Functional Analysis of the C-Terminal Region of γ-Glutamyl Kinase of <I>Saccharomyces cerevisiae</I>
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概要
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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.
著者
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Takagi Hiroshi
Graduate School Of Biological Sciences Nara Inst. Of Sci. And Technol.
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Kaino Tomohiro
Graduate School Of Biolosical Sciences Nara Institute Of Science And Technology
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TATEHASHI Yuki
Graduate School of Biological Sciences, Nara Institute of Science and Technology
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Tasaka Yumi
Graduate School Of Biological Sciences Nara Institute Of Science And Technology
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