Updated on 2024/02/15

写真a

 
TAMURA Takashi
 
Organization
Institute of Global Human Resorce Development Professor
Position
Professor
External link

Degree

  • Ph.D. ( Kyoto University )

Research Interests

  • redox

  • Selenium

  • 放線菌の二次代謝酵素

  • 量子酵素化学

  • レドックス生物化学

  • セレン生物化学

Research Areas

  • Life Science / Bioorganic chemistry

  • Life Science / Applied biochemistry

  • Life Science / Food sciences

Education

  • Kyoto University    

    - 1993

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  • Kyoto University   農学研究科   農芸化学

    - 1993

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    Country: Japan

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  • Okayama University    

    - 1988

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  • Okayama University   農学部   農芸化学

    - 1988

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    Country: Japan

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Research History

  • - 岡山大学環境生命科学研究科 教授

    2013

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  • - Professor,Graduate School of Environmental and life Science,Okayama University

    2013

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  • - 理化学研究所播磨研究所 研究員

    2010

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  • - 東京大学生産技術研究所 研究員

    2010

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  • - Researcher

    2010

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  • Associate Professor,Graduate School of Natural Science and Technology,Okayama University

    2004 - 2013

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  • 岡山大学自然科学研究科 准教授

    2004 - 2013

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Professional Memberships

 

Papers

  • Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation. International journal

    Koki Okada, Yu Morimoto, Yukine Shiraishi, Takashi Tamura, Shigeki Mayama, Takashi Kadono, Masao Adachi, Kentaro Ifuku, Michiko Nemoto

    Marine biotechnology (New York, N.Y.)   2023.12

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    Language:English   Publishing type:Research paper (scientific journal)  

    Nitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500 μg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development.

    DOI: 10.1007/s10126-023-10273-w

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  • Author Correction: Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota. International journal

    Titouan Jaunet-Lahary, Tatsuro Shimamura, Masahiro Hayashi, Norimichi Nomura, Kouta Hirasawa, Tetsuya Shimizu, Masao Yamashita, Naotaka Tsutsumi, Yuta Suehiro, Keiichi Kojima, Yuki Sudo, Takashi Tamura, Hiroko Iwanari, Takao Hamakubo, So Iwata, Kei-Ichi Okazaki, Teruhisa Hirai, Atsuko Yamashita

    Nature communications   14 ( 1 )   6053 - 6053   2023.9

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  • Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota. International journal

    Titouan Jaunet-Lahary, Tatsuro Shimamura, Masahiro Hayashi, Norimichi Nomura, Kouta Hirasawa, Tetsuya Shimizu, Masao Yamashita, Naotaka Tsutsumi, Yuta Suehiro, Keiichi Kojima, Yuki Sudo, Takashi Tamura, Hiroko Iwanari, Takao Hamakubo, So Iwata, Kei-Ichi Okazaki, Teruhisa Hirai, Atsuko Yamashita

    Nature communications   14 ( 1 )   1730 - 1730   2023.4

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    An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

    DOI: 10.1038/s41467-023-36883-5

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  • Recombinant expression using the tetrathionate hydrolase promoter in Acidithiobacillus ferrooxidans.

    Tadayoshi Kanao, Tomoki Kunihisa, Shuji Ohgimoto, Megumi Ito, Chisa Murakami, Hisayuki Nakayama, Takashi Tamura, Kazuo Kamimura

    Journal of bioscience and bioengineering   135 ( 3 )   176 - 181   2023.3

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    In the iron- and sulfur-oxidizing acidophilic chemolithoautotrophic bacterium, Acidithiobacillus ferrooxidans, tetrathionate hydrolase gene (Af-tth) is highly expressed during tetrathionate growth. The expression levels of Af-tth were specifically determined by quantitative reverse transcription-polymerase chain reaction and the expression ratios of S0/Fe2+ and S4O62-/Fe2+ were found to be 68 ± 21 and 181 ± 5, respectively. The transcriptional start site was identified by primer extension. Promoter regions of Af-tth were cloned into the expression shuttle vector pMPJC and GFP gene was under the direction of the regions. Green fluorescence was observed by UV irradiation in recombinant A. ferrooxidans harboring the plasmid colonies grown on tetrathionate. Furthermore, His-tagged Af-Tth was synthesized in the recombinant cells grown on tetrathionate. Recombinant, His-tagged Af-Tth in an active form, was rapidly purified through metal-affinity column chromatography, although recombinant Af-Tth was synthesized in the inclusion bodies of Escherichia coli and acid-refolding treatment was necessary to recover the activity. The specific activity of purified Af-Tth from recombinant A. ferrooxidans (2.2 ± 0.37 U mg-1) was similar to that of acid-refolded Af-Tth from recombinant E. coli (2.5 ± 0.18 U mg-1). This method can be applied not only to heterologous expression but also to homologous expression of target genes for modification or specific mutation in A. ferrooxidans cells.

    DOI: 10.1016/j.jbiosc.2022.12.005

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  • Characterization and application of l-methionine γ-lyase Q349S mutant enzyme with an enhanced activity toward l-homocysteine

    Atsushi Okawa, Haruhisa Handa, Eri Yasuda, Masaki Murota, Daizo Kudo, Takashi Tamura, Tomoo Shiba, Kenji Inagaki

    Journal of Bioscience and Bioengineering   133 ( 3 )   213 - 221   2022.3

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    Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.jbiosc.2021.11.008

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  • A new l-arginine oxidase engineered from l-glutamate oxidase

    Yoshika Yano, Shinsaku Matsuo, Nanako Ito, Takashi Tamura, Hitoshi Kusakabe, Kenji Inagaki, Katsumi Imada

    PROTEIN SCIENCE   30 ( 5 )   1044 - 1055   2021.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY  

    The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 mu M. The linear relationship is maintained around 1 mu M of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

    DOI: 10.1002/pro.4070

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  • Structural basis for substrate specificity of L-methionine decarboxylase

    Atsushi Okawa, Tomoo Shiba, Masaya Hayashi, Yuki Onoue, Masaki Murota, Dan Sato, Junko Inagaki, Takashi Tamura, Shigeharu Harada, Kenji Inagaki

    PROTEIN SCIENCE   30 ( 3 )   663 - 677   2021.3

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    L-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B-6-dependent enzyme and catalyzes the non-oxidative decarboxylation of L-methionine to produce 3-methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand-free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.

    DOI: 10.1002/pro.4027

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  • Correction to: Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases. International journal

    Michiko Nemoto, Sayako Iwaki, Hisao Moriya, Yuki Monden, Takashi Tamura, Kenji Inagaki, Shigeki Mayama, Kiori Obuse

    Marine biotechnology (New York, N.Y.)   23 ( 1 )   157 - 157   2021.2

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  • Multiple mutations in RNA polymerase β-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory. International journal

    Saori Ogawa, Hitomi Shimidzu, Koji Fukuda, Naoki Tsunekawa, Toshiyuki Hirano, Fumitoshi Sato, Kei Yura, Tomohisa Hasunuma, Kozo Ochi, Michio Yamamoto, Wataru Sakamoto, Kentaro Hashimoto, Hiroyuki Ogata, Tadayoshi Kanao, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

    Bioscience, biotechnology, and biochemistry   2021.1

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    Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

    DOI: 10.1093/bbb/zbab011

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  • Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride

    Masaki Kitagawa, Nanako Ito, Yuya Matsumoto, Masaya Saito, Takashi Tamura, Hitoshi Kusakabe, Kenji Inagaki, Katsumi Imada

    Journal of Structural Biology: X   5   100044 - 100044   2021

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    DOI: 10.1016/j.yjsbx.2021.100044

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  • Structural basis of strict substrate recognition of l ‐lysine α‐oxidase from Trichoderma viride

    Hiroki Kondo, Masaki Kitagawa, Yuya Matsumoto, Masaya Saito, Marie Amano, Shigeru Sugiyama, Takashi Tamura, Hitoshi Kusakabe, Kenji Inagaki, Katsumi Imada

    Protein Science   2020.9

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    Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1002/pro.3946

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    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pro.3946

  • Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases. Reviewed International journal

    Michiko Nemoto, Sayako Iwaki, Hisao Moriya, Yuki Monden, Takashi Tamura, Kenji Inagaki, Shigeki Mayama, Kiori Obuse

    Marine biotechnology (New York, N.Y.)   2020.6

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    Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

    DOI: 10.1007/s10126-020-09976-1

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  • Novel method for l-methionine determination using l-methionine decarboxylase and application of the enzyme for l-homocysteine determination. Reviewed International journal

    Atsushi Okawa, Masaya Hayashi, Junko Inagaki, Toshihide Okajima, Takashi Tamura, Kenji Inagaki

    Bioscience, biotechnology, and biochemistry   0916-8451 (print) ( 1347-6947 (online) )   1 - 9   2020.1

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    For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10-200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC.Abbreviations: DTT: dithiothreitol; IPTG: isopropyl-β-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5'-phosphate.

    DOI: 10.1080/09168451.2020.1715781

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  • Antiviral effect of sinefungin on in vitro growth of feline herpesvirus type 1. Reviewed

    Yudai Kuroda, Haruka Yamagata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura, Ken Maeda

    The Journal of antibiotics   72 ( 12 )   981 - 985   2019.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media {LLC}  

    Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

    DOI: 10.1038/s41429-019-0234-4

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  • 量子化学が解き明かすピリドキサール酵素の反応多様性

    田村 隆

    生化学   91 ( 2 )   260 - 264   2019.4

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    File: 生化学PLP.pdf

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  • Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase. Reviewed International journal

    Shinya Sutoh, Yuko Uemura, Yuko Yamaguchi, Asako Kiyotou, Rena Sugihara, Makiko Nagayasu, Mihoko Kurokawa, Koreaki Ito, Naoki Tsunekawa, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

    Biochimica et biophysica acta. Proteins and proteomics   1867 ( 3 )   194 - 201   2019.3

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    Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was -172 mV, representing a less-oxidizing catalysis than the wild type DsbA (-122 mV), but one that is closer to yeast protein disulfide isomerase (-175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of -172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.

    DOI: 10.1016/j.bbapap.2018.12.005

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  • Integrated transcriptomic and proteomic analyses of a molecular mechanism of radular teeth biomineralization in Cryptochiton stelleri. Reviewed International journal

    Michiko Nemoto, Dongni Ren, Steven Herrera, Songqin Pan, Takashi Tamura, Kenji Inagaki, David Kisailus

    Scientific reports   9 ( 1 )   856 - 856   2019.1

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    Many species of chiton are known to deposit magnetite (Fe3O4) within the cusps of their heavily mineralized and ultrahard radular teeth. Recently, much attention has been paid to the ultrastructural design and superior mechanical properties of these radular teeth, providing a promising model for the development of novel abrasion resistant materials. Here, we constructed de novo assembled transcripts from the radular tissue of C. stelleri that were used for transcriptome and proteome analysis. Transcriptomic analysis revealed that the top 20 most highly expressed transcripts in the non-mineralized teeth region include the transcripts encoding ferritin, while those in the mineralized teeth region contain a high proportion of mitochondrial respiratory chain proteins. Proteomic analysis identified 22 proteins that were specifically expressed in the mineralized cusp. These specific proteins include a novel protein that we term radular teeth matrix protein1 (RTMP1), globins, peroxidasins, antioxidant enzymes and a ferroxidase protein. This study reports the first de novo transcriptome assembly from C. stelleri, providing a broad overview of radular teeth mineralization. This new transcriptomic resource and the proteomic profiles of mineralized cusp are valuable for further investigation of the molecular mechanisms of radular teeth mineralization in chitons.

    DOI: 10.1038/s41598-018-37839-2

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  • An Internal Sequence Region Catalytically Essential for the Human Selenophosphate Synthetase 2 Reviewed

    Muneaki Takahata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

    FREE RADICAL BIOLOGY AND MEDICINE   112   65 - 65   2017.11

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    DOI: 10.1016/j.freeradbiomed.2017.10.092

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  • Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico

    Takashi Tamura, Naoki Tsunekawa, Michiko Nemoto, Kenji Inagaki, Toshiyuki Hirano, Fumitoshi Sato

    SCIENTIFIC REPORTS   6   19742   2016.1

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    Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing delta-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.

    DOI: 10.1038/srep19742

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  • Molecular cloning and characterization of L-methionine gamma-lyase from Streptomyces avermitilis Reviewed

    Daizou Kudou, Eri Yasuda, Yoshiyuki Hirai, Takashi Tamura, Kenji Inagaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   120 ( 4 )   380 - 383   2015.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    A pyridoxal 5'-phosphate-dependent methionine gamma-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to alpha-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2015.02.019

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  • Reactivity of sorbose dehydrogenase from Sinorhizobium sp 97507 for 1,5-anhydro-D-glucitol Reviewed

    Toshio Araki, Tomoko Nakatsuka, Fuminao Kobayashi, Emi Watanabe-Ishimaru, Hirokazu Sanada, Takashi Tamura, Kenji Inagaki

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   79 ( 7 )   1130 - 1132   2015.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:TAYLOR & FRANCIS LTD  

    DOI: 10.1080/09168451.2015.1012148

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  • Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, l-lysine alpha-oxidase from Trichoderma viride* Reviewed

    Marie Amano, Haruka Mizuguchi, Tadahisa Sano, Hiroki Kondo, Kengo Shinyashiki, Junko Inagaki, Takashi Tamura, Tatsuya Kawaguchi, Hitoshi Kusakabe, Katsumi Imada, Kenji Inagaki

    JOURNAL OF BIOCHEMISTRY   157 ( 6 )   549 - 559   2015.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    L-Lysine alpha-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form alpha-keto-epsilon-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 <remove> resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

    DOI: 10.1093/jb/mvv012

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    Other Link: http://orcid.org/0000-0003-1342-8885

  • Draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin Reviewed

    Kenshiro Oshima, Masahira Hattori, Hitomi Shimizu, Koji Fukuda, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

    Genome Announcements   3 ( 4 )   e00715   2015

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.

    File: Microbiology Resource Announcements-2015-Oshima-e00715-15.full.pdf

    DOI: 10.1128/genomeA.00715-15

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  • Sulfur Transport System for the formation of iron-sulfur cluster

    Tamura Takashi, Asano Kaori

    VITAMINS   89 ( 2 )   80 - 82   2015

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  • Mechanism of N_2 fixation and H_2 production by nitrogenase

    Tamura Takashi

    VITAMINS   89 ( 8 )   409 - 412   2015

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    DOI: 10.20632/vso.89.8_409

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  • Glycerol kinase from Cellulomonas sp. NT3060 which has high resistance against preservatives Reviewed

    Sogabe Atsushi, Kitabayashi Masao, Morishima Kenichi, Furukawa Miyoko, Hatta Takashi, Fukuda Yasuhisa, Nishise Hiroshi, Oka Masanori, Tamura Takashi, Inagaki Kenji

    Seibutsu-kogaku Kaishi   92 ( 8 )   402 - 409   2014.8

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    Glycerol kinase is suitable for clinical diagnostic use in levels of neutral fat. Glycerol kinase from Cellulomonas sp. NT3060 shows good stability against several preservatives such as N-methylisothiazolone and imidazolidinylurea. We have cloned glpK gene from Cellulomonas sp. NT3060. This glpK gene is composed of 1,518 bp nucleotides and encodes 505 amino acid residues. The glpK gene was expressed in Escherichia coli and Cellulomonas sp. glycerol kinase was purified to homogeneity by column chromatography. The glycerol kinase exhibited high tolerance to various preservatives compared to other glycerol kinases. This tolerance was found to be favorable feature as diagnostic reagent for quantity of triglyceride.

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  • The Role of Amino Acid Residues in the Active Site of L-Methionine gamma-lyase from Pseudomonas putida Reviewed

    Mitsuki Fukumoto, Daizou Kudou, Shouko Murano, Tomoo Shiba, Dan Sato, Takashi Tamura, Shigeharu Harada, Kenji Inagaki

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   76 ( 7 )   1275 - 1284   2012.7

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    Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine gamma-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel beta-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

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  • Arg305 of Streptomyces L-glutamate oxidase plays a crucial role for substrate recognition Reviewed

    Tomohiro Utsumi, Jiro Arima, Chika Sakaguchi, Takashi Tamura, Chiduko Sasaki, Hitoshi Kusakabe, Shigetoshi Sugio, Kenji Inagaki

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   417 ( 3 )   951 - 955   2012.1

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    Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking simulation using.-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate. (C) 2011 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2011.12.033

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  • 2PT109 Structure of L-lysine α-oxidase from Trichoderma viride(The 50th Annual Meeting of the Biophysical Society of Japan)

    Sano Tadahisa, Kawaguchi Tatsuya, Amano Marie, Shinyashiki Kengo, Nakata Haruka, Tamura Takashi, Inagaki Kenji, Kusakabe Hitoshi, Imada Katsumi

    Seibutsu Butsuri   52   S123   2012

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    DOI: 10.2142/biophys.52.S123_1

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  • Quantum Chemical Mechanism of Hydrogeanses

    Tamura Takashi

    VITAMINS   86 ( 7 )   412 - 414   2012

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    DOI: 10.20632/vso.86.7_412

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    Other Link: http://search.jamas.or.jp/link/ui/2013003170

  • Selenite Reduction by the Thioredoxin System: Kinetics and Identification of Protein-Bound Selenide Reviewed

    Takashi Tamura, Kumi Sato, Kentaro Komori, Takeshi Imai, Mitsuhiko Kuwahara, Takahiro Okugochi, Hisaaki Mihara, Nobuyoshi Esaxi, Kenji Inagaki

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 6 )   1184 - 1187   2011.6

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    Selenite (SeO32-) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/auto-radiography using Se-75-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).

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  • Identification and Conformer Analysis of a Novel Redox-Active Motif, Pro-Ala-Ser-Cys-Cys-Ser, in Drosophila Thioredoxin Reductase by Semiempirical Molecular Orbital Calculation Reviewed

    Mitsuhiko Kuwahara, Takashi Tamura, Kentaro Kawamura, Kenji Inagaki

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 3 )   516 - 521   2011.3

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    Mammalian thioredoxin reductases (TrxRs) contain selenium as selenocysteine (Sec) in the C-terminal redox center -Gly-Cys-Sec-Gly-OH to reduce Trx and other substrates; a Sec-to-Cys substitution in mammalian TrxR yields an almost inactive enzyme. The corresponding tetrapeptide sequence in Drosophila melanogaster TrxR (Dm-TrxR), -Ser-Cys-Cys-Ser-OH, endows the orthologous enzyme with a catalytic competence similar to mammalian selenoenzymes, but implementation of the Ser-containing tetrapeptide sequence SCCS into the mammalian enzyme does not restore the activity of the Sec-to-Cys mutant form (turnover number < 2/min). MOPAC calculation suggested that the C-terminal hexapeptide Pro-Ala-Ser-Cys-Cys-Ser-OH functions as a redox center that alleviates the necessity for selenium in Dm-TrxR, and a mutant form of human lung TrxR that mimics this hexapeptide sequence showed improved catalytic turnover (17.4/min for DTNB and 13.2/min for E. coli trx) compared to the Sec-to-Cys mutant. MOPAC calculation also suggested that the dominant form of the Pro-containing hexapeptide is a C+ conformation, which perhaps has a catalytic advantage in facile reduction of the intramolecular disulfide bond between Cys497 and Cys498 by the N-terminal redox center in the neighboring subunit.

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  • Protein Involvement in Early Phase of Se Metabolism

    Tamura Takashi, Okugochi Takahiro

    VITAMINS   85 ( 7 )   346 - 348   2011

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    DOI: 10.20632/vso.85.7_346

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    Other Link: http://search.jamas.or.jp/link/ui/2011324915

  • Selenoproteins in response to ER stress

    Tamura Takashi

    VITAMINS   85 ( 1 )   27 - 28   2011

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    DOI: 10.20632/vso.85.1_27

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  • Selenoproteins as potential nutritional biomarkers for Se

    Tamura Takashi

    VITAMINS   84 ( 2 )   77 - 79   2010

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    DOI: 10.20632/vso.84.2_77

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    Other Link: http://search.jamas.or.jp/link/ui/2010133267

  • 9.高基質特異性L-アミノ酸オキシダーゼの基質認識機構の解析(第419回研究協議会研究発表要旨,ビタミンB研究委員会)

    内海 友宏, 有馬 二朗, 田村 隆, 稲垣 賢二

    ビタミン   84 ( 5 )   274 - 275   2010

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  • Identification and Purification of an L-Alanine Dehydrogenase Homolog that Catalyzes the Formation of L-Ornithine Lactam from Sinefungin-Producing Streptomyces incarnatus NRRL8089 Reviewed

    FUKUDA Koji, TAMURA Takashi, SATO Kumi, INAGAKI Kenji

    Actinomycetologica   24 ( 2 )   63 - 65   2010

  • Thioredoxin Reductase 1 Is Important for Selenoprotein Biosynthesis in HeLa Cells Reviewed

    Kurokawa S, Mihara H, Yokoyama I, Mochizuki M, Yodoi J, Tamura T, Kurihara T, Esaki N

    Biomedical research on trace elements   19 ( 1 )   84 - 87   2008.4

  • 3.好熱好酸性古細菌Sulfolobus tokodaii由来シスタチオニンγ-シンターゼ(第410回研究協議会研究発表要旨,ビタミンB研究委員会)

    柳谷 昌彦, 田村 隆, 稲垣 賢二

    ビタミン   82 ( 1 )   61 - 61   2008

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  • 1.放線菌Streptomyces avermitilis由来L-メチオニンγ-リアーゼの精製と性質(第413回研究協議会研究発表要旨,ビタミンB研究委員会)

    工藤 大蔵, 田村 隆, 稲垣 賢二

    ビタミン   82 ( 11 )   617 - 617   2008

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    DOI: 10.20632/vso.82.11_617_1

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  • 3. Cellulomonas属細菌由来のグリセロールキナーゼの特性と立体構造解析(第407回研究協議会研究発表要旨,ビタミンB研究委員会)

    稲垣 賢二, 田村 隆, 曽我部 敦

    ビタミン   81 ( 5 )   258 - 259   2007

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  • 1P036 Implication for reaction mechanism of bacterial NAD dependent isocitrate dehydrogenase based on crystal structure and quantum chemistry

    Imada K., Tamura T., Takenaka R., Kobayashi I., Inagaki K., Namba K.

    Seibutsu Butsuri   45   S40   2005

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    DOI: 10.2142/biophys.45.S40_4

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  • 16.硫黄酸化細菌A.thiooxidansのNAD^+依存型イソクエン酸脱水素酵素(ビタミンB研究委員会第399回研究協議会研究発表要旨)

    稲垣 賢二, 田村 隆

    ビタミン   79 ( 3 )   194 - 194   2005

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  • 8.抗腫瘍性B_6酵素L-メチオニンγ-リアーゼにおけるCys116の役割(第398回ビタミンB研究委員会研究発表要旨)

    工藤 大蔵, 田村 隆, 稲垣 賢二

    ビタミン   79 ( 1 )   43 - 44   2005

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  • Identification of thermoacidophilic bacteria and a new Alicyclobacillus genomic species isolated from acidic environments in Japan Reviewed

    K Goto, Y Tanimoto, T Tamura, K Mochida, D Arai, M Asahara, M Suzuki, H Tanaka, K Inagaki

    EXTREMOPHILES   6 ( 4 )   333 - 340   2002.8

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    Sixty strains of thermoacidophilic bacteria have been isolated from soil and water samples obtained from various acidic environments in Japan. An initial comparative sequence analysis of the hypervariable regions of the 16S rDNA revealed that all strains could be assigned to the Alicyclobacillus acidocaldarius-Alicyclobacillus genomic species 1 group, which could be further subdivided into three clusters (Clusters I-III). On the basis of phenotypic characteristics, chemotaxonomic profiles, and phylogenetic data of six selected strains, five strains were identified as either A. acidocaldarius or Alicyclobacillus genomic species 1; however, one strain (MIH 332) could not be determined to belong to either of these species. 16S rDNA sequence homology values between strain MIH 332 and the reference strains of A. acidocaldarius (ATCC 27009(T)) and Alicyclobacillus genomic species 1 (DSM 11984) were 98.8% and 99.1%, respectively, which were higher than the corresponding similarity between the reference strains (98.4%). On the other hand, DNA-DNA hybridization levels between strain MIH 332 and the reference strains were 39% and 44%, respectively, which were lower than the value between the reference strains (59% or 65%). However, the phenotype of strain MIH 332 was also similar to those of the reference strains, and a typical phenotype could not be found for the strain, thus indicating that the strain may be a new genomic species of A. acidocaldarius, for which the name Alicyclobacillus genomic species 2 is tentatively proposed. The results of this study suggest that A. acidocaldarius and its related species are widely distributed in acidic environments in Japan, with slight regional variations in morphological and genotypic characteristics.

