Development of protein sequence data mining method to design total sequence of artificial amino acid metabolism enzymes

• Project/Area Number: 18K14391
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 38030:Applied biochemistry-related
• Research Institution: University of Shizuoka
• Principal Investigator: Nakano Shogo 静岡県立大学, 食品栄養科学部, 助教 (80748541)
• Project Period (FY): 2018-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: L-アミノ酸酸化酵素 / 祖先型設計 / D-アミノ酸 / 光学分割 / 基質特異性 / アミノ酸酸化酵素 / L-アミノ酸 / タンパク質工学 / 人工タンパク質 / アミノ酸代謝関連酵素 / 祖先型設計法

Outline of Final Research Achievements

In this study, we try to develop a method to curate protein sequence data and increase succession rate for the design of highly functional artificial enzymes. By applying the method to the design of two enzymes belonging to L-amino acid oxidase family (L-arginine oxidase and L-amino acid oxidase), we can generate artificial enzymes bearing high stability, solubility and reactivity with ancestral sequence reconstruction.

Academic Significance and Societal Importance of the Research Achievements

データベースに登録されるアミノ酸配列データは今後も際限なく拡大していくと予測されている。本手法は拡大を続ける配列データベースから必要な情報を選抜し、高機能化した人工酵素配列の設計を可能にするなど、配列データベースの持つ潜在能力を引き出すことができる。また本研究で得られた人工酵素群の機能は、自然界のものより優れており、産業応用への展開が期待できるものも多かった。本成果は人工酵素は自然界由来の酵素より機能が劣るという、一般論を覆すものであると考えている。

