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Study on a novel dehydrochlorinase from BHC-degrading bacterium

Research Project

Project/Area Number 11660075
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 InstitutionTohoku University (2000)
The University of Tokyo (1999)

Principal Investigator

NAGATA Yuji  Tohoku University, Institute of Genetic Ecology, Associate Professor, 遺伝生態研究センター, 助教授 (30237531)

Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
KeywordsBHC / Environmental pollutant / Sphingomonas / Dehydrochlorinase / Dehalogenase / Cellular localization / periplasm
Research Abstract

γ-Hexachlorocyclohexane dehydrochlorinase (LinA) is a unique dehydrochlorinase that has no homologous sequence at the amino acid-sequence level, and for which the evolutionary origin is unknown. We here propose that LinA is a member of a novel structural superfamily of enzymes containing scytalone dehydratase, 3-oxo-Δ5-steroid isomerase, nuclear transport factor-2, and the β-subunit of naphthalene dioxygenase-all known structures with different functions. The three-dimensional structure of LinA is predicted by homology modeling. More than twenty mutants were prepared by site-directed mutagenesis. These mutants were expressed in E.coli, and their activities in crude extract were evaluated. Most of the features of the LinA mutants could be explained on the basis of the present LinA model, indicating its validity. We conclude that LinA catalyses the proton abstraction via the catalytic dyad H73-D25 by the similar mechanism as described for scytalone dehydratase. The results suggest that LinA and scytalone dehydratase evolved from a common ancestor. LinA may have evolved from an enzyme showing a dehydratase activity.
Stereochemical analysis of the reaction products formed during conversion of γ-HCH by LinA was investigated by GC-MS, NMR, CD and molecular modeling. LinA requires the presence of a 1, 2-biaxial HCl pair on a substrate molecule. LinA enantiotopologically differentiates two 1, 2-biaxial HCl pairs present on γ-HCH and gives rise to a single PCCH enantiomer 1, 3 (R), 4 (S), 5(S), 6 (R)-PCCH.Furthermore, LinA enantiomerically differentiates 1, 3 (S), 4 (R), 5 (R), 6 (S)-PCCH and 1, 3 (R), 4 (S), 5 (S), 6 (R)-PCCH.The proposed mechanism of enzymatic biotransformation of γ-HCH to 1, 2, 4-TCB by LinA consists of two 1, 2-anti conformationally dependent dehydrochlorinations followed by 1, 4-anti dehydrochlorination.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] Glatz,Z. et al: "Determination of haloalkane dehalogenase activity by capillary zone electrophoresis."Journal of Chromatography A. 895. 219-225 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Marek,J. et al: "Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26."Biochemistry. 39. 14082-14086 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Trantirek,L. et al: "Reaction mechanism and stereochemistry of γ-hexachiorocyciohexane dehydrochlorinase LinA."J.Biol.Chem.. (in press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Marvanova,S. et al: "Biochemical characterization of broad-specificity enzymes using multivariate experimental design and a colorimetric microplate assay : characterization of the haloalkane dehalogenase mutants."J.Microbiol. Methods. (in press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nagata,Y. et al: "Two different types of dehalogenase, LinA and LinB, which are involved in the γ-HCH degradation in Sphingomonas paucimobilis UT26, are localized in periplasmic space without molecular processing."J.Bacteriol.. 181. 5409-5413 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nagata,Y. et al: "Complete analyisis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26."J.Industial Microbiology & Biotechnology. 23. 380-390 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Glatz, Z., M.V.Marini, M.Wimmerova, J.Damborsky, and Y.Nagata: "Determination of haloalkane dehalogenase activity by capillary zone electrophoresis."Journal of Chromatography A. 895. 219-225 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Marek, J., J.Vevodova, I.K.Smatanova, Y.Nagata, L.A.Svensson, J.Newman, M.Takagi, and J.Damborsky: "Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26."Biochemistry. 39. 14082-14086 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Trantirek, L., K.Hynkova, Y.Nagata, A.G.Murzin, A.Ansorgova, V.Sklenar, and J.Damborsky: "Reaction mechanism and stereochemistry of γ-hexachlorocyclohexane dehydrochlorinase LinA."J.Biol.Chem.. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Marvanova, S., Y.Nagata, M.Wimmerova, J.Sykorova, K.Hynkova and J.Damborsky: "Biochemical characterization of broad-specificity. enzymes using multivariate experimental design and a colorimetric microplate assay : characterization of the haloalkane dehalogenase mutants."J.Microbiol.Methods. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nagata, Y., A.Futamura, K.Miyauchi and M.Takagi: "Two different types of dehalogenase, LinA and LinB, which are involved in the γ-HCH degradation in Sphingomonas paucimobilis UT26, are localized in periplasmic space without molecular processing."J.Bacteriol. 181. 5409-5413 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nagata, Y., K.Miyauchi, and M.Takagi: "Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26."Journal of Industrial Microbiology & Biotechnology. 23. 380-390 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Glatz,Z. et al: "Determination of haloalkane dehalogenase activity by capillary zone electrophoresis."Journal of Chromatography A. 895. 219-225 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Marek,J. et al: "Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26."Biochemistry. 39. 14082-14086 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Trantirek,L. et al: "Reaction mechanism and stereochemistry of γ-hexachlorocyclohexane dehydrochlorinase LinA."J.Biol.Chem.. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Marvanova,S. et al: "Biochemical characterization of broad-specificity enzymes using multivariate experimental design and a colorimetric microplate assay : characterization of the haloalkane dehalogenase mutants."J.Microbiol.Methods. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] I. Smatanov ら: "Crystallization and preliminary X-ray diffraction analysis of halsalkane dehalogenase Lin B from Sphingomonas paucimobilis UT26"Acta Crystallographica Section D, Biological Crystallography. D55. 1231-1233 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Nagata ら: "Two different types of dehalogenase LinA and LinB, which are involved in the γ-HCH degradation in S.paucimobilis UT26, are localized in ‥‥"J. Bacteriol.. 181. 5409-5413 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Ohtsubo ら: "PcpA, which is involved in the degradation of pentachloropherol in S.chlorophenolica ATCC39723, is a novel type of ‥‥"FEBS Lett.. 459. 395-398 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Nagata ら: "Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a ‥‥"Protein Expression and Purification. 17. 299-304 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K. Miyauchi ら: "Cloning and sequencing of a novel meta-cleavage dioxygenase gene whose product is involved in the degradation of γ-HCH in S.paucimobilis"J. Bacteriol.. 181. 6712-6719 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y. Nagata ら: "Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26"J. Industrial Microbiology & Biotechnology. 23. 380-390 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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