Elucidation of the maturation mechanism of subtilisins from hyperthermophiles and development of their potential use

• Project/Area Number: 21380065
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Osaka University
• Principal Investigator: KANAYA Shigenori 大阪大学, 大学院・工学研究科, 教授 (30273585)
• Co-Investigator(Renkei-kenkyūsha): KOGA Yuichi 大阪大学, 大学院・工学研究科, 准教授 (30379119)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥18,720,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥4,320,000); Fiscal Year 2011: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000); Fiscal Year 2010: ¥6,760,000 (Direct Cost: ¥5,200,000、Indirect Cost: ¥1,560,000); Fiscal Year 2009: ¥8,450,000 (Direct Cost: ¥6,500,000、Indirect Cost: ¥1,950,000)
• Keywords: タンパク質工学 / 微生物酵素 / 構造生物学 / サチライシン / セリンプロテアーゼ / 超好熱菌 / 成熟化 / プロペプチド / プリオン / タンパク工学 / ジェリーロール / X線結晶構造解析 / セルピン

Research Abstract

The maturation mechanisms of subtilisins, Tk-subtilisin and Tk-SP, from the hyperthermophilic archaeon Thermococcus kodakarensis were analyzed. The results indicate that both proteins are activated(matured) upon autoprocessing and degradation of N-terminal propeprides, folding of Tk-subtilisin is induced upon binding of the Ca^<2+> ions to the Ca^<2+>-binding loop, Tk-SP is matured in the absence of the Ca^<2+> ions, Tk-SP requires C-terminalβ-jelly roll domain for hyperstability. It was also shown that Tk-subtilisin is useful for degradation of abnormal prion proteins.

Research Products

• [Journal Article] An alternative mature form of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis identified in the presence of Ca^<2+> (2011)
  • Author(s): Sinsereekul, N., Foophow, T., Yamanouchi, M., Koga, Y., Takano, K. and Kanaya, S.
  • Journal Title: FEBS J Volume: 278 Pages: 1901-1911
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/2144
  • Peer Reviewed
• [Journal Article] Inhibition of chymotrypsin-and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcu skodakaraensis (2011)
  • Author(s): Tanaka, S., Koga, Y., Takano, K., and Kanaya, S.
  • Journal Title: Biochim. Biophys. Acta Volume: 1814 Pages: 299-307
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/2111
  • Peer Reviewed
• [Journal Article] An alternative mature form of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis identified in the presence of Ca2+ (2011)
  • Author(s): Sinsereekul, N., Foophow, T., Yamanouchi, M., Koga, Y., Takano, K., Kanaya, S.
  • Journal Title: FEBS J. Volume: 278 Pages: 1901-1911
  • Peer Reviewed
• [Journal Article] Inhibition of chymotrypsin-and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis. (2011)
  • Author(s): Tanaka, S., Koga, Y., Takano, K., Kanaya, S.
  • Journal Title: Biochim.Biophys.Acta Volume: 1814 Pages: 299-307
  • Peer Reviewed
• [Journal Article] Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis : requirement of a C-terminalβ-jelly roll domain for hyperstability (2010)
  • Author(s): Foophow, T., Tanaka, S., Angkawidjaja, C., Koga, Y., Takano, K., and Kanaya, S.
  • Journal Title: J. Mol. Biol Volume: 400 Pages: 865-877
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/20595040
  • Peer Reviewed
• [Journal Article] Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N-and C-terminal propeptides (2010)
  • Author(s): Foophow, T., Tanaka, S., Koga, Y., Takano, K., Kanava, S.
  • Journal Title: Protein Eng.Des.Sel. Volume: 23 Pages: 347-355
  • Peer Reviewed
• [Journal Article] Crystal structure of a subtilisin homologue, Tk-SP from Ther mococcus kodakaraensis : requirement of a C-terminal β-jelly roll domain for hyperstability. (2010)
  • Author(s): Foophow, T., Tanaka, S., An gkawidjaja, C., Koga, Y., Takano, K., Kanaya, S.
