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1998 Fiscal Year Final Research Report Summary

Structure-Function of Novel Carboxyl Proteinases from Microorganisms

Research Project

Project/Area Number 08044202
Research Category

Grant-in-Aid for international Scientific Research

Allocation TypeSingle-year Grants
SectionJoint Research
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, Professor, 繊維学部, 教授 (50081584)

Co-Investigator(Kenkyū-buntansha) HIRAGA Kazumi  Kyoto Institute of Technology, Department of Applied Biology, Assistant, 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)
KAY John  ウェールズ大学カーディフ校, 分子医生物科学部, 教授
DUNN Ben M  フロリダ大学, 医学部, 教授
DUNN B.M.  University of Florida College of Medicine, Professor
KAY Jhon  University of Wales College of Cardiff, Professor
Project Period (FY) 1996 – 1998
KeywordsProkaryote / Pepstatin / Carboxyl proteinase / Acid proteinase / Aspartic proteinase / Catalytic residue (s) / Amino acid sequence / Structure-Function Relationship
Research Abstract

In this study, we aimed to identify the catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryote cells. We focussed 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. Substrate Specificity and Subsite Structure of Kumamolysin
Substrate specificity of Kumamolysin was investigated by using two sets of synthetic substrates. The subsite structure of Kumamolysin was found to be different with those of aspartic proteinases. These data will be published in J.Biochem.
2. Identification of Catalytic Residues by Using [^<14>C] … More Stylene Oxide
In order to identify the catalytic residue(s) of XCP, [^<14>C] stylene oxide was used. It was found that the [^<14>C]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 [^<14>C] DCCD
In order to identify the catalytic residue(s) of PCP, chemical modification was carried out by using N.N'-dicyclohexylcafrbodiimide (DCCD) and specific inhibitor. tyrostatin, It was found that [^<14>C] 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
Bacillus coagulans 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. Less

  • Research Products

    (24 results)

All Other

All Publications (24 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
  • [Publications] M.Ito: "Idetification 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
  • [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
  • [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
  • [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
  • [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
  • [Publications] M.Ito,: "Substrate specificities of pepstatin-insensitive carboxyl proteinases from Gram-negative bacteria" J.Biochem.,. 120・4. 845-850 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Oda,: "Cloning and expression of an isovaleryl pepstatin-insensi-tive carboxyl proteinase gene from Xanthomonas sp. T-22" J.Biochem.,. 120・3. 564-572 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Kakikmori,: "Nucleotide sequence of the gene encoding pepstatin-insensi-tive acid protease B, Scytalidopepsin B, of Scytalidium lignicolum" Biosci.Biotech.Biochem.,. 60・7. 1210-1211 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [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
  • [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
  • [Publications] B.M.Dunn: "Aspartic Proteinases" M.James ed., Plenum Press, New York, 6 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [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
  • [Publications] M.Ito et al.: "Identification of carboyl residues in pepstation-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
  • [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
  • [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
  • [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
  • [Publications] M.Ito et al.: "Substrate specificity 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
  • [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
  • [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
  • [Publications] K.Oda: "Scytalidopepsin B" 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
  • [Publications] K.Oda: "Xanthomonapepsin" Handbook of Proteolytic Enzymes (ed.A.J.Baret et al.). Academic Press. 2 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Oda et al.: "Pepstatin-insensitive Carboxyl Proteinases from Prokaryotes Catalytic Residues and Substrate Specificities" 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
  • [Publications] B.M.Dunn et al.: "Comparison of the Specificity of the Aspartic Proteinases towards Internally Consistent Sets of oligopeptide Substrates" 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

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Published: 1999-12-08  

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