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Structure-Function Relationships and Molecular Evolutions of Novel Carboxyl Proteinases from Microorganisms

Research Project

Project/Area Number 09660089
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionFaculty of Textile Science, Kyoto Institute of Technology

Principal Investigator

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

Co-Investigator(Kenkyū-buntansha) HIRAGA Kazumi  Kyoto Institute of Technology Department of Applied Biology Faculty of Textile S, 繊維学部, 助手 (50252549)
Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1997: ¥2,600,000 (Direct Cost: ¥2,600,000)
KeywordsProkaryote / Pepstatin / Carboxyl proteinase / Acid proteinase / Aspartic proteinase / Catalytic residue (s) / Amino acid sequence / Structure-Function Relationship / 酵性プロテアーゼ
Research Abstract

In this study, we aimed to identify the catalytic residues of pepstatin-insensitive carboxylproteinases from prokaryote cells. We focused our studies on carboxyl proteinases from Pseudomonas sp. 101 (PCP). Xanthomonas sp. T-22 (XCP), Bacillus coagulans J-4 (J-4), and Bacillus novosp. MN-32 (kumamolysin). The primary structures of them does not have any similarities to those of aspartic proteinases (pepstatin-insensitive carboxyl proteinase) reported so far. Moreover, the well-conserved structure, -Asp*-Thr-Gly-(Asp* : catalytic residue) in the active center of aspartic proteinases was not observed, The following results were obtained.
1. Identification of Catalytic Residues by Using Site-directed Mutagenesis Technique
PCP (372 amino acid residues) and XCP (398 amino acid residues) have 52% identity to each other. Based on the high sequence identity, eight amino acid residues for catalytic residues (Asp or Glu) were poked up, and all of them were mutated to Ala residues. We analyzed these … More Ala mutants for both auto-catalytic processing ability and proteinase activity. Consequently, a pair of Dl70 and D328 for PCP.And a pair of D169 and D348 for XCP were identified as catalytic residues, respectively.
2. Identification of Catalytic Residues by Using [14C] Stylene Oxide
In order to identify the catalytic residue(s) of XCP.[14C]stylene oxide was used. It was found that the [14C]stylene oxide was bound to E75 and D110 residues of XCP, respectively. Of them, E75 residue (corresponding to E80 residue of PCP) and its vicinities were conserved in PCP.Based on these data. E80 residue of PCP and E75 residue of XCP were thought to be involved in their catalytic function, as a substrate binding site, respectively.
3. Identification of Catalytic Residues by Using [14C] DCCD
In order to identify the catalytic residue(s) of PCP, chemical modification was carried out by using N.N'-dicyclohexylcarbodiimide (DCCD) and specific inhibitor, tyrostatin. It was found that [14C] DCCD was bound to D140 and E222 residues of PCP, respectively. Of them, E222 residue (corresponding to E235 residue of XCP) and its vicinities were found out to be conserved in XCP.Furthermore, E222A mutant had no any activity, whereas XE235A (corresponding to E222A for PCP) had proteinase activity. Based on these data. E222 residue of PCP and E235 of XCP were thought to be involved in their catalytic function. probably as a substrate binding site, respectively.
4. Cloning of Carboxyl Proteinase J-4 Gene from Bacillus coagulans
Bacilluscoagulans J-4 carboxyl proteinase. designated as J-4, is characterized as alcohol resistant and insensitive to pepstatin. Most of the gene has been cloned, sequenced. After getting the whole gene. we will try to construct a high expression system and determine the catalytic residues by site-directed mutagenesis.
5. Construction of High Expression System of Kumamolysin
Bacillus novosp. MN-32 carboxyl proteinase. designated as Kumamolysin. is characterized as thermostable enzyme. We succeeded in constructing the high expression system for this enzyme. Less

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • Research Products

    (29 results)

All Other

All Publications (29 results)