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  • Role of tyrosine 114 of L-methionine y-lyase from Pseudomonas putida

    H Inoue, K Inagaki, N Adachi, T Tamura, N Esaki, K Soda, H Tanaka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   64 ( 11 )   2336 - 2343   2000.11

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    L-Methionine gamma -lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of gamma -family pyridoxal 5'-phosphate dependent enzymes. A mutant form of L-methionine gamma -lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in k(cat) for alpha,gamma -elimination of L-methionine, while the K-m remained the same as the wild type enzyme. The Y114F mutant had the reduced k(cat) by only 28- and 16-fold for substrates with an electron-withdrawing group at the gamma -position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of k(cat) for alpha,beta -elimination and deamination substrates. The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that gamma -elimination process for L-methionine is the rate-limiting determination step in alpha,gamma -elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine gamma -lyase is important in gamma -elimination of the substrate.

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  • Crystal structure of the pyridoxal 5 '-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida

    H Motoshima, K Inagaki, T Kumasaka, M Furuichi, H Inoue, T Tamura, N Esaki, K Soda, N Tanaka, M Yamamoto, H Tanaka

    JOURNAL OF BIOCHEMISTRY   128 ( 3 )   349 - 354   2000.9

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    L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 Angstrom resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.

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  • Noncompetitive, reversible inhibition of aminoacylase-1 by a series of L-alpha-hydroxyl and L-alpha-fluoro fatty acids: Ligand specificity of Aspergillus oryzae and porcine kidney enzymes

    T Tamura, Y Oki, A Yoshida, T Kuriyama, H Kawakami, H Inoue, K Inagaki, H Tanaka

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   379 ( 2 )   261 - 266   2000.7

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    L-Lactate and L-beta-phenyllactate have been identified in the culture broth of Streptomyces sp. KY-11 as reversible noncompetitive inhibitors of Aspergillus oryzae aminoacylase-l and porcine kidney aminoacylase I. A series of alpha-hydroxyl acids (DL-R-CH(OH)-COOH, R = Et, n-pro, n-butyl, n-pentyl, n-hexyl) also inhibited the two enzymes in reversible noncompetitive kinetics, and the inhibition potency (-log K-i) increased with the increased hydrophobicity of the R group. The two eukaryotic enzymes showed distinct preferences to the ligand alpha-alkyl group, and the fungus enzyme was inhibited by L-beta-phenyllactate (R = benzyl) 10(3)-fold more potently than the mammalian enzyme. L-alpha-Fluoro-beta-phenyl-propionate and its D-isomer were used to show that the L-configuration of the alpha-substituent was important for potent inhibition of both the enzymes. The fungus aminoacylase-1 steeply decreased the affinity to alpha-fluoro-and alpha-hydroxy-n-caproate as pH was raised from 7 to 11, whereas the mammalian enzyme retained the affinity to these ligands under alkaline conditions. These results suggest that A. oryzae aminoacylase-1 has an acidic residue that interacts with OH or -F, while the mammalian enzyme would have a basic residue that recognizes the alpha-substituents. (C) 2000 Academic Press.

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  • Purification of Anti-Escherichia Coli O-157 components produced by Enterococcus faecalis THIO, an isolate from Malaysian fermentation food, tempeh

    Ohhira, I, T. Tamura, H. Kurokawa, H. Nakaue, K. Inagaki, H. Tanaka

    Milk Science   49 ( 2 )   81 - 88   2000

  • Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida Reviewed

    H Inoue, K Inagaki, SI Eriguchi, T Tamura, N Esaki, K Soda, H Tanaka

    JOURNAL OF BACTERIOLOGY   179 ( 12 )   3956 - 3962   1997.6

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    A 15-kb region of Pseudomonas putida chromosomal DNA containing the mde operon and an upstream regulatory gene (mdeR) has been cloned and sequenced. The mde operon contains two structural genes involved in L-methionine degradative metabolism: the already-identified mdeA, which encodes L-methionine gamma-lyase (H. Inoue, K. Inagaki, M. Sugimoto, N. Esaki, K. Soda, and fi, Tanaka, J. Biochem, (Tokyo) 117:1120-1125, 1995), and mdeB, which encodes a homologous protein to the homodimeric-type E1 component of pyruvate dehydrogenase complex, A rho-independent terminator was present just downstream of mdeB, and open reading frames corresponding to other components of alpha-keto acid dehydrogenase complex were not found, When MdeB was overproduced in Escherichia coli, the cell extract showed the E1 activity with high specificity for alpha-ketobutyrate rather than pyruvate. These results suggest that MdeB plays an important role in the metabolism of alpha-ketobutyrate produced by MdeA from L-methionine, Accordingly, MdeB encodes a novel E1 component, alpha-ketobutyrate dehydrogenase E1 component, of an unknown alpha-keto acid dehydrogenase complex in P. putida, In addition, we found that the mdeR gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been identified as a member of the leucine-responsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon.

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  • 構造生物学

    共立出版  2007 

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  • 核酸系抗生物質シネフンギンの動物ウイルスに対する抗ウイルス活性評価

    黒田雄大, 根本理子, 稲垣賢二, 田村隆, 前田健, 前田健

    日本獣医学会学術集会講演要旨集   162nd   2019

  • [NiFeSe]型ヒドロゲナーゼのOne-Step精製を目的としたゲノム改変

    小沼瞳, 山神将大, 袴塚響, 田嶋智之, 高口豊, 根本理子, 稲垣賢二, 田村隆

    日本農芸化学会西日本支部大会およびシンポジウム講演要旨集   2019   2019

  • 放線菌Streptomyces sp.590由来の抗腫瘍性酵素L‐メチオニン脱炭酸酵素の遺伝子クローニング,組換え発現,均一精製および性質検討

    林将也, 岡田茜, 山本久美子, 奥河内知美, 日下知香, 工藤大蔵, 根本理子, 稲垣純子, 広瀬侑, 岡島俊英, 田村隆, 左右田健次, 稲垣賢二

    ビタミン   92 ( 3 )   113‐116   2018.3

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    J-GLOBAL

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  • Structural and mechanistic insights into homocysteine degradation by a mutant of methionine gamma-lyase based on substrate-assisted catalysis

    Dan Sato, Tomoo Shiba, Shunsuke Yunoto, Kazuo Furutani, Mitsuki Fukumoto, Daizou Kudou, Takashi Tamura, Kenji Inagaki, Shigeharu Harada

    PROTEIN SCIENCE   26 ( 6 )   1224 - 1230   2017.6

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    Methionine gamma-lyse (MGL) catalyzes the alpha, gamma-elimination of L-methionine and its derivatives as well as the alpha, beta-elimination of L-cysteine and its derivatives to produce alpha-keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site-directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51-Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5'-phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate-assisted catalysis.

    DOI: 10.1002/pro.3158

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  • Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590. International journal

    Masaya Hayashi, Akane Okada, Kumiko Yamamoto, Tomomi Okugochi, Chika Kusaka, Daizou Kudou, Michiko Nemoto, Junko Inagaki, Yuu Hirose, Toshihide Okajima, Takashi Tamura, Kenji Soda, Kenji Inagaki

    Journal of biochemistry   161 ( 4 )   389 - 398   2017.4

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    l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

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  • L-メチオニン、L-ホモシステイン新規定量法へのL-メチオニン脱炭酸酵素の活用

    林 将也, 大川 敦司, 半田 暖尚, 根本 理子, 稲垣 純子, 菊池 可菜子, 木村 隆, 田村 隆, 稲垣 賢二

    ビタミン   91 ( 4 )   286 - 286   2017.4

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  • The hyperthermophilic cystathionine gamma-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights

    Dan Sato, Tomoo Shiba, Sae Mizuno, Ayaka Kawamura, Shoko Hanada, Tetsuya Yamada, Mai Shinozaki, Masahiko Yanagitani, Takashi Tamura, Kenji Inagaki, Shigeharu Harada

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   73   152 - 158   2017.3

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    Cystathionine gamma-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)dependent enzyme, catalyzes the formation of cystathionine from an l-homoserine derivative and l-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P2(1); unit-cell parameters a = 58.4, b = 149.3, c = 90.2 angstrom , beta = 108.9 degrees) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C222(1); unit-cell parameters a = 117.7, b = 117.8, c = 251.3 angstrom ) and in form II' at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 angstrom ) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 angstrom resolution for forms I, II and III', respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II', suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

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  • 海洋性細菌由来の新規キノン含有グリシンオキシダーゼ −組換え発現と性質検討−

    溝端佐津紀, 根本理子, 田村隆, 稲垣賢二

    2016年度ビタミンB研究委員会報告書   147 - 148   2017.2

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    91 ( 5-6 )   355 - 360   2017

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  • Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp NT3060

    Yasuhisa Fukuda, Asuka Abe, Takashi Tamura, Takahide Kishimoto, Atsushi Sogabe, Satoshi Akanuma, Shin-ichi Yokobori, Akihiko Yamagishi, Katsumi Imada, Kenji Inagaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   121 ( 5 )   497 - 502   2016.5

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    Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (T-m), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60 degrees C (t(1/2)). Three mutants showed increased stabilities compared to the wild type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2015.09.011

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  • Identification and characterization of the zosA gene involved in copper uptake in Bacillus subtilis 168

    Takahiro Fukuhara, Kazuo Kobayashi, Yousuke Kanayama, Shu-ichi Enomoto, Taeko Kondo, Naoki Tsunekawa, Michiko Nemoto, Naotake Ogasawara, Kenji Inagaki, Takashi Tamura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   80 ( 3 )   600 - 609   2016.3

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    DL-Penicillamine, a copper-specific metal chelator, remarkably suppressed the growth of Bacillus subtilis 168 when added to a synthetic medium under Cu2+ limitation. DNA microarray and screening of 2,602 knockout mutants showed that the zosA gene was de-repressed in the presence of 0.1% dl-penicillamine, and that the zosA mutant was sensitive to dl-penicillamine medium. The zosA mutant delayed the growth under Cu-limitation even without the chelator, and the sensitivity to dl-penicillamine was reversed by induction using 0.3mM IPTG and the Pspac promoter inserted directly upstream of the zosA gene. Furthermore, the zosA mutant showed elevated tolerance of excessive Cu2+ but not of excessive Zn2+ added to LB and synthetic media. Homology modeling of the ZosA protein suggested that the protein can fold itself into essential domains for constituting a metal transporting ATPase. Our study suggests that zosA is a candidate gene involved in copper uptake.

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  • Identification and characterization of the zosA gene involved in copper uptake in Bacillus subtilis 168

    Takahiro Fukuhara, Kazuo Kobayashi, Yousuke Kanayama, Shu-ichi Enomoto, Taeko Kondo, Naoki Tsunekawa, Michiko Nemoto, Naotake Ogasawara, Kenji Inagaki, Takashi Tamura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   80 ( 3 )   600 - 609   2016.3

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    DL-Penicillamine, a copper-specific metal chelator, remarkably suppressed the growth of Bacillus subtilis 168 when added to a synthetic medium under Cu2+ limitation. DNA microarray and screening of 2,602 knockout mutants showed that the zosA gene was de-repressed in the presence of 0.1% dl-penicillamine, and that the zosA mutant was sensitive to dl-penicillamine medium. The zosA mutant delayed the growth under Cu-limitation even without the chelator, and the sensitivity to dl-penicillamine was reversed by induction using 0.3mM IPTG and the Pspac promoter inserted directly upstream of the zosA gene. Furthermore, the zosA mutant showed elevated tolerance of excessive Cu2+ but not of excessive Zn2+ added to LB and synthetic media. Homology modeling of the ZosA protein suggested that the protein can fold itself into essential domains for constituting a metal transporting ATPase. Our study suggests that zosA is a candidate gene involved in copper uptake.

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  • 1.放線菌由来の低基質特異性L-アミノ酸オキシダーゼの性質と構造(第442回研究協議会研究発表要旨,ビタミンB研究委員会)

    村上 佳穂, 伊藤 菜々子, 今田 勝巳, 山田 美和, 礒部 公安, 田村 隆, 稲垣 賢二

    ビタミン   90 ( 1 )   39 - 40   2016.1

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  • 海洋性細菌が産生する新規なキノン含有アミノ酸酸化酵素の利用に向けた研究

    稲垣賢二, 赤地周作, 溝端佐津紀, 田村隆

    低炭素社会と食の安全・安心を統合した環境生命科学的研究 成果報告書   23 - 25   2016

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  • A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8

    Saho Kamada, Takahiro Okugochi, Kaori Asano, Ryuta Tobe, Hisaaki Mihara, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   80 ( 10 )   1970 - 1972   2016

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    Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-C-14] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The K-M and k(cat) for Sephs2-Sec60Cys were determined to be 26M and 0.352min(-1), respectively.

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  • A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8

    Saho Kamada, Takahiro Okugochi, Kaori Asano, Ryuta Tobe, Hisaaki Mihara, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   80 ( 10 )   1970 - 1972   2016

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    Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-C-14] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The K-M and k(cat) for Sephs2-Sec60Cys were determined to be 26M and 0.352min(-1), respectively.

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  • 大学生の朝食欠食習慣の統計解析と改善への新指針

    田村,隆, 揖斐, 隆之, 稲垣,賢二, 久保,康隆, 奥田,潔

    岡山大学農学部学術報告   105   1 - 5   2016

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  • 放線菌由来の低基質特異性L-アミノ酸オキシダーゼの性質と構造

    村上佳穂, 伊藤菜々子, 今田勝巳, 山田美和, 磯部公安, 田村 隆, 稲垣賢二

    2015年度ビタミンB研究委員会報告書   22 - 23   2016

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  • Quantum Chemistry in Elucidation of the Reaction Mechanisms of PLP-dependent Enzymes

    89 ( 5 )   281 - 285   2016

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  • Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, L-lysine α-oxidase from Trichoderma viride

    Amano Marie, Mizuguchi Haruka, Sano Tadahisa, Kondo Hiroki, Shinyashiki Kengo, Inagaki Junko, Tamura Takashi, Kawaguchi Tatsuya, Kusakabe Hitoshi, Imada Katsumi, Inagaki Kenji

    Vitamins   89 ( 10 )   495 - 498   2015.10

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    DOI: 10.20632/vso.89.10_495

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  • Rhodococcus sp.AIU Z‐35‐1由来L‐アミノ酸酸化酵素―組換え酵素の精製及び性質―

    村上佳穂, 橋本義輝, 小林達彦, 山田美和, 磯部公安, 根本理子, 田村隆, 稲垣賢二

    日本生物工学会大会講演要旨集   67th   275 - 275   2015.9

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  • 2-IV-24 海洋性細菌Marinomonas mediterranea由来グリシンオキシダーゼの大腸菌発現系の構築(一般演題要旨,ビタミン・バイオファクター研究のさらなる魅力〜大和まほろぱからの発信〜,第67回大会講演要旨)

    溝端 佐津紀, 赤地 周作, 田村 隆, 稲垣 賢二

    ビタミン   89 ( 4 )   263 - 263   2015.4

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  • 2-IV-20 組換えL-メチオニン脱炭酸酵素の精製および性質検討(一般演題要旨,ビタミン・バイオファクター研究のさらなる魅力〜大和まほろぱからの発信〜,第67回大会講演要旨)

    林 将也, 岡田 茜, 田村 隆, 稲垣 賢二

    ビタミン   89 ( 4 )   261 - 261   2015.4

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  • 放線菌Streptomyces incarnatus NRRL8089のドラフトゲノムシーケンス

    清水瞳, 大島健志朗, 服部正平, 稲垣賢二, 田村隆

    日本農芸化学会大会講演要旨集(Web)   2015   3A37P07 (WEB ONLY)   2015.3

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  • Rhodococcus属放線菌由来低基質特異性L‐アミノ酸酸化酵素の遺伝子クローニングと発現系の構築

    村上佳穂, 田港静, 橋本義輝, 小林達彦, 山田美和, 礒部公安, 田村隆, 稲垣賢二

    日本農芸化学会大会講演要旨集(Web)   2015   2C22P14 (WEB ONLY)   2015.3

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  • 海洋細菌Marinomonas mediterranea由来L‐リシンε‐オキシダーゼの大腸菌発現系の構築

    赤地周作, 松井大亮, 浅野泰久, 田村隆, 稲垣賢二

    日本農芸化学会大会講演要旨集(Web)   2015   2C22P15 (WEB ONLY)   2015.3

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  • Improvement of dye-mediated dehydrogenase activity of pyranose oxidase by site-directed mutagenesis

    荒木 俊雄, 中柄 朋子, 田村 隆, 稲垣 賢二

    岡山大学農学部学術報告   104   1 - 4   2015.2

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    Pyranose oxidase (EC 1.1.3.10 ; PROD) catalyzes the oxidation of aldopyranoses at the position C‒2to yield the corresponding 2‒keto-aldoses and H2O2 , using oxygen as an electron acceptor. The enzyme shows broad substrate specificity as well as reactivity for 1,5‒anhydro‒d‒glucitol (1,5‒AG), which is known as a clinical glycemic marker. It is considered that the reactivity of PROD for 1,5‒AG is useful in the development of an amperometric-type biosensor, which is a convenient diagnostic device for selfmonitoringblood glucose (SMBG). However, the levels of dissolved oxygen in blood affect biosensorsystems that are equipped with an artificial electron mediator. In the present study, we attempted to develop an O2‒insensitive oxidase that would improve the dye-mediated dehydrogenase activity. We performed site-directed mutagenesis on PROD isolated from basidiomycetous fungus No. 52, which generated 11 mutants. The amino acid substitution Q421A exhibited a significant decrease (8.8% of wild type) in its oxidase activity, whereas it maintained its dehydrogenase activity (67% of wild type). In this study, we characterized PROD mutants from basidiomycetous fungus No. 52, which showed improved dye-mediated dehydrogenase activity.

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  • 海洋細菌Marinomonas mediterranea由来L -リシンε-オキシダーゼの精製と性質

    稲垣賢二, 赤地周作, 田村 隆, 松井大亮, 岡崎誠司, 浅野泰久

    2014年度ビタミンB研究委員会報告書   88 ( 9 )   1 - 1   2015

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    DOI: 10.20632/vso.88.9_481

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  • 抗腫瘍性L-リシンα-オキシダーゼの異種発現系構築と基質複合体のX線結晶構造解析

    天野万里, 橋本義輝, 小林達彦, 近藤裕輝, 今田勝巳, 日下部均, 田村 隆, 稲垣賢二

    2014年度ビタミンB研究委員会報告書   89 ( 3 )   19 - 19   2015

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    DOI: 10.20632/vso.89.3_148

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  • 3P-026 In vitro SPS assay using Thermus thermophiles HB8 PPDK

    Asano Kaori, Tobe Ryuta, Mihara Hisaaki, Nemoto Michiko, Inagaki Kenji, Tamura Takashi

    67   277 - 277   2015

  • 2P-184 Identification of [NiFeSe]-hydrogenases from sulfate reducing bacteria using ICP-MS

    Mishima Ayaka, Imoto Satoshi, Morikawa Daichi, Nemoto Michiko, Inagaki Kenji, Tamura Takashi

    67   220 - 220   2015

  • 2-I-27 セレノリン酸合成酵素のPEPジキナーゼ共役法の確立(一般演題要旨,第66回大会講演要旨)

    田村 隆, 鎌田 早帆, 稲垣 賢二

    ビタミン   88 ( 4 )   240 - 240   2014.4

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  • 2-IV-21 L-メチオニンγ-リアーゼ基質複合体のX線結晶構造解析と基質認識に関わる残基の機能解析(一般演題要旨,第66回大会講演要旨)

    安田 江里, 古谷 和大, 佐藤 暖, 福本 充樹, 工藤 大蔵, 志波 智生, 原田 繁春, 田村 隆, 稲垣 賢二

    ビタミン   88 ( 4 )   261 - 261   2014.4

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  • 海洋細菌Marinomonas mediterranea由来L-リシンε-オキシダーゼの精製と性質検討、X線結晶構造解析

    赤地 周作, 岡崎 誠司, 松井 大亮, 田村 隆, 稲垣 賢二, 浅野 泰久

    ビタミン   88 ( 4 )   240 - 240   2014.4

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  • Characterization of L-Arginine Oxidase Made from L-Glutamate Oxidase

    中井 隆一郎, 藤野 志保子, 内海 友宏, 田村 隆, 日下部 均, 稲垣 賢二

    岡山大学農学部学術報告   103   5 - 9   2014.2

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    L‒Glutamate oxidase (LGOX) from Streptomyces sp. X‒119‒6 has strict substrate specificity towardL‒glutamate. Recently, we solved the X‒ray crystal structure of LGOX and this revealed that Arg305 inthe active site is the key residue involved in substrate recognition. Therefore, we created 19 mutantenzymes of R305X‒LGOX by saturation mutagenesis. One of them R305D‒LGOX, Arg305 substitutedwith Asp exhibited oxidase activity for L‒Arg. Optimum pH of R305D‒LGOX mutant enzyme was pH8.5. Interestingly, the activity of R305D‒LGOX toward L‒Arg was inhibited by phosphate. And furthermore,the substrate specificity of R305D‒LGOX was affected by using buffer. The results of inhibitionanalysis suggest, that phosphate is a competitive inhibitor of R305D‒LGOX when L‒Arg is used assubstrate. Kinetic analysis of R305D‒LGOX showed that Km value and kcat value of R305D‒LGOX towardl-Arg were 0.68 mM and 6.7 s-1 respectively. In this study, we showed that R305D‒LGOX mutantenzyme is a novel l-arginine oxidase and useful for l-arginine biosensor.

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  • 1P-053 Purification and properties of a new L-tyrosine oxidase made from L-glutamate oxidase.

    Fujino Shihoko, Tamura Takashi, Kusakabe Hitoshi, Inagaki Kenji

    66   30 - 30   2014

  • 1P-052 Construction of recombinant expression system of L-lysine α-oxidase in Streptomyces.

