Updated on 2024/02/01

写真a

 
MORI Koichi
 
Organization
Faculty of Interdisciplinary Science and Engineering in Health Systems Assistant Professor
Position
Assistant Professor
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Degree

  • Ph.D ( 1998.3   Okayama University )

  • 修士(工学) ( 1994.3   岡山大学 )

Research Interests

  • enzyme

  • protein

  • biotechnology

Research Areas

  • Life Science / Functional biochemistry

  • Life Science / Structural biochemistry

  • Manufacturing Technology (Mechanical Engineering, Electrical and Electronic Engineering, Chemical Engineering) / Biofunction and bioprocess engineering

Education

  • Okayama University   大学院自然科学研究科   生物資源科学専攻

    1994.4 - 1998.3

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    Country: Japan

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  • Okayama University   大学院工学研究科   生物応用工学専攻

    1992.4 - 1994.3

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    Country: Japan

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  • Okayama University   工学部   生物応用工学科

    1988.4 - 1992.3

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    Country: Japan

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Research History

  • Okayama University   Faculty of Interdisciplinary Science and Engineering in Health Systems   Assistant Professor

    2021.4

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    Country:Japan

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  • Okayama University   Graduate School of Interdisciplinary Science and Engineering in Health Systems   Assistant Professor

    2018.4 - 2021.3

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    Country:Japan

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  • Okayama University   Graduate School of Natural Science and Technology   Assistant Professor

    2007.4 - 2018.3

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    Country:Japan

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  • Okayama University   Graduate School of Natural Science and Technology   Research Associate

    2005.4 - 2007.3

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  • Okayama University   Faculty of Engineering, Department of Bioscience and Biotechnology   Research Associate

    2004.1 - 2005.3

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    Country:Japan

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  • Research Institute for Biological Sciences, Okayama   Postdoctoral Fellowships of Japan Society for the Promotion of Science

    2003.1 - 2003.12

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    Country:Japan

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  • Research Institute for Biological Sciences, Okayama

    2001.11 - 2002.12

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    Country:Japan

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  • Okayama University

    1998.4 - 2001.3

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    Country:Japan

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Professional Memberships

  • Japan Bioindustry Association

    2010.6

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  • The Society for Biotechnology, Japan

    2002.4

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  • The Vitamin Society of Japan

    1999.1

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  • Japan Society for Bioscience, Biotechnology, and Agrochemistry

    1995.11

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  • The Japanese Biochemichal Society

    1993.4

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Committee Memberships

  • 日本ビタミン学会   代議員  

    2023.11 - 2025.10   

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    Committee type:Academic society

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  • 日本ビタミン学会   代議員  

    2021.11 - 2023.10   

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  • 日本ビタミン学会   代議員  

    2019.11 - 2021.10   

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    日本ビタミン学会

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  • 日本ビタミン学会   代議員  

    2017.11 - 2019.10   

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    日本ビタミン学会

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Papers

  • Coenzyme B12-dependent eliminases: Diol and glycerol dehydratases and ethanolamine ammonia-lyase Reviewed International journal

    Tetsuo Toraya, Takamasa Tobimatsu, Koichi Mori, Mamoru Yamanishi, Naoki Shibata

    Methods in Enzymology   668   181 - 242   2022.6

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    Language:English   Publishing type:Part of collection (book)   Publisher:Elsevier  

    Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.

    DOI: 10.1016/bs.mie.2021.11.027

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  • Reactivating chaperones for coenzyme B12-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase Reviewed International journal

    Tetsuo Toraya, Takamasa Tobimatsu, Naoki Shibata, Koichi Mori

    Methods in Enzymology   668   243 - 284   2022.6

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    Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.

    DOI: 10.1016/bs.mie.2021.11.028

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  • Necessity of flanking repeats R1' and R8' of human Pumilio1 protein for RNA binding Reviewed International journal

    Kento Nakamura, Taishu Nakao, Tomoaki Mori, Serika Ohno, Yusuke Fujita, Keisuke Masaoka, Kazuki Sakabayashi, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    Biochemistry   60 ( 40 )   3007 - 3015   2021.9

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    Human Pumilio (hPUM) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1′ and R8′, however, remain unclear. Here, we report how the repeats contribute to overall RNA binding. We first prepared three mutants in which R1′ and/or R8′ were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1′ contributed more than R8′, unlike Drosophila PUM. We next investigated which amino acid residues of R1′ or R8′ were responsible for RNA binding. With detailed analysis of the protein tertiary structure, we found a hydrophobic core in each of the repeats. We therefore mutated all hydrophobic amino residues in each core to alanine. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1′ had a significant influence. In the present study, we demonstrated that the flanking R1′ and R8′ repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1′–R1 interactions may stabilize the whole hPUM structure.

    DOI: 10.1021/acs.biochem.1c00445

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  • Genome sequence analysis of new plum pox virus isolates from Japan Reviewed International journal

    Tomoaki Mori, Chiaki Warner, Serika Ohno, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    BMC Research Notes   14 ( 1 )   266   2021.7

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

    Objective
    To find mutations that may have recently occurred in Plum pox virus (PPV), we collected six PPV-infected plum/peach trees from the western part of Japan and one from the eastern part. After sequencing the full-length PPV genomic RNAs, we compared the amino acid sequences with representative isolates of each PPV strain.

    Results
    All new isolates were found to belong to the PPV-D strain: the six isolates collected from western Japan were identified as the West-Japan strain while the one collected from eastern Japan as the East-Japan strain. Amino acid sequence analysis of these seven isolates suggested that the 1407th and 1529th amino acid residues are characteristic of the West-Japan and the East-Japan strains, respectively. Comparing them with the corresponding amino acid residues of the 47 non-Japanese PPV-D isolates revealed that these amino acid residues are undoubtedly unique. A further examination of the relevant amino acid residues of the other 210 PPV-D isolates collected in Japan generated a new hypothesis regarding the invasion route from overseas and the subsequent diffusion route within Japan: a PPV-D strain might have invaded the western part of Japan from overseas and spread throughout Japan.

    DOI: 10.1186/s13104-021-05683-9

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  • Site-specific integration by recruitment of a complex of ΦC31 integrase and donor DNA to a target site by using a tandem, artificial zinc-finger protein Reviewed International journal

    Tatsuhiko Sumikawa, Serika Ohno, Takeharu Watanabe, Ryo Yamamoto, Miyu Yamano, Tomoaki Mori, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    Biochemistry   57 ( 50 )   6868 - 6877   2018.11

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    To solve the problem of uncontrolled therapeutic gene integration, which is a critical drawback of retroviral vectors for gene therapy, the integration sites of exogenous genes should be precisely controlled not to perturb endogenous gene expression. To accomplish this, we explored the possibility of site-specific integration using two six-finger artificial zinc-finger proteins (AZPs) tandemly conjugated via a flexible peptide linker (designated “Tandem AZP”). A Tandem AZP in which two AZPs recognize specific 19 bp targets in a donor and acceptor DNA was expected to site-specifically recruit the donor DNA to the acceptor DNA. Thereafter, an exogenously added integrase was expected to integrate the donor DNA into a specific site in the acceptor DNA (as it might be in the human genome). We demonstrated in vitro that in the presence of Tandem AZP, ΦC31 integrase selectively integrated a donor plasmid into a target acceptor plasmid not only at 30 °C (the optimum temperature of the integrase) but also at 37 °C (for future application in humans). We expect that with further improvement of our current system, a combination of Tandem AZP with integrase/recombinase will enable site-specific integration in mammalian cells and provide safer gene therapy technology.

    DOI: 10.1021/acs.biochem.8b00979

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  • Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein-staphylococcal nuclease hybrid Reviewed International journal

    Tomoaki Mori, Kento Nakamura, Keisuke Masaoka, Yusuke Fujita, Ryosuke Morisada, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    Biochemical and Biophysical Research Communications   479 ( 4 )   736 - 740   2016.10

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    Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms.

    DOI: 10.1016/j.bbrc.2016.09.142

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  • Catalytic roles of substrate-binding residues in coenzyme B12-dependent ethanolamine ammonia-lyase Reviewed International journal

    Koichi Mori, Toshihiro Oiwa, Satoshi Kawaguchi, Kyosuke Kondo, Yusuke Takahashi, Tetsuo Toraya

    Biochemistry   53 ( 16 )   2661 - 2671   2014.4

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

    Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. 1-OH of the substrate is hydrogen-bonded with Gluα287, Argα160, and Asnα193 and 2-NH2 with Gluα287, Glnα162, and Aspα362. The active site somewhat resembles that of diol dehydratase. All five residues were important for the high-affinity binding of the substrate and for catalysis. The -COO– group at residue α287 was absolutely required for activity and coenzyme Co–C bond cleavage, and there was a spatially optimal position for it, suggesting that Gluα287 contributes to Co–C bond homolysis, stabilizes the transition state for the migration of NH2 from C2 to C1 through partial deprotonation of spectator OH, and functions as a base in the elimination of ammonia. A positive charge and/or the hydrogen bond at position α160 and the hydrogen bonds at positions α162 and α193 with the substrate are important for catalysis and for preventing a radical intermediate from undergoing side reactions. Argα160 would stabilize the trigonal transition state in NH2 migration by electrostatic catalysis and hydrogen bonding with spectator OH. Asnα193 would contribute to maintaining the appropriate position and direction of the guanidinium group of Argα160, as well. Hydrogen bond acceptors were necessary at position α162, but hydrogen bond donors were rather harmful. Glnα162 might stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3+. The activity was very sensitive to the position of -COO– at α362. Aspα362 would assist Co–C bond homolysis indirectly and stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3+ and electrostatic interaction.

