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1991 Fiscal Year Final Research Report Summary

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
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

  • Research Products

    (16 results)

All Other

All Publications (16 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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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

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Published: 1993-03-16  

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