Kaino Tomohiro, Tasaka Yumi, Tatehashi Yuki, Takagi Hiroshi
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
Biosci Biotechnol Biochem. 2012;76(3):454-61. doi: 10.1271/bbb.110682.
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(1)-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 Saccharomyces cerevisiae 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 Escherichia coli and S. cerevisiae 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 E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.
γ-谷氨酰激酶（GK）是微生物脯氨酸合成中的限速酶。大多数微生物GK包含一个N端激酶结构域和一个C端假尿苷合酶及古肌苷转糖基酶（PUA）结构域。相比之下，高等真核生物拥有一种双功能的Δ(1)-吡咯啉-5-羧酸合成酶，它由一个不含PUA的GK结构域和一个γ-谷氨酰磷酸还原酶（GPR）结构域组成。在此，为了研究酿酒酵母GK的包括PUA结构域在内的C端区域的作用，我们构建了多种截短的酵母GK和GK/GPR融合蛋白，其中C端区域被删除。在大肠杆菌和酿酒酵母中进行的互补试验以及对重组蛋白的酶活性分析表明，在一个255个氨基酸残基的激酶结构域和一个106个氨基酸残基的PUA结构域之间的一个67个氨基酸残基的连接序列对于GK活性至关重要。似乎C端区域的67个或更多氨基酸残基而非PUA结构域本身对于GK活性的充分展现是必需的。此外，GK/GPR融合蛋白在大肠杆菌中具有功能，但与野生型GK相比稳定性和镁结合能力有所下降。这些结果表明酿酒酵母GK的C端区域参与激酶结构域的折叠和/或稳定性。