    DOI: 10.1021/bi500223k

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  • Sandwiched zinc-finger nucleases demonstrating higher homologous recombination rates than conventional zinc-finger nucleases in mammalian cells Reviewed International journal

    Tomoaki Mori, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    Bioorganic & Medicinal Chemistry Letters   24 ( 3 )   813 - 816   2014.2

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    We previously reported that our sandwiched zinc-finger nucleases (ZFNs), in which a DNA cleavage domain is inserted between two artificial zinc-finger proteins, cleave their target DNA much more efficiently than conventional ZFNs in vitro. In the present study, we compared DNA cleaving efficiencies of a sandwiched ZFN with those of its corresponding conventional ZFN in mammalian cells. Using a plasmid-based single-strand annealing reporter assay in HEK293 cells, we confirmed that the sandwiched ZFN induced homologous recombination more efficiently than the conventional ZFN; reporter activation by the sandwiched ZFN was more than eight times that of the conventional one. Western blot analysis showed that the sandwiched ZFN was expressed less frequently than the conventional ZFN, indicating that the greater DNA-cleaving activity of the sandwiched ZFN was not due to higher expression of the sandwiched ZFN. Furthermore, an MTT assay demonstrated that the sandwiched ZFN did not have any significant cytotoxicity under the DNA-cleavage conditions. Thus, because our sandwiched ZFN cleaved more efficiently than its corresponding conventional ZFN in HEK293 cells as well as in vitro, sandwiched ZFNs are expected to serve as an effective molecular tool for genome editing in living cells.

    DOI: 10.1016/j.bmcl.2013.12.096

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  • Essential roles of nucleotide-switch and metal-coordinating residues for chaperone function of diol dehydratase-reactivase Reviewed International journal

    Koichi Mori, Koji Obayashi, Yasuhiro Hosokawa, Akina Yamamoto, Mayumi Yano, Toshiyuki Yoshinaga, Tetsuo Toraya

    Biochemistry   52 ( 48 )   8677 - 8686   2013.12

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    Diol dehydratase-reactivase (DD-R) is a molecular chaperone that reactivates inactivated holodiol dehydratase (DD) by cofactor exchange. Its ADP-bound and ATP-bound forms are high-affinity and low-affinity forms for DD, respectively. Among DD-Rs mutated at the nucleotide-binding site, neither the Dα8N nor Dα413N mutant was effective as a reactivase. Although Dα413N showed ATPase activity, it did not mediate cyanocobalamin (CN-Cbl) release from the DD·CN-Cbl complex in the presence of ATP or ADP and formed a tight complex with apoDD even in the presence of ATP, suggesting the involvement of Aspα413 in the nucleotide switch. In contrast, Dα8N showed very low ATPase activity and did not mediate CN-Cbl release from the complex in the presence of ATP, but it did cause about 50% release in the presence of ADP. The complex formation of this mutant with DD was partially reversed by ATP, suggesting that Aspα8 is involved in the ATPase activity but only partially in the nucleotide switch. Among DD-Rs mutated at the Mg2+-binding site, only Eβ31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R β subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R β and DD β subunits, Gluβ97 of DD would coordinate to Mg2+. The complex of Eβ97Q DD with CN-Cbl was not activated by wild-type DD-R. No complex was formed between this mutant and wild-type DD-R, indicating that the coordination of Gluβ97 to Mg2+ is essential for subunit swapping and therefore for (re)activation.

    DOI: 10.1021/bi401290j

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  • Redesign of coenzyme B12 dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols Reviewed International journal

    Mamoru Yamanishi, Koichiro Kinoshita, Masaki Fukuoka, Takuya Saito, Aya Tanokuchi, Yuuki Ikeda, Hirokazu Obayashi, Koichi Mori, Naoki Shibata, Takamasa Tobimatsu, Tetsuo Toraya

    FEBS Journal   279 ( 5 )   793 - 804   2012.3

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    Coenzyme B12 dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co–C bond. Bachovchin et al. [Biochemistry16, 1082–1092 (1977)] reported that glycerol bound in the GS conformation, in which the pro-S-CH2OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the GS conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. kinact of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. kcat/kinact showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols.

    DOI: 10.1111/j.1742-4658.2012.08470.x

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  • Diol dehydratase-reactivating factor is a reactivase – evidence for multiple turnovers and subunit swapping with diol dehydratase Reviewed International journal

    Koichi Mori, Yasuhiro Hosokawa, Toshiyuki Yoshinaga, Tetsuo Toraya

    FEBS Journal   277 ( 23 )   4931 - 4943   2010.12

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    Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co–C bond. The damaged cofactor remains tightly bound to the active site. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg2+. Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzyme·cyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1 : 1 and 1 : 2 complexes with the reactivase in the presence of ADP and Mg2+. Upon complex formation, one β subunit was released from the (αβ)2 tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD β subunit and the reactivase β subunit, suggests that subunit displacement or swapping takes place upon formation of the enzyme·reactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.

    DOI: 10.1111/j.1742-4658.2010.07898.x

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  • Coenzyme B12-dependent diol dehydratase is a potassium ion-requiring calcium metalloenzyme: evidence that the substrate-coordinated metal ion is calcium Reviewed International journal

    Tetsuo Toraya, Susumu Honda, Koichi Mori

    Biochemistry   49 ( 33 )   7210 - 7217   2010.8

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    The X-ray analyses of coenzyme B12-dependent diol dehydratase revealed two kinds of electron densities that correspond to metal ions in the active site. One is directly coordinated by substrate [Shibata, N., et al. (1999) Structure 7, 997−1008] and the other located near the adenine ring of the coenzyme adenosyl group [Masuda, J., et al. (2000) Structure 8, 775−788]. Both have been assigned as potassium ions, although the coordination distances of the former are slightly shorter than expected. We examined the possibility that the enzyme is a metalloenzyme. Apodiol dehydratase was strongly inhibited by incubation with EDTA and EGTA in the absence of substrate. The metal analysis revealed that the enzyme contains ∼2 mol of tightly bound calcium per mole of enzyme. The calcium-deprived, EDTA-free apoenzyme was obtained by the EDTA treatment, followed by ultrafiltration. The activity of the calcium-deprived apoenzyme was dependent on Ca2+ when assayed with 1 mM substrate. The Km for Ca2+ evaluated in reconstitution experiments was 0.88 μM. These results indicate that the calcium is essential for catalysis. Ca2+ showed a significant stabilizing effect on the calcium-deprived apoenzyme as well. It was thus concluded that the substrate-coordinated metal ion is not potassium but calcium. The potassium ion bound near the adenine ring would be the essential one for the diol dehydratase catalysis. Therefore, this enzyme can be considered to be a metal-activated metalloenzyme.

    DOI: 10.1021/bi100561m

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  • Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates Reviewed International journal

    Naoki Shibata, Hiroko Tamagaki, Naoki Hieda, Keita Akita, Hirofumi Komori, Yasuhito Shomura, Shin-ichi Terawaki, Koichi Mori, Noritake Yasuoka, Yoshiki Higuchi, Tetsuo Toraya

    Journal of Biological Chemistry   285 ( 34 )   26484 - 26493   2010.8

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    N-terminal truncation of the Escherichia coli ethanolamine ammonia-lyase β-subunit does not affect the catalytic properties of the enzyme (Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., Yamanishi, M., Mori, K., and Toraya, T. (2010) J. Biochem. 147, 83–93). The binary complex of the truncated enzyme with cyanocobalamin and the ternary complex with cyanocobalamin or adeninylpentylcobalamin and substrates were crystallized, and their x-ray structures were analyzed. The enzyme exists as a trimer of the (αβ)2 dimer. The active site is in the (β/α)8 barrel of the α-subunit; the β-subunit covers the lower part of the cobalamin that is bound in the interface of the α- and β-subunits. The structure complexed with adeninylpentylcobalamin revealed the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Argα160 contributes to substrate binding most likely by hydrogen bonding with the O1 atom. The modeling study implies that marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme Co–C bond. The coenzyme adenosyl radical in the productive conformation was modeled by superimposing its adenine ring on the adenine ring-binding site followed by ribosyl rotation around the N-glycosidic bond. A major structural change upon substrate binding was not observed with this particular enzyme. Gluα287, one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co–C bond.

    DOI: 10.1074/jbc.m110.125112

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  • Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli Reviewed International journal

    Keita Akita, Naoki Hieda, Nobuyuki Baba, Satoshi Kawaguchi, Hirohisa Sakamoto, Yuka Nakanishi, Mamoru Yamanishi, Koichi Mori, Tetsuo Toraya

    Journal of Biochemistry   147 ( 1 )   83 - 93   2010.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

    The methods of homologous high-level expression and simple large-scale purification for coenzyme B12-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer α6β6. Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B12, the binding of 1 mol of the inhibitor per mol of the αβ unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme.

    DOI: 10.1093/jb/mvp145

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  • Studies on reactivating factors for coenzyme B12-dependent enzymes Invited

    Koichi Mori

    Vitamins   83 ( 3 )   95 - 110   2009.3

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    Authorship:Lead author   Language:Japanese   Publisher:The Vitamin Society of Japan  

    Adenosylcobalamin-dependent enzymes tend to inactivate holoenzyme accompanying the modification of the coenzyme. We identified reactivating factors for inactivated holoenzymes of adenosylcobalamin-dependent diol dehydratase (DD), glycerol dehydratase (GD), and ethanolamine ammonia-lyase (EAL), i.e., DDR, GDR, and EALR, respectively. DDR hydrolyzes ATP to ADP and induces its conformational change. Then, DDR facilitates the dissociation of the damaged coenzyme from the inactivated holoDD through formation of tight DD-DDR-ADP complex. This complex is dissociated into apoDD and DDR by replacing ADP on DDR with ATP, and then active holoenzyme is reconstituted. Crystal structures of DDR allow us to construct a model of DD-DDR complex. DD should be bind to DDR with concomitant displacement of a DDR β subunit by a DD β subunit. It induces steric repulsion between DD α and DDR α subunits that would lead to the release of a damaged coenzyme from inactivated holoDD. GDR reactivates inactivated holoGD by similar mechanism.