    Amano Marie, Mizuguchi Haruka, Saitoh Yuki, Hashimoto Yoshiteru, Kobayashi Michihiko, Kusakabe Hitoshi, Tamura Takashi, Inagaki Kenji

    66   30 - 30   2014

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  • 海洋細菌Marinomonas mediterranea由来L‐リシンε‐オキシダーゼの性質検討

    赤地周作, 田村隆, 松井大亮, 岡崎誠司, 浅野泰久, 稲垣賢二

    生化学   85 ( 7 )   603   2013.7

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  • 2-II-11 共役法によるセレノリン酸合成酵素の機能解析(一般演題要旨,第65回大会講演要旨)

    鎌田 早帆, 田村 隆, 奥河内 貴大, 稲垣 賢二

    ビタミン   87 ( 4 )   245 - 245   2013.4

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  • 1-III-4 Trichoderma viride由来抗腫瘍性酵素L-Lysine α-oxidaseのX線結晶構造(一般演題要旨,第65回大会講演要旨)

    天野 万里, 新屋敷 健悟, 佐野 宰久, 川口 辰也, 日下部 均, 今田 勝巳, 田村 隆, 稲垣 賢二

    ビタミン   87 ( 4 )   207 - 207   2013.4

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  • 海洋細菌Marinomonas mediterranea由来L‐リシンε‐オキシダーゼの精製,性質検討及び結晶化

    赤地周作, 松井大亮, 岡崎誠司, 浅野泰久, 田村隆, 稲垣賢二

    日本農芸化学会中四国支部講演会講演要旨集   35th   27   2013.1

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  • 1P-073 Characterization of substrate specificity modified enzymes (R305D & R305L) made from L-glutamate oxidase

    Nakai Ryuichiro, Fujino Shihoko, Tamura Takashi, Sano Tadahisa, Imada Katsumi, Kusakabe Hitoshi, Inagaki Kenji

    65   36 - 36   2013

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  • 2P-062 Disulfide Isomerase Activity of E.coli dsbA [CXXC] Mutants

    Suto Shinya, Kiyoto Asako, Inagaki Kenji, Tamura Takashi

    65   119 - 119   2013

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  • L-グルタミン酸オキシダーゼより作成したL-ヒスチジンオキシダーゼの構造解析とL-アルギニンオキシダーゼの特性解析

    稲垣賢二, 中井隆一郎, 田村隆, 今田勝巳, 日下部均

    ビタミンB研究委員会報告書   87 ( 1 )   51 - 52   2013

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  • マンガン酸化系を構成する2種の細菌の相互作用解析

    田村隆, 周藤慎也, 稲垣賢二

    特別経費(プロジェクト分)-大学の特性を生かした多様な学術研究機能の充実-成果報告書   89   2013

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  • 抗腫瘍性酵素 L-リシンα-オキシダーゼのX線結晶構造解析

    稲垣賢二, 天野万里, 田村隆, 佐野宰久, 今田勝己, 日下部均

    ビタミン   87 ( 8 )   463 - 464   2013

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    DOI: 10.20632/vso.87.8_463

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  • 2-V-16 セレノリン酸合成酵素SPS1/SPS2の相互作用(一般演題要旨,日本ビタミン学会第64回大会講演要旨)

    田村 隆, 奥河内 貴大, 鎌田 早帆, 高畑 宗明, 稲垣 賢二

    ビタミン   86 ( 4 )   289 - 289   2012.4

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  • Purification and Characterization of Thermostable Amidase from Thermus sp.O-3-1

    小林 史明, 青峰 弘起, 水無 渉, 湯 不二夫, 田村 隆, 稲垣 賢二

    岡山大学農学部学術報告   101   1 - 6   2012.2

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    The gene encoding a thermostable amidase (EC 3.5.1.4) from thermophilic bacterium Thermus sp.O-3-1, was cloned and expressed in Escherichia coli JM109. The cloned amidase gene (ami) is 930 bp and encodes a protein composed of 310 amino acids. The protein is predicted to have a molecular mass of 33,089 Da. The amidase from Thermus sp.O-3-1 was purified by heat treatment and DEAE Toyopearl 650M column chromatography. The molecular mass of the native enzyme was estimated to be about 70 kDa by gel filtration chromatography, indicating that the enzyme has a homodimeric structure. The purified enzyme was stable up to 80°C and within a pH range from 7.0 to 10.0. The optimum temperature and pH for enzyme activity were 90°C, and 9.0, respectively. The enzyme was strongly inhibited by the metal-chelating compound EDTA. The activity of the EDTA-treated enzyme was reactivated by the addition of Co(2+), Ni(2+) and Mn(2+) ions. Therefore the enzyme was predicted to be metalloenzyme. Finally,as a result of investigation into substrate specificity, the purified enzyme was suggested to be D-amino acid specific amidase, as it showed higher activity toward D-Leu-pNA than L-Leu-pNA.好熱性細菌Thermus sp.O-3-1 由来の耐熱性アミダーゼ遺伝子を大腸菌中にクローニングし,その塩基配列を決定した.ami 遺伝子は930 bp からなり,310アミノ酸をコードしていた.本酵素の分子量は33,089 Daであると予想された.Thermus sp.O-3-1 由来アミダーゼを大腸菌で生産させ,熱処理とDEAE-トヨパール650M陰イオン交換カラム等により精製した.ゲル濾過クロマトグラフィーとSDS-PAGE の結果から本酵素は分子質量33 kDa のサブユニット2分子からなるダイマー構造を有していることが明らかとなった.精製酵素の熱安定性は80℃まで,pH 安定性は7.0~10.0であり,安定性の高い酵素であった.最適温度は90℃,最適 pH は9.0であった.EDTA により活性が著しく阻害され,Co(2+)やNi(2+),Mn(2+)によって活性の回復,向上が見られたため,本酵素は金属酵素であることが示唆された.基質特異性の検討の結果,L-Leu-pNA よりもD-Leu-pNA に対して高い活性を示したため,本酵素がD-アミノ酸基質に特異性を持つアミダーゼであることが判明した.本酵素は耐熱性を有するユニークなD-アミノ酸アミダーゼであり,今後産業利用が期待される.

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  • 2Dp10 Molecular Evolution of [NiFeSe]H2ase based on Phylogenetic Tree and Modeling

    Tamura Takashi, Tsunekawa Naoki, Hirano Toshiyuki, Sato Fumitoshi, Inagaki Kenji

    64   52 - 52   2012

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  • 3Cp04 Analysis of the substrate recognition mechanism in high substrate specificity L-glutamate oxidase and characterization of substrate specificity modified enzymes

    Nakai Ryuichiro, Tamura Takashi, Kusakabe Hitoshi, Imada Katsumi, Inagaki Kenji

    64   123 - 123   2012

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  • L-メチオニンγ-リアーゼC116H変異酵素の構造と機能解析

    福本充樹, 工藤大蔵, 原田繁春, 田村隆, 稲垣賢二

    ビタミンB研究委員会報告書   85 ( 8 )   446 - 447   2012

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  • 2-I-28 ヒト由来セレノシステインβリアーゼ遺伝子の発現系構築と機能解析(一般演題要旨,日本ビタミン学会第63回大会講演要旨)

    田村 隆, 佐藤 久美, 稲垣 賢二

    ビタミン   85 ( 4 )   249 - 249   2011.4

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  • 2-IV-9 好熱好酸性アーキアSulfolobus tokodaii由来シスタチオニンγ-シンターゼのPhe97残基の機能解析(一般演題要旨,日本ビタミン学会第63回大会講演要旨)

    山田 哲也, 福本 充樹, 柳谷 昌彦, 工藤 大蔵, 田村 隆, 倉光 成紀, 稲垣 賢二

    ビタミン   85 ( 4 )   247 - 247   2011.4

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  • Purification and characterization of L-methionine decarboxylase from Streptomyces sp. 590

    前村 知美, 内富 久美子, 日下 知香, 稲垣 純子, 田村 隆, 左右田 健次, 稲垣 賢二

    Scientific Reports of the Faculty of Agriculture, Okayama University   100 ( 100 )   3 - 7   2011.2

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    L-Methionine decarboxylase [EC 4.1.1.57] catalyzes the decarboxylation of L-methionine and is a pyridoxal 5'-phosohate(PLP)-dependent enzyme. L-Methionine decarboxylase has been purified 630-fold by DEAE-Toyopearl 650M, Phenyl-Toyopearl 650M and Sephacryl S-300 column chromatographies from Streptomyces sp.590. The enzyme has a dimeric structure with identical subunits of Mr 60,000. This enzyme shows optimum activity at pH7.0 and 45°C, and is stable between pH5.7 and pH9.0. L-Methionine decarboxylase has antitumor activity against RERF-LC-AI and HeLa cells. Ten N-terminal amino acid sequence of L-methionine decarboxylase was determined, and the sequence showed no homology with other reported proteins.

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  • Structural Characterization of L-Glutamate Oxidase from Streptomyces sp. X-119-6

    Arima Jiro, Sasaki Chiduko, Sakaguchi Chika, Mizuno Hiroshi, Tamura Takashi, Kashima Akiko, Kusakabe Hitoshi, Sugio Shigetoshi, Inagaki Kenji

    Vitamins   85 ( 1 )   23 - 24   2011.1

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    DOI: 10.20632/vso.85.1_23

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  • 食品分析用小型うま味センサーの開発

    稲垣賢二, 田村隆, 中井隆一郎

    うま味研究会助成金による成果報告書   2011

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  • ヒト由来セレノシステインβ-リアーゼ様タンパク質の発現, 活性確認と機能解析

    田村隆, 佐藤久美, 稲垣賢二

    ビタミン   85 ( 3 )   155 - 155   2011

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  • 3Ea13 Random Mutation of DsbA and redox potentials

    Kiyoto Asako, Tamura Takashi, Yoshida Takamasa, Inagaki Kenji

    63   229 - 229   2011

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  • 2Dp21 Structure and metal analysis of thermostable amidase from Thermus sp. O-3-1

    Kobayashi Fumiaki, Aomine Hiroki, Ohtaki Akashi, Odaka Masafumi, Yohda Masafumi, Mizunashi Wataru, Yu Fujio, Tamura Takashi, Inagaki Kenji

    63   140 - 140   2011

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  • Introduction of MOPAC simulation for experimental biochemists

    TAMURA Takashi

    82 ( 9 )   863 - 867   2010.9

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  • Production improvement of antifungal, antitrypanosomal nucleoside sinefungin by rpoB mutation and optimization of resting cell system of Streptomyces incarnatus NRRL 8089.

    Koji Fukuda, Takashi Tamura, Hideyuki Ito, Sayaka Yamamoto, Kozo Ochi, Kenji Inagaki

    Journal of bioscience and bioengineering   109 ( 5 )   459 - 65   2010.5

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    Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the beta-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM L-Arg. D-Arg or L-ornithine did not enhance the sinefungin production, and >50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of L-Arg and ATP.

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  • Production improvement of antifungal, antitrypanosomal nucleoside sinefungin by rpoB mutation and optimization of resting cell system of Streptomyces incarnatus NRRL 8089.

    Koji Fukuda, Takashi Tamura, Hideyuki Ito, Sayaka Yamamoto, Kozo Ochi, Kenji Inagaki

    Journal of bioscience and bioengineering   109 ( 5 )   459 - 65   2010.5

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    Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the beta-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM L-Arg. D-Arg or L-ornithine did not enhance the sinefungin production, and >50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of L-Arg and ATP.

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  • 1-III-6 Pseudomonas putida由来L-メチオニン γ-リアーゼの活性中心残基への変異導入による基質特異性の改変(一般演題,日本ビタミン学会第62回大会発表要旨)

    福本 充樹, 工藤 大蔵, 田村 隆, 稲垣 賢二

    ビタミン   84 ( 4 )   165 - 165   2010.4

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  • 2-IV-30 キイロショウジョウバエ由来チオレドキシン還元酵素のレドックス機能解析(一般演題,日本ビタミン学会第62回大会発表要旨)

    桑原 光彦, 田村 隆, 稲垣 賢二

    ビタミン   84 ( 4 )   214 - 214   2010.4

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  • 2-IV-28 放射性同位元素^<64>Cuを用いたBacillus sbutilis 168の銅代謝解析(一般演題,日本ビタミン学会第62回大会発表要旨)

    田村 隆, 金山 洋介, 榎本 秀一, 小林 和夫, 小笠原 直毅, 稲垣 賢二

    ビタミン   84 ( 4 )   213 - 213   2010.4

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  • 2-IV-29 セレノリン酸合成酵素SPS1はSPS2を抑制する(一般演題,日本ビタミン学会第62回大会発表要旨)

    奥河内 貴大, 田村 隆, 高畑 宗明, 三原 久明, 江崎 信芳, 稲垣 賢二

    ビタミン   84 ( 4 )   214 - 214   2010.4

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  • Identification of Ornithine-lactam Converted from Arginine in Streptomyces incarnatus NRRL8089

    福田 康二, 田村 隆, 稲垣 賢二

    岡山大学農学部学術報告   99 ( 1 )   1 - 5   2010.2

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    Sinefungin is a nucleoside antibiotic, in which a molecule of L-ornithine is linked to the 5&#039; end of adenosine through a C-C bond. The antibiotic was isolated from the culture broth of Streptomyces incarnatus. For the purpose of detecting intermediate of sinefungin biosynthesis, resting cell suspensions were incubated with supplemental L-arginine, and L-ornithine. 50mM Arginine was converted to a compound X that has low polarity. 50mM ornithine was not converted and remained in reaction solution. Compound X was purified using HPLC, and analyzed using (1)H-NMR and FAB-MS. These analyses showed that a compound X is &quot;ornithine-lactam&quot; (Mw=114), which has a structure of circularized ornithine. These results indicated that S. incarnatus has an enzyme that converts arginine to ornithine-lactam. Such an enzyme has never been reported, and suggested that it may be relevant to sinefungin biosynthesis.シネフンギンは抗真菌,抗マラリア活性を有する核酸系抗生物質であり,放線菌 S. incarnatus により生合成される.シネフンギンはアデノシンとオルニチンがCンC結合した構造であり,無細胞抽出液での取り込み実験からLンアルギニンと ATP から生合成されると推測される.Lンアルギニン,Lンオルニチンを S. incarnatus の休止菌体反応系への投与を行いシネフンギン中間体の探索を行った.その結果50ヒアルギニンは24時間以内に低極性化合物へと変換された.一方50ヒオルニチンは変換されず反応液中に残存した.HPLC で化合物を精製し,1HンNMR,FABンMS での分析の結果オルニチン環状モノペプチド,「オルニチンラクタム」(分子量114)であることを明らかにした.この結果は S. incarnatus がアルギニンからオルニチンラクタムへの変換酵素を有する事を示唆する.このような酵素の報告例はこれまでになく,ニ次代謝酵素であることが示唆され,シネフンギン生合成との関連性に興味が持たれる.

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  • Purification and Characterization of Cystathionine γ-Synthase from Thermoacidophilic Archaea Sulfolobus tokodaii

    篠崎 舞, 柳谷 昌彦, 兼田 翔一郎, 工藤 大蔵, 遠藤 祐一, 田村 隆, 倉光 成紀, 稲垣 賢二

    岡山大学農学部学術報告   99 ( 1 )   7 - 12   2010.2

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    The gene encoding a cystathionine γ-synthase from Sulfolobus tokodaii was cloned and expressed in Escherihia coli Rosetta-gami (DE3). Cystathionine γ-synthase [EC 2. 5. 1. 48] from Sulfolobus tokodaii (stCGS) was purified by heat treatment, DEAE- Toyopearl 650M and Sephacryl S-300 column chromatographies from E. coli transformants. stCGS shows optimum activity at pH 7.0, and is stable between pH5.0 and pH9.0. The optimum temperature of stCGS is above 100℃, and the enzyme showed the remaining activity of almost 100% up to 70℃. The K(m) and V(max) with O-phospho-L- homoserine as a substrate are 0.82 mM and 2.42 U/mg. To analyze the role of Phe 97 in the active site of stCGS, we constructed F97Y, R99C, and F97Y-R99C mutant enzymes. Although native stCGS has no activity toward l-methionine, F97Y mutant enzyme gained the elimination activity toward L-methionine.好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニンγンシンターゼ(stCGS)遺伝子を pET-11a に組み込み pET-stCGS を構築した.このベクターでE. coli Rosettaンgami(DE3)を形質転換し,本遺伝子を発現させ,精製及び性質検討を行った.大腸菌で発現したシスタチオニンγンシンターゼの活性が無細胞抽出液で確認できた.S. tokodaii シスタチオニンγンシンターゼを70℃熱処理 DEAEントヨパールイオン交換カラム等により単一精製した.精製酵素の最適温度は100℃以上であり,熱安定性は60分間処理で70℃までほぼ100オの残存活性を示した.また,最適pHについてはリン酸緩衝液やブリトンンロビンソン広域緩衝液の場合はpH7.0の時が最も活性が高く,トリス塩酸緩衝液の場合はpH9.0が最適であった.pH安定性についてはpH5.0~9.0において安定であった.O-ホスホ-l-ホモセリンに対するKm,Vmaxは,それぞれ0.82mM,2.42U/㎎であった.アポ酵素のホロ化実験により,本酵素活性がPLP に依存していることが明らかとなった.更に本酵素の脱離反応での基質特異性の検討を行った.変異酵素を用いた実験により,stCGSの基質特異性には,活性中心に存在するPhe97を含む領域が深く関わっていることが示唆された.

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  • 中性脂肪測定用酵素グリセロールキナーゼの構造特性解析と耐熱化

    稲垣賢二, 福田靖久, 田村隆, 岸本高英, 曽我部敦

    2009年度ビタミンB研究委員会報告書   83 ( 11 )   632 - 633   2010

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    DOI: 10.20632/vso.83.11_632_2

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  • 3P-1075 Purification and crystallization of antitumor enzyme, an L-lysine α-oxidase from Trichoderma viride

    SHINYASHIKI Kengo, NAKATA Haruka, IMADA Katsumi, TAMURA Takashi, INAGAKI Kenji

    22   76 - 76   2010

  • 2P-1053 Expression of E. colt DsbA[CRIC] mutant has redox potential of Disulfide Isomerase

    KIYOTOU Asako, TAMURA Takashi, YOSHIDA Takamasa, INAGAKI Kenji

    22   22 - 22   2010

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  • Structural characterization of l-glutamate oxidase from Streptomyces sp X-119-6

    Jiro Arima, Chiduko Sasaki, Chika Sakaguchi, Hiroshi Mizuno, Takashi Tamura, Akiko Kashima, Hitoshi Kusakabe, Shigetoshi Sugio, Kenji Inagaki

    FEBS JOURNAL   276 ( 14 )   4318 - 4327   2009.7

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    L-Glutamate oxidase (LGOX) from Streptomyces sp. X- 119- 6, which catalyzes the oxidative deamination of L-glutamate, has attracted increasing attention as a component of amperometric L-glutamate sensors used in the food industry and clinical biochemistry. The precursor of LGOX, which has a homodimeric structure, is less active than the mature enzyme with an alpha(2)beta(2)gamma(2) structure; enzymatic proteolysis of the precursor forms the stable mature enzyme. We solved the crystal structure of mature LGOX using molecular replacement with a structurally homologous model of l- amino acid oxidase ( LAAO) from snake venom: LGOX has a deeply buried active site and two entrances from the surface of the protein into the active site. Comparison of the LGOX structure with that of LAAO revealed that LGOX has three regions that are absent from the LAAO structure, one of which is involved in the formation of the entrance. Furthermore, the arrangement of the residues composing the active site differs between LGOX and LAAO, and the active site of LGOX is narrower than that of LAAO. Results of the comparative analyses described herein raise the possibility that such a unique structure of LGOX is associated with its substrate specificity.

    DOI: 10.1111/j.1742-4658.2009.07103.x

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  • 2-I-16 亜セレン酸の還元代謝に関わるセレン-Thioredoxin付加体の同定(一般研究発表,日本ビタミン学会第61回大会研究発表要旨)

    佐藤 久美, 田村 隆, 古森 健太郎, 戸部 隆太, 三原 久明, 江崎 信芳, 稲垣 賢二

    ビタミン   83 ( 4 )   191 - 191   2009.4

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  • 2-II-24 放線菌由来L-グルタミン酸オキシダーゼの構造特性と基質認識機構の解析(一般研究発表,日本ビタミン学会第61回大会研究発表要旨)

    内海 友宏, 田村 隆, 坂口 智香, 有馬 二郎, 日下部 均, 稲垣 賢二

    ビタミン   83 ( 4 )   208 - 208   2009.4

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  • Expression, Purification and Properties of Alanine Racemase from Thermus thermophillus HB8

    柳谷 昌彦, 上前 智, 白神 智行, 田村 隆, 稲垣 賢二

    岡山大学農学部学術報告   98 ( 1 )   1 - 7   2009.2

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    An alanine racemase (EC 5.1.1.1) from an extreme thermophilic bacterium Thermus thermophilus HB8, was purified and characterized, and its gene was cloned. The cloned alanine racemase gene (alr) was expressed in Escherichia coli JM 109. The alr gene is composed of a 1080 bp and encoded a 360 aminoacid, and was predicted to have a molecular weight of 38,596. The enzyme was purified by heat shock at 70°C for 10min and DEAE Toyopearl 650M column chromatography. The purified enzyme had an optimum pH9.0∼10.0 and an optimum temperature of 55°C∼60°C. Enzyme activity was retained 100% after incubation of the enzyme at 70°C for 10min. Alanine racemase from Thermus thermophilus HB8 is a monomeric enzyme with a molecular mass of 39 kDa.高度好熱性細菌 Thermus thermophilus HB8 由来アラニンラセマーゼ遺伝子を大腸菌中にクローニングし、発現させた後に、精製及び性質検討を行った。alr遺伝子は1080bpからなり360アミノ酸残基 HB8をコードしていたので、本酵素は38,596Daの分子量であると予想された。alr遺伝子の [ G + C ] 含量は、72%であり、Tm値は98.8℃であった。T.thermophilus HB8由来アラニンセマーゼを中等度好熱性細菌 Geobacillus stearothermophilus 及び赤痢菌 Shigella sonnei 由来アラニンラセマーゼと一次配列の比較をしたところ、G.stearothermophilus 由来のものと33%、赤痢菌由来の酵素を28%の相同性を示した。T.thermophilus HB8 由来アラニンラセマーゼを、70℃で10分間の熱処理後、DEAE-トーヨーパール陰イオン交換カラム等により精製した。精製酵素の最適温度は、d-アラニンからl-アラニンへの反応で55℃、l-アラニンからd-アラニンへの反応では60℃であり、最適pHは、9.0〜10.0であった。また、70℃で30分インキュベーションを行った後にも、活性の低下は見受けられず耐熱性を示した。更に、本酵素は分子量38,000モノマー酵素であると推定され、その反応機構に興味が持たれる。

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  • Cuckoo Fungus Mimics Termite Eggs by Producing the Cellulose-Digesting Enzyme beta-Glucosidase

    Kenji Matsuura, Toshihisa Yashiro, Ken Shimizu, Shingo Tatsumi, Takashi Tamura

    CURRENT BIOLOGY   19 ( 1 )   30 - 36   2009.1

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    Insects and fungi share a long history of association in various habitats, including the wood-decomposition niche [7]. Fungal mimicry of termite eggs is one of the most striking evolutionary consequences of insect-fungus association [2, 3]. Termites of the genus Reticulitermes often harbor fungal sclerotia, called "termite balls," along with eggs in nursery chambers, whereby the fungus gains a competitor-free habitat in termite nests. Sophisticated morphological and chemical camouflage are needed for the fungus to mimic termite eggs [3]. However, the mechanism of chemical egg mimicry by the fungus is unknown. Here, we show that the fungus mimics termite eggs chemically by producing the cellulose-digesting enzyme beta-glucosidase. We found that the termite egg-recognition pheromone consists of beta-glucosidase and lysozyme. Both enzymes are major salivary compounds in termites and are also produced in termite eggs. Termite balls were tended by termites only when the fungus produced beta-glucosidase. Our results demonstrated that the overlap of the cellulose digestion niche between termites and the fungus sharing the same chemicals provided the opportunity for the origin of termite egg mimicry by the fungus. This suggests that pheromone compounds might have originally evolved within other life history contexts, only later gaining function in chemical communication.

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  • Cuckoo Fungus Mimics Termite Eggs by Producing the Cellulose-Digesting Enzyme beta-Glucosidase

    Kenji Matsuura, Toshihisa Yashiro, Ken Shimizu, Shingo Tatsumi, Takashi Tamura

    CURRENT BIOLOGY   19 ( 1 )   30 - 36   2009.1

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    Insects and fungi share a long history of association in various habitats, including the wood-decomposition niche [7]. Fungal mimicry of termite eggs is one of the most striking evolutionary consequences of insect-fungus association [2, 3]. Termites of the genus Reticulitermes often harbor fungal sclerotia, called "termite balls," along with eggs in nursery chambers, whereby the fungus gains a competitor-free habitat in termite nests. Sophisticated morphological and chemical camouflage are needed for the fungus to mimic termite eggs [3]. However, the mechanism of chemical egg mimicry by the fungus is unknown. Here, we show that the fungus mimics termite eggs chemically by producing the cellulose-digesting enzyme beta-glucosidase. We found that the termite egg-recognition pheromone consists of beta-glucosidase and lysozyme. Both enzymes are major salivary compounds in termites and are also produced in termite eggs. Termite balls were tended by termites only when the fungus produced beta-glucosidase. Our results demonstrated that the overlap of the cellulose digestion niche between termites and the fungus sharing the same chemicals provided the opportunity for the origin of termite egg mimicry by the fungus. This suggests that pheromone compounds might have originally evolved within other life history contexts, only later gaining function in chemical communication.