  • Journal Title: J.Mol.Biol. Volume: 400 Pages: 865-877
  • Peer Reviewed
• [Journal Article] Sybtukusub-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides. (2010)
  • Author(s): Foophow, T., Tanaka, S., Koga, Y., Takano, K., Kanaya, S.
  • Journal Title: Protein Eng.Des.Sel. Web掲載(doi : 10. 1093/protein/gzp092)
  • Peer Reviewed
• [Journal Article] Identification of the interactions critical for propeptide-catalyzed folding of Tk-subtilisin (2009)
  • Author(s): Tanaka, S., Matsumura, H., Koga, Y., Takano, K., and Kanaya, S.
  • Journal Title: J. Mol. Biol Volume: 394 Pages: 306-319
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/19766655
  • Peer Reviewed
• [Journal Article] Requirement of a unique Ca^<2+>-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon (2009)
  • Author(s): Takeuchi, Y., Tanaka, S., Matsumura, H., Koga, Y., Takano, K., and Kanaya, S.
  • Journal Title: Biochemistry Volume: 48 Pages: 10637-10643
  • URL: http://www.ncbi.nlm.nih.gov/pubmed/19813760
  • Peer Reviewed
• [Journal Article] Identification of the interactions critical for propeptide-catalyzed folding of Tk-subtilisin. (2009)
  • Author(s): Tanaka, S., Matsumura, H., Koga, Y., Takano, K., Kanaya, S.
  • Journal Title: J.Mol.Biol. 394 Pages: 306-319
  • Peer Reviewed
• [Journal Article] Requirement of a unique Ca2+-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon. (2009)
  • Author(s): Takeuchi, Y., Tanaka, S., matsumura, H., Koga, Y., Takano, K., Kanaya, S.
  • Journal Title: Biochemistry 48 Pages: 10637-10643
  • Peer Reviewed
• [Presentation] Unique maturation mechanism of Tk-subtilisin from a hyperthermophilic archaeon (2012)
  • Author(s): 金谷茂則
  • Organizer: 2012年度日本農芸化学会
  • Place of Presentation: 京都女子大学(京都)(招待講演)
  • Year and Date: 2012-03-25
• [Presentation] Unique maturation mechanism of Tk-subtilisin from a hyperthermophilic archaeon (2012)
  • Author(s): 金谷茂則
  • Organizer: 2011年度日本農芸化学会(シンポジウム)
  • Place of Presentation: 京都女子大学(京都)
• [Presentation] Mechanism for Accelerating Maturation of Pro-Tk-subtilisin with the Mutation at the C-terminus of Propeptide (2011)
  • Author(s): Ryo Uehara, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
  • Organizer: The 2nd SeleCa Minisymposium
  • Place of Presentation: RWTH Aachen, Germany
  • Year and Date: 2011-12-13
• [Presentation] 超好熱菌由来Tk-subtilisinの耐熱化機構 (2011)
  • Author(s): 上原了、田中俊一、古賀雄一、高野和文、金谷茂則
  • Organizer: 農芸化学会関西支部第469回講演会
  • Place of Presentation: 京都府立大学合同講義棟(京都)
  • Year and Date: 2011-05-28
• [Presentation] Unique folding and stabilization mechanisms of subtilisin-like serine proteases from a hyperthermophilic archaeon (2011)
  • Author(s): Shigenori Kanaya
  • Organizer: International Conference on Enzyme Science and Technology