    DOI: 10.20632/vso.83.3_95

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    Other Link: https://www.jstage.jst.go.jp/article/vso/83/3/83_KJ00005489097/_article/-char/en

  • Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis Reviewed International journal

    Ken-ichi Ogura, Shin-ichi Kunita, Koichi Mori, Takamasa Tobimatsu, Tetsuo Toraya

    FEBS Journal   275 ( 24 )   6204 - 6216   2008.12

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    The X-ray structure of the diol dehydratase–adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Serα224, and of 6-NH2, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ε-NH2 group of Lysβ135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Serα224 and Lysβ135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sα224A, Sα224N and Kβ135E mutants were 19–2% as active as the wild-type enzyme, whereas the Kβ135A, Kβ135Q and Kβ135R mutants retained 58–76% of the wild-type activity. The presence of a positive charge at the β135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme–cobalamin interaction. The Sα224A and Sα224N mutants showed a kcat/kinact value that was less than 2% that of the wild-type, whereas for Lysβ135 mutants this value was in the range 25–75%, except for the Kβ135E mutant (7%). Unlike the wild-type holoenzyme, the Sα224N and Sα224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Serα224 is important for cobalt–carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Sα224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5′-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.

    DOI: 10.1111/j.1742-4658.2008.06745.x

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  • Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol Reviewed International journal

    Tetsuo Toraya, Naohisa Tamura, Takeshi Watanabe, Mamoru Yamanishi, Naoki Hieda, Koichi Mori

    Journal of Biochemistry   144 ( 4 )   437 - 446   2008.10

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    The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co–C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5′-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg2+ and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5′-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H2S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.

    DOI: 10.1093/jb/mvn086

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  • Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases Reviewed International journal

    Hideki Kajiura, Koichi Mori, Naoki Shibata, Tetsuo Toraya

    FEBS Journal   274 ( 21 )   5556 - 5566   2007.11

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    Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme–reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase–DDR complex was cross-formed, although the diol dehydratase–GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme–reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase.

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  • Release of a damaged cofactor from a coenzyme B12-dependent enzyme: X-ray structures of diol dehydratase-reactivating factor Reviewed International journal

    Naoki Shibata, Koichi Mori, Naoki Hieda, Yoshiki Higuchi, Mamoru Yamanishi, Tetsuo Toraya

    Structure   13 ( 12 )   1745 - 1754   2005.12

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    The crystal structures of ADP bound and nucleotide-free forms of molecular chaperone-like diol dehydratase-reactivating factor (DDR) were determined at 2.0 and 3.0 Å, respectively. DDR exists as a dimer of heterodimer (αβ)2. The α subunit has four domains: ATPase domain, swiveling domain, linker domain, and insert domain. The β subunit, composed of a single domain, has a similar fold to the β subunit of diol dehydratase (DD). The binding of an ADP molecule to the nucleotide binding site of DDR causes a marked conformational change of the ATPase domain of the α subunit, which would weaken the interactions between the DDR α and β subunits and make the displacement of the DDR β subunit by DD through the β subunit possible. The binding of the DD β subunit to the DDR α subunit induces steric repulsion between the DDR α and DD α subunits that would lead to the release of a damaged cofactor from inactivated holoDD.

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  • Recognition of phospholipids in Streptomyces phospholipase D Reviewed International journal

    Yoshiko Uesugi, Koichi Mori, Jiro Arima, Masaki Iwabuchi, Tadashi Hatanaka

    Journal of Biological Chemistry   280 ( 28 )   26143 - 26151   2005.7

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    To investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces phospholipase D (PLD), we constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. By comparing the activities of chimeras, six candidate residues related to transphosphatidylation activity were shown. Based on the above result, we constructed several mutants to identify the key residues involved in the recognition of phospholipids. By kinetic analysis, we identified that Gly188 and Asp191 of PLD from Streptomyces septatus TH-2, which are not present in the highly conserved catalytic HXKXXXXD (HKD) motifs, are key amino acid residues related to the transphosphatidylation activity. To investigate the role of two residues in the recognition of phospholipids, the effects of these residues on binding to substrates were analyzed by surface plasmon spectroscopy. The result suggests that Gly188 and Asp191 are involved in the recognition of phospholipids in correlation with the N-terminal HKD motif. Furthermore, this study also provides experimental evidence that the N-terminal HKD motif contains the catalytic nucleophile, which attacks the phosphatidyl group of the substrate.

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  • Crystallization and preliminary X-ray analysis of molecular chaperone-like diol dehydratase-reactivating factor in ADP-bound and nucleotide-free forms Reviewed International journal

    Koichi Mori, Naoki Hieda, Mamoru Yamanishi, Naoki Shibata, Tetsuo Toraya

    Acta Crystallographica Section F Structural Biology and Crystallization Communications   61 ( 6 )   603 - 605   2005.6

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    Adenosylcobalamin (coenzyme B12) dependent diol dehydratase (EC 4.2.1.28) catalyzes the conversion of 1,2-diols and glycerol to the corresponding aldehydes. It undergoes mechanism-based inactivation by glycerol. The diol dehydratase-reactivating factor (DDR) reactivates the inactivated holoenzymes in the presence of adenosylcobalamin, ATP and Mg2+ by mediating the release of a damaged cofactor. This molecular chaperone-like factor was overexpressed in Escherichia coli, purified and crystallized in the ADP-bound and nucleotide-free forms by the sandwich-drop vapour-diffusion method. The crystals of the ADP-bound form belong to the orthorhombic system, with space group P212121 and unit-cell parameters a = 83.26, b = 84.60, c = 280.09 Å, and diffract to 2.0 Å. In the absence of nucleotide, DDR crystals were orthorhombic, with space group P212121 and unit-cell parameters a = 81.92, b = 85.37, c = 296.99 Å and diffract to 3.0 Å. Crystals of both forms were suitable for structural analysis.

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  • Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling Reviewed International journal

    Tomofumi Negishi, Takafumi Mukaihara, Koichi Mori, Hiroko Nishikido, Yuko Kawasaki, Hiroyuki Aoki, Michiko Kodama, Hatsuho Uedaira, Yoshiko Uesugi, Masaki Iwabuchi, Tadashi Hatanaka

    Biochimica et Biophysica Acta (BBA) - General Subjects   1722 ( 3 )   331 - 342   2005.4

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    To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.

    DOI: 10.1016/j.bbagen.2005.01.009

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  • Repeat-length-independent broad-spectrum shuffling, a novel method of generating a random chimera library in vivo Reviewed International journal

    Koichi Mori, Takafumi Mukaihara, Yoshiko Uesugi, Masaki Iwabuchi, Tadashi Hatanaka

    Applied and Environmental Microbiology   71 ( 2 )   754 - 760   2005.2

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    We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL+ gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb+ background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.

    DOI: 10.1128/aem.71.2.754-760.2005

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  • Identification of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase. Reviewed International journal

    Koichi Mori, Reiko Bando, Naoki Hieda, Tetsuo Toraya

    Journal of Bacteriology   186 ( 20 )   6845 - 6854   2004.10

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    The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

    DOI: 10.1128/jb.186.20.6845-6854.2004

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  • A mutant phospholipase D with enhanced thermostability from Streptomyces sp. Reviewed International journal

    Tadashi Hatanaka, Tomofumi Negishi, Koichi Mori

    Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics   1696 ( 1 )   75 - 82   2004.1

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    To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329–441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.

    DOI: 10.1016/j.bbapap.2003.09.013

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  • Inhibition of Streptomyces chromofuscus phospholipase D activity by dichloro-(2,2':6',2"-terpyridine)-platinum (II) dihydrate Reviewed International journal

    Megumi Kubota-Akizawa, Tomofumi Negishi, Koichi Mori, Tadashi Hatanaka

    Journal of Enzyme Inhibition and Medicinal Chemistry   17 ( 5 )   329 - 332   2002.1

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    To determine the catalytic site of Streptomyces chromofuscus phospholipase D (PLD), which lacks an HKD motif, we examined the effects of inhibitors on the hydrolytic activity of the PLD by comparing it with cabbage and Streptomyces PLDs, which have two HKD motifs. We showed that dichloro-(2,2':6',2"-terpyridine)-platinum (II) dihydrate, a His- and Cys-directed chemical modifier, had inhibitory effects on the activities of all types of PLD examined. On the other hand, N -ethylmaleimide, a thiol-directed modifier had no such effects on PLD activity. These results suggest that the His residue plays an important role in the activity of Streptomyces chromofuscus PLD.

    DOI: 10.1080/1475636021000033252

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  • Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase Reviewed International journal

    Hideki Kajiura, Koichi Mori, Takamasa Tobimatsu, Tetsuo Toraya

    Journal of Biological Chemistry   276 ( 39 )   36514 - 36519   2001.9

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    Adenosylcobalamin-dependent glycerol dehydratase undergoes mechanism-based inactivation by its physiological substrate glycerol. We identified two genes (gdrAB) ofKlebsiella pneumoniae for a glycerol dehydratase-reactivating factor (Tobimatsu, T., Kajiura, H., Yunoki, M., Azuma, M., and Toraya, T. (1999) J. Bacteriol. 181, 4110–4113). Recombinant GdrA and GdrB proteins formed a tight complex of (GdrA)2(GdrB)2, which is a putative reactivating factor. The purified factor reactivated the glycerol-inactivated and O2-inactivated glycerol dehydratases as well as activated the enzyme-cyanocobalamin complex in vitro in the presence of ATP, Mg2+, and adenosylcobalamin. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins in the presence of ATP and Mg2+ through intermediate formation of apoenzyme. The factor showed extremely low ATP-hydrolyzing activity and formed a tight complex with apoenzyme in the presence of ADP. Incubation of the enzyme-cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin. The resulting tight inactive complex of apoenzyme with the factor dissociated upon incubation with ATP, forming functional apoenzyme and a low affinity form of factor. Thus, it was established that the reactivation of the inactivated holoenzymes takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme-factor complex. We propose that the glycerol dehydratase-reactivating factor is a molecular chaperone that participates in reactivation of the inactivated enzymes.

