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Structure-Function Relationships of Pepstatin-insensitive Caroboxyl Protease produced by Pseudomonas sp. No. 101

Research Project

Project/Area Number 02660124
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 応用微生物学・発酵学
Research InstitutionCollege of Agriculture, University of Osaka Prefecture

Principal Investigator

ODA Kohei  University of Osaka Prefecture Department of Agricultural Chemistry College of Agriculture Associated Professor, 農学部, 助教授 (50081584)

Project Period (FY) 1990 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
KeywordsAcid protease / Carboxyl protease / Aspartic protease / Pepstatin-insensitive / S-PI-insensitive / Scytalidium type / Pepsin type / Structure-Function Relationship
Research Abstract

It is well known that carboxyl proteases are commonly inhibited by pepstatin^<1)>, DAN^<2)> and EPNP^<3)>, and their catalytic residues are composed of two aspartic acid residues. Thus, carboxyl proteases are termed aspartic proteases. These enzymes are highly homologous in both the primary and tertiary structures.
We have isolated novel carboxyl proteases from fungi, bacteria and also thermophilic bacteria based on their insensitivities to pepstatin, DAN and EPNP. These enzymes were tentatively named pepstatin-insensitve carboxyl proteases. In one of our studies, the primary structure of carboxyl protease B(consisting of 204 amino acids)from a fungus Scytalidium lignicolum has been established, one of the catalytic residues of which was clarified to be Glu-53. This is the first report on glutamic protease. It seemed probable that the pepstatin-insensitive carboxyl proteases are not aspartic proteases but glutamic proteases. To confirm this possibility, we focussed our studies on a peps … More tatin-insensitive carboxyl protease from Pseudonionas sp. No. 101(PCP), which is the the first carboxyl protease-isolated from prokaryote cells. The primary structure of PCP has been determined to be a single polypeptide composed of 372 amino acid residues with one disulfide bridge. PCP does not have any homologous structure to those of aspartic proteases reported so far. Moreover, the well-conserved structure, -Asp*-Thr-Gly- in the active center of aspartic proteases was not observed.
In this study, the following results were obtained.
1. Identification of Catalytic Residues In our attempt to use inhibitor in the study of active center, we had isolated a novel inhibitor, tyrostatin(N-isovaleryl-tyrosyl-leucyl-tyrosinal, Ki = 2.5 nM)from Kitasatosporia sp. No. 55. Based on the chemical structure, we succeeded in synthesizing a competitive inhibitor, available for probing the catalytic residues of PCP(Carbobenzoxy-Tyr-O-CH_2-Epoxide).
2. Analysis of PCP Gene We determined the whole DNA sequence of the PCP gene(abaout 3 kbp). it was elucidated that PCP is composed of prepro part protein(215 amino acid residues)and mature protein(372 amino acid residues). Primary structure of the mature protein was identical to that chemically determined previously. It was suggested that the propart protein plays important roles in the activation as well as secretion through the double layer of the cell.
Accordingly, it is ready now to study the structure-function relationships, especially the catalytic residues on both side of protein and DNA level.
1)pepstatin, pepsin inhibitor ; 2)DAN, diazoacetyl-DL-norleucine methylester ; 3)EPNP, 1.2-epoxy-3(P-nitrophenoxy)propane. Less

Report

(3 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (32 results)

All Other

All Publications (32 results)

  • [Publications] K.Oda,Y.Fukada,S.Murao K.Uchida and M.Kainosho: "A Novel Proteinase Inhibitor,Inhibiting Some Pepstatin-insensitive Carboxyl Proteinases" Agric.Biol.Chem.53. 405-415 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] D.Tsuru,A.Naotuka,R.Kobayashi,T.Yoshimoto,K.Oda and S.Murao: "Inactivation of Scytalidium lignicolum Acid Proteinase B with 1,2-epoxy-3-(4'-azido-2'-nitrophenoxy)propane" Agric.Biol.Chem.53. 2751-2756 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Oda,H.Nakatani and B.M.Dunn: "Substrate Specificity,and Kinetic Properties of Pepstatin-insensitive Carboxyl Proteinase from Pseudomonas sp.No.101" Biochim.Biophys.Acta.(1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Oda et al.: "Complete Amino Acid Sequence of Pseudomonas sp.No.101 Pepstatin-insensitive Carboxyl Proteinase" J.Biochem.(Tokyo).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Oda et al.: "cloning and Sequencing of Pepstatin-insensitive Carboxyl Proteinase Gene from Pseudomonas sp.No.101" J.Biochem.(Tokyo).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Oda et al.: "The Role of Prepro Region of Pepstatin-insensitive Carboxyl Proteinase Gene from Pseudomonas sp.No.101" J.Biochem.(Tokyo).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Y.Yoshinaka,Y.Kato and K.Oda: "Retroviral Protease Maturation and Morphogenesis(ed.by L.H.Pearl) "Retrovira Protease:Substrate Specificity and Inhibitors"" Stockton Press,N.Y., 9 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Oda and S.Murao: "The Aspartic Proteinases:Genetic,Structures and Mechanisms (ed.by B.M.Dunn) "Pepstatin-insensitive Carboxyl Proteinases"" Plenum Publishing Corporation,N.Y., 7 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Oda, Y. Fukuda, S. Murao, K. Uchida and M. Kainosho: "A Novel Proteinase Inhibitor, Tyrostatin, Inhibiting Some Pepstatin-insensitive Carboxyl Proteinases" Agric. Biol. Chem.53. 405-415 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] D. Tsuru, A. Naotuka, R. Kobayashi, T. Yoshimoto, K. Oda and S. Murao: "Inactivation of Scytalidium lignicolum Acid Protease B with 1, 2-Epoxy-3-(4'-azido-2'-nitrophenoxy)propane" Agric. Biol. Chem.53. 2751-2756 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Oda H. Nakatani and B. M. Dunn: "Substrate Specificity, and Kinetic Properties of Pepstatin-insensitive Carboxyl Proteinase from Pseudomonas sp. No. 101" Biochem. Biophys. Acta. (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Oda et al: "Complete Amino Acid Sequence of Pepstatin-insensitive Carboxyl Proteinase from Pseudomonas sp. No. 101" J. Biochem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Oda et al: "Cloning and Sequencing of Pepstatin-insensitve Carboxyl Proteinase Gene from Pseudomonas sp. No. 101" J. Biochem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] "The Role of Prepro Region of Pepstatin-insensitive Carboxyl Proteinase Gene from Pseudomonas sp. No. 101" J. Biochem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Y. Yoshinaka, Y. Kato and K. Oda: "Retroviral Proteases Maturation and Morphogenesis" "Retroviral Protease : Substrate Specificity and Inhibitors". Stockton Press, N. Y.(1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Oda and S. Murao: "The aspartic Proteinases : Genetic, Structures and Mechanisms" "Pepstatin-insensitive Carboxyl Proteinases". Plenum Publishing Corporation, N. Y.(1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Oda,Y.Fukuda,S.Murao,K.Uchida and M.Kainosho: "A Novel Proteinase Inhibitor,Inhibiting Some Pepstatininsensitive Carboxyl Proteinases" Agric.Biol.Chem.53. 405-415 (1989)