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  • 微生物の水素発酵に関わるギ酸脱水素酵素の発現制御機構の解明

    田村隆, 稲垣賢二

    岡山大学大学院自然科学研究科プロジェクト「CO2循環・削減型社会実現に向けての融合的研究」2008年度成果報告書   13 - 16   2009

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  • Conflicting Two Hypotheses on Reductive Selenite Assimilation in Escharichia coli

    Takashi Tamura

    Biomed. Res. Trace Elements   20 ( 3 )   226 - 231   2009

  • Enhanced Production of the Fluorinated Nucleoside Antibiotic Nucleocidin by a rifR-Resistant Mutant of Streptomyces calvus IFO13200

    Koji Fukuda, Takashi Tamura, Yuko Segawa, Yuta Mutaguchi, Kenji Inagaki

    Actinomycetologica   23 ( 2 )   51 - 55   2009

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    DOI: 10.3209/saj.SAJ230205

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  • 2Dp12 Gene cloning and purification of novel thermostable amidase

    Kobayashi Fumiaki, Aomine Hiroki, Mizunashi Wataru, Nakamura Tetsuji, Yu Fujio, Tamura Takashi, Inagaki Kenji

    21   50 - 50   2009

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  • 2Dp06 Bisulfide Isomerase Activity of E. coli DsbA[CDIC] mutant

    Kiyotou Asako, Tamura Takashi, Yoshida Takamasa, Inagaki Kenji

    21   48 - 48   2009

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  • 1Bp22 Sinefungin high-producer strain of S. incarnatus induced by rpoB mutation

    Yamashiro Daichi, Fukuda Koji, Tamura Takashi, Ochi Kozo, Inagaki Kennji

    21   11 - 11   2009

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  • 糸状菌Trichoderma viride由来L-リジンα-オキシダーゼ 完全長cDNAの単離と大腸菌発現系構築

    中田 春香, 田村 隆, 稲垣 純子, 日下部 均, 稲垣 賢二

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   81回・31回   1T8 - 3   2008.11

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  • The role of cysteine 116 in the active site of the antitumor enzyme L-methionine gamma-lyase from Pseudomonas putida

    Daizou Kudou, Shintaro Misaki, Masao Yamashita, Takashi Tamura, Nobuyoshi Esaki, Kenji Inagaki

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   72 ( 7 )   1722 - 1730   2008.7

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    The cysteinyl residue at the active site Of L-methionine gamma-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k(cat) toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the gamma-elimination reaction of L-methionine and in substrate recognition in the MGLs.

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  • 亜セレン酸還元代謝の「定説」を疑う!!

    田村 隆, 高畑 宗明, 古森 健太郎, 三原 久明, 江崎 信芳, 稲垣 賢二

    Biomedical research on trace elements   19 ( 2 )   164 - 164   2008.6

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  • 2-III-26 好熱好酸性アーキアSulfolobus tokodaii strain 7由来シスタチオニンγシンターゼの機能解析(一般研究発表,新世紀ビタミン学の展望と先進的展開を目指して,日本ビタミン学会第60回大会発表要旨)

    工藤 大蔵, 柳谷 昌彦, 田村 隆, 倉光 成紀, 稲垣 賢二

    ビタミン   82 ( 4 )   291 - 291   2008.4

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  • 2-IV-16 ヒト肺由来セレノリン酸合成酵素アイソザイムのドメイン機能解析(一般研究発表,新世紀ビタミン学の展望と先進的展開を目指して,日本ビタミン学会第60回大会発表要旨)

    田村 隆, 高畑 宗明, 三原 久明, 江崎 信芳, 稲垣 賢二

    ビタミン   82 ( 4 )   301 - 301   2008.4

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  • 2-IV-14 Drosophila TrxRのC末端レドックスモチーフの同定(一般研究発表,新世紀ビタミン学の展望と先進的展開を目指して,日本ビタミン学会第60回大会発表要旨)

    桑原 光彦, 田村 隆, 稲垣 賢二

    ビタミン   82 ( 4 )   300 - 300   2008.4

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  • Selenite assimilation into formate dehydrogenase H depends on thioredoxin reductase in Escherichia coli

    Muneaki Takahata, Takashi Tamura, Katsumasa Abe, Hisaaki Mihara, Suguru Kurokawa, Yoshihiro Yamamoto, Ryuhei Nakano, Nobuyoshi Esaki, Kenji Inagaki

    JOURNAL OF BIOCHEMISTRY   143 ( 4 )   467 - 473   2008.4

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    Escherichia coli growing under anaerobic conditions produce H(2) and CO(2) by the enzymatic cleavage of formate that is produced from pyruvate at the end of glycolysis. Selenium is an integral part of formate dehydrogenase H (FDH(H)), which catalyses the first step in the formate hydrogen lyase (FHL) system. The genes of FHL system are transcribed only under anaerobic conditions, in the presence of a sigma(54)-dependent transcriptional activator Fh1A that binds formate as an effector molecule. Although the formate addition to the nutrient media has been an established procedure for inducing high FDHH activity, we have identified a low-salt nutrient medium containing &lt;0.1% NaCl enabled constitutive, high expression of FDH(H) even without formate and D-glucose added to the medium. The novel conditions allowed us to study the effects of disrupting genes like trxB (thioredoxin reductase) or gor (glutathione reductase) on the production of FDH(H) activity and also reductive assimilation of selenite (SeO(3)(2-)) into the selenoprotein. Despite the widely accepted hypothesis that selenite is reduced by glutathione reductase-dependent system, it was demonstrated that trxB gene was essential for FDH(H) production and for labelling the FDH(H) polypeptide with (75)Se-selenite. Our present study reports for the first time the physiological involvement of thioredoxin reductase in the reductive assimilation of selenite in E. coli.

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  • Selenite assimilation into formate dehydrogenase H depends on thioredoxin reductase in Escherichia coli

    Muneaki Takahata, Takashi Tamura, Katsumasa Abe, Hisaaki Mihara, Suguru Kurokawa, Yoshihiro Yamamoto, Ryuhei Nakano, Nobuyoshi Esaki, Kenji Inagaki

    JOURNAL OF BIOCHEMISTRY   143 ( 4 )   467 - 473   2008.4

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    Escherichia coli growing under anaerobic conditions produce H(2) and CO(2) by the enzymatic cleavage of formate that is produced from pyruvate at the end of glycolysis. Selenium is an integral part of formate dehydrogenase H (FDH(H)), which catalyses the first step in the formate hydrogen lyase (FHL) system. The genes of FHL system are transcribed only under anaerobic conditions, in the presence of a sigma(54)-dependent transcriptional activator Fh1A that binds formate as an effector molecule. Although the formate addition to the nutrient media has been an established procedure for inducing high FDHH activity, we have identified a low-salt nutrient medium containing &lt;0.1% NaCl enabled constitutive, high expression of FDH(H) even without formate and D-glucose added to the medium. The novel conditions allowed us to study the effects of disrupting genes like trxB (thioredoxin reductase) or gor (glutathione reductase) on the production of FDH(H) activity and also reductive assimilation of selenite (SeO(3)(2-)) into the selenoprotein. Despite the widely accepted hypothesis that selenite is reduced by glutathione reductase-dependent system, it was demonstrated that trxB gene was essential for FDH(H) production and for labelling the FDH(H) polypeptide with (75)Se-selenite. Our present study reports for the first time the physiological involvement of thioredoxin reductase in the reductive assimilation of selenite in E. coli.

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  • Purification, characterization and crystal structure of isoamylase from thermophilic bacteria Rhodothermus marinus

    立花 亜紀子, 田村 隆, 今田 勝巳, 木下 実紀, 難波 啓一, 堤 紀子, 橋田 みよ子, 坂口 博脩, 稲垣 賢二

    岡山大学農学部学術報告   97 ( 97 )   1 - 7   2008.2

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    The isoamylase gene from Rhodothermus marinus was cloned into and expressed in Escherichia coliTop 10. As a result of characterization of purified R. marinus isoamylase. the enzyme had an optimumpH of 4.0 and optimum temperature of 70℃. Thermal inactivation studies of the purified R. marinusisoamylase revealed the enzymatic activity to be uninfluenced after one hour incubation at 60℃. Theseresults suggest that R. marinus isoamylase has high thermostability. The crystallization and crystalstructure analysis of R. marinus isoamylase was performed. The three-dimensional structure at 1.9Åresolution was determined in complex with the panose. R. marinus isoamylase is composed of threedomains N, A and C, and, has a (β/α)8-barrel in domain A. The secondary structural alignments of theR. marinus isoamylase and P. amyloderamosa isoamylase was carried out. They have the four active-siteconsensus regions characteristic of the α-amylase family. And the essential residue of the α-amylasefamily (D359, E395, and D467) was conserved in these enzymes. R. marinus isoamylase has shorter loopsthan P. amyloderamosa isoamylase. And R. marinus isoamylase had no Ca2+ binding site. These resultsare thought to be factors of thermostability of R. marinus isoamylase.Rhodothermus marinus 由来イソアミラーゼ遺伝子を組み込んだプラスミド pBX2を使用し,大腸菌 Top10株を形質転換し,16時間の前培養,24時間の本培養後,菌体破砕し,得られた無細胞抽出液を熱処理(80℃,10 min),50オ硫安分画,陰イオン交換カラムクロマトグラフィー(DEAEントヨパール),ハイドロキシアパタイトカラムクロマトグラフィーに供して本酵素の精製を行った.本精製酵素の性質検討を行った結果,本酵素の最適反応温度は70℃,pH4であり,また本酵素は60℃で1時間処理しても活性が低下することが無く,Pseudomonas amyloderamosa 由来イソアミラーゼよりも高い耐熱性を有することが判明した.本酵素の結晶化・X線結晶構造解析を行った結果,本酵素は P. amyloderamosa 由来イソアミラーゼと同様Nドメイン・AドメインCドメインの3つのドメインから構成されており,活性残基(D359,E395,D467)など活性中心付近のアミノ酸残基も P. amyloderamosa 由来イソアミラーゼと同様,高度に保存されていた.本酵素の熱安定性が P. amyloderamosa 由来イソアミラーゼよりも高い要因として,P. amyloderamosa 由来イソアミラーゼよりもループの長さが全体的に短いことと,カルシウムイオン結合サイトの欠如が挙げられた.今後さらに構造解析を進めることにより,本酵素の熱安定性機構,反応機構など更なる知見が得られることが期待される.

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  • Structure and quantum chemical analysis of NAD(+)-dependent isocitrate dehydrogenase: Hydride transfer and co-factor specificity

    Katsumi Imada, Takashi Tamura, Ryo Takenaka, Issei Kobayashi, Keiichi Namba, Kenji Inagaki

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   70 ( 1 )   63 - 71   2008.1

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    The crystal structure of Acidithiobacillus thiooxidans isocitrate dehydrogenase complexed with NAD(+) and citrate has been solved to a resolution of 1.9 angstrom. The protein fold of this NAD(+)-dependent enzyme shares a high similarity with those of NADP(+-)dependent bacterial 1CDHs. The NAD(+) and the citrate are clearly identified in the active site cleft with a well-defined electron density. Asp-357 is the direct cofactor-specificity determinant that interacts with 2'-OH and 3'-OH of the adenosine ribose. The adenosine ribose takes a C2'-endo puckering conformation as previously reported for an NAD(+-)specific isopropylmalate dehydrogenase. The nicotinamide moiety of NAD(+) has the amide NH2 group oriented in cis conformation with respect to the C4 carbon of the nicotinamide ring, slanted toward the bound citrate molecule with a dihedral angle of -21 degrees. The semi-empirical molecular orbital calculation suggests that the pro-R hydrogen atom at C4 of NADH would bear the largest negative charge when the amide NH2 group is in such conformation, suggesting that the amide group has a catalytically significant role in stabilizing the transition state as NADH is being formed during the hydride transter catalysis.

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  • 臨床診断・生化学検査に貢献する酵素の構造と機能解析

    田村 隆, 稲垣賢二

    生物工学会誌   86 ( 4 )   177 - 179   2008

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  • 2Fa07 Antitumor enzyme L-Lysine α-oxidase from filamentous fungi

    NAKATA Haruka, TAMURA Takashi, INAGAKI Junko, KUSAKABE Hitoshi, INAGAKI Kenji

    日本生物工学会大会講演要旨集   20   159 - 159   2008

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  • 1Fp11 Thermostabilization of glycerol kinase by ancestral mutation

    Fukuda Yasuhisa, Tmura Takashi, Abe Asuka, Kishimoto Takahide, Sogabe Atsushi, Inagaki Kenji

    20   155 - 155   2008

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  • The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

    Kenji Matsuura, Takashi Tamura, Norimasa Kobayashi, Toshihisa Yashiro, Shingo Tatsumi

    PLOS ONE   2 ( 8 )   813   2007.8

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    Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites.

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  • 2-II-7 Q494R型チオレドキシン還元酵素の基質特異性(ビタミン学の原点・栄養学への21世紀的回帰, 日本ビタミン学会第59回大会)

    桑原 光彦, 田村 隆, 奥山 祐子, 稲垣 賢二

    ビタミン   81 ( 4 )   154 - 154   2007.4

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  • 2-II-6 分子軌道計算に基づくTrxRの精密分子設計とアレニウスプロット解析(ビタミン学の原点・栄養学への21世紀的回帰, 日本ビタミン学会第59回大会)

    田村 隆, 奥山 祐子, 中山 恵美, 稲垣 賢二

    ビタミン   81 ( 4 )   153 - 153   2007.4

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  • Structure of the antitumour enzyme L-methionine gamma-lyase from pseudomonas putida at 1.8 angstrom resolution

    Daizou Kudou, Shintaro Misaki, Masao Yamashita, Takashi Tamura, Tomoaki Takakura, Takayuki Yoshioka, Shigeo Yagi, Robert M. Hoffman, Akio Takimoto, Nobuyoshi Esaki, Kenji Inagaki

    JOURNAL OF BIOCHEMISTRY   141 ( 4 )   535 - 544   2007.4

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    L-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multi-functional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 angstrom resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug.

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  • 抗腫瘍性酵素L-リジンα-オキシダーゼの遺伝子クローニング

    中田 春香, 田村 隆, 稲垣 純子, 日下部 均, 稲垣 賢二

    ビタミン   81 ( 4 )   148 - 148   2007.4

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  • キイロショウジョウバエ由来のチオレドキシン還元酵素のC末端テトラペプチド配列は、ヒト肺由来のチオレドキシン還元酵素では酸化還元活性を示さない

    桑原 光彦, 田村 隆, 河村 健太郎, 稲垣 賢二

    岡山大學農學部學術報告 = Scientific report of the Faculty of Agriculture, Okayama University   ( 96 )   1 - 5   2007.2

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    ほ乳類チオレドキシン還元酵素はC末端配列-Gly-Cys-SeCys-Gly(end)の後ろから2番目にセレノシステイン(SeCys)残基を持つ。SeCysをシステインに変換すると酵素の活性は大きく低下するので、SeCys残基が触媒活性に必須であることが分かる。これに対してキイロショウジョウバエのチオレドキシン還元酵素(Dm-rxR)のC末端配列にはセレンが含まれず、システイン残基の対が2つのセリンに挟まれた配列-Ser-Cys-Cys-Ser(end)を持つ。それでもDm-rxRはほ乳類のセレン含有酵素と同程度の触媒能を示す。われわれはヒト肺チオレドキシン還元酵素にDm-rxRのC末端テトラペプチド配列を導入してその効果を調べた。しかし、酵素活性はまったく上昇せず、Dm-rxRのC末端のテトラペプチド配列-Ser-Cys-Cys-SerだけではCys残基のチオール基を活性化する効果はなかった。そこで、分子軌道計算MOPACを用いて酸化還元機能を担うためのC末端配列モチーフを探索した。その結果、テトラペプチドにさらに2つ先のプロリンまでを含めたPro-X-Ser-Cys-Cys-Ser(end)により初めて酸化還元モチーフとして機能する可能性が示唆された。Proを含むこの配列モチーフはミツバチや蚊などほかの昆虫のrxRでも保存されていた。

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  • 大腸菌の亜セレン酸代謝はチオレドキシン還元酵素に依存する

    高畑宗明, 田村 隆, 阿部勝正, 三原久明, 江崎信芳, T.C. Stadtman, 稲垣賢二

    Trace Nutrient Res., 24, 42-48 (2007)   24   42 - 48   2007

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  • The C-terminal Tetrapeptide Sequence of Drosophila Thioredoxin Reductase Does not Function as a Redox-active Motif in the Human Lung Counterpart

    Kuwahara, M, Tamura, T, Kawamura, K, Inagaki, K

    Sci. Rep. Fac. Agric. Okayama Univ.   96 1-5   1 - 5   2007

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  • 2S3PM5 Structures and Functions of Enzymes used in Biochemical Diagnosis

    TAMURA Takashi, INAGAKI Kenji

    19   34 - 34   2007

  • 2B09-2 Expression and Characterization of Homoserine kinase from Thermoacidphillic Archaeon, Sulfolobus tokodaii strain 7

    YANAGITANI Masahiko, KUDOU Daizou, KURAMITU Seiki, TAMURA Takasi, INAGAKI Kenzi

    19   61 - 61   2007

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  • 1C11-3 Production and characterization of mutant enzyme C49K of antitumor enzyme L-methionine gamma-lyase

    KUDOU Daizou, TAMURA Takashi, TAKIMOTO Akio, TAKAKURA Tomoaki, INAGAKI Kenji

    19   79 - 79   2007

  • 1C11-4 Relationship between the structure of L-Glutamate oxidase and its enzymological characteristics

    ARIMA Jiro, SASAKI Chiduko, SAKAGUCHI Chika, TAMURA Takashi, KUSAKABE Hitoshi, SUGIO Shigetoshi, INAGAKI Kenji

    19   79 - 79   2007

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  • Performance characterization of recombinant L-glutamate oxidase in a micro GOT/GPT sensing system

    Sanjay Upadhyay, Naoto Ohgami, Hitoshi Kusakabe, Hiroshi Mizuno, Jiro Arima, Takashi Tamura, Kenji Inagaki, Hiroaki Suzuki

    SENSORS AND ACTUATORS B-CHEMICAL   119 ( 2 )   570 - 576   2006.12

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    Large-scale production Of L-glutamate oxidase (L-GlOx) from Streptomyces sp. X-119-6 was conducted with the use of an Escherichia coli expression system. The active alpha(2)beta(2)gamma(2)-subunit structure of the enzyme was obtained by proteolysis of a precursor form using metalloendopeptidase. The performance of this recombinant enzyme was tested in an amperometric sensing system to determine the L-glutamate concentration. To this end, a thin-film, three-electrode system was formed. Hydrogen peroxide produced by an enzymatic reaction was detected by using a platinum working electrode. The sensitivity of the L-glutamate sensor was 220 nA/mM, and the lower detection limit was 3 mu M (S/N = 3). Up to 800 mu M, a linear relationship was observed between the output current and the L-glutamate concentration. Furthermore, we used a microanalysis system to determine the activities of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) and, ultimately, to test whether the recombinant enzyme can additionally be used to analyze enzyme activities. A polydimethylsiloxane (PDMS) flow channel with a mixing compartment was also used. The substrate solution and the sample solution for either GOT or GPT were mixed as they were being removed from the flow channel. As time elapsed, the L-glutamate concentration in the mixed solution increased because of the enzymatic reaction of GOT or GPT; consequently, a constant increase in current was observed. The relation between the slope of the response curve and the enzyme activity was linear up to 127 U/l for GOT and to 88 U/l for GPT. The results of this series of experiments demonstrate that recombinant L-GlOx can be used to construct micro sensors and microanalysis systems. (c) 2006 Elsevier B.V. All rights reserved.

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  • The crystal structure of hypothetical methyltransferase from Thermus thermophilus HB8

    C Sasaki, Sugiura, I, A Ebihara, T Tamura, S Sugio, K Nagaki

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   64 ( 2 )   552 - 557   2006.8

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  • 2-III-25 放線菌由来L-グルタミン酸オキシダーゼの大腸菌での大量精製系の確立(一般研究発表,日本ビタミン学会 第58回大会研究発表要旨)

    坂口 智香, 水野 寛士, 田村 隆, 日下部 均, 稲垣 賢二

    ビタミン   80 ( 4 )   253 - 253   2006.4

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  • 1-II-6 抗腫瘍性酵素L-methionine γ-lyaseの活性中心におけるAsp241の機能解析(一般研究発表,日本ビタミン学会 第58回大会研究発表要旨)

    工藤 大蔵, 岬 真太郎, 瀧本 明生, 田村 隆, 稲垣 賢二

    ビタミン   80 ( 4 )   208 - 208   2006.4

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  • 2-III-14 大腸菌の亜セレン酸同化はチオレドキシン還元酵素に依存する(一般研究発表,日本ビタミン学会 第58回大会研究発表要旨)

    田村 隆, 高畑 宗明, 阿部 勝正, 三原 久明, 江崎 信芳, 稲垣 賢二

    ビタミン   80 ( 4 )   247 - 247   2006.4

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  • Characterization of monofunctional catalase KatA from radioresistant bacterium Deinococcus radiodurans

    Kobayashi, I, T Tamura, H Sghaier, Narumi, I, S Yamaguchi, K Umeda, K Inagaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   101 ( 4 )   315 - 321   2006.4

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    Catalase plays a key role in protecting cells against toxic reactive oxygen species. Here we report on the cloning, purification and characterization of a catalase (KatA, DR1998) from the extremely radioresistant bacterium Deinococcus radiodurans. The size of purified D. radiodurans KatA monomer was 65 kDa while gel filtration revealed that the size of the enzyme was 240 kDa, suggesting that KatA formed a homotetramer in solution. Purified KatA displayed a final specific activity of 68,800 U/mg of protein. The catalase activity of KatA was inhibited by sodium azide, sodium cyanide and 3-amino-1,2,4-triazole. The absorption spectrum of KatA exhibited a Soret band at 408 nm. The position of the spectral peak remained unchanged following reduction of KatA with dithionite. No peroxidase activity was found for KatA. These results demonstrate that D. radiodurans KatA is a typical monofunctional heme-containing catalase. The stability of KatA with respect to H2O2 stress was superior to that of commercially available Aspergillus niger and bovine liver catalases. The relative abundance of KatA in cells in addition to the H2O2 resistance property may play a role in the survival strategy of D. radiodurans against oxidative damage.

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  • Analysis of Decolorization Mechanism by Azo Dyes Decolizing Microorganism

    馬場 直子, 坂口 博脩, 田口 隆章, 早瀬 伸樹, 田村 隆, 稲垣 賢二

    岡山大学農学部学術報告   95 ( 95 )   1 - 5   2006.2

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    The mehcanism of azo dyes decolorization by Candida sp. MK-1, Aeromonas sp. B-5 and Actinobacillus sp. B-11 were analyzed. The maximal decolorization activity was observed at pH 7.5 and 30℃ on Candida sp. MK-1, at alkaline and at 35℃ on Aeromonas sp. B-5 and Actinobacillus sp. B-11. The HPLC analysis of the supernatant of the Acid Red 27 detected in the blank. The retention time of this peak matched that of a reference standard compound of 4-amino-1-naphthalenesulfonate, produced by reductive cleavage of Acid Red 27. The decolorization of azo dyes with cell free extract of Candida sp. MK-a was promoted by the addition of several coenzymes or lawsone. The remarkable promotion of decolorization was observed by the addition of glutamate dehydrogenase with α-ketoglutarate and NH4+. Therefore, it was suggested that Candida sp. MK-1 azoreductase catalyzed decolorization of azo dye by NADPH dependent reductive cleavage.