  • Place of Presentation: Kusadasi(Turkey)
• [Presentation] Unique folding and stabilization mechanisms of subtilisin-like serine proteases from a hyperthermophilic archaeon (2011)
  • Author(s): Shigenori Kanaya
  • Organizer: International Conference on Enzyme Science and Technology (ICEST2011)
  • Place of Presentation: Kusadasi, Turkey(招待講演)
• [Presentation] Enzymatic degradation of PrPSc with a hyperthermstable protease (2011)
  • Author(s): Yuichi Koga, Haruki Otaguro, Mutsuo Aranishi, Kouta Yuzaki, Shun-ichi Tanaka, Akikazu Sakudo, Kazufumi Takano, Shigenori Kanaya
  • Organizer: Asian Pacific Prion Symposium (APPS) 2011
  • Place of Presentation: ホテルマロウド軽井沢(長野県北佐久郡軽井沢町)
• [Presentation] Accelerated maturation of Tk-subtilisin by the mutation at the C-terminus of propeptide (2011)
  • Author(s): Ryo Uehara, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
  • Organizer: 第11回GCOE国際会議
  • Place of Presentation: 阪大吹田キャンパス銀杏会館(大阪)
• [Presentation] 超好熱性プロテアーゼTk-subtilisinの成熟化速度制御 (2011)
  • Author(s): 上原了、田中俊一、古賀雄一、高野和文、金谷茂則
  • Organizer: 第11回日本蛋白質科学会
  • Place of Presentation: ホテル阪急エキスポパーク(大阪)
• [Presentation] An alternative mature form of subtilisin homologue, Tk-SP, from Ther mococcus kodakaraensis identified in the presence of Ca2+ (2011)
  • Author(s): Nitat Sinsereekul, Tita Foophow, Mai Yamanouchi, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
  • Organizer: 第11回日本蛋白質科学会
  • Place of Presentation: ホテル阪急エキスポパーク(大阪)
• [Presentation] 異常プリオン蛋白質(PrPSc)の超好熱菌由来プロテアーゼによる分解 (2011)
  • Author(s): 古賀雄一、田中俊一、作道章一、高野和文、金谷茂則
  • Organizer: 第38回日本防菌防黴学会
  • Place of Presentation: 千里ライフサイエンスセンター(大阪府豊中市)
• [Presentation] Role of the Ca2+ ions for stability of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis (2011)
  • Author(s): Nitat Sinsereekul, Tita Foophow, Clement Angkawidjaja, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
  • Organizer: 第84回日本生化学会
  • Place of Presentation: 国立京都国際会館(京都)
• [Presentation] Thermococcus kodakaraensis由来プロテアーゼTk2168の構造機能解析 (2011)
  • Author(s): 大田黒晴樹、平田あずみ、中堀美賀子、田中俊一、古賀雄一、高野和文、金谷茂則
  • Organizer: 第84回日本生化学会
  • Place of Presentation: 国立京都国際会館(京都)
• [Presentation] 超好熱性プロテアーゼTk-subtilisinの成熟化加速機構の解析 (2011)
  • Author(s): 上原了、上田泰徳、古賀雄一, 高野和文, 金谷茂則
  • Organizer: 第63回日本生物工学会
  • Place of Presentation: 東京農工大(東京都小金井市)
• [Presentation] 超好熱始原菌由来サチライシンの成熟化における挿入配列の役割 (2010)
  • Author(s): 上原了, 田中俊一, 古賀雄一, 高野和文, 金谷茂則
  • Organizer: 第5回産業用酵素シンポジウム
  • Place of Presentation: 長浜
  • Year and Date: 2010-11-06
• [Presentation] Degradation of abnormal prion protein by a new protease from a hyperthermophile (2010)
  • Author(s): Yuichi Koga, Shun-ichi Tanaka, Akikazu Sakudo, Kazuyoshi Ikuta, Kazufumi Takano, and Shigenori Kanaya.
