Functional Analysis of the C-Terminal Region of γ-Glutamyl Kinase of Saccharomyces cerevisiae

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γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ<SUP>1</SUP>-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of <I>Saccharomyces cerevisiae</I> GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in <I>Escherichia coli</I> and <I>S. cerevisiae</I> and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in <I>E. coli</I>, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of <I>S. cerevisiae</I> GK is involved in the folding and/or the stability of the kinase domain.

収録刊行物

  • Bioscience, biotechnology, and biochemistry

    Bioscience, biotechnology, and biochemistry 76(3), 454-461, 2012-03-23

    公益社団法人 日本農芸化学会

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各種コード

  • NII論文ID(NAID)
    10030750315
  • NII書誌ID(NCID)
    AA10824164
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    09168451
  • NDL 記事登録ID
    023553671
  • NDL 請求記号
    Z53-G223
  • データ提供元
    CJP書誌  NDL  J-STAGE 
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