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  • Mechanism of reactivation of coenzyme B12-dependent diol dehydratase by a molecular chaperone-like reactivating factor Reviewed International journal

    Koichi Mori, Tetsuo Toraya

    Biochemistry   38 ( 40 )   13170 - 13178   1999.10

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    The mechanism of reactivation of diol dehydratase by its reactivating factor was investigated in vitro by using enzyme·cyanocobalamin complex as a model for inactivated holoenzyme. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins through intermediate formation of apoenzyme. The factor showed extremely low but distinct ATP-hydrolyzing activity. It formed a tight complex with apoenzyme in the presence of ADP but not at all in the presence of ATP. Incubation of the enzyme·cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin, leaving the tight apoenzyme-reactivating factor complex. Although the resulting complex was inactive even in the presence of added adenosylcobalamin, it dissociated by incubation with ATP, forming the apoenzyme, which was reconstitutable into active holoenzyme with added coenzyme. Thus, it was established that the reactivation of the inactivated holoenzyme by the factor in the presence of ATP and Mg2+ takes place in two steps:  ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme·factor complex. ATP plays dual roles as a precursor of ADP in the first step and as an effector to change the factor into the low-affinity form for diol dehydratase. The enzyme-bound adenosylcobalamin was also susceptible to exchange with free adeninylpentylcobalamin, although to a much lesser degree. The mechanism for discrimination of adenine-containing cobalamins from adenine-lacking cobalamins was explained in terms of formation equilibrium constants of the cobalamin·enzyme·reactivating factor ternary complexes. We propose that the reactivating factor is a new type of molecular chaperone that participates in reactivation of the inactivated enzymes.

    DOI: 10.1021/bi9911738

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  • A reactivating factor for coenzyme B12-dependent diol dehydratase Reviewed International journal

    Tetsuo Toraya, Koichi Mori

    Journal of Biological Chemistry   274 ( 6 )   3372 - 3377   1999.2

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    Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicide inactivation by glycerol, a physiological substrate. The coenzyme is modified through irreversible cleavage of its cobalt-carbon bond, resulting in inactivation of the enzyme by tight binding of the modified coenzyme to the active site. Recombinant DdrA and DdrB proteins of K. oxytoca were co-purified to homogeneity from cell-free extracts of Escherichia coli overexpressing theddrAB genes. They existed as a tight complex,i.e. a putative reactivating factor, with an apparent molecular weight of 150,000. The factor consists of equimolar amounts of the two subunits with M r of 64,000 (A) and 14,000 (B), encoded by the ddrA and ddrB genes, respectively. Therefore, its subunit structure is most likely A2B2. The factor not only reactivated glycerol-inactivated and O2-inactivated holoenzymes but also activated enzyme-cyanocobalamin complex in the presence of free adenosylcobalamin, ATP, and Mg2+. The reactivating factor mediated ATP-dependent exchange of the enzyme-bound cyanocobalamin for free 5-adeninylpentylcobalamin in the presence of ATP and Mg2+, but the reverse was not the case. Thus, it can be concluded that the inactivated holoenzyme becomes reactivated by exchange of the enzyme-bound, adenine-lacking cobalamins for free adenosylcobalamin, an adenine-containing cobalamin.

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  • Characterization, sequencing, and expression of the genes encoding a reactivating factor for glycerol-inactivated adenosylcobalamin-dependent diol dehydratase Reviewed International journal

    Koichi Mori, Takamasa Tobimatsu, Tetsuya Hara, Tetsuo Toraya

    Journal of Biological Chemistry   272 ( 51 )   32034 - 32041   1997.12

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    Diol dehydratase undergoes suicide inactivation by glycerol during catalysis involving irreversible cleavage of the Co-C bond of adenosylcobalamin. In permeabilized Klebsiella oxytoca and Klebsiella pneumoniae cells, the glycerol-inactivated holoenzyme or the enzyme-cyanocobalamin complex is rapidly activated by the exchange of the inactivated coenzyme or cyanocobalamin for free adenosylcobalamin in the presence of ATP and Mg2+ (Honda, S., Toraya, T., and Fukui, S. (1980) J. Bacteriol. 143, 1458–1465; Ushio, K., Honda, S., Toraya, T., and Fukui, S. (1982) J. Nutr. Sci. Vitaminol. 28, 225–236). Permeabilized Escherichia coli cells co-expressing the diol dehydratase genes with two open reading frames in the 3′-flanking region were capable of reactivating glycerol-inactivated diol dehydratase as well as activating the enzyme-cyanocobalamin complex in situ in the presence of free adenosylcobalamin, ATP, and Mg2+. These open reading frames, designated as ddrA and ddrB genes, were identified as the genes of a putative reactivating factor for inactivated diol dehydratase. The genes encoded polypeptides consisting of 610 and 125 amino acid residues with predicted molecular weights of 64,266 and 13,620, respectively. Co-expression of the open reading frame in the 5′-flanking region was stimulatory but not obligatory for conferring the reactivating activity upon E. coli. Thus, the product of this gene was considered not an essential component of the reactivating factor.

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  • A protein factor is essential for in situ reactivation of glycerol-inactivated adenosylcobalamin-dependent diol dehydratase Reviewed International journal

    Koichi Mori, Takamasa Tobimatsu, Tetsuo Toraya

    Bioscience, Biotechnology, and Biochemistry   61 ( 10 )   1729 - 1733   1997.10

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    The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co–C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg2+. β,γ-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg2+, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the α-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3′-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.

    DOI: 10.1271/bbb.61.1729

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Presentations

  • Cleavage of Influenza RNA Using Artificial RNA-cleaving Enzyme International conference

    Tomoaki Mori, Kento Nakamura, Keisuke Masaoka, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    Experimental Biology 2021  2021.4 

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    Event date: 2021.4.27 - 2021.4.30

    Language:English  

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    Other Link: https://doi.org/10.1096/fasebj.2021.35.S1.01768

  • Development of novel artificial RNA-cleaving enzymes for inactivating RNA viruses International conference

    Tomoaki MoriKento, NakamuraKeisuke, MasaokaKoich MoriTakamasa Tobimatsu, Takashi Sera

    ACS Spring 2021  2021.4  American Chemical Society

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    Event date: 2021.4.5 - 2021.4.30

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  • 新規人工RNA切断酵素の開発

    中城遥, 安福和也, 星ひかる, 森友明, 森光一, 飛松孝正, 世良貴史

    第35回中国四国ウイルス研究会  2020.9.20 

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    Event date: 2020.9.19 - 2020.9.20

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:出雲市  

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  • 人工RNA切断酵素を用いたインフルエンザRNA切断

    森友明, 中村健人, 正岡敬祐, 森光一, 飛松孝正, 世良貴史

    第35回中国四国ウイルス研究会  2020.9.19 

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    Event date: 2020.9.19 - 2020.9.20

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:出雲市  

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  • 人工RNA切断酵素によるインフルエンザウイルスの複製阻害

    木口芙巳, 樋口新, 森友明, 森光一, 飛松孝正, 世良貴史

    第35回中国四国ウイルス研究会  2020.9.19 

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    Event date: 2020.9.19 - 2020.9.20

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:出雲市  

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  • RNAウイルス不活性化に向けた人工RNA結合タンパク質のセレクション系の構築

    浮田康平, 原知明, 戸川剛志, 森友明, 森光一, 飛松孝正, 世良貴史

    第35回中国四国ウイルス研究会  2020.9.19 

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    Event date: 2020.9.19 - 2020.9.20

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:出雲市  

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  • The action of coenzyme B12-dependent diol dehydratase on 3,3,3-trifluoro-1,2-propanediol results in unexpected formation of acetaldehyde accompanying defluorination International coauthorship

    Koichi Mori, Dong Jiang, Bernard T. Golding, Tetsuo Toraya

    2019.9.16 

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    Event date: 2019.9.16 - 2019.9.18

    Language:Japanese   Presentation type:Oral presentation (general)  

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  • クレブシラオキシトカpduオペロンの大腸菌での発現とその解析

    飛松孝正, 斉藤拓也, 柴田千尋, 世良貴史, 森光一, 虎谷哲夫

    第455回ビタミンB研究協議会  2019.3.9  ビタミンB研究委員会

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    Event date: 2019.3.9

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:富山市  

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  • 補酵素B12関与diol dehydratase βおよびγサブユニットのN末領域による低溶解性化能の解析

    池田渓太, 平田佳久, 森光一, 世良貴史, 虎谷哲夫, 飛松孝正

    2017年度生命科学系学会合同年次大会(第40回日本分子生物学会年会,第90回日本生化学会大会)  2017.12.7  日本分子生物学会, 日本生化学会

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    Event date: 2017.12.6 - 2017.12.9

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:神戸  

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  • 人工RNA結合タンパク質のデザイン法の開発

    門家拓哉, 仲尾太秀, 森光一, 飛松孝正, 世良貴史

    第11回バイオ関連化学シンポジウム  2017.9  日本化学会

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    Event date: 2017.9.7 - 2017.9.9

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:東京  

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  • 人工DNA結合タンパク質を用いた位置特異的遺伝子導入システムの開発