  • Organizer: PRION 2010
  • Place of Presentation: Salzburg(Austria)
• [Presentation] 超好熱菌由来プロテアーゼによる異常プリオン蛋白質分解 (2010)
  • Author(s): 古賀雄一、田中俊一、作道章一、高野和文、金谷茂則
  • Organizer: 日本生物工学会
  • Place of Presentation: フェニックス・シーガイア・リゾート(宮崎)
• [Presentation] Degradation of abnormal prion protein by a new protease from a hyperthermophile (2010)
  • Author(s): Yuichi Koga, Shun-ichi Tanaka, Akikazu Sakudo, Kazuyoshi Ikuta, Kazufumi Takano, Shigenori Kanaya
  • Organizer: PRION 2010
  • Place of Presentation: Salzburg, Austria
• [Presentation] 超好熱菌由来プロテアーゼによる異常プリオン蛋白質分解 (2010)
  • Author(s): 古賀雄一, 田中俊一, 作道章一, 高野和文, 金谷茂則
  • Organizer: 日本生物工学会
  • Place of Presentation: フェニックス・シーガイア・リゾート、宮崎
• [Presentation] Tk-subtilisinの成熟化における安定化機構 (2010)
  • Author(s): 上原了, 田中俊一, 古賀雄一, 高野和文, 金谷茂則
  • Organizer: 日本生物工学会
  • Place of Presentation: フェニックス・シーガイア・リゾート、宮崎
• [Presentation] Studies on the maturation process of subtilisin-like serine protease, Tk-SP, from hyperthermophilic archaeon Thermococcus kodakaraensis (2010)
  • Author(s): Nitat Sinsereekul, Tita Foophow, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya
  • Organizer: 日本分子生物学会・生化学会合同年会
  • Place of Presentation: 神戸国際会議場、神戸市
• [Presentation] 超好熱古細菌由来セリンプロテアーゼTk-SP65の特性解析 (2010)
  • Author(s): 平田あずみ、中堀美賀子、田中俊一、古賀雄一、高穿和文、金谷茂則
  • Organizer: 日本分子生物学会・生化学会合同年会
  • Place of Presentation: 神戸国際会議場、神戸市
• [Presentation] 超好熱始原菌由来サチライシンの成熟化における挿入配列の役割 (2010)
  • Author(s): 上原了, 田中俊一, 古賀雄一, 高野和文, 金谷茂則
  • Organizer: 日本分子生物学会・生化学会合同年会
  • Place of Presentation: 神戸国際会議場、神戸市
• [Presentation] 高アルカリ性、高温でのTk-subtilisinの成熟化促進機構の解析 (2009)
  • Author(s): 上田泰徳、田中俊一、古賀雄一、高野和文、金谷茂則
  • Organizer: 日本生化学会
  • Place of Presentation: 神戸国際会議場(神戸市)
• [Presentation] Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis with a C-terminal jelly roll β-barrel domain (2009)
  • Author(s): Tita Foophow, Shun-ichi Tanaka, Clement Angkawidjaja, Yuichi Koga, Kazufumi Takano, Shigenori Kanaya.
  • Organizer: 日本生化学会
  • Place of Presentation: 神戸国際会議場(神戸市)
• [Patent(Industrial Property Rights)] プロテアーゼの製造方法、並びにプロテアーゼ溶液およびプロテアーゼのプロ体溶液 (2009)
  • Inventor(s): 金谷茂則、田中俊一、竹内勇希、高野和文、古賀雄一
  • Industrial Property Rights Holder: 大阪大学
  • Filing Date: 2009-08-21
  • Overseas
• [Patent(Industrial Property Rights)] 新規なプロテアーゼおよびその利用 (2009)
  • Inventor(s): 金谷茂則、チタフーパオ、高野和文、古賀雄一
  • Industrial Property Rights Holder: 大阪大学
  • Filing Date: 2009-07-30
• [Patent(Industrial Property Rights)] 新規なプロテアーゼおにびその利用 (2009)
  • Inventor(s): 金谷茂則、チタフーパオ、高野和文、古賀雄一
  • Industrial Property Rights Holder: 大阪大学
  • Filing Date: 2009-07-30
  • Overseas
• [Patent(Industrial Property Rights)] プロテアーゼの製造方法、並びにプロテアーゼ溶液およびプロテアーゼのプロ体溶液 (2009)
  • Inventor(s): 金谷茂則、田中俊一、竹内勇希、高野和文、古賀雄一
  • Industrial Property Rights Holder: 大阪大学
  • Filing Date: 2009-08-21
  • Overseas