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1990 Fiscal Year Final Research Report Summary

Biosynthetic Studies of C-P Compounds by the Use of a New Gene Modification Technique

Research Project

Project/Area Number 01470127
Research Category

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

Allocation TypeSingle-year Grants
Research Field 製造化学・食品
Research InstitutionUniversity of Tokyo

Principal Investigator

SETO Haruo  Inst. of Applied Microbilogy, Univ. of Tokyo, Professor, 応用微生物研究所, 教授 (10013335)

Co-Investigator(Kenkyū-buntansha) NAGAOKA Kozo  Discovery Res. Lab., Pharmaceutical Res. Center, Meiji Seika Kaisha, Director, 薬品総合研究所・バイオテクノロジー研究所, 所長
SHIMAZU Akira  Inst. of Applied Microbiology, Univ. of Tokyo, Associate Professor, 応用微生物研究所, 助手 (50092234)
HAYAKAWA Yoichi  Inst. of Applied Microbiology, Univ. of Tokyo, Associate Professor, 応用微生物研究所, 助教授 (20208606)
Project Period (FY) 1989 – 1990
KeywordsBialaphos / C-P compounds / C-P bond forming enzymes / Biosynthesis / Gene modification techniques / In vitro derived mutation / Carboxyphosphonoenplpyruvate
Research Abstract

The purpose of this research is to study the biosynthesis of bialaphos produced by Streptomyces hygroscopicus, more specifically the formation mechanisms of very unique C-P bonds, by the use of a new technique (in vitro derived mutation) which we have developed recently.
One C-P bond forming mechanism in the biosynthesis of bialaphos, which consists of the reaction between phosphoenolpyruvate and phosphonoformic acid to give phosphinopyruvic acid, was considered to consist of at least two reactions from the chemical point of view. Investigation of this mechanism, however, was not feasible due to the lack of suitable blocked mutants. Therefore, we attempted to prepare new types of blocked mutants by introducing mutation through the use of in vitro derived mutation. As a result, we could obtain a new mutant. Cosynthesis experiments using this mutant and another one which had been obtained by a conventional method revealed the involvement of a new biosynthetic intermediate in this reaction. We isolated this metabolite and determined its structure to be carboxyphosphonoenolpyruvate. Furthermore, we purified the new enzyme which converted it to phosphinopyruvate, and cloned the corresponding gene.
We utilized the same technique to investigate another C-P bond forming reaction, which transformed phosphoenolpyruvate to phosphonopyruvate. This reaction is ubiquitous among organisms which produce C-P compounds. As a result, we succeeded in preparation of blocked mutants unable to catalyze this reaction and we cloned the gene corresponding to this reaction. Furthermore, we could express this gene in another microorganism, Streptomyces lividans. This success enabled us to investigate the C-P bond formation mechanism in more detail. Therefore, it should be emphasized that the technique we have developed is a very useful method to study the biosynthetic mechanism of microbial metabolites.

  • Research Products

    (13 results)

All Other

All Publications (13 results)

  • [Publications] T.Hidaka: "Studies on the biosynthesis of bialaphos (SFー1293).9.Biochemical mechanism of CーP bond formation in bialaphos:discovery of phosphoenolpruvate phosphomutase which catalyzes the formation of phosphonopyruvate from phosphoenolpyruvate." J.Antibiotics. 42. 491-494 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Hidaka: "Biosynthetic mechanism of CーP bond formation;Isolation of carboxyphosphonoenolpyruvate and its conversion to phosphinopyruvate." J.Am.Chem.Soc.111. 8012-8013 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Hidaka: "Carboxyphosphonoenolpyruvate phosphomutase,the novel enzyme catalyzing CーP bond formation." J.Bacteriol.172. 3066-3072 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.W.Shimotohno: "Purification and characterization of citrate synthase from Streptomyces hygroscopicus SFー1293 and comparison of its properties with those of 2ーphosphinomethylmalic acid symthase." Agric.Biol.Chem.54. 463-470 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Hidaka: "Biochemical mechanism of CーP bond formation of of bialaphos:Use of gene manipulation for the analysis of the CーP bond formation step." Agric.Biol.Chem. 54. 2121-2125 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Hidaka: "Comparison of two CーP bond forming enzymes involved in the biosynthesis of bialaphos." Agric.Biol.Chem.54. 2467-2468 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Koyama(Ed): "Trends in Actinomycetolgy in Japan" Society for Actinomycetes,Japan, (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T. Hidaka, M. Mori, S. Imai, O. Hara, K. Nagaoka and H. Seto :"Studies on the biosynthesis of bialaphos (SF-1293). 9. Biochemical mechanism of C-P bond formation in bialaphos : discovery of phosphoenolpyruvate phosphomutase which catalyzes the formation of phosphonopyruvate from phosphoenolpyruvate." J. Antibiotics. 42. 491-494 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Hidaka, S. Imai and H. Seto :"Biosynthetic mechanism of C-P bond formation ; Isolation of carboxyphosphonoenolpyruvate and its conversion to phosphinopyruvate." J. Am. Chem. Soc.111,. 8012-8013 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Hidaka, S. Imai, O. Hara, H. Anzai, T. Murakami, K. Nagaoka and H. Seto :"Carboxyphosphonoenolpyruvate phosphomutase, the novel enzyme catalyzing C-P bond formation." J. Bacteriol.172,. 3066-3072 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. W. Shimotohno, S. Imai, T. Murakami and H. Seto :"Purification and characterization of citrate synthase from Streptomyces hygroscopicus SF-1293 and comparison of its properties with those of 2-phosphinomethylmalic acid synthase." Agric. Biol. Chem.54,. 463-470 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Hidaka, O. Hara, S. Imai, H. Anzai, T. Murakami, K. Nagaoka and H. Seto :"Biochemical mechanism of C-P bond formation of of bialaphos : Use of gene manipulation for the analysis of the C-P bond formation step." Agric. Biol. Chem.54,. 2121-2125 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Hidaka and H. Seto :"Comparison of two C-P bond-forming enzymes involved in the biosynthesis of bialaphos." Agric. Biol. Chem.54. 2467-2468 (1990)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1993-08-12  

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