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Studies on electron-transferring mechanism in an nitrite-reducing system containing Cu-proteins

Research Project

Project/Area Number 09660075
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 InstitutionThe University of Tokyo

Principal Investigator

NISHIYAMA Makoto  The University of Tokyo, Biotechnology Research Center, Associate professor, 生物生産工学研究センター, 助教授 (00208240)

Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,700,000 (Direct Cost: ¥2,700,000)
Keywordsnitrite reductase / electron transfer / nitirc oxide reductase / copper protein / heme-containing protein / 蛋白質工学
Research Abstract

We planned to elucidate the denitrification mechanism of
Alcaligenes faecalis S-6 by characterizing enzymes and genes involved in the denitrification process. Since a cytochrome c is often served as an electron donor in this process, a protein which reacted with heme-staining reagent was partially purified from A.faecalis S-6, and its N-terminal amino acid sequence was determined. The sequence was highly similar to those of small subunits of nitric oxide reductases (NOR) from several denitrifying bacteria. DNA fragments containing the gene coding for the subunit was cloned by oligonucleotide-probing method. Nucleotide sequencing revealed that the cloned fragments contained nine open reading frames. Base on the homology analysis we tentatively designated the open reading frames as dnr, orf1, orf2, norO, norP, norC, norB, norQ, and norD.Just upstream of the norO, norC, and norQ, short palin-drome sequences similar to FNR-recognition sequence with a motif of TTGATxxxxATCAA were found. Considering that FNR was a transcriptional activater for genes expressed under the anaerobic conditions, this suggested, that expression of the genes was controled in the stage of transcription in the absence of oxygen, and these genes were transcribed as at least three different operons. We constructed several plasmids for the expression of nor genes and introduced them into Escherichia coli JM105. E.coli cells carrying the plasmids were cultured for 30 h after IPTG induction, and their membrane fractions were subjected to SDS-PAGE and heme staining. Although no accumulation of specific proteins was not seen by CBB staining, production of NORB (38 kDa) and NORC (17 kDa) was detected by heme staining.However, no NOR activity was detected in any fraction of E.coli cells. This suggested the presence of some other genes or their products involved in the activation of NOR proteins.

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • Research Products

    (4 results)

All Other

All Publications (4 results)

  • [Publications] S.Sasaki et al.: "Application of nitrite reductase from Alcaligenes faecalis S-6 for nitrite measurement." Bioscnsors & Bioelectronics. 13(1). 1-5 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] S.Sasaki et al.: "Application of nitrite reductase from Alcaligenes faecalis S-6 for nitrite muasurement" Biosensors & Bioelectronics. 13-1. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] S.Sasaki et al.: "Application of nitrite reductase from Alcaligenes faecalis S-6 for nitrite measurement." Biosensors & Bioelectronics. 13(1). 1-5 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] C.A.Peters-Libeu: "Site-directed mutants of pseudoazurin:explanation of increased redox potentials from X-ray structures and from calculation of redox potential difference" Biochemistry. 36. 13160-13179 (1997)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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