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  • 1F10-2 Purification and characterization of L-methionine γ-lyase from Streptomyces avermitilis

    INAGAKI Kenji, KUDOU Daizou, HIRAI Yoshiyuki, TAMURA Takashi

    18   92 - 92   2006

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  • 2-B-13 NAD依存型イソクエン酸脱水素酵素の量子酵素化学(第57回大会一般研究発表)(第57回大会研究発表要旨)

    田村 隆, 今田 勝巳, 竹中 涼, 小林 一聖, 稲垣 賢二

    ビタミン   79 ( 4 )   251 - 251   2005.4

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  • 化学合成独立栄養細菌Acidithiobacillus thiooxidans 由来アコニターゼの遺伝子解析と大腸菌での発現

    金原陽平, 田村 隆, 徳田千束, 中村淳雄, 松川寛和, 稲垣賢二

    岡山大学農学部学術報告   94   1 - 7   2005

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    Acidihiobacillus hiooxidans由来アコニターゼ遺伝子を大腸菌中にクローニングし、発現させ、精製及び性質検討を行った。A. hiooxidansアコニターゼはEscherichia coli JM109株で発現させ、活性が無細胞抽出液で確認された。646アミノ酸から成っており、イソクエン酸脱水素酵素遺伝子(icd)の上流に位置していた。またブタ心筋由来アコニターゼと大腸菌アコニターゼA型と高いアミノ酸配列相同性があり、ブタ心筋アコニターゼで触媒反応、基質の結合に関与するとされている27アミノ酸のうち25アミノ酸の保存が確認された。A. hiooxidansアコニターゼはDEAE-oyopearl 650M等により精製された。精製酵素は至適pH7.5、至適温度60℃であった。また40℃で1時間インキュベートを行った後にも、活性の低下は見受けられなかった。pH6.0-8.0で1時間インキュベートした後も、100%の活性を維持していた。更に本酵素は、分子量66000のモノマー酵素であることが明らかになった。なお、本論文中に記載したA. hiooxidansのアコニターゼ遺伝子を含む塩基配列はDDBJデータベースに登録されており、アクセション番号AB196983で検策できる。

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    Other Link: http://agriknowledge.affrc.go.jp/RN/2010710742

  • ヒト肺がん細胞のセレノリン酸合成酵素Sps1,Sps2の機能解析

    田村 隆, 山本真平, 高畑宗明, 田中英彦, 稲垣賢二

    微量栄養素研究   22   147 - 152   2005

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  • Discovery of a New Enzyme That Contains Biofactor Selenium and Characterization of Selenium Metabolism in Human Lung Adenocarcinoma Cells

    TAMURA Takashi

    VITAMINS   79 ( 3 )   143 - 153   2005

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    Selenium is an essential trace element for mammals, birds, and some bacteria. Its remarkable biological effects in eukaryotes may be related to unique functions of various selenoproteins. A^<75>Se-labeled protein was purified from a human lung adenocarcinoma cell line, NCI-H441 . A homodimer of 57-kDa subunits contained selenium in the form of selenocysteine. The selenoprotein contained FAD as a prosthetic group and catalyzes NADPH-dependent reduction of insulin in the presence of thioredoxin. The subunit composition and catalytic properties were identical to those of mammalian thioredoxin reductase (TrxR). This was the first report on the biochemical relevance between selenium and Trx-mediated metabolic regulation. Interestingly, human lung adenocarcinoma cells produced the selenoenzyme TrxR over hundred times that of normal mammalian cells. A Iabile selenium donor compound monoselenophosphate, required for selenoprotein biosynthesis, is synthesized from selenide and ATP by selenophosphate synthetase (SPS).

    DOI: 10.20632/vso.79.3_143

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  • 2C13-5 Large scale purification and crystal structure of L-glutamate oxidase

    INAGAKI Kenji, SAKAGUCHI Chika, TAMURA Takashi, KUSAKABE Hitoshi, KASHIMA Akiko, SASAKI Chizuko, SUGIO Shigetoshi

    17   109 - 109   2005

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  • Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation

    T Tamura, S Yamamoto, M Takahata, H Sakaguchi, H Tanaka, TC Stadtman, K Inagaki

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   101 ( 46 )   16162 - 16167   2004.11

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    A labile selenium donor compound monoselenophosphate is synthesized from selenide and ATP by selenophosphate synthetase (SPS). In the present study, Sps1 and Sps2 were cloned from a cDNA library prepared from human lung adenocarcinoma cells (NCI-H441). The human lung Sps1 has been cloned as an ORF of 1,179 bp, identical in sequence to that of the recently revised human liver Sps1. The in-frame TGA codon of the lung Sps2 was genetically altered to TGT (Cys) to obtain the Sps2Cys gene. Expression of the recombinant plasmids containing Spsi or Sps2Cys was highly toxic to Escherichia coli host cells grown aerobically. Accordingly, the human lung Sps homologs were characterized by an in vivo complementation assay using a selD mutant strain. An added selenium source and a low salt concentration (0.1-0.25% NaCl) in the medium were required for reproducible and sensitive in vivo complementation. Sps2Cys effectively complemented the selD mutant, and the resulting formate dehydrogenase H activity was as high as that of WT E. coli MC4100. In contrast, only a weak complementation of the selD mutant by the Sps1 gene was observed when cells were grown in selenite media. Better complementation with added L-selenocysteine suggested involvement of a selenocysteine lyase for mobilization of selenium. Based on this apparent substrate specificity of the Sps1 and Sps2 gene products we suggest that the Sps1-encoded enzyme depends on a selenium salvage system that recycles L-selenocysteine, whereas the Sps2 enzyme can function with a selenite assimilation system.

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  • 2-II-26 超好熱好酸性古細菌Sulfolobus tokodaii由来シスタチオニンγ-シンターゼの精製および性質検討(第56回大会一般研究発表)

    遠藤 祐一, 田村 隆, 倉光 成紀, 田中 英彦, 稲垣 賢二

    ビタミン   78 ( 4 )   246 - 246   2004.4

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  • Saccharomyces cerevisiae 由来D-アミノ酸アセチルトランスフェラーゼの精製と性質.

    田代(山田, 百合子, 田村隆, 田中英彦, 稲垣賢二

    岡山大学農学部学術報告   93 ( 1 )   13 - 18   2004

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    Some properties of D-amino scid acetyltransferase purified from Saccharomyces cerevisiae were investigated. The enzyme was purified to homogeneity by ammonium sulfate fractionation, column chromatographies on DEAE-Toyopearl 650M, Sephacryl S-200, QAE-Toyopearl 550C and affinity chromatography with D-glutamate as a ligand. The molecular weight was estimated to be about 53,000 by gel filtration. Relative molecular mass studies indicated that the enzyme was a monomer structure. The purified enzyme had an optimum pH of 8.4 and an optimum temperature of 40C. The Km values of the purified enzyme determined with tryptophan and acetyl-CoA were 4.5 * 10 -3M, respectively. The 20 residues of N-terminal amino acid sequence were analyzed.

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  • 2A11-4 Redox potentials of DsbA combinatorial mutant library

    TAMURA Takashi, UEMURA Yuko, NAGAYASU Makiko, INAGAKI Kenji

    16   106 - 106   2004

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  • Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp X-119-6

    J Arima, T Tamura, H Kusakabe, M Ashiuchi, T Yagi, H Tanaka, K Inagaki

    JOURNAL OF BIOCHEMISTRY   134 ( 6 )   805 - 812   2003.12

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    L-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha(2)beta(2)gamma(2). The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha(2)beta(2)gamma(2) and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.

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  • Cosynthesis of monofluoroacetate and 4-fluorothreonine by resting cells of blocked mutants of Streptomyces cattleya NRRL8057

    T Tamura, Y Sawamoto, T Kuriyama, K Oba, H Tanaka, K Inagaki

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   23 ( 2-6 )   257 - 263   2003.9

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    Streptomyces cattleya NRRL 8057 produces monofluoroacetate and 4-fluorothreonine from inorganic fluoride. Mutants blocked in fluorometabolite production were prepared by chemical mutagenesis, and cosynthesis experiments with these blocked mutants were carried out by suspending cells of one blocked mutant in the supernatant broth of another blocked mutant. The harvest age of the cells, pH of the buffer, potassium fluoride concentration and glycerol supplementation were optimized for the monofluoroacetate production by a resting-cell suspension of S. cattleya. Successful cosynthesis with pairs of the mutants characterized four distinctive blocked sites in the order N-82, N-44, N-43 and N-47. Additional preparation of blocked mutants by UV irradiation and their cosynthesis assay confirmed that U-303, U-304, U-400 and U-500 were blocked in later steps than N-47. O'Hagan et al. recently proposed that fluoroacetaldehyde, the hypothetical precursor of monofluoroacetate and 4-fluorothreonine, derives from 5'-fluoro-5-deoxyadenosine, the first fluorinated metabolite synthesized from S-adenoSyl-L-methionine and inorganic fluoride by the novel enzyme 'fluorinase'. We were able to detect fluorinase activity in crude extracts of wild type and N-47 mutant strains, but not in the other mutant strains whose blocked steps flanked that of N-47. (C) 2003 Elsevier B.V. All rights reserved.

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  • Purification and substrate characterization of alpha-ketobutyrate decarboxylase from Pseudomonas putida

    H Inoue, A Nishito, S Eriguchi, T Tamura, K Inagaki, H Tanaka

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   23 ( 2-6 )   265 - 271   2003.9

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    alpha-Ketobutyrate decarboxylase encoded in the L-methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of M-r 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg2+ Mn2+, and Co2+. The enzyme showed high activity for alpha-ketobutyrate and alpha-keto-n-valerate rather than pyruvate, but the alpha-keto acids with increasing length of the side chain as well as branching, such as alpha-keto-n-caproate and alpha-keto-3-methylvalerate, were not used by the enzyme. The K-m values for alpha-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the k(cat)/K-m value(10.69 s(-1) mM(-1))for alpha-ketobutyrate was 29-fold greater than that for pyruvate. Thus, alpha-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of alpha-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes. (C) 2003 Elsevier B.V. All rights reserved.

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  • ヒト由来チオレドキシン還元酵素のカルボキシル末端配列の酸化還元状態の半経験的分子軌道計算

    田村 隆, 田中 英彦, 稲垣 賢二

    岡山大學農學部學術報告 = Scientific report of the Faculty of Agriculture, Okayama University   ( 92 )   17 - 20   2003.2

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    ヒトのチオレドキシン還元酵素(TrxR)のカルボキシル末端配列の酸化還元に伴うエンタルピー変化を計算した。半経験的分子軌道計算WinMOPAC 3.5Proを用いてモデルペプチド、N-Ac-Ser-Ile-Leu-Gln-Ala-Gly-X 1-X2-Glyの生成熟を算出した。X1、X2のアミノ酸配列は-SeCys-Cys-、-Cys-SeCys-、-SeCys-SeCys-、-Cys-Cys-のいずれかで、それぞれ酸化状態と還元状態を計算し、そのエンタルピー差を求めた。ハミルトニアンAM1とPM3は同じ傾向の計算結果を示し、セレノスルフィドまたはジセレニドを形成するペプチドは酸化状態でより安定化するのに対して-Cys-Cys-の配列を持つペプチドは還元形の方が安定であることを示した。ほ乳類のTrxRのSeCys498をCysに置換すると酵素活性が1%程度にまで低下することが報告されている。これは、変異酵素が-Cys497-Cys498-間で酸化的架橋を形成にくいからと考察されていたが、今回の分子軌道計算の結果は、この仮説を支持している。

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  • Semiemperical molecular orbital calculation for the redox property of C-terminal active site sequence of human thioredoxin reductase

    T. Tamura, H. Tanaka, K. Inagaki

    Sci. Rep. Fac. Agric. Okayama Univ   2003

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  • 好酸性細菌Acidocella facilis 22Mの制限修飾系遺伝子のクローニングと塩基配列決定

    山岡誠司, 田村 隆, 竹信尚典, 古城俊之, 田中英彦, 稲垣賢二

    岡山大学農学部学術報告   92   9 - 15   2003

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    CGATCGを認識するAfa22MI制限修飾系遺伝子を好酸性細菌Acidocella facilis 22Mより、クローニングし、塩基配列を決定した。その結果、C5-シトシンメチラーゼに特徴的なモチーフが保存されたM.Afa22MI遺伝子、その上流に、M.Afa22MI遺伝子とは逆方向に、very short patch repair endonuclease様タンパク質遺伝子(Afa22MI vsr)、M.Afa22MI遺伝子の下流にM.Afa22MI遺伝子と同方向に、制限酵素(R.Afa22MI)遺伝子と推定されるオープンリーディングフレームが見出された。M.Afa22MIの推定アミノ酸配列は、Xanthomonas oryzae pv. oryzae由来M.XorIIの配列と全体で約63%、variable region内で約53%の高い配列類似性を示した。また、Afa22MI vsrの推定アミノ酸配列も、M.XorIIに付随するXorII vsrの配列と約66%の高い類似性を示した。

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  • Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans

    H Inoue, T Tamura, N Ehara, A Nishito, Y Nakayama, M Maekawa, K Imada, H Tanaka, K Inagaki

    FEMS MICROBIOLOGY LETTERS   214 ( 1 )   127 - 132   2002.8

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    An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M-r 45 000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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  • 1.シネフンギン生合成酵素の遺伝子クローニング

    田中 英彦, 田村 隆

    ビタミン   76 ( 4 )   241 - 241   2002.4

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  • 抗がん酵素L-Methionine γ-lyaseの精密立体構造

    山下 真生, 田村 隆, 瀧本 明生, 光島 健二, 江崎 信芳, 左右田 健次, 田中 英彦, 稲垣 賢二

    ビタミン   76 ( 3 )   177 - 177   2002.3

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  • An in vitro screening method for DNA cytosine-C-5-methylase inhibitor

    T Tamura, A Kataoka, LY Shu, A Ashida, H Tanaka, K Inagaki

    NATURAL PRODUCT LETTERS   16 ( 1 )   25 - 27   2002.2

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    A specific inhibitor of DNA cytosine C-5-methylases would be useful for studying genomic imprinting, X-chromosome inactivation, carcinogenesis, and regulation of tissue-specific gene expression, for these physiological phenomena appears to be regulated through DNA methylation in promoter sequences. This paper reports a novel convenient in vitro assay method for screening DNA cytosine C-5-Methylase inhibitor. Our method uses a commercially available Hae III methylase (cytosine C-5 methylase), its corresponding Hae III endonuclease, and lambda DNA as their substrate.

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  • 好酸性細菌 Acidocella facilis 由来菌体外酸性エステラーゼ遺伝子のクローニング

    高橋竜也, 田中 登, 田村 隆, 向井 千佳, 磯部公安, 若尾 紀夫, 田中英彦, 稲垣賢二

    岡山大学農学部学術報告   91   7 - 14   2002

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    好酸性従属栄養細菌Acidocella facilis AIU409株は、基質としてソルビタンモノエステルであるTweenを含む培地中で培養された時に、菌体外に熱安定性の高い酸性エステラーゼを生産する。エステラーゼをコードする遺伝子(estA)をA. facilis AIU409株のゲノムライブラリーから単離し、大腸菌MV1184にクローニングし、その遺伝子の全塩基配列を決定した。その結果、estAの構造遺伝子が、1881塩基対であることが明らかになった。酸性エステラーゼ遺伝子のオープンリーディングフレーム(ORF)は、627アミノ酸残基(計算された分子量は64、140ダルトン)をコードしていた。ロー囚子非依存性の転写終結シグナルが終止コドンであるTGAのすぐ下流に存在していた。そのN末端予想アミノ酸配列より、酸性エステラーゼの前駆体は、N末端に23個のアミノ酸残基から成るシグナルペプチドを有していた。また、リパーゼのコンセンサス配列であるG-X-S-X-Sの配列が存在することが明らかとなっ。酸性エステラーゼの予想アミノ酸配列から計算された分子量は61,486であり、これはSDS-PAGEによって予想されていた分子量より、やや低い値であった。また、Acidocella facilis酸性エステラーゼの予想アミノ酸配列は、Aeromonas hydrophila由来のacyltrans-ferase12、13)やXenorhabdus luminescens由来のリパーゼ14)と高い相同性を示した。

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  • L-グルタミン酸オキシダーゼを用いたグルタミン酸センサーの開発及びGOT/GTPセンシングへの応用

    有馬二朗, 田村 隆, 篠原寛明, 日下部均, 田中英彦, 稲垣賢二

    岡山大学農学部学術報告   2002

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  • Improvement of sinefungin-producing strain of Streptomyces incarnatus by conferring rifampicin-resistance through ultraviolet light irradiation and protoplast regeneration

    Tamura, T, L.-Y. Shu, H. Tanaka, K. Inagaki

    Sci. Rep. Fac. Agric. Okayama Univ.   2002

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  • Selenium for Cancer Prevention and Human Health

    Foods & food ingredients journal of Japan   198, 24-38 ( 198 )   24 - 38   2002

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  • Mammalian thioredoxin reductases

    T Tamura, TC Stadtman

    PROTEIN SENSORS AND REACTIVE OXYGEN SPECIES, PT A, SELENOPROTEINS AND THIOREDOXIN   347   297 - 306   2002

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  • 2-III-15 ヒト肺がん細胞由来のチオレドキシン還元酵素遺伝子の点突然変異

    田村 隆, 長谷川 雅哉, 稲垣 賢二, 田中 英彦

    ビタミン   75 ( 4 )   243 - 243   2001.4

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  • 2-III-17 硫黄酸化細菌Thiobacillus thiooxidans由来イソクエン酸脱水素酵素と同遺伝子の解析

    江原 渚, 井上 宏之, 田村 隆, 田中 英彦, 稲垣 賢二

    ビタミン   75 ( 4 )   244 - 244   2001.4

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  • 2-III-13 放線菌由来L-グルタミン酸オキシダーゼ : クローニング,発現,及び諸性質検討

    有馬 二朗, 崎川 智代, 田村 隆, 芦内 誠, 八木 年晴, 日下部 均, 田中 英彦, 稲垣 賢二

    ビタミン   75 ( 4 )   242 - 243   2001.4

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  • 2. Pseudomonas putida L-Methionine γ-lyaseのTyrosine114の役割

    井上 宏之, 稲垣 賢二, 田村 隆, 江崎 信芳, 左右田 健次, 田中 英彦

    ビタミン   75 ( 3 )   154 - 155   2001.3

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  • Purification and characterization of an acid trehalase from Acidobacterium capsulatum

    K Inagaki, N Ueno, T Tamura, H Tanaka

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   91 ( 2 )   141 - 146   2001.2

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    We purified an acid trehalase (EC 3.2.1.28, a,alpha,alpha'-trehalose glucohydrolase) from an acidophilic bacterium, Acidobacterium capsulatum. The enzyme was homogeneous based on polyacrylamide gel electrophoresis, and was composed of a single polypeptide chain with a molecular mass of 57 kDa. Maximum trehalase activity was observed at pH 2.5. The acid trehalase exhibited an apparent K-m of 1.0 mM for trehalose at 30 degreesC and pH 3.0, The trehalase was located in the periplasmic space. The activity of the enzyme was activated by 1.0 mM MnCl2 or CoCl2, and inhibited by 1.0 mM PbCl2, HgCl2, NiCl2, p-chloromercuribenzoate, N-ethylmaleimide, monoiodoacetate, or EDTA. The enzyme showed high specificity for trehalose. It was found that an equimolar mixture of alpha -D-glucose and beta -D-glucose was formed on hydrolysis of trehalose by the trehalase.

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  • セレンは猛毒性・発癌性の栄養素?

    田村 隆

    バイオアクティブ   11,15-18   2001

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  • Molecular cloning of thioredoxin reductase isozymes, TrxR1 and TrxR2, from human lung adenocarcinoma cell line, NCI-H441

    Proceedings Symposium on Trace Nutrients Research   18   149 - 153   2001

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  • Streptomyces sp X-119-6 由来L-グルタミン酸オキシダーゼのクローニング及び発現

    有馬二朗, 崎川智代, 田村 隆, 芦内 誠, 八木年春, 日下部均, 田中英彦, 稲垣賢二

    微量栄養素研究   2001

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  • 116 19F-nmrを用いるモノフルオロ酢酸生産のCosynthesis実験

    田村 隆, 澤本 有香子, 田中 英彦, 稲垣 賢二

    日本生物工学会大会講演要旨集   13   106 - 106   2001

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  • 709 放線菌由来L-グルタミン酸オキシダーゼを利用したL一GluセンサーとGOT・GPTセンシングへの応用

    有馬 二朗, 田村 隆, 崎川 智代, 金原 陽平, 篠原 寛明, 日下部 均, 田中 英彦, 稲垣 賢二

    日本生物工学会大会講演要旨集   13   289 - 289   2001

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  • Purification and some characteristics of a monomeric alanine racemase from an extreme thermophile, Thermus thermophilus

    TK Seow, K Inagaki, T Nakamura, R Maeda, T Tamura, H Tanaka

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   90 ( 3 )   344 - 346   2000.9

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    We purified to homogeneity an alanine racemase (EC 5.1.1.1) from Thermus thermophilus HB8, an extreme thermophile. Interestingly, the enzyme possessed a monomeric structure with a molecular weight of about 38,000. The enzyme was most active at pH 8 and 75 degreesC, and remained active after incubation at 80 degreesC for 30 min.

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  • A colorimetric screening method for microorganisms having methionine gamma-lyase activity

    LL Choo, T Tamura, RA Rahim, AM Ali, HM Ghazali, K Inagaki, H Tanaka

    ASIA-PACIFIC JOURNAL OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY   8 ( 1 )   95 - 97   2000.6

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    This paper reports a convenient screening method for detecting methionine gamma -lyase activity in microorganisms which can grow on DL-methionine as the sole nitrogen source. L-Methionine gamma -lyase has been known to have the most versatile catalytic properties among other pyridoxal-5'-phosphate-dependent enzymes, but only known from a limited number of biological sources. For an efficient screening of the enzyme, we employed 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to detect the enzyme reaction product methanethiol, which spontaneously reduces DTNB and develops a yellow coloration on and around colonies. The yellow color has emerged and faded away within 72 h, suggesting that the reduced DTNB may be oxidized by spontaneous and/or enzymatic formation of disulfide. This method, despite reduction in specificity compared to the standard assay, allowed us to select candidate microorganisms out of hundreds of other similar colonies merely by observing the yellow coloration.

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  • 1-II-9 L-メチオニンγ-リアーゼの立体構造

    稲垣 賢二, 田村 隆, 田中 英彦, 本島 浩之, 熊坂 崇, 古市 真木雄, 山本 雅貴, 田中 信夫, 江崎 信芳, 左右田 健次

    ビタミン   74 ( 4 )   197 - 197   2000.4

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  • Preparation of NAD:penicillamine ADP transferase high producing Bacillus sphaericus.

    Izumi Takuya, Tamura Takashi, Inagaki Kenji, Soda Kenji, Tanaka Hidehiko

    12   34 - 34   2000

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  • Purification and characterization of NAD:penicillamine ADP transferase from Bacillus sphaericus: A novel NAD-dependent enzyme catalyzing phosphoramide bond formation

    Jun Yanagidani, Takashi Tamura, Kenji Inagaki, Kenji Soda, Hidehiko Tanaka

    Journal of Biological Chemistry   274 ( 2 )   795 - 800   1999.1

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    A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide. The phosphoramide product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. The enzyme showed high substrate specificity to NAD (K(m) = 13.0 mM) and L-penicillamine (K(m) = 6.5 mM)
    other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released. The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B. sphaericus.

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  • Purification and characterization of NAD:penicillamine ADP transferase from Bacillus sphaericus: A novel NAD-dependent enzyme catalyzing phosphoramide bond formation

    Jun Yanagidani, Takashi Tamura, Kenji Inagaki, Kenji Soda, Hidehiko Tanaka

    Journal of Biological Chemistry   274 ( 2 )   795 - 800   1999.1

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    A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide. The phosphoramide product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. The enzyme showed high substrate specificity to NAD (K(m) = 13.0 mM) and L-penicillamine (K(m) = 6.5 mM)
    other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released. The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B. sphaericus.

