Principles for assembling cascade enzymes

• Project/Area Number: 25248038
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bio-related chemistry
• Research Institution: Kyoto University
• Principal Investigator: MORII Takashi 京都大学, エネルギー理工学研究所, 教授 (90222348)
• Project Period (FY): 2013-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥46,540,000 (Direct Cost: ¥35,800,000、Indirect Cost: ¥10,740,000); Fiscal Year 2016: ¥11,180,000 (Direct Cost: ¥8,600,000、Indirect Cost: ¥2,580,000); Fiscal Year 2015: ¥11,310,000 (Direct Cost: ¥8,700,000、Indirect Cost: ¥2,610,000); Fiscal Year 2014: ¥11,310,000 (Direct Cost: ¥8,700,000、Indirect Cost: ¥2,610,000); Fiscal Year 2013: ¥12,740,000 (Direct Cost: ¥9,800,000、Indirect Cost: ¥2,940,000)
• Keywords: 核酸関連化学 / タンパク質 / 単分子配置 / 酵素 / 代謝 / DNAナノテクノロジー / 受容体 / 人工代謝系 / DNAナノ構造体 / 代謝反応 / 酵素反応 / 多段階反応 / 分子コンビナート / クラスター効果

Outline of Final Research Achievements

Understanding the effect of spatial organization on the efficiency of multi-enzyme systems reveals a principle that efficiently drives multi-enzymatic cascade. DNA nanostructures provide useful scaffolds for the spatial organization of enzymes to form artificial enzyme cascades. We have developed methods to use sequence-specific DNA binding proteins as adaptors to stably locate the enzymes at specific positions on DNA scaffold. Our protein-adaptor-based method successfully assembled recombinant enzymes in high loading yields with distinctly controlling the number of enzyme molecules and maintaining the catalytic activities of enzymes. An artificial enzyme cascade of xylose reductase (XR) and xylitol dehydrogenase (XDH) has been constructed by coassembling the enzymes on DNA origami. Further extension of this artificial metabolic pathway was achieved by using modular adaptors equipped with the protein-tag to successfully construct XR/XDH/xylulose kinase.

Research Products

• [Journal Article] Construction of a library of structurally diverse ribonucleopeptides with catalytic groups (2017)
  • Author(s): T. Tamura, S. Nakano, E. Nakata, T. Morii
  • Journal Title: Bioorg. Med. Chem. Volume: 25 Issue: 6 Pages: 1881-1888
  • DOI: 10.1016/j.bmc.2017.02.007
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Latent pH-responsive ratiometric fluorescent cluster based on self-assembled photoactivated SNARF derivatives (2016)
  • Author(s): E. Nakata, Y. Yukimachi, Y. Uto, H. Hori, T. Morii
  • Journal Title: Sci. Tech. Adv. Mater. Volume: 17 Issue: 1 Pages: 431-436
  • DOI: 10.1080/14686996.2016.1204888
  • NAID: 120006323761
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds (2015)
  • Author(s): E. Nakata, H. Dinh, T.A. Ngo, M. Saimura, T. Morii
  • Journal Title: Chem. Commun. Volume: 51 Issue: 6 Pages: 1016-1019
  • DOI: 10.1039/c4cc08167f
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Peptidosteroid Tweezers Revisited: DNA Binding Through an Optimised Design (2014)
  • Author(s): Carrette, Lieselot L. G.; Morii, Takashi; Madder, Annemieke
  • Journal Title: Eur. J. Org. Chem. Volume: 14 Issue: 14 Pages: 2883-2891
  • DOI: 10.1002/ejoc.201301854
  • Peer Reviewed
• [Journal Article] Phosphorylation regulates fibrillation of an aggregation core peptide in the second repeat of microtubule-binding domain of human tau (2014)
  • Author(s): M. Inoue, S. Kaida, S. Nakano, C. Annoni, E. Nakata, T. Konno, T. Morii