    河田隆宏, 住川達彦, 王野瀬里香, 森友明, 森光一, 飛松孝正, 世良貴史

    第11回バイオ関連化学シンポジウム  2017.9  日本化学会

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    Event date: 2017.9.7 - 2017.9.9

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:東京  

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  • サンドイッチ型ジンクフィンガーヌクレアーゼを用いた大腸菌ゲノム編集

    梶谷貴大, 森友明, 森光一, 飛松孝正, 世良貴史

    第11回バイオ関連化学シンポジウム  2017.9  日本化学会

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    Event date: 2017.9.7 - 2017.9.9

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:東京  

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  • 補酵素B12関与アミノプロパンジオールデヒドラターゼの変異型酵素の機能解析

    仙波和崇, 飛松孝正, 世良貴史, 森光一

    日本ビタミン学会第69回大会  2017.6.10  日本ビタミン学会

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    Event date: 2017.6.9 - 2017.6.10

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:横浜  

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  • 翻訳制御に基づいた人工RNA結合タンパク質のセレクション

    戸川剛志, 原知明, 前田遥香, 森下しおみ, 森友明, 森光一, 飛松孝正, 世良貴史

    日本化学会第97春季年会  2017.3  日本化学会

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    Event date: 2017.3.16 - 2017.3.19

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:横浜  

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  • 人工RNA制限酵素を用いたインフルエンザRNA切断

    森友明, 中村健人, 正岡敬祐, 森光一, 飛松孝正, 世良貴史

    日本化学会第97春季年会  2017.3  日本化学会

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    Event date: 2017.3.16 - 2017.3.19

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:横浜  

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  • 人工RNA結合タンパク質の創出

    門家拓哉, 仲尾太秀, 佐藤根妃奈, 中村健人, 森光一, 飛松孝正, 世良貴

    日本化学会第97春季年会  2017.3  日本化学会

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    Event date: 2017.3.16 - 2017.3.19

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:横浜  

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  • 翻訳制御スイッチを用いたセレクションシステムの構築

    前田遥香, 原知明, 戸川剛志, 森下しおみ, 森友明, 森光一, 飛松孝正, 世良貴史

    日本化学会第97春季年会  2017.3  日本化学会

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    Event date: 2017.3.16 - 2017.3.19

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:横浜  

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  • 人工RNAヌクレアーゼを用いたRNA切断

    森友明, 中村健人, 正岡敬祐, 藤田裕介, 森貞亮祐, 阪林和貴, 森光一, 飛松孝正, 世良貴史

    第10回バイオ関連化学シンポジウム  2016.9  日本化学会

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    Event date: 2016.9.7 - 2016.9.9

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:金沢  

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  • Site-selective cleavage of target RNA by artificial RNA nucleases International conference

    Tomoaki Mori, Keisuke Masaoka, Yusuke Fujita, Ryosuke Morisada, Kazuki Sakabayashi, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    The International Chemical Congress of Pacific Basin Societies 2015  2015.12 

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    Event date: 2015.12.15 - 2015.12.20

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  • Structural analysis of RNA-binding protein to generate artificial RNA-binding proteins International conference

    Taishu Nakao, Kento Nakamura, Yusuke Fujita, Keisuke Masaoka, Tomoaki Mori, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    The International Chemical Congress of Pacific Basin Societies 2015  2015.12 

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    Event date: 2015.12.15 - 2015.12.20

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  • Zinc-finger-based artificial transcription factors and their applications International conference

    Kazuhiro Ofuji, Nao ji Nishida, Tomoaki Mori, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    The International Chemical Congress of Pacific Basin Societies 2015  2015.12 

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    Event date: 2015.12.15 - 2015.12.20

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  • Genome editing in Escherichia coli by sandwiched zinc-finger nuclease International conference

    Tsubasa Kai, Kaho Shimizu, Serika Ohno, Tomoaki Mori, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

    The International Chemical Congress of Pacific Basin Societies 2015  2015.12 

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    Event date: 2015.12.15 - 2015.12.20

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  • Klebsiella oxytoca pduオペロンを発現させた大腸菌組換え体の機能解析

    斉藤拓也, 荒木優貴乃, 柴田千尋, 世良貴史, 森光一, 虎谷哲夫, 飛松孝正

    BMB2015(第38回日本分子生物学会年会,第88回日本生化学会大会 合同大会)  2015.12  日本分子生物学会, 日本生化学会

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    Event date: 2015.12.3 - 2015.12.4

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:神戸  

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  • カラム不要でシンプルな活性のあるタンパク質の究極の精製法:単量体酵素への拡張

    北川優輔, 世良貴史, 森光一, 飛松孝正

    BMB2015(第38回日本分子生物学会年会,第88回日本生化学会大会 合同大会)  2015.12  日本分子生物学会, 日本生化学会

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    Event date: 2015.12.3 - 2015.12.4

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:神戸  

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  • 先導・革新的人工核酸結合タンパク質を用いたウイルス不活性化技術の確立と社会実装

    世良貴史, 森光一, 森友明, 木村光宏

    アグリビジネス創出フェア2015  2015.11 

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    Event date: 2015.11.18 - 2015.11.20

    Language:Japanese  

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  • 人工DNA結合タンパク質を用いた位置特異的な遺伝子導入法の開発

    仲尾太秀, 住川達彦, 河村知明, 森友明, 森光一, 飛松孝正, 世良貴史

    第9回バイオ関連化学シンポジウム  2015.9 

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    Event date: 2015.9.10 - 2015.9.12

    Language:Japanese   Presentation type:Oral presentation (general)  

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  • サンドイッチ型ジンクフィンガーヌクレアーゼを用いた大腸菌ゲノム編集

    甲斐翼, 清水香穂, 王野瀬里香, 森友明, 森光一, 飛松孝正, 世良貴史

    第9回バイオ関連化学シンポジウム  2015.9 

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    Event date: 2015.9.10 - 2015.9.12

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  • ビタミンB12補酵素関与エタノールアミンアンモニアリアーゼの基質結合アミノ酸残基の機能解析

    森光一, 大岩敏宏, 高橋佑典, 近藤恭介, 世良貴史, 虎谷哲夫

    日本ビタミン学会第65回大会  2013.5  日本ビタミン学会

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    Event date: 2013.5.17 - 2013.5.18

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:東京  

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  • ATPの遷移状態アナログを用いたジオールデヒドラターゼ再活性化因子の作用機作の解析

    森光一, 山本裕史, 虎谷哲夫

    特定領域研究「生体超分子の構造形成と機能制御の原子機構」第3回ワークショップ  2007.7 

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    Event date: 2007.7.11 - 2007.7.12

    Language:Japanese   Presentation type:Poster presentation  

    Venue:熱海  

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  • Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol International conference

    Tetsuo Toraya, Naohisa Tamura, Takeshi Watanabe, Mamoru Yamanishi, Naoki Hieda, Koichi Mori

    Gordon Research Conference on Vitamin B12 & Corphins  2007.7 

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    Event date: 2007.7.1 - 2007.7.6

    Language:English   Presentation type:Poster presentation  

    Venue:Biddeford  

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  • 補酵素B12関与ジオールデヒドラターゼの低溶解性化決定部位を用いたタンパク質精製法:単量体酵素への拡張

    飛松孝正, 北川優輔, 森光一, 世良貴史

    第446回ビタミンB研究協議会  2016.11.5  ビタミンB研究委員会

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    Venue:岐阜  

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  • 人工RNA切断酵素を用いた標的RNA切断

    星ひかる, 森友明, 中村健人, 正岡敬祐, 藤田裕介, 森光一, 飛松孝正, 世良貴史

    酵素工学研究会 第76回講演会  2016.10.7  酵素工学研究会

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    Venue:東京  

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  • Discovery and enzymatic characterization of a novel coenzyme B12-dependent dehydratase from Mesorhizobium loti

    Kazutaka Semba, Takeshi Sugihara, Takashi Sera, Takamasa Tobimatsu, Koichi Mori

    BMB2015 Biochemistry and Molecular Biology  2015.12.3 

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  • サンドイッチ型ジンクフィンガーヌクレアーゼを用いた大腸菌ゲノム編集

    甲斐翼, 清水香穂, 王野瀬里香, 森友明, 森光一, 飛松孝正, 世良貴史

    酵素工学研究会 第74回講演会  2015.10.16 

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  • サンドイッチ型ジンクフィンガーヌクレアーゼを用いた大腸菌ゲノム編集

    甲斐翼, 清水香穂, 王野瀬里香, 森友明, 森光一, 飛松孝正, 世良貴史

    日本化学会第95春季年会  2015.3.27  日本化学会

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  • 人工DNA結合タンパク質を用いた位置特異的遺伝子導入法の開発

    仲尾太秀, 住川達彦, 河村知明, 森友明, 森光一, 飛松孝正, 世良貴史

    日本化学会第95春季年会  2015.3.27  日本化学会

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  • B12補酵素関与エタノールアミンアンモニアリアーゼにおける活性部位アミノ酸残基の役割

    森光一

    第438回ビタミンB研究協議会  2014.11.22  ビタミンB研究委員会

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    Venue:大阪  

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  • Functional analysis of active-site residues of coenzyme B12-dependent ethanolamine ammonia-lyase

    Toshihiro Oiwa, Yusuke Takahashi, Kyosuke Kondo, Koichi Mori, Tetsuo Toraya

    The 84th Annual Meeting of the Japanese Biochemical Society  2011.9.22 

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  • 大腸菌YgfD蛋白質のB12補酵素関与メチルマロニルCoAムターゼの再活性化因子としての機能解析

    田尻麻衣, 塚田浩之, 田中佑樹, 灰垣佑輝, 安原麻衣, 森光一, 虎谷哲夫

    日本ビタミン学会第63回大会  2011.6.5  日本ビタミン学会

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    Venue:広島  

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  • YgfD as a reactivating-factor for coenzyme B12-dependent methylmalonyl-CoA mutase

    Mai Tajiri, Hironobu Tsukada, Yuuki Tanaka, Yuuki Haigaki, Mai Yasuhara, Koichi Mori, Tetsuo Toraya

    2010.12.7 

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  • B12補酵素関与ジオールデヒドラターゼはカリウムイオン依存性のカルシウムメタロエンザイムである:基質結合金属イオンの再同定

    虎谷哲夫, 本多進, 森光一

    ビタミンB研究委員会第422回研究協議会  2010.11.27  ビタミンB研究委員会

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  • Application of Coenzyme B12-dependent Enzyme System to Synthesis of a Raw Material for Polytrimethylene Terephthalate Invited

    Koichi Mori

    BioJapan2010  2010.9.29 

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  • B12補酵素関与エタノールアミンアンモニアリアーゼの立体構造と変異導入に基づく触媒機構の解析

    虎谷哲夫, 川口智史, 稗田直樹, 秋田敬太, 森光一, 柴田直樹, 樋口芳樹

    日本ビタミン学会第62回大会  2010.6.12  日本ビタミン学会

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    Venue:盛岡  

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  • Roles of active-site rsidues of coenzyme B12-dependent ethanolamine ammonia-lyase.