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  • 1. Thiobacillus thiooxidansのイソクエン酸脱水素酵素の性質と遺伝子クローニング

    稲垣 賢二, 井上 宏之, 田村 隆, 田中 英彦

    ビタミン   72 ( 10 )   571 - 571   1998.10

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  • Gene Cloning and Characterization of an Acidic Xylanase from Acidobacteriu

    Kenji Inagaki, Ken Nakahira, Kazuhisa Mukai, Takashi Tamura, Hidehiko Tanaka

    Bioscience, Biotechnology and Biochemistry   62 ( 6 )   1061 - 1067   1998.1

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    The gene xynA encoding an acid endo-β-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues. The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10. XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E. coli transformants. The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively. The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65°C, and is stable pH between 3.0 and 8.0. The Km and Vmax with oat spelt xylan as a substrate at pH 5.0 and 30°C are 3.5 mg/ml and 403 μmol/min/mg. © 1998 Taylor &amp
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  • Gene Cloning and Characterization of an Acidic Xylanase from Acidobacteriu

    Kenji Inagaki, Ken Nakahira, Kazuhisa Mukai, Takashi Tamura, Hidehiko Tanaka

    Bioscience, Biotechnology and Biochemistry   62 ( 6 )   1061 - 1067   1998.1

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    The gene xynA encoding an acid endo-β-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues. The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10. XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E. coli transformants. The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively. The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65°C, and is stable pH between 3.0 and 8.0. The Km and Vmax with oat spelt xylan as a substrate at pH 5.0 and 30°C are 3.5 mg/ml and 403 μmol/min/mg. © 1998 Taylor &amp
    Francis Group, LLC.

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  • Alanine racemase from an acidophile, acidiphilium organovorum: Purification and characterization

    Teck Keong Seow, Kenji Inagaki, Takashi Tamura, Kenji Soda, Hidehiko Tanaka

    Bioscience, Biotechnology and Biochemistry   62 ( 2 )   242 - 247   1998

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    An alanine racemase (EC 5.1.1.1) from an acidophilic heterotrophic bacterium, Acidiphilium organovorum 13H, was purified and characterized. The enzyme had a dimeric structure with identical subunits of M, 33,000 each. Although A. organovorum 13H is an acidophile, the enzyme had its maximum velocity at pH 9, corresponding to its location in the cytoplasm. Activity was maximum between 50 and 60°C. For an enzyme from a mesophile, it was stable to heat, showing no loss of activity after a 30-min incubation at 65°C. The enzyme needed pyridoxal 5′. phosphate (PLP) as a cofactor for its activity, as seen from the loss of activity upon dialysis against PLP-free buffer containing hydroxylamine and its absorption maximum at 420 nm. Activity was ihhibited by common inhibitors of PLP-dependent enzymes. PLP content studies found that 1 mole of enzyme contained 2 moles of PLP. The enzyme catalyzed the symmetric reversible racemization of alanine exclusively. © 1998, Taylor &amp
    Francis Group, LLC. All rights reserved.

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  • Alanine racemase from an acidophile, acidiphilium organovorum: Purification and characterization

    Teck Keong Seow, Kenji Inagaki, Takashi Tamura, Kenji Soda, Hidehiko Tanaka

    Bioscience, Biotechnology and Biochemistry   62 ( 2 )   242 - 247   1998

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    An alanine racemase (EC 5.1.1.1) from an acidophilic heterotrophic bacterium, Acidiphilium organovorum 13H, was purified and characterized. The enzyme had a dimeric structure with identical subunits of M, 33,000 each. Although A. organovorum 13H is an acidophile, the enzyme had its maximum velocity at pH 9, corresponding to its location in the cytoplasm. Activity was maximum between 50 and 60°C. For an enzyme from a mesophile, it was stable to heat, showing no loss of activity after a 30-min incubation at 65°C. The enzyme needed pyridoxal 5′. phosphate (PLP) as a cofactor for its activity, as seen from the loss of activity upon dialysis against PLP-free buffer containing hydroxylamine and its absorption maximum at 420 nm. Activity was ihhibited by common inhibitors of PLP-dependent enzymes. PLP content studies found that 1 mole of enzyme contained 2 moles of PLP. The enzyme catalyzed the symmetric reversible racemization of alanine exclusively. © 1998, Taylor &amp
    Francis Group, LLC. All rights reserved.

    DOI: 10.1271/bbb.62.242

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  • Pseudomonas putidaのL-メチオニン分解系オペロンの解析

    井上宏之, 田村 隆, 稲垣賢二, 田中英彦

    岡山大学農学部学術報告   87   53 - 58   1998

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  • 染料脱色微生物の検索, 単離及び, 脱色機構の解析

    稲垣賢二, 川口将和, 田口隆章, 田村 隆, 田中英彦

    岡山大学農学部学術報告   87   47 - 51   1998

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  • Determination of methylation specificity of sequence-specific DNA methyltransferases using matrix assisted laser desorption/ionization time-of-flight mass spectrometry

    Takashi Tamura, Yoshinori Araki, Seiji Yamaoka, Kenji Inagaki, Hidehiko Tanaka

    Nucleic Acids Research   25 ( 20 )   4162 - 4164   1997.10

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    We describe here a sensitive and straightforward method for characterizing the methylation specificity of type II DNA methyltransferase (MTase) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA substrate, prepared by ligation of a commercially available oligonucleotide, was modified by the subject MTase, and was derivatized to a mixture of single-stranded oligonucleotides through endonuclease treatment, heat-denaturation and limited digestion by 3'-terminus-specific phosphodiesterase I. MALDI-TOF mass spectrometry was used to determine the mass differences between the digestion products, and the methylated nucleotide was explicitly identified by the mass increase of 14 Da due to the base modification. The method was applicable to the three representative MTases M.EcoRI, M.BamHI and M.HaeIII.

    DOI: 10.1093/nar/25.20.4162

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  • Determination of methylation specificity of sequence-specific DNA methyltransferases using matrix assisted laser desorption/ionization time-of-flight mass spectrometry

    Takashi Tamura, Yoshinori Araki, Seiji Yamaoka, Kenji Inagaki, Hidehiko Tanaka

    Nucleic Acids Research   25 ( 20 )   4162 - 4164   1997.10

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    We describe here a sensitive and straightforward method for characterizing the methylation specificity of type II DNA methyltransferase (MTase) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA substrate, prepared by ligation of a commercially available oligonucleotide, was modified by the subject MTase, and was derivatized to a mixture of single-stranded oligonucleotides through endonuclease treatment, heat-denaturation and limited digestion by 3'-terminus-specific phosphodiesterase I. MALDI-TOF mass spectrometry was used to determine the mass differences between the digestion products, and the methylated nucleotide was explicitly identified by the mass increase of 14 Da due to the base modification. The method was applicable to the three representative MTases M.EcoRI, M.BamHI and M.HaeIII.

    DOI: 10.1093/nar/25.20.4162

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  • Determination of methylation specificity of sequence-specific DNA methyltransferases using matrix assisted laser desorption/ionization time-of-flight mass spectrometry

    Takashi Tamura, Yoshinori Araki, Seiji Yamaoka, Kenji Inagaki, Hidehiko Tanaka

    Nucleic Acids Research   25 ( 20 )   4162 - 4164   1997.10

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    We describe here a sensitive and straightforward method for characterizing the methylation specificity of type II DNA methyltransferase (MTase) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA substrate, prepared by ligation of a commercially available oligonucleotide, was modified by the subject MTase, and was derivatized to a mixture of single-stranded oligonucleotides through endonuclease treatment, heat-denaturation and limited digestion by 3'-terminus-specific phosphodiesterase I. MALDI-TOF mass spectrometry was used to determine the mass differences between the digestion products, and the methylated nucleotide was explicitly identified by the mass increase of 14 Da due to the base modification. The method was applicable to the three representative MTases M.EcoRI, M.BamHI and M.HaeIII.

    DOI: 10.1093/nar/25.20.4162

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  • ゴ-ルドラッシュ時代を迎えたセレンの生化学研究 (特集 最近の生命科学)

    田村 隆, 田中 英彦

    化学工業   48 ( 9 )   720 - 726   1997.9

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  • 5.ペニシラミンの微生物代謝の酵素化学 : NADの新しい生理機能 : ビタミンB研究委員会第357回会議研究発表要旨

    左右田 健次, 柳谷 純, 田村 隆, 稲垣 賢二, 田中 英彦

    ビタミン   71 ( 7 )   312 - 313   1997.7

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  • 2-III-4 ADP-penicillamine合成酵素の精製と反応生成物の同定 : 第49回大会一般研究発表要旨

    柳谷 純, 田村 隆, 稲垣 賢二, 田中 英彦, 左右田 健次

    ビタミン   71 ( 4 )   210 - 210   1997.4

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  • 1-II-7 Pseudomonas putidaのメチオニン誘導性の新規α-ケト酪酸脱水素酵素E1について : 第49回大会一般研究発表要旨

    井上 宏之, 西藤 晃, 田村 隆, 稲垣 賢二, 田中 英彦

    ビタミン   71 ( 4 )   179 - 179   1997.4

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  • 476 Isolation and Characterisation of Alanine Racemase from Extreme Thermophile, Thermus thermophilus :

    Keong Seow Teck, Inagaki Kenji, Nakamura Takeshi, Maeda Ritsuko, Tamura Takashi, Tanaka Hidehiko

    9   164 - 164   1997

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  • Pseudomonas putidaのα-ケト酪酸デヒドロゲナーゼE1成分の大腸菌内での発現と諸性質

    井上 宏之, 江里口 真一, 西藤 晃, 田村 隆, 稲垣 賢二, 田中 英彦

    日本分子生物学会年会プログラム・講演要旨集   19   417 - 417   1996.8

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  • ヒト肺腫瘍細胞に高発現するセレン酵素, チオレドキシン還元酵素の諸性質

    田村 隆, 田中 英彦, STADTMAN Thressa C.

    日本分子生物学会年会プログラム・講演要旨集   19   558 - 558   1996.8

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  • 3 - isopropylmalic acid dehydrogenase of Thiobacillus thiooxidans. Purification and property.

    松波秀行, 川口洋, 中山ゆみ, 田村隆, 稲垣賢二, 田中英彦

    日本分子生物学会年会プログラム・講演要旨集   19th   131   1996.7

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  • Isocitrate dehydrogenase of Thiobacillus thiooxidans. Purification and property.

    西藤晃, 松波秀行, 中山ゆみ, 川口洋, 田村隆, 稲垣賢二, 田中英彦

    日本分子生物学会年会プログラム・講演要旨集   19th   131   1996.7

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  • Pseudomonas putidaのL-メチオニン分解系オペロンの転写調節因子の解析 : 微生物

    井上 宏之, 田村 隆, 稲垣 賢二, 江崎 信芳, 左右田 健次, 田中 英彦

    日本農藝化學會誌   70   104 - 104   1996.3

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  • Bacillus属由来ペニシラミン分解酵素の反応生成物の同定 : 酵素

    柳谷 純, 田村 隆, 稲垣 賢二, 田中 英彦, 左右田 健次

    日本農藝化學會誌   70   156 - 156   1996.3

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  • 新規セレン酵素,チオレドキシン還元酵素の精製と諸性質 : 動物

    田村 隆, 田中 英彦, Stadtman Thressa C.

    日本農藝化學會誌   70   205 - 205   1996.3

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  • A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity

    Takashi Tamura, Thressa C. Stadtman

    Proceedings of the National Academy of Sciences of the United States of America   93 ( 3 )   1006 - 1011   1996.2

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    We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 μM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine
    hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se- carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent K(m) values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 μM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

    DOI: 10.1073/pnas.93.3.1006

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  • A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity

    Takashi Tamura, Thressa C. Stadtman

    Proceedings of the National Academy of Sciences of the United States of America   93 ( 3 )   1006 - 1011   1996.2

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    We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 μM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine
    hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se- carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent K(m) values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 μM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

    DOI: 10.1073/pnas.93.3.1006

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  • Antimicrobial activity against methicillin-resistant Staphylococcus aureus in the culture broth of Enterococcus faecalis TH 10, an isolate from Malaysian fermentation food, Temph

    OHHIRA Iichiro, TAMURA Takashi, FUJII Nozomi, INAGAKI Kenji, TANAKA Hidehiko

    JAPANESE JOURNAL OF DAIRY AND FOOD SCIENCE   45(4), 93-96 ( 4 )   A - 93-A-96   1996

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    Bioassay-directed screening for antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) led us to the culture broth of Enterococcus faecalis TH 10, which was isolated from a Malaysian fermentation food, Temph. The anti-MRSA component was readily extracted with ethyl acetate from the culture broth at pH 3, and the extract retained the activity when incubated overnight with various proteolytic enzymes. In addition, the extract showed poor antimicrobial activity against various lactic acid bacteria, including Enterococcus faecalis. These properties strongly suggest that the active compound is distinct from bacteriocin, an extracellularly released peptide or protein which shows potent activity against species closely related to the bacteriocin-producing strain. E. faecalis has been reported to produce bacteriocin which has strong hemolytic activity on mammalian erythrocytes, but the active component of the TH 10 strain did not mediate the lysis of rabbit and human erythrocytes.

    DOI: 10.11465/milkscience.45.A-93

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  • Purification and Characterization of Xylanase from Acidobacterium capsulatum.

    Nakahira Ken, Mukai Kazuhisa, Tamura Takashi, Inagaki Kenji, Tanaka Hidehiko

    8   211 - 211   1996

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  • Antimicrobial activity against methicillin-resistant Staphylococcus aureus in the culture broth of Enterococcus faecalis TH 10, an isolate from Malaysian fermentation food, Temph

    OHHIRA Iichiro, TAMURA Takashi, FUJII Nozomi, INAGAKI Kenji, TANAKA Hidehiko

    JAPANESE JOURNAL OF DAIRY AND FOOD SCIENCE   45 ( 4 )   93 - 93-A-96   1996

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    Bioassay-directed screening for antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) led us to the culture broth of Enterococcus faecalis TH 10, which was isolated from a Malaysian fermentation food, Temph. The anti-MRSA component was readily extracted with ethyl acetate from the culture broth at pH 3, and the extract retained the activity when incubated overnight with various proteolytic enzymes. In addition, the extract showed poor antimicrobial activity against various lactic acid bacteria, including Enterococcus faecalis. These properties strongly suggest that the active compound is distinct from bacteriocin, an extracellularly released peptide or protein which shows potent activity against species closely related to the bacteriocin-producing strain. E. faecalis has been reported to produce bacteriocin which has strong hemolytic activity on mammalian erythrocytes, but the active component of the TH 10 strain did not mediate the lysis of rabbit and human erythrocytes.

    DOI: 10.11465/milkscience.45.A-93

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  • Incorporation of Fluoride Anion into Organofluoro Compounds by Microorganisms

    ESAKI Nobuyoshi, TAMURA Takashi, WADA Masaru, SODA Kenji

    6 ( 3 )   117 - 118   1995.12

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  • Synthesis and Biochemical Studies of Selenocysteine-Containing Peptides

    Tamura Takashi

    Scientific reports of the Faculty of Agriculture, Okayama University   84   69 - 72   1995.2

  • Synthesis of fluoroacetate from fluoride, glycerol, and β- hydroxypyruvate by Streptomyces cattleya

    T. Tamura, M. Wada, N. Esaki, K. Soda

    Journal of Bacteriology   177 ( 9 )   2265 - 2269   1995

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    Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]β-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U- 14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C
    about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]β-hydroxypyruvates to show that C-2 and C-3 of β-hydroxy pyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through β-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.

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  • The mutual sparing effects of selenium and vitamin E in animal nutrition may be further explained by the discovery that mammalian thioredoxin reductase is a selenoenzyme

    T Tamura, Gladyshev, V, SY Liu, TC Stadtman

    BIOFACTORS   5 ( 2 )   99 - 102   1995

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    The recent discovery that mammalian thioredoxin reductase is a selenoprotein furnishes an additional explanation of the mutual sparing roles of selenium and vitamin E in cellular antioxidant systems. Thioredoxin reductases isolated from human lung adenocarcinoma cells, human Jurkat T-cells and HeLa cells contain selenocysteine which is located in a C- terminal tripeptide, -Cys-SeCys-Gly.

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  • Synthesis of fluoroacetate from fluoride, glycerol, and β- hydroxypyruvate by Streptomyces cattleya

    T. Tamura, M. Wada, N. Esaki, K. Soda

    Journal of Bacteriology   177 ( 9 )   2265 - 2269   1995

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    Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]β-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U- 14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C
    about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]β-hydroxypyruvates to show that C-2 and C-3 of β-hydroxy pyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through β-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.

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  • The mutual sparing effects of selenium and vitamin E in animal nutrition may be further explained by the discovery that mammalian thioredoxin reductase is a selenoenzyme

    T Tamura, Gladyshev, V, SY Liu, TC Stadtman

    BIOFACTORS   5 ( 2 )   99 - 102   1995

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    The recent discovery that mammalian thioredoxin reductase is a selenoprotein furnishes an additional explanation of the mutual sparing roles of selenium and vitamin E in cellular antioxidant systems. Thioredoxin reductases isolated from human lung adenocarcinoma cells, human Jurkat T-cells and HeLa cells contain selenocysteine which is located in a C- terminal tripeptide, -Cys-SeCys-Gly.

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  • Synthesis and characterization of the selenium analog of glutathione disulfide

    Takashi Tamura, Tadao Oikawa, Akira Ohtaka, Nobutaka Fujii, Nobuyoshi Esaki, Kenji Soda

    Analytical Biochemistry   208 ( 1 )   151 - 154   1993

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    We synthesized the selenium analog of glutathione disulfide by a liquid phase method and named it glutaselenone (i.e., γ-L-glutamyl-L-selenocysteinylglycine) diselenide. The selenol of selenocysteine was protected by the p-methoxybenzyl group, which was removed by acidolysis with trifluoroacetic acid in the presence of thioanisol. The overall yield of the final product, glutaselenone diselenide, was about 9% based on the starting compound, Se-(p-methoxybenzyl)-L-selenocysteine. Glutaselenone diselenide showed a broad absorption band between 270 and 400 nm and circular dichroism bands around 270 nm (positive) and 330 nm (negative), which were attributable to diselenide bond. © 1993 Academic Press, Inc.

    DOI: 10.1006/abio.1993.1021

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  • グルタチオンのセレンアナログ、グルタセレノンの触媒するグルタチオンペルオキシダーゼ反応の機構 Reviewed

    江崎 信芳, 田村 隆, 中里 仁, 左右田 健次

    Biomed. Res. Trace Elements,4,87-88   1993

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  • Synthesis and characterization of the selenium analog of glutathione disulfide

    Takashi Tamura, Tadao Oikawa, Akira Ohtaka, Nobutaka Fujii, Nobuyoshi Esaki, Kenji Soda

    Analytical Biochemistry   208 ( 1 )   151 - 154   1993

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    We synthesized the selenium analog of glutathione disulfide by a liquid phase method and named it glutaselenone (i.e., γ-L-glutamyl-L-selenocysteinylglycine) diselenide. The selenol of selenocysteine was protected by the p-methoxybenzyl group, which was removed by acidolysis with trifluoroacetic acid in the presence of thioanisol. The overall yield of the final product, glutaselenone diselenide, was about 9% based on the starting compound, Se-(p-methoxybenzyl)-L-selenocysteine. Glutaselenone diselenide showed a broad absorption band between 270 and 400 nm and circular dichroism bands around 270 nm (positive) and 330 nm (negative), which were attributable to diselenide bond. © 1993 Academic Press, Inc.

    DOI: 10.1006/abio.1993.1021

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Presentations

  • Tagging [NiFeSe] hydrogenase in Desulfovibrio alaskensis G20

    Extremophile2018  2018 

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  • Invention of Protein Thermal Refolding (PTR)

    Extremophile2018  2018 

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  • 放線菌のrpoB遺伝子改変を目的としたマーカーレスゲノム改変技術

    日本農芸化学会中四国支部第47回講演会  2017 

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  • 新規[NiFeSe]ヒドロゲナーゼの探索とアフィニティタグ配列導入の検討

    日本農芸化学会関西・中四国・西日本支部合同大会  2017 

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  • 酸や熱ショックがS.incarnatusのシネフンギン生産に及ぼす影響

    日本放線菌学会  2017 

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  • 海洋性細菌Marinomonas mediterranea由来キノン含有新規グリシンオキシダーゼの基質特異性と分子構造

    日本農芸化学会中四国支部第47回講演会  2017 

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  • Rhodococcus属由来低基質特異性L-アミノ酸オキシダーゼの性質検討及び応用

    日本農芸化学会中四国支部第47回講演会  2017 

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  • L-グルタミン酸オキシダーゼから1アミノ酸置換で作成した新規L-アルギニンオキシダーぜの精製と特性解析

    日本農芸化学会中四国支部大会 第46回講演会  2016 

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  • Crystal structure of recombinant L-lysine α-oxidase in complex with its substrate

    ICC05-AEM2016  2016 

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  • Bacillus subtilis 168の銅取込みに関わるzosA遺伝子の機能解析

    日本ビタミン学会第68回大会  2016 

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  • 核酸系抗生物質の生産性向上を目的とした転写マシナリーの最適化検討

    日本農芸化学会中四国支部大会 第46回講演会  2016 

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  • 放線菌S. incarnatusのRNAP/dsDNA/RNA複合体の分子動力学モデリング

    第31回日本放線菌学会  2016 

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  • ZosA, a zinc-uptake P-type ATPase transporter, plays a role in copper import in Bacillus subtilis 168

    ICC05-AEM2016  2016 

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  • ジスルフィド架橋導入sfGFPを用いた蛍光回復過程の経時変化

    第39回日本分子生物学会年会  2016 

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  • Thermus thermophilus HB8のペリプラズムフォールディングシステムTthA0610/1422系の発現と機能解析

    第10回「高度好熱菌丸ごと一匹プロジェクト」連携研究会  2011 

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  • セレノリン酸合成酵素SPS1はSPS2を抑制する

    日本ビタミン学会  2010 

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  • 新規耐熱性アミダーゼの酵素精製と性質検討

    日本農芸化学会2010年度大会  2010 

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  • グリセロールキナーゼの構造安定化

    日本農芸化学会2010年度大会  2010 

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  • Why Sps1 and Sps2 differ in their catalysis? : Identification of an internal sequence region catalytically essential for the human lung selenophosphate synthetase 2

    第9回国際セレン生物医学シンポジウム  2010 

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  • 細胞抽出液を用いたシネフンギン生合成基質の検討

    日本生化学会 中国四国支部大会  2010 

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  • 放線菌 Streptomyes incarnatus 無細胞抽出液でのシネフンギン生合成

    日本農芸化学会2010年度大会  2010 

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  • 高基質特異性L-アミノ酸オキシダーゼの基質認識の機構解析

    日本農芸化学会2010年度大会  2010 

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  • キイロショウジョウバエ由来チオレドキシン還元酵素のレドックス機能解析

    日本ビタミン学会  2010 

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  • Thermus thermophilus HB8のTthA0610/1422系の機能解析とsuperfolder GFPの設計

    第9回「高度好熱菌丸ごと一匹プロジェクト」連携研究会  2010 

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  • 放射性同位元素64Cuを用いたBacillus subtilis168の銅代謝解析

    日本ビタミン学会  2010 

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  • メチオニンγリアーゼの部位特異的変異導入と基質特異性改変

    日本ビタミン学会  2010 

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  • 含フッ素核酸系化合物の酵素合成を目的とするフルオリナーゼ改変計画

    文部科学省 次世代IT基盤構築のための研究開発 第1回「イノベーション基盤シミュレーションソフトウェアの研究開発」プロジェクト シンポジウム -ものづくりを変革するシミュレーション技術の挑戦-  2009 

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  • MOPAC Calculation of Low Barrier Hydrogen Bonds and their Application to Molecular Design of Enzyme

    Joint International Open Symposium on Molecular Science of Fluctuations toward Biological Functions  2009 

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  • 微量必須元素セレンの生理機能とバイオマーカーとしての可能性

    第2回高度医療都市を創出する未来技術国際シンポジウム  2009 

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  • チオレドキシン還元酵素に依存する亜セレン酸代謝の分子機構解析