  • Journal Title: Bioorg. Med. Chem。 Volume: 22 Issue: 22 Pages: 6471-6480
  • DOI: 10.1016/j.bmc.2014.09.032
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells (2013)
  • Author(s): Shigeki Kiyonaka, Taketoshi Kajimoto, Reiko Sakaguchi, Daisuke Shinmi, Mariko Omatsu-Kanbe, Hiroshi Matsuura, Hiromi Imamura, Takenao Yoshizaki, Itaru Hamachi, Takashi Morii, Yasuo Mori
  • Journal Title: Nature Methods Volume: 10 Issue: 12 Pages: 1232-1238
  • DOI: 10.1038/nmeth.2690
  • Peer Reviewed
• [Journal Article] Toxicity inspired cross-linking for probing DNA-peptide interactions (2013)
  • Author(s): Carrette, Lieselot L. G.; Morii, Takashi; Madder, Annemieke
  • Journal Title: Bioconjugate Chemistry Volume: 24（12） Issue: 12 Pages: 2008-2014
  • DOI: 10.1021/bc400327q
  • Peer Reviewed
• [Journal Article] A protein adaptor to locate a functional protein dimer on molecular switchboard (2013)
  • Author(s): Tien Anh Ngo, Eiji Nakata, Masayuki Saimura, Tsutomu Kodaki, Takashi Morii
  • Journal Title: Methods Volume: 0 Issue: 2 Pages: 1-9
  • DOI: 10.1016/j.ymeth.2013.10.014
  • Peer Reviewed
• [Presentation] A Bioorganic Chemistry Approach to Understanding Molecular Recognition in Protein-Nucleic Acid Complexes (2017)
  • Author(s): 森井 孝
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
  • Invited
• [Presentation] 蛍光タンパク質を基本骨格とした一酸化窒素センサーの構築 (2017)
  • Author(s): 田嶋 竣介・中田 栄司・才村 正幸・森井 孝
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
• [Presentation] DNA オリガミを利用したナノリポソーム (2017)
  • Author(s): 小西 宏明・中田 栄司・仲野 瞬・森井 孝
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
• [Presentation] ATP sensors selected from a fluorophore librar (2017)
  • Author(s): S. NAKANO, T. TAMURA, Y-T. CHANG, T. MORII
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
• [Presentation] Topologically interlocked DNA nanostructures inside a DNA origami (2017)
  • Author(s): A. RAJENDRAN, S. PARK, E. NAKATA, Y. KWON, T. MORII
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
• [Presentation] Activity of spatially organized enzyme on DNA scaffold (2017)
  • Author(s): H. DINH, E. NAKATA, T. MORII
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
• [Presentation] Orthogonal modular adaptors for assembling multiple enzymes on DNA scaffold (2017)
  • Author(s): T. NGUYEN, E. NAKATA, M. SAIMURA, T. MORII
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
• [Presentation] Catalytic activity of an RNP receptor library with structural diversity (2017)
  • Author(s): T. TAMURA, S. NAKANO, T. MORII
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
• [Presentation] Enzymatic cascade reactions on the DNA scaffold (2017)
  • Author(s): E. NAKATA, H. DINH, T. NGUYEN, M. SAIMURA, T. MORII
  • Organizer: 日本化学会 第97春季年会2017
  • Place of Presentation: 横浜市、慶應義塾大学
  • Year and Date: 2017-03-16
• [Presentation] Evaluation of orthogonal modular adaptors for assembling proteins on DNA scaff old (2016)
  • Author(s): T.M. Nguyen, E. Nakata, M. Saimura, T. Morii
  • Organizer: The 43rd International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: kumamoto city, 100th Anniversary Hall, Kumamoto University
  • Year and Date: 2016-09-27
  • Int'l Joint Research
• [Presentation] Activity of the assembled enzyme on DNA scaff old (2016)
  • Author(s): H.T.T. Dinh, E. Nakata, T.A. Ngo, T. Morii