  • [Publications] S.Narutaki: "Subsite preferences of pepstatin-insensitive carboxyl proteinases from bacteria" J.Biochem.125・1. 75-81 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Ito: "Identification of carboxyl residues in pepstatin-insensi-tive carboxyl proteinase from Pseudomonas sp. 101 that participate in catalysis and substrate binding" J.Biochem.125・1. 210-216 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] 小田耕平: "LINCL病の原因遺伝子はシュードモナスのプロテアーゼ・ホモログ" バイオサイエンスとインダストリー. 156・12. 31-32 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Shibata: "Substrate specificity of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4" J.Biochem. 124・3. 642-647 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] H.Kondo: "Substrate specificities and kinetic properties of protei-nase A from Yeast Saccharomyces cerevisiae and the development of a novel substrate" J.Biochem. 124・1. 141-147 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] N.Oda: "Nucleotide Sequence of the gene encoding the precursor protein of pepstatin-insensitive acid protease B, Scytali-dopepsin B, from Scytalidium lignicolum" Biosci.Biotech.Biochem.62・8. 1637-1639 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] K.Oda: "Handbook of Proteolytic Enzymes" A.J.Barrett et al., ed. Academic Press, 4 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] K.Oda: "Aspartic Proteinases" M.James ed., Plenum Press, New York, 5 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Narutaki et al.: "Subsite preferences of pepstatin-insensitive carboxyl proteinases from bacteria" J.Biochem.125. 75-81 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Ito et al.: "Identification of carboxyl residues in pepstatin-insensitive carboxyl proteinase from Pseudomonas sp.101 that participates in catalysis and sustrate binding" J.Biochem.125. 210-216 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Shibata et al.: "Substrate specificity of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4" J.Biochem.124. 642-647 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] H.Kondo et al.: "Substrate specificities and kinetic properties of proteinase A from yeast Saccharomyces cerevisiae and the development of a novel substrate" J.Biochem.124. 141-147 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] N.Oda et al.: "Nucleotide sequence of the gene encoding the precursor protein of pepstatin-insensitive acid protease B,Scytalidopepsin B,from Scytalidium lignicolum" Biosci.Biotech.Biochem.62. 1637-1639 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Ito et al.: "Substrate specificities of pepstatin-insensitive carboxyl proteinases from Gram-negative bacteria" J.Biochem.120. 845-850 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] K.Oda et al.: "Cloning and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Xanthomonas sp.T-22" J.Biochem.120. 564-572 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Kakimori et al.: "Nucleotide sequence of the gene encoding pepstatin-insensitive acid protease B,Scytalidopepsin B,of Scytalidium lginicolum" Biosci.Biotech.Biochem.60. 1210-1211 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] K.Oda: "Scytalidopepsin B" In : Handbook of Proteolytic Enzymes (ed.A.J.Barret et al.). Academic Press., 2 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] K.Oda: "Xanthomonapepsin" In : Handbook of Proteolytic Enzymes (ed.A.J.Barret et al.). Academic Press., 2 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] K.Oda et al.: "Pepstatin-insensitive Carboxyl Proteinases from Prokaryotes Catalytic Residues and Substrate Specificities" In : Aspartic Proteinases (ed.by M.N.G.James). Plenum Press.New York, 5 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] B.M.Dunn et al.: "Comparison of the Specificity of the Aspartic Proteinases towards Internally Consistent Sets of oligopeptide Substrates" In : Aspartic Proteinases (ed.by M.N.G.James). Plenum Press.New York, 6 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] S.Narutaki: "Subsite preferences of pepstatin-insensitive carboxyl proteinases from bacteria" J.Biochem.,. 125・1. 75-81 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] M.Ito: "Identification of carboxyl residues in pepstatin-insensi-tive carboxyl proteinase from Pseudomonas sp.101 that participate in catalysis and substrate binding" J.Biochem.125・1. 210-216 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] 小田耕平: "LINCL病の原因遺伝子はシュードモナスのプロテアーゼ・ホモログ" バイオサイエンスとインダストリー. 156・12. 31-32 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] M.Shibata,: "Substrate specificity of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4" J.Biochem.,. 124・3. 642-647 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] H.Kondo,: "Substrate specificities and kinetic properties of protei-nase A from Yeast Saccharomyces cerevisiae and the development of a novel substrate" J.Biochem.,. 124・1. 141-147 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] N.Oda,: "Nucleotide Sequence of the gene encoding the precursor protein of pepstatin-insensitive acid protease B, Scytali-dopepsin B, from Scytalidium lignicolum" Biosci.Biotech.Biochem.,. 62・8. 1637-1639 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Oda: "Handbook of Proteolytic Enzymes" A.J.Barrett et al., ed. Academic Press, 4 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Oda: "Aspartic Proteinases" M.James ed., Plenum Press, New York, 5 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] M.N.G.James, ed: "Structure and Functions of Aspartic Proteinases:Retroviral and Cellular Enzymes,K.Oda et al.,Pepstatin-insensitive Carboxyl Proteinases from Prokaryote:Substrate Specificities and Catalytic Residues" Plenum Publishing Corporation, (5) (1998)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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