    日本農芸化学会中四国支部第23回講演会  2009 

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  • 新規耐熱性アミダーゼのスクリーニング,遺伝子クローニングと発現

    日本農芸化学会中四国支部第23回講演会  2009 

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  • 触媒ドメインの構造解析に基づくほ乳類SPS1とSPS2の機能的差異

    日本農芸化学会2009年度大会  2009 

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  • 放線菌Streptomyces calvusによるヌクレオシジン生産の検討

    日本農芸化学会2009年度大会  2009 

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  • Lメチオニンγリアーゼの活性中心に存在するAsp241の機能

    日本農芸化学会2009年度大会  2009 

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  • セレノリン酸合成酵素Sps1はSps2抑制型アンチザイム機能を持つ

    第82回日本生化学会大会  2009 

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  • Pseudomonas putida由来L-メチオニンγリアーゼの活性中心に存在するCys116及びAsp241残基への変異導入

    第82回日本生化学会大会  2009 

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  • Streptomyces sp x119-6由来L-グルタミン酸オキシダーゼの基質認識に関わるアミノ酸残基の解析

    第82回日本生化学会大会  2009 

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  • A novel active-site sequence Cys-Asp-Ile-Cys altered E. coli DsbA into a potent protein disulfide isomerase

    The 4th International Congress on Stress Responses in Biology and Medicine  2009 

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  • 祖先型変異導入によるグリセロールキナーゼの熱安定化

    第82回日本生化学会大会  2009 

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  • セレン輸送体はモノチオール型ではなくジチオール型蛋白質である

    第82回日本生化学会大会  2009 

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  • 放線菌由来フッ素導入酵素フルオリナーゼの植物発現ベクターの構築

    2009年度日本農芸化学会関西・中四国・西日本支部、日本栄養・食糧学会九州・沖縄支部および日本食品科学工学会西日本支部合同大会  2009 

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  • 好熱好酸性アーキアSulfobus tokodaii由来シスタチオニン合成酵素の活性中心残基

    2009年度日本農芸化学会関西・中四国・西日本支部、日本栄養・食糧学会九州・沖縄支部および日本食品科学工学会西日本支部合同大会  2009 

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  • 耐熱性ジスルフィド形成蛋白質TthA0610, TthA1422の発現と機能解析

    2009年度日本農芸化学会関西・中四国・西日本支部、日本栄養・食糧学会九州・沖縄支部および日本食品科学工学会西日本支部合同大会  2009 

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  • 亜セレン酸の還元代謝に関わるセレンThioredoxin付加体の同定

    日本ビタミン学会第61回大会  2009 

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  • Bacillus subtilis 168金属輸送ATPase ZosA の機能解析

    第50回日本生化学会中国・四国支部例会  2009 

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  • 有機電子論では解けない酵素化学の問題をパソコンによる分子軌道計算で解く

    日本農芸化学会2009年度大会  2009 

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  • Thermus thermophilus HB8のジスルフィド架橋形成酵素群

    日本農芸化学会2009年度大会  2009 

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  • 抗腫瘍性酵素メチオニンγ-リアーゼの活性中心残基への変 異導入による基質特異性の改変

    酵素補酵素研究会  2009 

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  • 補酵素を必要としない親電子触媒DsbAへのコンビナトリアル変異導入

    酵素補酵素研究会  2009 

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  • 放線菌由来Lグルタミン酸オキシダーゼの構造特性と基質認識機構の解析

    日本ビタミン学会第61回大会  2009 

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  • rpoB遺伝子改変によるStreptomyces incarnatusのシネフンギン高生産株

    日本生物工学会  2009 

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  • 新規耐熱性アミラーゼの遺伝子クローニングと酵素精製

    日本生物工学会  2009 

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  • 新規な活性中心配列を持つDsbA[cdic]のジスルフィドイソメラーゼ活性

    日本生物工学会  2009 

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  • 糸状菌Trichoderma viride 由来L-リジンαオキシダーゼ

    第31回日本分子生物学会年会・第81回日本生化学会大会  2008 

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  • 放線菌Streptomyces incarnatus休止菌体反応系によるシネフンギン生産の検討

    日本農芸化学会2008年度大会  2008 

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  • キイロショウジョウバエのチオレドキシン還元酵素のC末端レドックス配列

    日本農芸化学会2008年度大会  2008 

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  • E. coli 亜セレン酸同化はチオレドキシン還元酵素に依存する

    第49回日本生化学会中国・四国支部例会  2008 

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  • 放線菌由来L-グルタミン酸オキシダーゼの基質認識機構の解析

    日本農芸化学会2008年度大会  2008 

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  • 放線菌Streptomyces avermitilis由来メチオニンγリアーゼの精製および特性解析

    日本農芸化学会2008年度大会  2008 

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  • 高度好熱菌Thermus thermophilus HB8由来TthA0610発現プラスミドの構築

    日本農芸化学会2008年度大会  2008 

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  • 亜セレン酸還元代謝の「定説」を疑う!!

    日本微量元素学会大会  2008 

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  • チオレドキシン還元酵素のCXC変異とArg導入変異の相乗効果検討

    日本農芸化学会中四国支部例会  2008 

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  • 実験しながら計算もする量子酵素化学

    スーパーコンピューティング産業応用協議会 バイオworking group/東京大学生産技術研究所 革新的シミュレーション研究センター  2008 

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  • Drosophilla TrxRのC末端配列を模倣したヒトTrxRの速度論解析

    第31回日本分子生物学会年会・第81回日本生化学会大会  2008 

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  • ヒト肺細胞由来セレノリン酸合成酵素のドメイン機能解析

    第31回日本分子生物学会年会・第81回日本生化学会大会  2008 

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  • 放線菌Streptomyces sp.590株由来のL-メチオニン脱炭酸酵素の性質検討

    第31回日本分子生物学会年会・第81回日本生化学会大会  2008 

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  • Selenite Assimilation into Formate Dehydrogenase H depends on Thioredoxin Reductase in Escherichia coli

    Second International Interdisciplinary Conference on Vitamines, Coenzymes, and Biofactors  2008 

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  • 糸状菌Trichoderma viride由来の抗腫瘍酵素L-リジンαオキシダーゼ 遺伝子の単離と解析

    第60回 生物工学会大会  2008 

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  • Thermus thermophilus HB8のTthA0610, TthA1422発現系の構築と機能解析 - ペリプラズム酵素群のシステムバイオロジー構築に向けて -

    Thermus thermophilus HB8丸ごと一匹研究会  2008 

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  • 放線菌Streptomyces cattleya由来flA遺伝子のタバコ培養細胞への導入

    2008年度日本放線菌学会大会  2008 

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  • シネフンギン生産金Streptomyces incarnatusが生産する核酸系代謝産物

    2008年度日本放線菌学会大会  2008 

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  • Thioredoxin system is the specific gateway for selenite assimilation

    The special seminar in Redox Research Center in Nebraska University  2008 

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  • L-Glutamate oxidase from Streptomyces sp.

    Second International Interdisciplinary Conference on Vitamines, Coenzymes, and Biofactors  2008 

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  • 祖先型変異導入によるグリセロールキナーゼの耐熱化

    日本生物工学会  2008 

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  • パソコン量子化学計算に基づく酵素の反応機構解析と精密分子設計

    日本農芸化学会大会  2008 

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  • Thermus thermophilus HB8のペリプラズム酵素TthA0610, TthA1422の機能解析

    第31回日本分子生物学会年会・第81回日本生化学会大会  2008 

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  • 亜セレン酸代謝に関わるチオレドキシン還元系

    日本農芸化学会2008年度大会  2008 

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  • 基質結合によるグリセロールキナーゼの構造変化の分子機構

    第8回 日本蛋白質科学会年会  2008 

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  • Bacillus subtilis 168 由来zosAが銅イオン取り込み遺伝子であることを示す4つのエビデンス

    日本農芸化学会中四国支部第21回公演会  2008 

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  • ほ乳類セレノリン酸合成酵素のドメイン機能解析

    日本農芸化学会2008年度大会  2008 

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  • L-グルタミン酸オキシダーゼの立体構造と機能の相関関係

    日本生物工学会大会  2007 

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  • L-グルタミン酸オキシダーゼの構造と基質認識機構の解析

    第30回日本分子生物学会年会 第80回日本生化学会大会合同大会  2007 

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  • チオレドキシン還元系に依存する大腸菌の亜セレン酸代謝

    第30回日本分子生物学会年会 第80回日本生化学会大会合同大会  2007 

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  • 抗腫瘍性酵素L-メチオニンγリアーゼの変異酵素C49Kの作製および性質検討

    日本生物工学会大会  2007 

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  • 臨床診断・生化学検査に貢献する酵素の構造と機能解析

    日本生物工学会  2007 

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  • Bacillus subtilis 168のP型トランスポーター zosA遺伝子の機能解析

    日本農芸化学会中四国・西日本支部合同大会  2007 

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  • ヒト肺細胞由来セレノリン酸合成酵素のドメイン機能解析

    第30回日本分子生物学会年会 第80回日本生化学会大会合同大会  2007 

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  • L-グルタミン酸オキシダーゼの基質結合部位と取込み経路のドッキングスタディによる解析

    酵素補酵素研究会2007  2007 

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  • 超好熱好酸性古細菌Sulfolobus tokodaii strain 7由来ホモセリンキナーゼの大腸菌での発現と性質検討

    日本生物工学会大会  2007 

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  • 亜セレン酸代謝に関わるチオレドキシン還元反応系

    日本農芸化学会  2007 

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  • ヒト肺由来チオレドキシン還元酵素のCXC型酵素

    日本農芸化学会中四国・西日本支部合同大会  2007 

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  • TthA0610はジスルフィド架橋形成酵素DsbAのホモログ ? 〜耐熱性の蛋白質リフォールディング酵素を求めて〜

    「高度好熱菌丸ごと一匹プロジェクト」第6回連携研究会  2007 

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  • 大腸菌の亜セレン酸同化は,チオレドキシン還元系に依存する

    微量栄養素研究会  2007 

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  • 放線菌Streptomyces incarnatusのrif変異導入によるシネフンギン生産性の向上

    日本放線菌学会大会  2007 

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  • 放線菌由来L-グルタミン酸オキシダーゼ 結晶構造と成熟化機構の解析

    日本農芸化学会  2007 

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  • 抗腫瘍性酵素メチオニンγ-リアーゼ活性中心に存在するCys残基の機能解析

    日本農芸化学会  2007 

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  • Bacillus subtilis 168の銅吸収P型トランスポーター遺伝子の機能解析

    日本放線菌学会大会  2007 

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  • 分子軌道計算に基づくTrxRの精密分子設計とアレニウスプロット解析

    日本ビタミン学会  2007 

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  • Q494R型チオレドキシン還元酵素の基質特異性

    日本ビタミン学会  2007 

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  • MOPAC計算に基づくチオレドキシン還元酵素の分子設計

    日本農芸化学会  2007 

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  • 大腸菌の亜セレン酸同化はチオレドキシン還元酵素に依存する

    日本ビタミン学会  2006 

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  • 超好熱好酸性細菌Sulfolobus tokodaii由来シスタチオニンγシンターゼの精製,性質検討および立体構造解析

    日本農芸化学会2006年度大会  2006 

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  • 放線菌Streptomyces avermitilis由来のメチオニンγリアーゼの発現系構築と性質

    日本農芸化学会2006年度大会  2006 

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  • Thermus thermophilus HB8由来アラニンラセマーゼの構造と機能解析

    日本農芸化学会2006年度大会  2006 

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  • 好熱性細菌Rhodothermus marinus由来耐熱性イソアミラーゼの大腸菌クローンからの精製及び性質検討

    日本農芸化学会2006年度大会  2006 

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  • 放線菌由来,cyclo(Leu-Phe)酸化酵素遺伝子のクローニングと発現

    日本農芸化学会2006年度大会  2006 

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  • 放線菌由来メチオニン脱炭酸酵素の精製と性質

    日本農芸化学会2006年度大会  2006 

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  • 硫黄酸化細菌A.thiooxidans由来イソクエン酸脱水素酵素の基質および補酵素認識に関与する残基の解析

    日本農芸化学会2006年度大会  2006 

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  • ヒト肺由来チオレドキシン還元酵素のC末端配列のセリン残基導入効果

    日本農芸化学会2006年度大会  2006 

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  • MOPAC計算によるTrxRの精密分子設計とアレニウスプロット解析

    日本農芸化学会2006年度大会  2006 

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  • 大腸菌DsbAランダム変異酵素ライブラリーの酸化還元電位測定

    日本農芸化学会2006年度大会  2006 

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  • 放線菌Streptomyces incarnatusのrif変異によるSinefungin生産性の向上

    日本農芸化学会中四国支部創立5周年記念第16回講演会(支部大会)  2006 

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  • Thioredoxin reductase-dependent assimilation of selenite into FDHH in Escherichia coli

    Selenium 2006  2006 

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  • Assimilation of selenite into FDHH in Escherichia coli

    国際生化学分子生物学会合同会議  2006 

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  • Molecule design of thioredoxin reductase based on semiempirical molecule orbital calculation, WinMOPAC

    国際生化学分子生物学会合同会議  2006 

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  • 新規な活性中心配列を持つDsbA[CDIC]の触媒作用

    日本生化学会支部大会  2006 

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  • Combinatorial mutagenesis of the active-site dipeptide of DsbA

    国際生化学分子生物学会合同会議  2006 

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  • 抗腫瘍性酵素L-methionineγ-lyaseの活性中心におけるAsp241の機能解析

    日本ビタミン学会  2006 

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  • 放線菌由来L-グルタミン酸オキシダーゼの大腸菌での大量発現系の確立

    日本ビタミン学会  2006 

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  • ヒト肺がん細胞のセレノリン酸合成酵素Sps1, Sps2の機能解析

    第22回微量栄養素研究会シンポジウム  2005 

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  • NAD依存型イソクエン酸脱水素酵素の量子酵素化学

    日本ビタミン学会  2005 

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  • 抗腫瘍性酵素L-mehtioine γ-lyaseの活性中心近傍に存在するアミノ酸残基の機能解析

    日本農芸化学会2005年度大会  2005 

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  • Neomycin耐性陽性選択ベクターpTRA502の開発

    日本農芸化学会2005年度大会  2005 

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  • 量子化学計算に基づくチオレドキシン還元酵素の精密分子設計

    日本農芸化学会2005年度大会  2005 

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  • Crystallography and quantum enzyme chemistry of NAD-dependent isocitrate dehydrogenase from Acidithiobacillus thiooxidans

    Pacifichem2005  2005 

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  • Selenophosphate synthetase genes from lung adenocarcinoma cells International

    Interdisciplinary Conference on Vitamins, Coenzymes, and Biofactors 2005  2005 

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  • Molecular cloning of thioredoxin reductase genes from human lung adenocarcinoma cells, NCI-H441

    International Interdisciplinary Conference on Vitamins, Coenzymes, and Biofactors 2005  2005 

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  • ヒト肺がん細胞のチオレドキシン還元酵素の高発現と点突然変異

    日本農芸化学会2004年度大会  2004 

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  • DsbAコンビナトリアル変異ライブラリーの酸化還元電位評価

    2004年度日本生物工学会大会  2004 

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  • 量子化学計算に基づくチオレドキシン還元酵素C末端配列の分子設計と蛋白質工学

    平成16年度日本農芸化学会中四国支部大会  2004 

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  • ヒト肺がん細胞NCI-H441で発現するセレノリン酸合成酵素の機能解析

    第45回日本生化学会中国・四国支部例会  2004 

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  • バイオファクターセレンを含む新規酵素の発見と肺がん細胞のセレン代謝特性の解明

    日本ビタミン学会平成16年度奨励賞受賞講演  2004 

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  • ヒト肺がん細胞からのセレノリン酸合成酵素遺伝子Sps1, Sps2のクローニングとin vivo 系での諸性質の検討

    第77回日本生化学会大会  2004 

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  • 量子化学計算に基づく次世代蛋白質工学の確立

    第27回日本分子生物学会年会  2004 

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  • 放射線抵抗性細菌Deinococcus radiodurance 由来カタラーゼの精製と性質検討

    第27回日本分子生物学会年会  2004 

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  • イオウ酸化細菌Acidithiobacillus thiooxidans 由来のイソクエン酸脱水素酵素の結晶構造解析

    日本農芸化学会2004年度大会  2004 

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  • 超好熱性好酸性古細菌Sulfolobus tokodaii 由来シスタチオニンγ-シンターゼと推定される蛋白質の大腸菌での発現系構築,精製と性質検討

    日本農芸化学会2004年度大会  2004 

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  • 抗腫瘍性酵素メチオニンγ-リアーゼのプロテアーゼ切断部位の同定と血中安定性向上のためのPEG化条件の検討

    日本農芸化学会2004年度大会  2004 

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  • 高度好熱性細菌Thermus thermophilus HB8由来アラニンラセマーゼ-大腸菌クローンからの精製と結晶化-.

    日本農芸化学会2004年度大会  2004 

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  • 分子軌道計算に基づくチオレドキシン還元酵素の分子設計

    日本農芸化学会2004年度大会  2004 

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  • ヒト肺腫瘍細胞由来セレノリン酸合成酵素の大腸菌in vivoアッセイ

    日本農芸化学会2004年度大会  2004 

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  • チオレドキシン還元酵素Gln494への変異導入効果

    日本農芸化学会2004年度大会  2004 

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  • The molecular design of thioredoxin reductase based on the semiempirical molecular orbital calculation, MOPAC

    The 1st Pacific-Rim International Conference on Protein Science  2004 

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  • 放射線耐性菌Deinococcus radiodurans由来カタラーゼの精製と性質検討

    日本農芸化学会2004年度大会  2004 

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  • 大腸菌のファージ感受性を向上させるDsbA[CDIC]の酸化還元電位

    日本農芸化学会2004年度大会  2004 

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  • L-methioine g-lyase の活性部位残基Cys116の役割

    日本農芸化学会2003年度大会  2003 

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  • チオレドキシン還元酵素C末端配列の量子酵素化学

    平成15年度日本農芸化学会西日本支部、中四国支部、日本栄養・食糧学会西日本支部,日本食品科学工学会西日本支部鹿児島合同大会  2003 

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  • 放線菌 L-グルタミン酸オキシダーゼの成熟化機構の解析

    日本農芸化学会2003年度大会  2003 

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  • 高度好熱性細菌Thermus thermophilus HB8由来アラニンラセマーゼの大量発現系構築と性質検討

    日本農芸化学会2003年度大会  2003 

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  • Bacillus subtilis 168の銅欠乏下でのykvW遺伝子の発現

    日本農芸化学会2003年度大会  2003 

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  • Aconitase from chemolithoautotrophic bacterium Acidithiobacillus thiooxidans ミgene analysis and expression in Escherichia coli

    第76回日本生化学会大会  2003 

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  • 発現を指標とする放線菌遺伝子クローニング用ベクターpTRA502の構築

    平成15年度日本農芸化学会西日本支部、中四国支部、日本栄養・食糧学会西日本支部,日本食品科学工学会西日本支部鹿児島合同大会  2003 

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  • ヒト肺由来チオレドキシン還元酵素の発現における大腸菌宿主へのレアコドン添加効果

    平成15年度日本農芸化学会西日本支部、中四国支部、日本栄養・食糧学会西日本支部,日本食品科学工学会西日本支部鹿児島合同大会  2003 

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  • チオレドキシン還元酵素のカルボキシル基末端配列の酸化還元状態の半経験的分子軌道計算

    第26回日本分子生物学年会  2003 

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  • ヒト肺腫瘍細胞NCI-H441由来セレノリン酸合成酵素アイソザイムの機能解析

    第26回日本分子生物学年会  2003 

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  • 超好熱性酸性古細菌Sulfolobus tokodaii由来シスタチオニンγシンターゼ遺伝子の解析と大腸菌での発現

    第26回日本分子生物学年会  2003 

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  • Characterization of Bacillus subtilis 168 ykvW as Cu uptake ATPase under DL-penicillamine-induced Cu-deficiency

    第76回日本生化学会大会  2003 

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  • Crystal structure of the L-methionine gamma-lyase from Pseudomonas putida.

    3rd International Symposium on Vitamin B6, PQQ, Carbonyl Catalysis and Quinoproteins  2002 

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  • DyeMatrix担体を用いるNAD+:penicillamine ADP転移酵素の精製

    日本農芸化学会2002年度大会  2002 

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  • 放線菌由来L-グルタミン酸オキシダーゼのプロセッシング

    日本農芸化学会2002年度大会(仙台,2002,3)  2002 

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  • ヒト肺腫瘍細胞のセレノリン酸合成酵素の遺伝子クローニング

    日本農芸化学会2002年度大会  2002 

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  • Streptomyces cattleya モノフルオロ酢酸非生産株のCosynthesis実験

    日本農芸化学会2002年度大会  2002 

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  • M13ファージ感染アッセイ法を用いるDsbAランダム変異の作成

    日本農芸化学会2002年度大会  2002 

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  • 抗腫瘍性酵素L-メチオニン γ-Lyaseの構造安定化の試み,

    日本農芸化学会2002年度大会  2002 

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  • リファンピシン耐性を指標とするStreptomyces incarnatusのシネフンギン高生産株の取得,

    2002年度日本放線菌学会大会  2002 

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  • 放線菌Streptomyces cattleyaの形質転換法の検討

    第74回日本生化学会大会  2001 

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  • 硫黄酸化細菌Thiobacillus thiooxidans由来イソクエン酸脱水素酵素の基質,補酵素認識機構の解析

    第74回日本生化学会大会  2001 

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  • ヒト肺がん細胞のセレノリン酸合成酵素の遺伝子クローニング

    日本農芸化学会2001年度関西・西日本・中四国支部合同大会  2001 

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  • シネフンギン生合成酵素の遺伝子クローニング

    日本農芸化学会2001年度関西・西日本・中四国支部合同大会  2001 

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  • 放線菌由来L-グルタミン酸オキシダーゼを利用したL-GluセンサーとGOT・GTPセンシングへの応用

    日本生物工学会大会  2001 

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  • 19F-nmrを用いるモノフルオロ酢酸生産のCosynthesis実験

    日本生物工学会大会  2001 

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  • イソクエン酸脱水素酵素の基質特異性の変換

    日本農芸化学会2001年度関西・西日本・中四国支部合同大会  2001 

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  • 放線菌由来L-グルタミン酸オキシダーゼの分子形態の解析

    日本農芸化学会2001年度関西・西日本・中四国支部合同大会  2001 

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  • 半経験的分子軌道計算を用いるチオレドキシン還元酵素の変異設計

    日本農芸化学会2001年度関西・西日本・中四国支部合同大会  2001 

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  • Streptomyces cattleya フッ素代謝欠損株を用いるCosynthesis実験

    日本農芸化学会2001年度関西・西日本・中四国支部合同大会  2001 

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  • ヒト肺がん細胞由来のチオレドキシン還元酵素の点突然変異

    第74回日本生化学会大会  2001 

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  • 部位特異的変異導入法によるL-Methionine γ-Lyaseの機能解析

    第74回日本生化学会大会  2001 

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  • 硫黄酸化細菌Thiobacillus thiooxidans 由来イソクエン酸脱水素酵素と同遺伝子の解析

    日本ビタミン学会第53回大会  2001 

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  • ヒト肺がん細胞由来のチオレドキシン還元酵素遺伝子の点突然変異

    日本ビタミン学会第53回大会  2001 

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  • ヒト肺腫瘍細胞のセレン酵素チオレドキシン還元酵素遺伝子のアイソザイム

    日本農芸化学会2001年度大会  2001 

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  • 放線菌由来L-グルタミン酸オキシダーゼの大腸菌クローン株からの精製および性質

    日本農芸化学会2001年度大会  2001 

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  • 放線菌由来L-グルタミン酸オキシダーゼのクローニングと大腸菌での発現

    日本農芸化学会2001年度大会  2001 

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  • 活性炭を用いたシネフンギンの抽出法

    日本農芸化学会2001年度大会  2001 

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  • Molecular cloning of thioredoxin reductase isozymes TrxR1 and TrxR2 from human lung adenocarcinoma cell line, NCI-H441

    第18回微量栄養素研究会シンポジウム  2001 

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  • Streptomyces sp. X-119-6由来L-グルタミン酸オキシダーゼのクローニング及び発現

    第18回微量栄養素研究会シンポジウム  2001 

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  • Streptomyces cattleya モノフルオロ酢酸非生産株の相補実験

    日本農芸化学会2001年度大会  2001 

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  • 酸性環境から分離した高温性好酸性芽胞形成細菌に関する研究

    日本農芸化学会2001年度大会  2001 

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  • 放線菌由来L-グルタミン酸オキシダーゼ: クローニング,発現,及び諸性質検討

    日本ビタミン学会第53回大会  2001 

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  • チオレドキシン還元酵素のアミノ酸置換効果の量子化学計算

    第24回日本分子生物学会年会  2001 

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  • 高度好熱性細菌Thermus thermophilus のアラニンラセマーゼ 遺伝子クローニングと大腸菌での発現

    日本農芸化学会2001年度大会  2001 

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  • L-メチオニンγ-リアーゼのシステイン残基のアラニン残基への変換

    日本農芸化学会2001年度大会  2001 

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  • Thiobacillus thiooxidans 由来イソクエン酸脱水素酵素 -クローン株からの精製と性質-

    日本農芸化学会2000年度大会  2000 

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  • ヒト肺腫瘍細胞の含セレン酵素チオレドキシン還元酵素遺伝子のクローニング

    日本農芸化学会平成12年度関西支部大会  2000 

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  • NAD+: penicillamine ADP transferase高生産性Bacillus sphaericusの作成

    日本生物工学会2000年度大会  2000 

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  • L-メチオニンg-リアーゼの立体構造

    日本ビタミン学会2000年度大会  2000 

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  • 好酸性細菌 Acidocella facilis 22M由来のAfa22Mlメチラーゼ遺伝子の構造解析

    日本農芸化学会2000年度大会  2000 

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  • NAD:penicillamine ADP転換酵素の微生物分布

    日本農芸化学会2000年度大会  2000 

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  • Streptomyces cattleya モノフルオロ酢酸非生産株の調製とその宿主ベクター系の検討

    日本農芸化学会平成12年度関西支部大会  2000 

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  • Streptomyces cattleyaの宿主ベクター系の検討

    日本農芸化学会2000年度大会  2000 

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Awards

  • 日本生物工学会西日本支部学生賞(受賞者 清遠亜紗子)

    2009  

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    Country:Japan

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  • 日本生物工学会西日本支部学生賞 (受賞者 福田康二)

    2009  

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    Country:Japan

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  • 優秀審査員表彰

    2009  

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    Country:Japan

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  • 日本ビタミン学会奨励賞

    2004  

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Research Projects

  • Development of Thermal Refolding and application for antibody production

    Grant number:15K14695  2015.04 - 2018.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    Takashi Tamura

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    Grant amount:\3900000 ( Direct expense: \3000000 、 Indirect expense:\900000 )

    In this research, a novel technique was developed which enabled the refolding of proteins at high temperatures with catalysis by thermostable protein disulfide isomerase. For this purpose a novel reporter protein sfGFP-2SS was invented, which emits green fluorescence depending on restoration of correct S-S bonds to be introduced. The fluorescence can be once vanished by erroneous S-S crosslink upon oxidation under denaturation, but green fluorescence was restored by disulfide isomerase from Thermus thermophilus HB8 in the programmed thermal cycling with redox buffer GSH/GSSG. The invention of Protein Thermal Refolding technique, termed as PTR, was established as a protein version of PCR, leading denatured proteins with abbrenet disulfide bonds to the correct structure with high expectation for recombinant antibody production.