    Satoshi Kawaguchi, Keisuke Maeda, Koichi Mori, Tetsuo Toraya

    The 82nd Annual Meeting of the Japanese Biochemical Society  2009.10.23 

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  • Re-identification of substrate-coordinated metal ion in coenzyme B12-dependent diol dehydratase.

    Tetsuo Toraya, Susumu Honda, Koichi Mori

    The 82nd Annual Meeting of the Japanese Biochemical Society  2009.10.22 

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  • Coenzyme B12-dependent diol dehydratase is a potassium ionrequiring calcium-metaloenzyme: Re-identification of the substratecoordinated metal ion as calcium International conference

    Tetsuo Toraya, Susumu Honda, Koichi Mori

    Gordon Research Conference on Vitamin B12 & Corphins  2009.8.5 

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    Venue:Oxford  

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  • Crystal structure and action mechanism of coenzyme B12-dependent ethanolamine ammonia-lyase International conference

    Tetsuo Toraya, Naoki Hieda, Satoshi Kawaguchi, Keita Akita, Nobuyuki Baba, Koichi Mori, Naoki Shibata, Hiroko Tamagaki, Yoshiki Higuchi

    14th International Conference on Biological Inorganic Chemistry  2009.7.29 

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    Venue:Nagoya  

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  • エタノールアミンアンモニアリアーゼのタンパク質工学的改変と結晶構造解析および変異導入

    虎谷哲夫, 稗田直樹, 秋田敬太, 川口智史, 馬場伸之, 森光一, 柴田直樹, 玉垣裕子, 樋口芳樹

    日本ビタミン学会第61回大会  2009.5.31  日本ビタミン学会

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    Venue:京都  

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  • B12補酵素関与エタノールアミンアンモニアリアーゼの性質とタンパク質工学的改変と結晶構造解析

    虎谷哲夫, 稗田直樹, 秋田敬太, 川口智史, 馬場伸之, 森光一, 柴田直樹, 玉垣裕子, 樋口芳樹

    ビタミンB研究委員会第415回研究協議会  2009.2.14  ビタミンB研究委員会

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    Venue:東京  

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  • Effect of diol dehydratase-reactivating factor for formation of 3-hydroxypropionaldehyde by diol dehydratase

    Koichi Mori, Seiki Yamada, Tetsuo Toraya

    Annual Meeting 2008, The Society for Biotechnology, Japan  2008.8.28 

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  • Preparations, properties, and active-site structure of Escherichia coli ethanolamine ammonia-lyase International conference

    Tetsuo Toraya, Keita Akita, Naoki Hieda, Nobuyuki Baba, Hisahiro Sakamoto, Koichi Mori, Naoki Shibata, Hiroko Tamagaki, Yoshiki Higuchi

    33rd FEBS Congress & 11th IUBMB Conference  2008.6.30  Hellenic Society for Biochemistry and Molecular Biology (HSBMB), Federation of European Biochemical Societies (FEBS), International Union of Biochemistry and Molecular Biology (IUBMB)

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    Venue:Athens  

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  • ビタミンB12補酵素関与酵素の再活性化蛋白質に関する研究 Invited

    森光一

    日本ビタミン学会第60回大会(奨励賞受賞講演)  2008.6.14  日本ビタミン学会

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    Venue:仙台  

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  • エタノールアミンアンモニアリアーゼのX線結晶構造解析

    柴田直樹, 玉垣裕子, 小森博文, 庄村康人, 稗田直樹, 秋田敬太, 森光一, 虎谷哲夫, 樋口芳樹

    第8回日本蛋白質科学会年会  2008.6.10  日本蛋白質科学会

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    Venue:東京  

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  • B12補酵素関与ジオールデヒドラターゼとその再活性化因子の変異型蛋白質の機能解析

    森光一, 虎谷哲夫

    特定領域研究「生体超分子構造」第4回公開シンポジウム  2007.12.19 

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    Venue:豊中  

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  • Molecularbasis for specificities of reactivating factors for coenzyme B12-dependent diol and glycerol dehydratases.

    Hideki Kajiura, Koichi Mori, Naoki Shibata, Tetsuo Toraya

    Biochemistry and Molecular Biology 2007 (BMB2007)  2007.11.11 

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  • Preparation and property of ethanolamine-ammonia lyase with a truncated beta subunit.

    Naoki Hieda, Koichi Mori, Tetsuo Toraya

    Biochemistry and Molecular Biology 2007 (BMB2007)  2007.11.11 

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  • B12補酵素関与ジオールおよびグリセロールデヒドラターゼの再活性化因子特異性はどのようにして決まるか

    梶浦英樹, 森光一, 細川康宏, 柴田直樹, 虎谷哲夫

    2007年度酵素・補酵素研究会  2007.11.9 

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    Venue:倉敷  

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  • 不飽和1,2-ジオール類によるジオールデヒドラターゼの自殺不活性化とその機構

    田村直久, 渡辺丈士, 稗田直樹, 山西守, 森光一, 虎谷哲夫

    日本ビタミン学会第59回大会  2007.5.25  日本ビタミン学会

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    Venue:佐世保  

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  • ATPの遷移状態アナログを用いたジオールデヒドラターゼ再活性化因子の作用機作の解析

    山本裕史, 細川康宏, 森光一, 虎谷哲夫

    日本ビタミン学会第59回大会  2007.5.25  日本ビタミン学会

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    Venue:佐世保  

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  • B12補酵素関与エタノールアミンアンモニアリアーゼの再活性化因子の同定

    虎谷哲夫, 森光一, 坂東礼子, 稗田直樹

    ビタミンB研究委員会第407回研究協議会  2007.2.17  ビタミンB研究委員会

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    Venue:東京  

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  • B12補酵素関与ジオールデヒドラターゼの再活性化機構:酵素-再活性化因子複合体の生成と役割

    森光一, 虎谷哲夫

    特定領域研究「生体超分子の構造形成と機能制御の原子機構」第3回公開シンポジウム  2006.12.12 

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    Language:Japanese  

    Venue:つくば  

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  • B12補酵素関与ジオールデヒドラターゼの再活性化機構:酵素-再活性化因子複合体の生成と役割

    森光一, 細川康宏, 大林浩二, 好永利幸, 虎谷哲夫

    平成18年度 酵素・補酵素を楽しむ会  2006.10.7 

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    Venue:別府  

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  • Roles of Eβ97 of coenzyme B12-dependent diol dehydratase and Eβ31, Tα105, Dα166, and Dα183 in the Mg2+-binding site of diol dehydratase-reactivating factor for reactivation of inactivated holoenzyme

    Yasuhiro Hosokawa, Toshiyuki Yoshinaga, Koji Obayashi, Akina Yamamoto, Mayumi Yano, Koichi Mori, Tetsuo Toraya

    第79回日本生化学会大会(20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress)  2006.6.19  日本生化学会

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    Language:Japanese  

    Venue:京都  

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  • B12補酵素関与ジオールデヒドラターゼの再活性化因子:結晶構造と精密作用機作

    虎谷哲夫, 柴田直樹, 森光一, 稗田直樹, 樋口芳樹, 山西守

    第402回ビタミンB研究委員会  2005.11.26  ビタミンB研究委員会

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    Venue:京都  

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  • X-ray structure and action mechanism of molecular chaperone-like reactivating factor for coenzyme B12-dependent diol dehydratase

    Tetsuo Toraya, Naoki Shibata, Koichi Mori, Naoki Hieda, Yoshiki. Higuchi, Mamoru Yamanishi

    International Interdisciplinary Conference on Vitamins, Coenzymes, and Biofactors 2005  2005.11.10 

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    Language:Japanese  

    Venue:淡路  

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  • Mechanism of action of reactivating factor for adenosylcobalamin-dependent diol dehydratase: Evidence for displacement of DdrB by diol dehydratase and catalytic turnovers

    Koichi Mori, Yasuhiro Hosokawa, Toshiyuki Yoshinaga, Tetsuo Toraya

    International Interdisciplinary Conference on Vitamins, Coenzymes, and Biofactors 2005  2005.11.10 

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    Language:Japanese  

    Venue:淡路  

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  • Roles of Dα8, Dα413, Gα556, and Gα557 in the ATPase domain of the reactivating factor for coenzyme B12-dependent diol dehydratase

    Koji Obayashi, Toshiyuki Yoshinaga, Koichi Mori, Tetsuo Toraya

    第78回日本生化学会大会  2005.10.20  日本生化学会

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    Venue:神戸  

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  • Characterization and action of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase

    Naoki Hieda, Reiko Bando, Koichi Mori, Tetsuo Toraya

    第78回日本生化学会大会  2005.10.20  日本生化学会

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    Venue:神戸  

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  • Characterization of the complexes between coenzyme B12-dependent diol dehydratase and its reactivating factor

    Koichi Mori, Yasuhiro Hosokawa, Toshiyuki Yoshinaga, Tetsuo Toraya

    第78回日本生化学会大会  2005.10.20  日本生化学会

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    Venue:神戸  

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  • How a damaged cofactor is released from a coenzyme B12 dependent enzyme: Crystal structures of diol dehydratase-reactivating factor

    Naoki Shibata, Koichi Mori, Naoki Hieda, Yoshiki Higuchi, Mamoru Yamanishi, Tetsuo Toraya

    第78回日本生化学会大会  2005.10.20  日本生化学会

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    Venue:神戸  

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  • Crystallization and structure analysis of molecular chaperone-like reactivating factor for coenzyme B12-dependent diol dehydratase International conference

    Tetsuo Toraya, Naoki Shibata, Koichi Mori, Naoki Hieda, Yoshiki Higuchi, Mamoru Yamanishi

    30th FEBS Congress-9th IUBMB Conference  2005.7.3 

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    Venue:Budapest  

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  • Streptomyces phospholipase Dの転移反応に関わるアミノ酸残基について

    上杉佳子, 森光一, 岩渕雅樹, 畑中唯史

    日本農芸化学会2005年度大会  2005.3.29  日本農芸化学会

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    Venue:札幌  

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  • Stereochemical aspects of catalysis, glycerol inactivation, and reactivation of coenzyme B12-dependent diol and glycerol dehydratases

    Tetsuo Toraya, Koichiro Kinoshita, Masaki Fukuoka, Yuka Nakanishi, Naoki Shibata, Koichi Mori, Takamasa Tobimatsu, Mamoru Yamanishi

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University  2005.3.18 

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    Language:Japanese  

    Venue:牛窓  

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  • Characterization of complexes between adenosylcobalamin-dependent diol dehydratase and its reactivating factor

    Koichi Mori, Toshiyuki Yoshinaga, Tetsuo Toraya

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University  2005.3.18 

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    Language:Japanese  

    Venue:牛窓  

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  • Characterization of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase

    Naoki Hieda, Reiko Bando, Koichi Mori, Tetsuo Toraya

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University  2005.3.8 

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    Language:Japanese  

    Venue:牛窓  

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  • Presence and role of Ca2+ in the reactivating factor for adenosylcobalamin-dependent diol dehydaratase

    Shoshiro Hirayama, Yasuhiro Hosokawa, Koichi Mori, Tetsuo Toraya

    第77回日本生化学会大会  2004.10.14  日本生化学会

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    Language:Japanese  

    Venue:横浜  

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  • Improvement of in vivo DNA shuffling system

    Yoshiko Uesugi, Koichi Mori, Masaki Iwabuchi, Tadashi Hatanaka

    The 1st Pacific-Rim International Conference on Protein Science  2004.4.15 

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    Venue:横浜  

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  • Phospholipase Dの転移反応に関わるアミノ酸残基の探索

    上杉佳子, 森光一, 岩渕雅樹, 畑中唯史

    日本農芸化学会2004年度大会  2004.3.30  日本農芸化学会

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    Venue:広島  

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  • ホスホリパーゼDの耐熱性に関わるアミノ酸残基の探索

    森光一, 岩渕雅樹, 畑中唯史

    日本生物工学会平成15年度大会  2003.9.17  日本生物工学会

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    Venue:熊本  

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  • ホスホリパーゼDにおける温度感受性に関わるアミノ酸残基の探索 (2)

    森光一, 岩渕雅樹, 畑中唯史

    日本農芸化学会2003年度大会  2003.4.2  日本農芸化学会

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    Language:Japanese  

    Venue:藤沢  

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  • ホスホリパーゼDにおける温度感受性に関わるアミノ酸残基の探索

    畑中唯史, 森光一, 岩渕雅樹

    日本農芸化学会2003年度大会  2003.4.2  日本農芸化学会

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    Venue:藤沢  

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  • 大腸菌ssb-3変異株を用いた新規in vivo DNAシャフリング系の開発とホスホリパーゼDへの応用

    森光一, 向原隆文, 岩淵雅樹, 畑中唯史

    日本生物工学会平成14年度大会  2002.10.28  日本生物工学会

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    Venue:大阪  

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  • ビタミンB12補酵素関与グリセロールデヒドラターゼ再活性化因子の精製と性質

    梶浦英樹, 森光一, 飛松孝正, 虎谷哲夫

    第74回日本生化学会大会  2001.11.25  日本生化学会

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    Venue:京都  

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  • グリセロールデヒドラターゼ再活性化因子の発見と作用機作

    梶浦英樹, 森光一, 飛松孝正, 虎谷哲夫

    第384回ビタミンB研究委員会  2001.11.20  ビタミンB研究委員会

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    Venue:名古屋  

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  • アデノシルコバラミン関与エタノールアミンアンモニアリアーゼの再活性化因子の作用機作

    森光一, 坂東礼子, 虎谷哲夫

    第73回日本生化学会大会  2000.10.13  日本生化学会

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    Venue:横浜  

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  • The mechanisms of action of coenzyme B12-dependent diol dehydratase and its reactivating factor International conference

    Tetsuo Toraya, Koichi Mori, Masahiro Kawata, Mamoru Yamanishi, Takamasa Tobimatsu, Masataka Eda, Kazunari Yoshizawa, Jun Masuda, Naoki Shibata, Yukio Morimoto, Noritake Yasuoka

    5th European Symposium on Vitamin B12 and B12-Proteins  2000.9.11 

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    Language:English   Presentation type:Symposium, workshop panel (public)  

    Venue:Marburg  

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  • ビタミンB12補酵素関与エタノールアミンアンモニアリアーゼの不活性化されたホロ酵素の再活性化。再活性化因子の遺伝子クローン化と確認

    森光一, 坂東礼子, 虎谷哲夫

    日本ビタミン学会第52回大会  2000.5.20  日本ビタミン学会

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    Venue:岡山  

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  • 再活性化能を増強した大腸菌ジオールデヒドラターゼ発現系の構築と補酵素アナログの活性測定

    虎谷哲夫, 山田盛輝, 森光一, 山西守

    日本ビタミン学会第51回大会  1999.6.4  日本ビタミン学会

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    Venue:静岡  

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▼display all

Industrial property rights

  • 1,3-プロパンジオールの製造方法

    虎谷哲夫, 飛松孝正, 山西守, 森光一, 梶浦英樹, 山田盛輝, 柚木路生, 東宗明, 原哲也, 安田信三

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    Applicant:株式会社日本触媒

    Application no:特願2003-337663  Date applied:2003.9.29

    Announcement no:特開2005-102533  Date announced:2005.4.21

    特許公開2005-102533

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  • ホスホリパーゼDの安定化方法

    畑中唯史, 森光一

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    Applicant:岡山県

    Application no:特願2003-313308  Date applied:2003.9.4

    Announcement no:特開2005-80519  Date announced:2005.3.31

    特許公開2005-80519

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  • 安定化型ホスホリパーゼD

    畑中唯史, 根岸智史, 森光一, 井上良計, 松村美和

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    Applicant:岡山県, 備前化成株式会社

    Application no:特願2002-259395  Date applied:2002.9.4

    Announcement no:特開2004-97011  Date announced:2004.4.2

    特許公開2004-97011

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  • 融合遺伝子の調製方法

    向原隆文, 根岸智史, 森光一, 畑中唯史

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    Applicant:岡山県

    Application no:特願2002-56519  Date applied:2002.3.1

    Announcement no:特開2003-250543  Date announced:2003.9.9

    Patent/Registration no:特許4267241  Date registered:2009.2.27  Date issued:2009.5.27

    Rights holder:岡山県

    特許公開2003-250543

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Awards

  • 化学素材研究開発振興財団記念基金「グラント」研究奨励金

    2010.9   (財) バイオインダストリー協会   ビタミンB12補酵素関与ジオールデヒドラターゼとその再活性化蛋白質を用いる高機能性繊維ポリトリメチレンテレフタレートの原料の酵素的生産

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    Award type:Award from publisher, newspaper, foundation, etc.  Country:Japan

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  • 日本ビタミン学会奨励賞

    2008.6   日本ビタミン学会   ビタミンB12補酵素関与酵素の再活性化蛋白質に関する研究

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    Award type:Award from Japanese society, conference, symposium, etc.  Country:Japan

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Research Projects

  • Basic research for the application of bacterial polyheadral organelle for bio-nano-reactor

    Grant number:15K14237  2015.04 - 2019.03

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

    Tobimatsu Takamasa

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

    Considerable number of bacteria have proteinaceous polyhedral organelle in which metabolic reactions are carried out minimize the loss of volatile metabolic intermediates. In this study, we have searched for the interaction regions between enzymes of the pdu organelle. Short N-terminal regions of medium and small subunit of diol dehydratase and PduP aldehyde dehydrogenase is shown to be required for the interaction between organelle enzymes.

