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2002 Fiscal Year Final Research Report Summary

Microbial carboxyl proteinases related to a fatal neurodegenerative disease: proposal for a novel catalytic mechanism

Research Project

Project/Area Number 13460043
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionKYOTO INSTITUTE OF TECHNOLOGY

Principal Investigator

ODA Kohei  Kyoto Institute of Technology Department of Applied Biology Professor, 繊維学部, 教授 (50081584)

Co-Investigator(Kenkyū-buntansha) HIRAGA Kazumi  Kyoto Institute of Technology Department of Applied Biology Assistant Professor, 繊維学部, 助手 (50252549)
OYAMA Hiroshi  Kyoto Institute of Technology Department of Applied Biology Lecturer, 繊維学部, 講師 (50221700)
Project Period (FY) 2001 – 2002
Keywordsfatal neurodegenerative disease / Batten disease / pepstatin / catalytic mechanism / three-dimensional structure / carboxyl proteinase / serine-carboxyl proteinase / inhibitor
Research Abstract

We carrited out enzymatic and molecular biological investigations on microbial pepstatin-insensitive carboxyl proteinases (PSCP, XSCP, kumamolisin, and J-4) and their mammalian homologue, the CLN2 gene product (CLN2 protein), which is related to the fatal neurodegenerative disease. Compared these results with the data from structural analyses of PSCP and kumamolisin, we clarified their structures and novel catalytic mechanism and discussed on their molecular evolution, too.
SUMMARY IN 2001:
(i) Conserved serine residues
The conserved serine residues among five enzymes (PSCP, XSCP, kumamolisin, J-4, and the CLN2 protein) were elucidated to be involved in the catalytic residues by their site-directed mutagenesis studies.
(ii) Structural analysis of PSCP and its catalytic mechanism
Structural analysis has revealed that a novel type of catalytic triad composed of Ser287, GIu80, and Asp84 residues is involved in its catalytic function. They were confirmed to be the catalytic residues of PSCP by … More site-directed mutagenesis and inhibitor studies.
(iii) New inhibitors for kumamolisin and its application
Novel type of inhibitors were designed and synthesized, and they were used for structural analysis of kumamolisin.
SUMMARY IN 2002:
(i) Structural analysis of prepro-PSCP
S287A mutant was expressed as the insoluble prepro-protein, and the refolded enzyme did not show any autocatalytic processing- and proteinase-activity. These results strongly suggested that Ser287 residue was involved in the catalytic residues. Structural analysis of the S287A mutant is now in progress.
(ii) Catalytic residues in kumamolisin
Based on the structure of kumamolisin, Glu32 and Trp129 residues were suggested to be an unique catalytic residues in kumamolisin molecule. The k_<cat>/K_m values of the E32A and W129A mutants were about 6 and 4 % of that of the control, indicating that both residues were involved in the catalytic function.
(iii) Structural analysis of the CLN2 protein
The CLN2 protein was elucidated to have a substrate-binding site consisting of six subsites (S3-S3') by kinetics analysis. Structural analysis of the CLN2 protein expressed in silkworm pupae is now in progress. Less

  • Research Products

    (17 results)

All Other

All Publications (17 results)

  • [Publications] Wlodawer, A., et al.: "Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes"Nature Structural Biology. 8. 442-446 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Wlodawer, A., et al.: "Inhibitor complexes of the Pseudomonas serine-carboxyl proteinase"Biochemistry. 40. 15602-15611 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Fujiwara, S., et al.: "Effect of pressure on the activity and spectroscopic properties of carboxyl proteinases : Apparent correlation of pepstatin-insensitivity and pressure response"Eur. J. Biochem.. 268. 645-655 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Oyama, H., et al.: "A CLN2-related and thermostable serine-carboxyl proteinase, kumamolysin : cloning, expression, and identification of catalytic serine residue"J. Biochem. (Tokyo). 131. 757-765 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Comellas-Bigler, M., et al.: "The 1.4 Å crystal structure of kumamolysin : a thermostable serine-carboxyl-type proteinase"Structure (Camb). 10. 865-876 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 小田耕平: "プロテアーゼの新ファミリー:セリン-カルボキシルプロテアーゼの構造と機能"生化学. 74. 1459-1463 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsuruoka, N., et al.: "Collagenolytic serine-carboxyl proteinase from Alicyclobacillus sendaiensis strain NTAP-1 : purification, characterization, gene cloning, and heterologous expression"Appl. Environ. Microbiol.. 69. 162-169 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Oda, K.: "Sedolisin (3 pages), Sedolisin B (3 pages), and kumamolisin (4 pages)"Handbook of Proteolytic Enzymes 2^<nd> edition (edited by Alan J.Barrett, et al.)(Academic Press, NY). 10 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Wlodawer, A., et al.: "Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes"Nature Structural Biology. 8. 442-446 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Wlodawer, A., et al.: "Inhibitor complexes of the Pseudomonas serine-carboxyl proteinase"Biochemistry. 40. 15602-15611 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Oyama, H., et al.: "A CLN2-related and thermostable serine-carboxyl proteinase, kumamolysin: cloning, expression, and identification of catalytic serine residue"J Biochem. (Tokyo). 131. 757-765 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Comellas-Bigler, M., et al.: "The 1.4 A crystal structure of kumamolysin: a thermostable serine-carboxyl-type proteinase"Structure (Camb). 10. 865-876 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kohei Oda: "A novel family of proteinases: structure and function of serine-carboxyl proteinases"Seikagaku. 74. 1459-1463 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tsuruoka, N., et al.: "Collagenolytic serine-carboxyl proteinase from Alicyclobacillus sendaiensis strain _NTAP-1: purification, characterization, gone cloning, and heterologous expression"Appl. Environ. Microbiol.. 69. 162-169 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Oda, K.: "Sedolisin in "Handbook of Proteolytic Enzymes 2nd edition (edited by Alan J. Barrett, et al.)""Academic Press, NY. 3 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Oda, K.: "Sedolisin B in "Handbook of Proteolytic Enzymes 2nd edition (edited by Alan J. Barrett, et al.)""Academic Press, NY. 3 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Oda, K.: "kumamolisin in "Handbook of Proteolytic Enzymes 2nd edition (edited by Alan J. Barrett, et al.)""Academic Press, NY. 4 (2003)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2004-04-14  

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