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  • Structural characterization of L-amino acid oxidase with high substrate specificity and industrial applications

    Grant number:24560962  2012.04 - 2015.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    INAGAKI KENJI, IMADA Katsumi, TAMURA Takashi, INAGAKI Junko

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    Grant amount:\5460000 ( Direct expense: \4200000 、 Indirect expense:\1260000 )

    We determined the crystal structure of two L-amino acid oxidases with high substrate specificity, L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 and L-lysine α-oxidase (LysOX) from Trichoderma viride. and we analyzed the molecular mechanism of the substrate recognition of L-amino acid oxidases with high substrate specificity. X-ray crystal structure of LGOX revealed that Arg305 in the active site is the key residue involved in substrate recognition. We created 19 mutant enzymes by substitution of Arg305. Some of them, R305D-LGOX, R305L-LGOX, and R305H-LGOX lost oxidase activity for L-Glu, and exhibited a new oxidase activity for L-Arg, L-His, and L-Tyr, respectively. These progress and findings will provide a great contribution to further industrial application of these enzymes.

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  • Development of thermostable protein-refolding catalysis and characterization of the kinetic properties

    Grant number:21580112  2009 - 2011

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    TAMURA Takashi, KURAMITSU Seiki

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    Grant amount:\4810000 ( Direct expense: \3700000 、 Indirect expense:\1110000 )

    The present study assessed the contribution of sctive site sequence of Escherichia coli DsbA by screening a library of mutant dsbA genes with the genetically randomized active-site sequence CXXC. Alleles of 24 dsbA mutants gave significant variation in the cellular functions, and a mutant dsbA gene that encodes Cys-Asp-Ile-Cys sequence enhanced pahge sensitivity by 40-fold more than the wild-type dsbA. The redox potential of DsbA[CDIC] was determined to be. 171. 8 mV, which suggested less oxidizing catalyst than the wild type DsbA(-125. 9 mV) and the redox potential was close to that of yeast protein disulfide isomerase(175 mV). It was suggested that DsbA[CDIC] improved the net yield of the correctly folded periplasmic proteins through the disulfide isomerase catalysis. Thermostable homologs were also characterized for application of folding catalysis at high temperature.

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  • Studies on Isozymes of Human Lung Selenophosphate Synthetase

    Grant number:18580122  2006 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    TAMURA Takashi, INAGAKI Kenji

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    Grant amount:\4060000 ( Direct expense: \3400000 、 Indirect expense:\660000 )

    必須微量元素セレンは, 抗酸化作用, 免疫賦活化作用, ホルモン調節作用, がん予防効果など, 多くの有益な生理活性を示す微量必須ミネラルである。高齢化社会におけるQuality of Lifeの維持に関心が高まる中で, セレンの持つ多彩で有益な生理活性に注目が集まっている。ところが必要摂取量の僅か10倍程度の摂取で過剰障害が表れるリスクがあるので, セレンの有益な生理活性を社会全体で享受するためには, セレン代謝に関わる知的基盤の確立が必要不可欠である。本研究はセレン同化経路の解明に焦点を当て, 1)亜セレン酸の還元代謝にチオレドキシン還元酵素が関わることを初めて明らかにし, 2)ヒトのセレノリン酸合成酵素の触媒機能ドメインを新たに発見し, 3)これまで機能不明とされたセレノリン酸合成酵素アイソザイムSps1がSps2の機能を抑制するアンチザイムとして作用することを初めて明らかにすることができた。

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  • Structural and functional analysis of anti-tumor microbial enzymes and their application for development of next generation anti-tutor enzymes.

    Grant number:18580076  2006 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    INAGAKI Kenji, TAMURA Takashi, INAGAKI Junko

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    Grant amount:\3890000 ( Direct expense: \3500000 、 Indirect expense:\390000 )

    A highly potent recombinant L-methionine γ-lyase from Pseudomonas putida (MGL_Pp. EC 4.4.1.11) has been characterized physicochemically and pharmacokinetically in vivo and in vitro as a potent anticancer agent that can deplete L-methionine from plasma. The detailed structure of MGL_Pp and the function of the active site residue have so far not been cleared to improve as a next generation antitumor enzyme. We demonstrated that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method at 1.8A resolution. Detailed information of the overall structure of MGL_Pp supplied clear pictures of the N-terminal domain and the substrate-and PLP-binding pockets. It was found that a hydrogen bond network was formed around a cofactor pyridoxal 5'-phosphate in the MGL active site, which is specific for MGLs. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug. The 3D structure of MGL_Pp suggested that Cys116 might be involved in the hydrogen bond network at the active site. We found that Cys116 plays an important role in the γ-elimination reaction of L-methionine and for the substrate recognition in the MGLs. We also discovered that the substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a change of the substrate specificity, suggesting that the His residue may be directly involved in the γ-elimination reaction. With a low purity, it was found that MGL_Pp was degraded between Cys49-Phe50 and the degraded enzyme lost the activity. Conjugation of MGL_Pp with polyethylene glycol (PEG) will importantly reduce proteolysis. Recently, the complete nucleotide sequence of the linear chromosome of S. avermitilis has been determined. The gene encoding L-methionine γ-lyase from Streptomyces avermitilis was cloned and expressed in Escherichia coli to characterize the enzymological properties of the gene product. MGL_Sa. for the first time over the world.

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  • Structure and fanctional analysis of NAD^+-dependent isocitrate dehydrogenase from the chemolithotroph Aci dithiobacillus thiooxidans

    Grant number:14580648  2002 - 2004

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    INAGAKI Kenji, TAMURA Takashi, IMADA Katsumi, TANAKA Hidehiko

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    Grant amount:\3500000 ( Direct expense: \3500000 )

    1)Functional analysis of Isocitrate Dehydrogenase from Acidithiobacillus thiooxidans
    Isocitrate dehydrogenase(ICDH) catalyzes the oxidative decarboxylation of D-isocitrate to 2-oxoglutarate and CO_2 with NAD^+ or NADP^+ as cofactor in the TCA cycle. ICDH from Acidithiobacillus thiooxidans is NAD^+-dependent, although most bacteria contain NADP^+-dependent ICDH. ICDH and 3-isopropylmalate dehydrogenase(IPMDH) which generally shows NAD^+-dependency belong to a class of protein family, decarboxylating dehydrogenases, that lack a typical βαβ nucleotide-binding fold which is commonly present in most dehydrogenases. Although these enzymes show high sequence homology and similarity in their 3-D structure, amino acid sequence of substrate-binding site is different. These enzymes also provide an attractive model system to study the coenzyme and substrate recognition. So we tried to build a product system which produces amount of enzyme (ICDH) by use of E.coli JM109 transformed with pkk-ICDH. And we purified ICDH by centrifugation, heat-shock, DEAE-Toyopearl and Sephacryl S-200HR. As a result of purification we obtained purified enzyme by 57.8-fold. We also analyzed the enzyme with thermostability, pH stability etc of At-ICDH. In result, ICDH from Acidithiobacillus thiooxidans is superior to that of ICDH from yeast.
    2)Crystallography and Quantum Enzyme Chemistry of At-ICDH
    Bacterial ICDHs normally require NADP as the cofactor, but At-ICDH is specific to NAD. We obtained bi-pyramidal crystals of ICDH-NAD complex with a space group P43212 and the unit cell of a=b=125.99Å, c=268.35Å. The structure was solved by SAD method using Os-derivative crystals and refined to 1.9Å resolution. Although most of enzyme-bound nicotinamide cofactors found in PDB have the amide NH2 group in trans to the C4 carbon of nicotinamide ring, we determined in At-ICDH the nitrogen was in cis to C4 carbon slanting by 23° toward substrate. Electro-potentials of hydrogen atoms on nicotinamide ring were computed along with the amide rotation using MNDO Hamiltonian. A hydrogen atom on C4 atom had the greatest negative charge when amide NH2 group was in the cis conformation with dihedral angle of 23°. Negative charge induced by the amide NH2 appeared to suggest a catalytic significance in stabilizing the transition state during the hydride transfer catalysis.

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  • Preparation and characterization of sinefungin synthetase

    Grant number:14560069  2002 - 2003

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    TANAKA Hidehiko, INAGAKI Kenji, TAMURA Takashi

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    Grant amount:\3600000 ( Direct expense: \3600000 )

    Sinefungin is a nucleoside antibiotic, in which a molecule of L-methionine is linked to the 5'-end of adenosine through a C-C bond. The antibiotic, isolated from the culture broth of Streptomyces incarnates NRRL8089, has a strong inhibitory effect on various fungi and trypanosome, and is expected to be useful as an anti-malaria drug as well. It has been reported that the cell-free extract prepared from a variant of S. incarnates produced sinefungin from L-arginine and ATP in the presence of pyridoxal-5'-phosphate, but the enzyme(s) involved in sinefungin-production have not been characterized clue to the instability and low expression of the responsible enzymes in the producer strain. In the present study we have constructed a new expression-vector of pTRA502, which derives from pKU110 shuttle vector. The present study was undertaken to identify genes involved in the sinefungin biosynthesis. We have constructed an original cloning vector of pTRA502 by replacing thiostrepton resistant gene with that of neomycin resistant gene of pKU110. Limited digestion of genome DNA was carried out with Sau3AI restrictions enzyme to obtain genetic fragments of about 5-6 kb. Optimal conditions were examined by restriction enzyme concentrations, enzyme concentration and incubation time. There was a problem that the collection of DNA was low in the process to extract DNA. The amount of collection and purity was improved by using the kit of glass beads. Shot-gun cloning is now being carried out with pTRA502 vector. The transformants were examined for the sinefungin producing ability by a capillary electrophoresis.

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  • Dynamism of activated-selenium species: Structural biological analysis of mechanism of biosynthesis of selenium-containing proteins

    Grant number:13480192  2001 - 2002

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    ESAKI Nobuyoshi, TAMURA Takashi, MIHARA Hisaaki, KURIHARA Tatsuo

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    Grant amount:\17100000 ( Direct expense: \17100000 )

    Selenocysteine lyase (SCL) is an enzyme which specifically catalyzes the decomposition of selenocysteine to form alanine and selenium. The enzyme is proposed to be involved in an initial step of the biosynthesis of selenoproteins containing selenocysteine residues because it has an activity to deliver selenide for selenophosphate synthetase. Escherichia coli contains its homologous counterpart enzyme, cysteine desulfurase (CSD). Unlike SCL, CSD acts on both cysteine and selenocysteine as substrates. A conserved cysteine residue important for the decomposition of cysteine has been demonstrated to be not essential for the decomposition of selenocyteine. Mouse SCL has eight cysteine residues. We constructed mutant enzymes C101S, C129S, C177S, C304S, C346, C364S, C375, and C390S by site-directed mutagenesis. The mutant enzymes were purifued and characterized. The C375S and C375A mutants lost the selenocysteine lyase activity. Therefore, Cys375 is essential for the selenocysteine lyase activity of the enzyme, indicating that the mechanism of selenocysteine lyase reaction by SCL is different from that catalyzed by CSD.

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  • 新規抗腫瘍性酵素の開発と癌治療への応用

    Grant number:13218081  2001

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas (C)  Grant-in-Aid for Scientific Research on Priority Areas (C)

    稲垣 賢二, 瀧本 明生, 田村 隆

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    Grant amount:\3800000 ( Direct expense: \3800000 )

    多くの腫瘍細胞に対して抗腫瘍性を有する酵素L-メチオニンγ-リアーゼを遺伝子工学的手法により改変し、様々な物理的要因に対して耐性を付与することを試みた。酵素の改変にあたりまず酵素-PLP複合体の結晶構造解析を行い本酵素活性部位の詳細な立体構造情報を得た。血中における本酵素の不安定要因として1)低酵素濃度下における酵素高次構造の崩壊2)低補酵素濃度下におけるアポ酵素からのPLPの脱離3)酸化条件における酵素の酸化失活4)プロテアーゼによる消化を想定し,得られた立体構造情報に基づいて,酵素サブユニット内及びサブユニット間における新規のS-S結合の導入,イオン性相互作用の強化,プロテアーゼ,酸化反応のターゲットとなるアミノ酸残基の置換を行った変異酵素を遺伝子工学的手法により作製し,これらの要因に対する耐性を酵素に付与することを試みた。作製した種々の変異酵素の内,特にF128C, S248C(S-S導入), K6H(プロテアーゼ耐性)は野生型酵素と比較して最大で約1.3倍の高い活性を有していた。L341H(PLP-アポ酵素間の親和性強化)は活性については野生型酵素の約1/10と低いものであったが、アポ酵素とPLPとの親和性は約1.1倍に増加していた。更に酵素活性の発現に重要であるとされる活性部位のCysの置換体C116S, T(活性部位の酸化耐性)は基質メチオニンに対する特異性が低下しており,本残基を含めた基質認識ネットワークの改変により本酵素の基質特異性を更に高められる可能性が示唆された。これらの変異酵素に対する種々の安定性評価試験を行い,そこから得られた知見に基づき,抗がん剤としての利用により適した酵素への改変が可能であると考えられる。

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  • Purification and Molcular Cloning of Sinefungin synthetase fom Streptomyces incarnatus NRRL8089.

    Grant number:11680636  1999 - 2001

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    INAGAKI Kenji, TAMURA Takashi, TANAKA Hidehiko

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    Grant amount:\3300000 ( Direct expense: \3300000 )

    Sinefungin is a nucleoside antibiotic, in which a molecule of L-omithine is linked to the 5'-end of adenosine through a CC bond. The antibiotic, isolated from the culture broth of Streptomyces incarnatus NRRL 8089, has a strong inhibitory effect on various fungi and trypanosome, and is expected to be useful as an anti-malaria drug as well. It has been reported that the cell-free extract prepared from a variant of incarnatus produced sinefungin from L-arginine and ATP in the presence of pyridoxal-5'-phosphate, but the enzymes involved in sinefungin-production have not been characterized due to the instability and low expression in the producer strain. The present study was undertaken to characterize the enzymes responsible for sinefungin production.
    In the present research, we have first established DNA methylse inhibition assay for detecting sinefungin. Then, the sinefungin producer strain underwent mutagenesis by ultraviolet light irradiation on the protoplast, and a high-producer strain was selected among the mutants that acquired rifampicin-resistance. Because the enzyme activity of sinefungin synthetase was still not detected in the crude cell extract, we attempted to clone the genes involved in sinefungin production. The genome DNA was partially digested, ligated to a vector plasmid pKU110, and transformed in Streptomyces lividans TK24. Three transformants (#19, #20, and #21) were screened from over 200 of transformants as the candidate strain that produces sinefungin. The transformants were cultivated in 2 L of the medium, and sinefungin was isolated from the culture broth by means of charcoal column chromatography, cation-exchange column chromatography, and anion-exchange column chromatography. The strain #19 produced sinefungin more than the strains #20 and #21. We attempted to isolate the recombinant plasmids from these sinefungin-producing transformants, but the conventional isolation protocol did not yield plasmid. We examined the presence of the thiostrepton-resistance marker sequence, which is located in the vector plasmid, by polymerase chain reaction, but the corresponding DNA fragment was not amplified. It was concluded that the genes of sinefungin production from S. incarnatus were integrated to the genome DNA of S. lividans. Two references were published in the present research.

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  • チオレドキシン還元酵素のセレノシステイン残基の触媒機能

    Grant number:11780449  1999 - 2000

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Young Scientists (A)  Grant-in-Aid for Encouragement of Young Scientists (A)

    田村 隆

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    Grant amount:\2100000 ( Direct expense: \2100000 )

    C末端から2番目にセレノシステイン残基を持つチオレドキシン還元酵素のアイソザイム(TrxR1,Trx2,Trx3)は哺乳類の細胞においては抗酸化機構やRedox制御によるシグナル伝達機構を構成する酵素として注目されている。本研究はチオレドキシン還元酵素のセレノシステイン残基の触媒機能の解明を目的として、ヒト肺腫瘍細胞からRT-PCRにより本酵素の三つのアイソザイムの遺伝子クローニングを行なった。
    PCR条件検討の結果、TrxR1(1,500bp)は通常のPCR法で特異的に増幅され、TrxR2(1,566bp)とTrxR3(1,575bp)はタッチダウンPCR(アニーリング温度75.0℃〜65.4℃,-0.4℃/cycle)により微弱なシグナルとして検出できた。正常肺細胞のmRNAからはTrxR1の遺伝子のみが増幅可能でTrxR2とTrxR3は検出されなかった。肺がん細胞では正常細胞の百倍以上もTrxR活性が高いことが知られていたが、アイソザイムの同定など詳細なことは明らかにされていなかった。この研究で得られた結果はDNAチップによる肺がんの早期診断を行なう上で有用な知見として役立つと期待される。
    ヒト肺がん細胞のTrxR1遺伝子にはすでに報告のあるヒト胎盤のTrxR1と同様にC末端から2番目にセレノシステイン残基を持つことが示された。DNAシークエンスの結果から肺がん細胞のTrxR1には3カ所の点突然変異が見つかったが、このうち2カ所は正常肺のTrxR1にも同定された。がんに特異的とされる変異箇所は一カ所のみでアミノ酸としてメチオニンがスレオニンに変換されていた。グルタチオン還元酵素の立体構造をモデルにTrxR1の立体構造を推定すると、興味深いことにその変異箇所は活性中心のジスルフィド結合にかぶさるヘリックス中に位置することが示された。活性中心を覆うメチオニンのスレオニンへの変異は酵素の酸化ストレス耐性を向上させる効果を示すことがスブチリシンのランダム突然変異の研究において報告例がある。肺がん細胞でTrxR1活性が正常細胞よりも100倍以上も高いのは酵素の発現量が増加しただけでなく、このようなセレノシステイン残基と相互作用する箇所で点突然変異が起こり、酵素の安定性が向上しているという可能性が示された。

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  • ANALYSIS OF SUBSTRATE RECOGNITION MECHANISM OF ISOPROPYLMALATE DEHYDROGENASE

    Grant number:10680612  1998 - 2000

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    TANAKA Hidehiko, TAMURA Takashi, INAGAKI Kenji

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    Grant amount:\2900000 ( Direct expense: \2900000 )

    1) Structure of 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans at 2.0 Åresolution.
    3-Isopropylmalate dehydrogenase (IPMDH) gene was cloned and sequenced from Thiobacillus ferrooxidans (Tf). We have determined the crystal structures of Tf-IPMDH and the substrate binding mechanism have been solved. IPMDH shows a marked similarity to isocitrate dehydrogenase (ICDH) in its structural framework and catalytic mechanism, and is classified into a unique group of decarboxylating dehydrogenases. The difference between their substrates is the γ moiety attached to 2R-malate.
    The structure shows a fully closed conformation and the substrate-binding site is quite similar to that of ICDH except for a region around the γ-isopropyl group. The γgroup is recognized by a unique hydrophobic pocket, which includes Glu88, Leu91 and Leu92. The Cβ and Cγ atoms of Glu88 interact with the γ-isopropyl moiety of 3-isopropylmalate (IPM) and are central to the recognition of substrate.
    2) Mutagenesis of substrate recognition site of Tf-IPMDH.
    We have changed γmoiety recognition site of Tf-IPMDH (Ala72〜Leu92) to that of Ec-ICDH (Pro102〜Val116). We analyzed the substrate specificity of the mutant enzyme. We have been investigated whether the higher-order structure for the mutant enzyme is stabilized because about ten amino acid residues of leuB gene are substituted. In result, The mutant enzyme's loop regions will be distorted slightly, but we expected the structure of the mutant enzyme was stabilized and began on this theme. The mutant enzyme was expressed as soluble protein in E.coli.
    3 )Structure of NAD dependent ICDH from Thiobacillus thiooxidans and coenzyme recognition mechanism.
    NAD dependent ICDH gene from Thiobacillus thiooxidans (Tt) was cloned and sequenced and the coenzyme recognition mechanism was analyzed. The amino acid sequence had a high degree of similarity to the NADP dependent ICDH and IPMDH.Tt-ICDH is 59.2%, and 22.6% identical to Ec-ICDH and Tf-IPMDH, respectively. It is expected that the tertiary structure of Tt-ICDH is similar to Ec-ICDH and Tf-IPMDH, because the regions composed a deduced secondary structure (α-helix and β-sheet) of Tt-ICDH are much alike.
    Tt-ICDH has conserved Asp357 which is significant of the NAD recognition in the coenzyme binding site alignment. On the other hand, Tt-ICDH lacked for a tyrosine residue which was conserved in NADP-ICDH and recognized phosphate of NADP+. Thus, it is revealed that this enzyme has similar coenzyme binding site to that of the IPMDH's.

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