXlab platform.
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The organization of the leuC, leuD and leuB genes of the extreme thermophile Thermus thermophilus.
Gene, 1998Co-Authors: Masatada Tamakoshi, Akihiko Yamagishi, Tairo OshimaAbstract:Abstract 3-Isopropylmalate dehydrogenase is encoded by leuB gene while leuC and leuB genes encode the large and small subunits of isopropylmalate isomerase in leucine biosynthetic pathway, respectively. Organization of the leuB , leuC and leuD genes of an extreme thermophile, Thermus thermophilus , was investigated by sequence analysis. Location of the genes was also tested by complementation analysis of leu deficiency of the thermophile and Escherichia coli . The order was the leuC , leuD , and leuB genes and, in contrast to a previous report, they did not overlap with each other. Sequence analysis of the leuC and leuD genes suggested that cysteine residues for iron–sulfur binding and other amino acid residues involved in isomerase activity, which have been inferred from analysis of a related protein, aconitase, were highly conserved.
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serial increase in the thermal stability of 3 isopropylmalate dehydrogenase from bacillus subtilis by experimental evolution
Protein Science, 1998Co-Authors: Satoshi Akanuma, Nobuo Tanaka, Akihiko Yamagishi, Tairo OshimaAbstract:We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type 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, Kazutaka Yamamoto, Akihiko Yamagishi, Gregory A Petsko, 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, Toshiharu Suzuki, T Tanimoto, 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.
Beate Averhoff - One of the best experts on this subject based on the ideXlab platform.
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shuffling genes around in hot environments the unique dna transporter of thermus thermophilus
Fems Microbiology Reviews, 2009Co-Authors: Beate AverhoffAbstract:Natural transformation permits the transport of DNA through bacterial membranes and represents a dominant mode for the transfer of genetic information between bacteria and between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal, or lateral, gene transfer, has been a major force for genome plasticity over evolutionary history, and is largely responsible for the spread of fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Here, we present a survey of the natural transformation machinery of the thermophile Thermus thermophilus HB27. A tentative model of the transformation machinery comprising of components similar to proteins of type IV pili and type II secretion systems is presented. A comparative discussion of the subunits and the structure of the DNA translocator and the underlying mechanism of transfer of free DNA in T. thermophilus highlights conserved and unique features of the DNA translocator in T. thermophilus. We hypothesize that the extraordinary broad substrate specificity and the high efficiency of the T. thermophilus DNA uptake system is of major importance for thermoadaptation and interdomain DNA transfer in hot environments.
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Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27.
FEBS Journal, 2006Co-Authors: Cornelia Schwarzenlander, Beate AverhoffAbstract:Horizontal gene transfer has been a major force for genome plasticity over evolutionary history, and is largely responsible for fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Recently, by performing a genome-wide mutagenesis approach with Thermus thermophilus HB27, we identified the first genes in a thermophilic bacterium for the uptake of free DNA, a process called natural transformation. Here, we present the first data on the biochemistry and bioenergetics of the DNA transport process in this thermophile. We report that linear and circular plasmid DNA are equally well taken up with a high maximal velocity of 1.5 µg DNA·(mg protein)−1·min−1, demonstrating an extremely efficient binding and uptake rate of 40 kb·s−1·cell−1. Uncouplers and ATPase inhibitors immediately inhibited DNA uptake, providing clear evidence that DNA translocation in HB27 is an energy-dependent process. DNA uptake studies with genomic DNA of Bacteria, Archaea and Eukarya revealed that Thermus thermophilus HB27 takes up DNA from members of all three domains of life. We propose that the extraordinary broad substrate specificity of the highly efficient Thermus thermophilus HB27 DNA uptake system may contribute significantly to thermoadaptation of Thermus thermophilus HB27 and to interdomain DNA transfer in hot environments.
Kentaro Miyazaki - One of the best experts on this subject based on the ideXlab platform.
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occurrence of randomly recombined functional 16s rrna genes in thermus thermophilus suggests genetic interoperability and promiscuity of bacterial 16s rrnas
Scientific Reports, 2019Co-Authors: Kentaro Miyazaki, Natsuki TomariguchiAbstract:Based on the structural complexity of ribosomes, 16S rRNA genes are considered species-specific and hence used for bacterial phylogenetic analysis. However, a growing number of reports suggest the occurrence of horizontal gene transfer, raising genealogical questions. Here we show the genetic interoperability and promiscuity of 16S rRNA in the ribosomes of an extremely thermophilic bacterium, Thermus thermophilus. The gene in this thermophile was systematically replaced with a diverse array of heterologous genes, resulting in the discovery of various genes that supported growth, some of which were from different phyla. Moreover, numerous functional chimeras were spontaneously generated. Remarkably, cold-adapted mutants were obtained carrying chimeric or full-length heterologous genes, indicating that horizontal gene transfer promoted adaptive evolution. The ribosome may well be understood as a patchworked supramolecule comprising patchworked components. We here propose the “random patch model” for ribosomal evolution.
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co enzyme specificity of 3 isopropylmalate dehydrogenase from thermits thermophilus hb8
Protein Engineering, 1994Co-Authors: Kentaro Miyazaki, Tairo OshimaAbstract:: The co-enzyme specificity of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus HB8, was changed from NAD to NADP by site-directed muta-genesis. Based on sequence comparison of 3-isopropylmalate dehydrogenases from various organisms with NAD- and NADP-dependent isocitrate dehydrogenases, Ser226, Ser253 and Ile279 of 3-isopropylmalate dehydrogenase were suggested as determining the co-enzyme specificity. These residues were replaced with the corresponding residues of NADP-dependent isocitrate dehydrogenases; Arg, Gly and Tyr respectively. The single-mutated enzymes, S226R and 1279Y, enhanced the activities towards NADP approximately 10- and approximately 3-fold respectively, whereas S253G reduced the activity. Among the multiple-mutated enzymes, the double-mutated S226R/I279Y increased the catalytic efficiency against NADP (approximately 5-fold) and shifted the specificity for NAD towards NADP most significantly (approximately 173-fold).
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chemical modification and site directed mutagenesis of tyr36 of 3 isopropylmalate dehydrogenase from thermus thermophilus hb8
Protein Engineering, 1994Co-Authors: Kentaro Miyazaki, Hideaki Moriyama, Nobuo Tanaka, Masahiro Sakurai, Shojiro Kadono, Tairo OshimaAbstract:: 3-Isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus HB8, was chemically modified with tetranitromethane which nitrated 1.5-2.0 Tyr residues per subunit. The nitration was biphasic and parallel to the loss of activity. The modified residue in the first phase was identified to be Tyr36, which is distantly located from the active site of the enzyme. The function of Tyr36 was investigated by site-specific replacement with Phe. The Michaelis constant for the substrate or co-enzyme was not altered by the replacement, whereas the catalytic constant decreased down to approximately 5%. X-ray analysis of the mutant enzyme revealed that Arg94 moved the largest distance among the active site residues, that is, the NH1 and NH2 of the guanidino group moved 1.11 and 1.32 A respectively. The results suggest that Arg94 is responsible for the enzyme catalysis.