  • Organizer: The 43rd International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: kumamoto city, 100th Anniversary Hall, Kumamoto University
  • Year and Date: 2016-09-27
  • Int'l Joint Research
• [Presentation] Screening of ribonucleopeptide receptor-based sensors by using fluorophore library (2016)
  • Author(s): S. Nakano, T. Tamura, Y.T. Chang, T. Morii
  • Organizer: The 43rd International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: kumamoto city, 100th Anniversary Hall, Kumamoto University
  • Year and Date: 2016-09-27
  • Int'l Joint Research
• [Presentation] The efficiency of cascade reactions by spatially organized enzymes on DNA scaffold (2016)
  • Author(s): E. Nakata, T.A. Ngo, M. Saimura, T. Morii
  • Organizer: The 43rd International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: kumamoto city, 100th Anniversary Hall, Kumamoto University
  • Year and Date: 2016-09-27
  • Int'l Joint Research
• [Presentation] Construction of a structurally diverse library of ribonucleopeptides (2016)
  • Author(s): T. Tamura, S. Nakano, T. Morii
  • Organizer: The 43rd International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: kumamoto city, 100th Anniversary Hall, Kumamoto University
  • Year and Date: 2016-09-27
  • Int'l Joint Research
• [Presentation] DNAナノ構造体に酵素を配置した分子スイッチボード (2016)
  • Author(s): 中田栄司，Ngo Anh Tien，Dinh Huyen，Nguyen Minh Thang，才村正幸，森井孝
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 金沢市、石川県立音楽堂もてなしドーム地下広場
  • Year and Date: 2016-09-07
• [Presentation] 蛍光タンパク質を基本骨格とした一酸化窒素センサー (2016)
  • Author(s): 田嶋竣介，中田栄司，才村正幸，森井孝
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 金沢市、石川県立音楽堂もてなしドーム地下広場
  • Year and Date: 2016-09-07
• [Presentation] 蛍光分子ライブラリーを利用したリボヌクレオペプチドセンサーの構築 (2016)
  • Author(s): 仲野瞬，田村友樹，Chang Young-Tae，森井孝
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 金沢市、石川県立音楽堂もてなしドーム地下広場
  • Year and Date: 2016-09-07
• [Presentation] 構造多様性を有するRNP リセプターライブラリーの構築 (2016)
  • Author(s): 田村友樹，仲野瞬，森井孝
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 金沢市、石川県立音楽堂もてなしドーム地下広場
  • Year and Date: 2016-09-07
• [Presentation] タンパク質の選択的な配置を目指したDNAナノチューブの構造制御 (2015)
  • Author(s): 西田 圭佑・DINH Huyen・NGO Anh Tien・中田 栄司・森井 孝
  • Organizer: 日本化学会第95春季年会
  • Place of Presentation: 千葉
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] DNAナノ構造体上に配置した酵素集合体の機能評価 (2015)
  • Author(s): NGO Anh Tien・○中田 栄司・才村 正幸・森井 孝
  • Organizer: 日本化学会第95春季年会
  • Place of Presentation: 千葉
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] タグタンパク質を融合したDNA結合アダプターによるDNAナノ構造体とタンパク質間の共有結合形成 (2015)
  • Author(s): 戸田 昂人・中田 栄司・才村 正幸・森井 孝
  • Organizer: 日本化学会第95春季年会
  • Place of Presentation: 千葉
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] 共有結合を介したDNAナノ構造体へのDNA結合アダプター融合タンパク質の配置 (2015)
  • Author(s): 佐々木 謙太・中田 栄司・才村 正幸・森井 孝
  • Organizer: 日本化学会第95春季年会
  • Place of Presentation: 千葉
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Orthogonal assembly of RuBisCO and Carbonic Anhydrase on a DNA nanoscaffold (2015)
  • Author(s): DINH, Huyen; NAKATA, Eiji; NGO, Tien Anh; ASHIDA, Hiroki; YOKOTA, Akiho; MORII, Takashi
  • Organizer: 日本化学会第95春季年会
  • Place of Presentation: 千葉
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Evaluation of the cluster effect by systematically assembling ATP-binding RNP receptors on DNA origami (2015)
  • Author(s): ANNONI, Chiara; NAKANO, Shun; YOSHIMURA, Yuki; NAKATA, Eiji; MORII, Takashi
