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Molecular dissection of Streptomyces trypsin on the recognition mechanism of structural protein substrates

Research Project

Project/Area Number 21750183
Research Category

Grant-in-Aid for Young Scientists (B)

Allocation TypeSingle-year Grants
Research Field Chemistry related to living body
Research InstitutionKyoto University

Principal Investigator

UESUGI Yoshiko  Kyoto University, 再生医科学研究所, 研究員 (30416416)

Project Period (FY) 2009 – 2010
Project Status Completed (Fiscal Year 2010)
Budget Amount *help
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2010: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2009: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Keywords酵素 / 基質認識 / 高分子基質 / セリンプロテアーゼ / 放線菌 / RIBSシャフリング / RIBS in vivo DNAシャフリング
Research Abstract

Serine proteases have recently received much attention in relation to degradation of recalcitrant animal proteins, such as collagen, keratin, blood clots, and amyloid prion proteins for beneficial use of industrial waste and medical applications. In this study, a serine protease from Streptomyces omiyaensis (SOT), which is a trypsin-like enzyme, was chosen as a model enzyme for clarifying the recognition mechanism of structural protein substrates in serine proteases. We constructed chimeras between SOT and SGT using an in vivo DNA shuffling system and several mutants to identify the key residues involved in topological specificities. By comparing substrate specificities of chimeras and mutants, we found that residues 71 and 72 of SOT contribute to topological specificity and collagen binding, respectively. Furthermore, we found that Lys217 and Ala221 of SOT, which is located in the C-terminal α-helix domain, as a crucial residues for fibrinolysis.

Report

(3 results)
  • 2010 Annual Research Report   Final Research Report ( PDF )
  • 2009 Annual Research Report
  • Research Products

    (7 results)

All 2011 2010 2009 Other

All Journal Article (4 results) (of which Peer Reviewed: 4 results) Presentation (2 results) Patent(Industrial Property Rights) (1 results)

  • [Journal Article] Highly potent fibrinolytic serine protease from Streptomyces.2011

    • Author(s)
      Yoshiko Uesugi, Hirokazu Usuki, Masaki Iwabuchi, Tadashi Hatanaka
    • Journal Title

      Enzyme and Microbial Technology 48

      Pages: 7-12

    • Related Report
      2010 Final Research Report
    • Peer Reviewed
  • [Journal Article] Highly potent fibrinolytic serine protease from Streptomyces.2011

    • Author(s)
      Yoshiko Uesugi, Hirokazu Usuki, Masaki Iwabuchi, Tadashi Hatanaka
    • Journal Title

      Enzyme and Microbial Technology

      Volume: 48 Pages: 7-12

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates.2009

    • Author(s)
      Yoshiko Uesugi, Hirokazu Usuki, Masaki Iwabuchi, Tadashi Hatanaka
    • Journal Title

      The FEBS Journal 276

      Pages: 5634-5646

    • Related Report
      2010 Final Research Report 2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Molecular dissection of Streptomyces trypsin on substrate recognition.

    • Author(s)
      Yoshiko Uesugi, Hirokazu Usuki, Jiro Arima, Masaki Iwabuchi, Tadashi Hatanaka
    • Journal Title

      Biochim Biophys Acta (In press.)

    • Related Report
      2010 Final Research Report
    • Peer Reviewed
  • [Presentation] 放線菌由来セリンプロテアーゼの血栓溶解に関わるアミノ残基2010

    • Author(s)
      上杉佳子
    • Organizer
      第83回日本生化学会大会
    • Place of Presentation
      神戸ポートアイランド(神戸)
    • Related Report
      2010 Annual Research Report 2010 Final Research Report
  • [Presentation] 放線菌由来セリンプロテアーゼの血栓溶解への応用2009

    • Author(s)
      上杉佳子
    • Organizer
      第82回日本生化学会大会
    • Place of Presentation
      神戸ポートアイランド(神戸)
    • Related Report
      2010 Final Research Report 2009 Annual Research Report
  • [Patent(Industrial Property Rights)] トリプシン様酵素2009

    • Inventor(s)
      畑中唯史、上杉佳子、西本幸史
    • Industrial Property Rights Holder
      畑中唯史、上杉佳子、西本幸史
    • Acquisition Date
      2009-08-06
    • Related Report
      2010 Final Research Report

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Published: 2009-04-01   Modified: 2016-04-21  

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