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  • B12酵素とその分子シャペロン様再活性化因子との超分子複合体の立体構造解析

    Grant number:18054023  2006 - 2007

    日本学術振興会  科学研究費助成事業  特定領域研究

    虎谷 哲夫, 森 光一

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    Grant amount:\6400000 ( Direct expense: \6400000 )

    我々は不活性化されたB_<12>補酵素関与ジオールデヒドラターゼ(DD)ホロ酵素を再活性化するDD再活性化因子(DDR)を発見し、生化学的および結晶構造学的解析を行って作用機作を明らかにしてきた。本研究では、DDおよびDDRの結晶構造解析の結果からDD-DDR複合体の形成に重要であると予測されるアミノ酸残基に変異を導入した変異型蛋白質の作成と機能解析、ATPアナログ結合型DDRの結晶構造解析、DD-DDR超分子複合体の結晶化、の3点について研究を行った。まず、変異型DDRと野生型DDを共発現した大腸菌の透過性菌体の系を用いて、グリセロールで不活性化されたDDの再活性化を調べた結果とDDおよびDDRの立体構造から、DD-DDR複合体中ではDDRのGluβ31に代わって、DDのGluβ97がMg^<2+>を介してDDRαサブユニットと相互作用し、DDRβサブユニットの解離が起こることが示唆された。また、グリセロールデヒドラターゼ(GD)およびGD再活性化因子(GDR)の精製蛋白質も用いて調べたところ、DDRはGDを再活性化できるが、GDRはDDを再活性化できないという再活性化因子の特異性は酵素との複合体形成能によって決定されていることが強く示唆された。これは立体構造に基づく両者の接触面積の計算結果からも支持された。よって、再活性化には安定な酵素一再活性化因子複合体の形成が不可欠であると結論できた。先に、ADP結合型DDRの立体構造を報告したが、再活性化の分子機構解明のためにはATP結合型の構造解析が必要である。今回は、ATP-y-S結合型DDRについて約3Åの分解能で結晶構造解析に成功した。さらに、DD-DDR超分子複合体の構造解析のために、複合体形成の過程で解離するDDRβサブユニットを系外へ除くことにより複合体を安定化し、結晶化を試みたが、結晶は得られなかった。

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  • ビタミンB12補酵素関与ジオールデヒドラターゼ再活性化因子の精密作用機構の解明

    Grant number:18770111  2006 - 2007

    日本学術振興会  科学研究費助成事業  若手研究(B)

    森 光一

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

    本年度計画していたATP結合型ジオールデヒドラターゼ再活性化因子(DDR)、およびジオールデヒドラターゼ(DD)・DDR複合体のX線結晶構造解析のうち、DD・DDR複合体については結晶を得るに至らなかったが、ATP結合型DDR(DDに対する低親和性型)に関しては、ヌクレオチド非結合型DDRの結晶にATPの非加水分解性アナログであるATPγSをソーキングし、その結晶を用いてSpring-8にてデータ測定を行った。以前に報告したADP結合型DDR(DDに対する低親和性型)の構造を用いて分子置換法で構造決定を行い、3.3Åの分解能でATPγS結合型DDRの構造を明らかにした。今後さらなる条件の改良によって分解能を向上させ、ADP結合型DDRとの構造比較を行う。また、前年度に発現系を構築した、DD・DDR複合体の形成に重要であると考えられるアミノ酸残基に変異を導入した変異型DDRを発現・精製し、再活性化やDD・DDR複合体の形成に対する影響を解析する予定であったが、精製のためにDDRαサブユニットのN末端に付加したHisタグや変異導入の影響により、蛋白質の発現においていくつかの問題が発生した。今回発現系を構築した変異型DDRはα/βサブユニットの相互作用に関係するアミノ酸残基に変異を導入しており、蛋白質の安定性の問題から迅速に精製する必要があり、精製用のタグの導入は不可欠である。そこで発現系の改良を行い、DDRαサブユニットのC末端側にHisタグを付加したところ、タグを付加していない天然型DDRと同様の発現を示したので、このプラスミドに部位特異的変異を導入し、変異型DDRの発現系を構築した。本年度中に結果を得ることは出来なかったが、今後、精製した変異型DDRを用いて機能解析を行う予定である。これらの実験で得られる結果は再活性化の詳細な分子機構の解明につながることが期待される。

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  • Structural biochemistry of radical-utilizing enzymes and their activating proteins

    Grant number:17370038  2005 - 2008

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

    TORAYA Tetsuo, TOBIMATSU Takamasa, MORI Koichi

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    Grant amount:\12360000 ( Direct expense: \11400000 、 Indirect expense:\960000 )

    ラジカル酵素は、生体内においてラジカルの高い反応性を制御しつつ利用することで化学的に困難な反応を触媒する。本研究では、B12関与酵素および他のラジカル酵素の結晶構造を解析し、各種の方法論を駆使して精密触媒機構を解明した。また、B12関与ラジカル酵素の再活性化蛋白質の結晶構造を解析し、それらの精密作用機作を明らかにした。さらに、両者の共発現により、ラジカル酵素の不安定性が克服でき、有用物質生産に有効であることを示した。

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Class subject in charge

  • Advanced Internship for Interdisciplinary Medical Sciences and Engineering (2023academic year) Year-round  - その他

  • Technical English for Interdisciplinary Medical Sciences and Engineering (2023academic year) Late  - その他

  • Research Works for Interdisciplinary Medical Sciences and Engineering (2023academic year) Year-round  - その他

  • Research Works for Interdisciplinary Medical Sciences and Engineering (2023academic year) Year-round  - その他

  • Advanced Molecular Enzymology (2023academic year) Late  - 木1~2

  • Experiment for Chemistry and Biotechnology 2 (2023academic year) Fourth semester  - 月5~8

  • Synthetic Chemistry Experiment 1 (2023academic year) Fourth semester  - 月5~8

  • Laboratory Work and Practice on Basic Engineering (2023academic year) 1st and 2nd semester  - 火5〜8

  • Mathematical and Data Sciences(Advanced) (2023academic year) Fourth semester  - 火1~2

  • Material Process Experiment 1 (2023academic year) Fourth semester  - 月5~8

  • 特別研究 (2023academic year) 通年  - その他

  • Biotechnology experiment 1 (2023academic year) Fourth semester  - 月5~8

  • Biotechnology experiment 2 (2023academic year) Third semester  - 火5〜8,金5〜8

  • Probability and Statistics 2 (2023academic year) Fourth semester  - 火1~2

  • Technical English for Interdisciplinary Medical Sciences and Engineering (2022academic year) Late  - その他

  • Research Works for Interdisciplinary Medical Sciences and Engineering (2022academic year) Year-round  - その他

  • Design of Artificial Biofunctional Molecules (2022academic year) Late  - 火3~4

  • Experiment for Chemistry and Biotechnology 2 (2022academic year) Fourth semester  - 月5~8

  • Synthetic Chemistry Experiment 1 (2022academic year) Fourth semester  - 月5~8

  • Laboratory Work and Practice on Basic Engineering (2022academic year) 1st and 2nd semester  - 火5〜8

  • Mathematical and Data Sciences(Advanced) (2022academic year) Fourth semester  - 火1~2

  • Material Process Experiment 1 (2022academic year) Fourth semester  - 月5~8

  • 特別研究 (2022academic year) 通年  - その他

  • Biotechnology experiment 1 (2022academic year) Fourth semester  - 月5~8

  • Biotechnology experiment 3 (2022academic year) Third semester  - 火5〜8,金5〜8

  • Biotechnology experiment 3 (2022academic year) Third semester  - 火5〜8,金5〜8

  • Probability and Statistics 2 (2022academic year) Fourth semester  - 火1~2

  • Enzyme Engineering (2022academic year) Fourth semester  - 水1〜2

  • Technical English for Interdisciplinary Medical Sciences and Engineering (2021academic year) Late  - その他

  • Research Works for Interdisciplinary Medical Sciences and Engineering (2021academic year) Year-round  - その他

  • Laboratory Work and Practice on Basic Engineering (2021academic year) 1st and 2nd semester  - 火5,火6,火7,火8

  • Radiation Safety Usage Engineering 2 (2021academic year) Second semester  - 金5,金6

  • Radiation Safety Engineering and Experiments (2021academic year) 1st and 2nd semester  - 金5,金6

  • 特別研究 (2021academic year) 通年  - その他

  • Biotechnology experiment 1 (2021academic year) Fourth semester  - 月5,月6,月7,月8,木5,木6,木7,木8

  • Biotechnology experiment 3 (2021academic year) Third semester  - 火5,火6,火7,火8,金5,金6,金7,金8

  • Biotechnology experiment 1 (2021academic year) Fourth semester  - 月5,月6,月7,月8,木5,木6,木7,木8

  • Biotechnology experiment 3 (2021academic year) Third semester  - 火5,火6,火7,火8,金5,金6,金7,金8

  • Probability and Statistics 2 (2021academic year) Fourth semester  - 火1,火2

  • Enzyme Engineering (2021academic year) Fourth semester  - 水1,水2

  • Technical English for Interdisciplinary Medical Sciences and Engineering (2020academic year) Late  - その他

  • Research Works for Interdisciplinary Medical Sciences and Engineering (2020academic year) Year-round  - その他

  • Design of Artificial Biofunctional Molecules (2020academic year) Late  - 火3,火4

  • Practical Interdisciplinary Medical Sciences and Engineering (2020academic year) Fourth semester  - その他

  • Laboratory Work and Practice on Basic Engineering (2020academic year) 1st and 2nd semester  - 火4,火5,火6,火7

  • Radiation Safety Usage Engineering 2 (2020academic year) Second semester  - 金5,金6

  • Radiation Safety Engineering and Experiments (2020academic year) 1st and 2nd semester  - 金5,金6

  • 特別研究 (2020academic year) 通年  - その他

  • Biotechnology experiment 1 (2020academic year) Fourth semester  - 月4,月5,月6,月7,木4,木5,木6,木7

  • Biotechnology experiment 3 (2020academic year) Third semester  - 火4,火5,火6,火7,金4,金5,金6,金7

  • Biotechnology experiment 1 (2020academic year) Fourth semester  - 月4,月5,月6,月7,木4,木5,木6,木7

  • Biotechnology experiment 3 (2020academic year) Third semester  - 火4,火5,火6,火7,金4,金5,金6,金7

  • Enzyme Engineering (2020academic year) Fourth semester  - 水1,水2

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