Research Products

• [Journal Article] Elucidation of Molecular Mechanism of a Selective PPARα Modulator, Pemafibrate, through Combinational Approaches of X-ray Crystallography, Thermodynamic Analysis, and First-Principle Calculations (2020)
  • Author(s): Kawasaki Mayu、Kambe Akira、Yamamoto Yuta、Arulmozhiraja Sundaram、Ito Sohei、Nakagawa Yoshimi、Tokiwa Hiroaki、Nakano Shogo、Shimano Hitoshi
  • Journal Title: International Journal of Molecular Sciences Volume: 21 Issue: 1 Pages: 361-361
  • DOI: 10.3390/ijms21010361
  • NAID: 120007132701
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Deracemization and Stereoinversion to Aromatic d-Amino Acid Derivatives with Ancestral l-Amino Acid Oxidase (2019)
  • Author(s): Nakano Shogo、Minamino Yuki、Hasebe Fumihito、Ito Sohei
  • Journal Title: ACS Catalysis Volume: 9 Issue: 11 Pages: 10152-10158
  • DOI: 10.1021/acscatal.9b03418
  • Peer Reviewed
• [Journal Article] Competitive Binding Assay with an Umbelliferone-Based Fluorescent Rexinoid for Retinoid X Receptor Ligand Screening (2019)
  • Author(s): Yamada Shoya、Kawasaki Mayu、Fujihara Michiko、Watanabe Masaki、Takamura Yuta、Takioku Maho、Nishioka Hiromi、Takeuchi Yasuo、Makishima Makoto、Motoyama Tomoharu、Ito Sohei、Tokiwa Hiroaki、Nakano Shogo、Kakuta Hiroki
  • Journal Title: Journal of Medicinal Chemistry Volume: 62 Issue: 19 Pages: 8809-8818
  • DOI: 10.1021/acs.jmedchem.9b00995
  • NAID: 120006882792
  • Peer Reviewed
• [Journal Article] Kinetics-based structural requirements of human immunoglobulin G binding peptides (2019)
  • Author(s): Kyohei Muguruma, Konomi Fujita, Akane Fukuda, Satoshi Kishimoto, Soichiro Sakamoto, Risako Arima, Mayu Ito, Mayu Kawasaki, Shogo Nakano, Sohei Ito, Kanade Shimizu, Akihiro Taguchi, Kentaro Takayama, Atsuhiko Taniguchi, Yuji Ito, Yoshio Hayashi
  • Journal Title: ACS Omega Volume: 4 Issue: 11 Pages: 14390-14397
  • DOI: 10.1021/acsomega.9b01104
  • Peer Reviewed / Open Access
• [Journal Article] Following the Evolutionary Track of a Highly Specific L-Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes (2019)
  • Author(s): Nakano Shogo、Niwa Masazumi、Asano Yasuhisa、Ito Sohei
  • Journal Title: Applied and Environmental Microbiology Volume: 10 Issue: 12 Pages: 00459-00459
  • DOI: 10.1128/aem.00459-19
  • Peer Reviewed
• [Journal Article] Development of analysis toolkit to visualize interaction energies generated by fragment molecular orbital calculations (2018)
  • Author(s): Takaki Tokiwa, Shogo Nakano, Yuta Yamamoto, Takeshi Ishikawa, Sohei Ito, Vladimir Sladek, Kaori Fukuzawa, Yuji Mochizuki, Hiroaki Tokiwa, Fuminori Misaizu, and Yasuteru Shigeta
  • Journal Title: J. Chem. Info. Model Volume: 59(1) Issue: 1 Pages: 25-30
  • DOI: 10.1021/acs.jcim.8b00649
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Dual conformation of the ligand induces the partial agonistic activity of retinoid X receptor α (RXRα) (2018)
  • Author(s): Miyashita Yurina、Numoto Nobutaka、Arulmozhiraja Sundaram、Nakano Shogo、Matsuo Naoya、Shimizu Kanade、Shibahara Osamu、Fujihara Michiko、Kakuta Hiroki、Ito Sohei、Ikura Teikichi、Ito Nobutoshi、Tokiwa Hiroaki
  • Journal Title: FEBS Letters Volume: 593 Issue: 2 Pages: 242-250
  • DOI: 10.1002/1873-3468.13301
  • Peer Reviewed
• [Journal Article] Improvement of ST0452 N-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering (2018)
  • Author(s): Honda Yuki、Nakano Shogo、Ito Sohei、Dadashipour Mohammad、Zhang Zilian、Kawarabayasi Yutaka
  • Journal Title: Applied and Environmental Microbiology Volume: 84 Issue: 24
  • DOI: 10.1128/aem.02213-18
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Computational Molecular Docking and X-ray Crystallographic Studies of Catechins in New Drug Design Strategies (2018)
  • Author(s): Nakano Shogo、Megro Shin-ichi、Hase Tadashi、Suzuki Takuji、Isemura Mamoru、Nakamura Yoriyuki、Ito Sohei
  • Journal Title: Molecules Volume: 23 Issue: 8 Pages: 2020-2020
  • DOI: 10.3390/molecules23082020
  • Peer Reviewed / Open Access
• [Journal Article] In Vitro and In Silico Studies of the Molecular Interactions of Epigallocatechin-3-O-gallate (EGCG) with Proteins That Explain the Health Benefits of Green Tea (2018)
  • Author(s): Saeki Koichi、Hayakawa Sumio、Nakano Shogo、Ito Sohei、Oishi Yumiko、Suzuki Yasuo、Isemura Mamoru
  • Journal Title: Molecules Volume: 23 Issue: 6 Pages: 1295-1295
  • DOI: 10.3390/molecules23061295
  • Peer Reviewed / Open Access
• [Journal Article] Binding of Catechins to Staphylococcal Enterotoxin A (2018)
  • Author(s): 2.Shimamura, Y., Utsumi, M., Hirai, C., Nakano, S., Ito, S., Tsuji, A., Ishii, T., Hosoya, T., Kan, T., Ohashi, N., Masuda. S
  • Journal Title: Molecules Volume: 23(5) Issue: 5 Pages: 1125-1125
  • DOI: 10.3390/molecules23051125
  • Peer Reviewed / Open Access
• [Journal Article] Benchmark analysis of native and artificial NAD+-dependent enzymes generated by a sequence based design method with or without phylogenetic data (2018)
  • Author(s): Shogo Nakano, Tomoharu Motoyama, Yurina Miyashita, Yuki Ishizuka, Naoya Matsuo, Hiroaki Tokiwa, Suguru Shinoda, Yasuhisa Asano, and Sohei Ito
  • Journal Title: Biochemistry Volume: 57(26) Issue: 26 Pages: 3722-3732
  • DOI: 10.1021/acs.biochem.8b00339
  • Peer Reviewed / Open Access
• [Journal Article] 新規アミノ酸配列解析手法の開発とタンパク質工学への応用 (2018)
  • Author(s): 中野 祥吾
  • Journal Title: 生物工学会誌 Volume: 96 Pages: 573-577
• [Journal Article] タンパク質の二次構造とアミノ酸の疎水性度に基づいた凝集に関連するホットスポットの合理的同定法 (2018)
  • Author(s): 松井 大亮, 中野 祥吾, Mohammad Dadashipour, 浅野 泰久
  • Journal Title: 酵素工学ニュース Volume: 80
• [Presentation] In silicoでタンパク質を創る (2019)
  • Author(s): 中野 祥吾
  • Organizer: 細胞を創る研究会12.0
  • Invited
• [Presentation] Reconstruction of ancestral L-amino acid oxidase to broaden substrate selectivity (2019)
  • Author(s): Shogo Nakano
  • Organizer: Enzyme Engineering XXV
  • Int'l Joint Research
• [Presentation] Filling a gap between computational and experimental researches through sequence-based protein design (2018)
  • Author(s): Shogo Nakano
  • Organizer: The 4th International Conference on Pharma and Food (ICPF) 2018
  • Int'l Joint Research / Invited
• [Presentation] 新規アミノ酸配列解析手法、INTMSAlign の開発と応用 (2018)
  • Author(s): 中野 祥吾
  • Organizer: 第１３８回日本薬学会大会
  • Invited
• [Patent(Industrial Property Rights)] 糖鎖結合性一本鎖抗体およびその作製方法 (2019)
  • Inventor(s): 隅田泰生,常盤広明,伊藤創平,中野祥吾,新地浩之
  • Industrial Property Rights Holder: 隅田泰生,常盤広明,伊藤創平,中野祥吾,新地浩之
  • Industrial Property Rights Type: 特許
  • Patent Publication Number: 2019-199472
  • Filing Date: 2019