Discovery of two novelproteinases andtheirbi rl emical resemph

2007 Fiscal Year Final Research Report Summary

• Project/Area Number: 18380068
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Kyoto Institute of Technology
• Principal Investigator: HIRAGA Kazumi Kyoto Institute of Technology, Graduate School of Science and Technology, Assistant Professor (50252549)
• Project Period (FY): 2006 – 2007
• Keywords: sedolisin / e olisin / elutamic nentidase / serine-carboxvl proteinase / structure and function / substrate specificity / reaction mechanism / proteinase inhibitor

Research Abstract

Sedolisin : 1) Structural analysis of CLN2 subsite : Combinatorial substrate library was designed and synthesized, and subsite-analysis is underway. 2) structure analysis of CLN2 : Procedures for expression and purification were investigated and an extremely efficient purification method was constructed. Large amount of CLN2 was prepared and the crystallization of CLN2 is undergoing.
Eqolisin family: 1) Elucidation of reaction mechanism: Glu136 and G1n53, presumed catalytic residues by structural analysis, were changed to Ala and their reaction kinetics revealed that these residues were the catalytic resideues. Based on the detailed analysis for substrate specificity using combinatorial library, synthetic inhibitor was developed (Y. Kataoka et al: FEBSE Letters, 579, 2991-2994 (2005)). Furthermore, enzyme-inhibitor complex was prepared and the obtained results from the structural analysis revealed the reaction mechanism (B. Pillai, et. al.: J. Mol Biol 365, 343-361 (2007)). 2) Distribution in nature: not completed. 3) Development of safety agrichemicals: Peptide inhibitors having inhibitory constant at subnano-molar-level were successively developed. The effect of the inhibitor for phytopathogenic microbials will be determined at lab scale.

Research Products

• [Journal Article] Crystal structure of Scytalidoglutamic peptidase with its first potent inhibitor provides insights into substrate specificity and catalysis. (2007)
  • Author(s): B. Pillai、他5名
  • Journal Title: J. Mol. Biol. 365 Pages: 343-361
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Crystal structure of Scytalidoglutamic peptidase with its first potent inhibitor provides insights into substrate specificity and catalysis (2007)
  • Author(s): B., Fillai, M. M., Cherney, K., Hiraga, K., Takada, K., Oda, M. N., G., James
  • Journal Title: T. Mal Biol 365 Pages: 343-361
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Processing,catalytic activity and crystal structures of kumolisin-As with an engineered active site. (2006)
  • Author(s): A. Okubo、他8名
  • Journal Title: FEBS-Journal 273 Pages: 2563-2576
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Synergetic effects of pressure and chemical denaturant on protein unfolding: stability of a serine-type carboxyl protease, kumamolisin. (2006)
  • Author(s): Y. Fujimoto、他5名
  • Journal Title: Biochim. Biophys. Acta. 1764 Pages: 364-371
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Processing, catalytic activity and crystal structures of kumolisin-As with an engineered active site (2006)
  • Author(s): A., Okubo, Mi, Li, M., Ashida, H., Oyama, A., Gustchina, K., Oda, B. M., Dunn, A., Wlodawer, T., Nakayama
  • Journal Title: FEBS-Journal 273 Pages: 2563-2576
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Synergetic effects of pressure and chemical denaturant on protein unfolding : stability of a serine-type carboxyl protease, kumamolisin (2006)
  • Author(s): Y., Fujimoto, H., Ikeuchi, T., Tada, H., Oyama, K., Oda, S., Kunugi
  • Journal Title: Biochim. Biophys. Acta 1764 Pages: 364-371
  • Description: 「研究成果報告書概要(欧文)」より