  • Organizer: 日本化学会第95春季年会
  • Place of Presentation: 千葉
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Spatially organized assembly of enzymes and receptors on the molecular switchboard (2015)
  • Author(s): 森井 孝
  • Organizer: Cutting-edge Technologies for New Drug Diacovery and Development
  • Place of Presentation: Dongguk University
  • Year and Date: 2015-02-05
  • Invited
• [Presentation] Spatially organized assembly of enzymes on the molecular switchboard (2015)
  • Author(s): 森井 孝
  • Organizer: Special Faculty Seminar
  • Place of Presentation: College of Pharmacy, Ewha University, Korea
  • Year and Date: 2015-02-03
  • Invited
• [Presentation] Site-specific and Covalent Modification of a Single Protein Molecule on DNA Nanoscaffold (2014)
  • Author(s): Eiji Nakata, Dinh Huyen,Tien Anh Ngo,Masayuki Saimura, Takashi Morii
  • Organizer: ISNAC2014 第41回国際核酸化学シンポジウム
  • Place of Presentation: 福岡
  • Year and Date: 2014-11-05
• [Presentation] Evaluation of the substrate selectivity of clustered ATP-binding RNA aptamers. (2014)
  • Author(s): Y. Yoshimura, C. Annoni, S. Nakano, E. Nakata, T. Morii
  • Organizer: ISNAC2014 第41回国際核酸化学シンポジウム
  • Place of Presentation: 福岡
  • Year and Date: 2014-11-05
• [Presentation] Application of DNA binding proteins for assembling proteins RuBisCO and Carbonic Anhydrase on DNA nanoscaffold (2014)
  • Author(s): H. Dinh, E. Nakata, T.A. Ngo, H. Ashida, A. Yokota, T. Morii
  • Organizer: ISNAC2014 第41回国際核酸化学シンポジウム
  • Place of Presentation: 福岡
  • Year and Date: 2014-11-05
• [Presentation] Assessment of both equilibrium and kinetic aspects of ATP-binding ribonucleopeptide (RNP) clusters arranged on DNA nanoscaffold (2014)
  • Author(s): C. Annoni, S. Nakano, E. Nakata, Y. Yoshimura, T. Morii
  • Organizer: ISNAC2014 第41回国際核酸化学シンポジウム
  • Place of Presentation: 福岡
  • Year and Date: 2014-11-05
• [Presentation] DNA origamiに機能性タンパク質を固定化した分子スイッチボード開発 (2014)
  • Author(s): 中田栄司・Ngo Tien Anh・Dinh Huyen・仲野瞬・才村正幸・小瀧努・森井孝
  • Organizer: 第63回高分子討論会
  • Place of Presentation: 長崎
  • Year and Date: 2014-09-26
• [Presentation] 集積化したRNAアプタマーの基質選択性評価 (2014)
  • Author(s): 吉村祐輝，アンノーニ キアラ，仲 瞬，中田栄司，森井孝
  • Organizer: 第8回バイオ関連化学シンポジウム
  • Place of Presentation: 岡山
  • Year and Date: 2014-09-11
• [Presentation] タグンパク質融合アダプーを用いたDNA ナノ構造体への 共有結合によるタンパク質ナノ構造体への配置 (2014)
  • Author(s): 戸田昂人，中田栄司，Ngo Anh Tien，Huyen Dinh，才村正幸，森井孝
  • Organizer: 第8回バイオ関連化学シンポジウム
  • Place of Presentation: 岡山
  • Year and Date: 2014-09-11
• [Presentation] DNAナノ構造体上に配置したATP結合性RNPリセプタ一の集積効果 (2014)
  • Author(s): Annoni Chiara，仲野瞬，森井孝
  • Organizer: 第8回バイオ関連化学シンポジウム
  • Place of Presentation: 岡山
  • Year and Date: 2014-09-11
• [Presentation] Application of DNA binding adaptors for assembling protein RuBisCO on DNA nano-scaffold (2014)
  • Author(s): Dinh Huyen，中田栄司，Ngo Anh Tien，蘆田弘樹，横田明穂，森井孝
  • Organizer: 第8回バイオ関連化学シンポジウム
  • Place of Presentation: 岡山
  • Year and Date: 2014-09-11
• [Presentation] Construction of organized assembly of enzymes on DNA origami (2014)
  • Author(s): T.A. Ngo, E. Nakata, M. Saimura, T. Kodaki, T. Morii
  • Organizer: 日本化学会第94春季年会
  • Place of Presentation: 名古屋大学 東山キャンパス
  • Year and Date: 2014-03-29
• [Presentation] Applications of sequence-specific DNA binding adaptors for assembling proteins on DNA origami (2014)