    • Related Report
      1991 Annual Research Report
  • [Publications] D.Tsuru,A.Naotuka,R.Kobayashi,T.Yoshimoto,K.Oda and S.Murao: "Inaction of Scytalidium lignicolum Acid Proteinase B with 1,2ーepoxyー3ー(4'ーazidoー2'ーnitophenoxy)propane" Aqric.Biol.Chem.53. 2751-2756 (1989)

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Oda,H.Nakatani and B.M.Dunn: "Subsrate Specificity,and Kinetic Properties of Pepstatininsensitive Carboxyl Proteinase from Pseudomonas sp.No.101" Biochim.Biophys.Acta.(1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Oda et al.: "Complete Amino Acid Sequence of Pseudomonas sp.No.101 Pepstatinーinsensitive Carboxyl Proteinase" J.Biochm.(Tokyo).

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Oda et al.: "Cloning and Sequencing of Pepstainーinsensitive Carboxyl Proteinase Gene from Pseudomonas sp.No.101" J.Biochem.(Tokyo).

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Oda et al.: "The Role of Prepro Region of Pepstatinーinsensitive Carboxyl Proteinase Gene from Pseudomonas ap.No.101" J.Biochem.(Tokyo).

    • Related Report
      1991 Annual Research Report
  • [Publications] Y.Yoshinaka,Y.Kato and K.Oda: "Retroviral Protease Maturation and Morphogenesis(ed.by L.H.Pearl)“Retrovira Protease:Substrate Specifictty and Inhibitors"" Stockton Press,N.Y., 9 (1990)

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Oda and S.Murao: "The Aspartic Proteinases:Genetic,Structures and Mechanisms(ed.by B.M.Dunn)“Pepstatinーinsensitive Carboxyl Proteinases"" Plenum Publishing Corporation,N.Y., 7 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] S.Murao and K.Oda: "Structure and Properties of Non Pepstatinーsensitive Carboxyl Proteinases" Proceedings of the 18th LinderstrmーLang Conference ″Aspartic Proteinases″. 39-39 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] 村尾 沢夫,小田 耕平: "放線菌由来のメタルプロテア-ゼインヒビタ-" 蛋白質・核酸・酵素. 34. 980-988 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] K.Oda,他4名: "A Novel Proteinase Inhibitor,Inhibiting Some Pepstatinー Insensitive Carboxyl Proteinaes" Agric.Biol.Chem.53. 405-415 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] D.Tsuru 他5名: "Inactivation of Scytalidium lignicolum Acid Proteinase B with 1,2ーEpoxyー3ー(4′ーazidoー2′ーnitrophenoxy)propane" Agric.Biol.Chem.53. 2751-2756 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] K.Oda et al.: "Complete amino Acid Sequence of Pseudomonas sp.No.101 Pepstatinーinsensitive Carboxyl Proteinase" J. Biochem.(Tokyo).

    • Related Report
      1990 Annual Research Report
  • [Publications] K.Oda et al.: "Cloning and Sequencing of Pseudomonas sp.No.101 Pepstatinーinsensitive Carboxyl Proteinase" Agric.Biol.Chem.

    • Related Report
      1990 Annual Research Report
  • [Publications] K.Morihara and K.Oda(ed.by G.Winkelmann): "Microbial Degradation of Natural Products (Microbial degradation of proteins" V.C.H.(F.R.G.), (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] K.Oda and S.Murao(ed.by B.M.Dunn): "Aspartic Proteinasea (Pepstatinーinsensitive carboxyl Proteinases" Prenum Publishing Corp.,New York., (1991)

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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