  • Author(s): H.T.T. Dinh, E. Nakta, A.T. Ngo, H. Ashida, A. Yokota, T. Morii
  • Organizer: 日本化学会第94春季年会
  • Place of Presentation: 名古屋大学 東山キャンパス
  • Year and Date: 2014-03-29
• [Presentation] DNAオリガミへの機能性タンパク質の１分子固定化 :共有結合型DNA結合アダプターの開発 (2014)
  • Author(s): 佐々木謙太，戸田昂人，中田栄司，森井孝
  • Organizer: 日本化学会第94春季年会
  • Place of Presentation: 名古屋大学 東山キャンパス
  • Year and Date: 2014-03-28
• [Presentation] DNAオリガミへの機能性タンパク質の1分子固定化:DNA結合アダプターとDNAオリガミとの共有結合形成 (2014)
  • Author(s): 戸田昂人，Ngo Anh Tien，Huyen Thi Thu Dinh，佐々木謙太，中田栄司，森井孝
  • Organizer: 日本化学会第94春季年会
  • Place of Presentation: 名古屋大学 東山キャンパス
  • Year and Date: 2014-03-28
• [Presentation] DNAオリガミを用いたRNAアプタマーの集積効果の評価 (2014)
  • Author(s): 吉村祐輝， ANNONI Chiara，仲野瞬，中田栄司，GELMI Maria LUISA，森井孝
  • Organizer: 日本化学会第94春季年会
  • Place of Presentation: 名古屋大学 東山キャンパス
  • Year and Date: 2014-03-27
• [Presentation] Construction of Protein Assemblies on Molecular Switchboard (2014)
  • Author(s): Takashi Morii
  • Organizer: Asian Chemical Biology Initiative 2014 Manila Meeting
  • Place of Presentation: Manila
  • Year and Date: 2014-01-25
  • Invited
• [Presentation] Site-specific covalent modification of DNA origami by functional proteins (2013)
  • Author(s): E. Nakata, H.T.T. Dinh, T.A. Ngo, M. Saimura, T. Morii
  • Organizer: The 40th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: Kanagawa University
  • Year and Date: 2013-11-14
• [Presentation] Development of a homodimeric adaptor for constructing an artificial protein assembly on molecular switchboard (2013)
  • Author(s): T.A. Ngo, E. Nakata, M. Saimura, T. Kodaki, T. Morii
  • Organizer: The 40th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: Kanagawa University
  • Year and Date: 2013-11-14
• [Presentation] Construction of a molecular switchboard by assembling ribonucleopeptide biosensors (2013)
  • Author(s): C. Annoni, F.F. Liew, S. Nakano, E. Nakata, M.L. Gelmi, T. Morii
  • Organizer: The 40th International Symposium on Nucleic Acids Chemistry
  • Place of Presentation: Kanagawa University
  • Year and Date: 2013-11-13
• [Presentation] DNA origami上に機能性タンパク質を固定化した分子スイッチボードの開発 (2013)
  • Author(s): 中田栄司，Ngo Tien Anh，仲野瞬，才村正幸，小瀧努，森井孝
  • Organizer: 第7回バイオ関連化学シンポジウム
  • Place of Presentation: 名古屋大学
  • Year and Date: 2013-09-27
• [Presentation] Development of an aritificial protein assembly on molecular switchboard (2013)
  • Author(s): T.A. Ngo, E. Nakata, M. Saimura, S. Nakano, T. Kodaki, T. Morii
  • Organizer: 第7回バイオ関連化学シンポジウム
  • Place of Presentation: 名古屋大学
  • Year and Date: 2013-09-27
• [Presentation] DNAナノ構造体による機能性タンパク質組織体の構築 (2013)
  • Author(s): 森井 孝
  • Organizer: 高分子討論会
  • Place of Presentation: 金沢大学
  • Year and Date: 2013-09-12
  • Invited
• [Presentation] Construction of Protein Assemblies on Molecular Switchboard (2013)
  • Author(s): 森井 孝
  • Organizer: A3RONA2013
  • Place of Presentation: Konan University
  • Year and Date: 2013-08-31
  • Invited
• [Presentation] Construction of a molecular switchboard by using monomeric and homodimeric protein adaptors (2013)
  • Author(s): T.A. Ngo, E. Nakata, M. Saimura, S. Nakano, T. Kodaki, T. Morii
  • Organizer: Gordon Reseach Conforence，Bioorganic Chemistry
  • Place of Presentation: Proctor Academy, Andover, NH, USA
  • Year and Date: 2013-06-09 – 2013-06-13
  • Invited
• [Remarks] 森井研ホームページ
  • URL: http://www.iae.kyoto-u.ac.jp/material/jp/index.html