The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichia coli.
The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichia coli.
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DOI：
10.1073/pnas.98.3.908
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发表时间：
2001-01
影响因子：
11.1
通讯作者：
Ling Qin;Takeshi Yoshida;Masayori Inouye
中科院分区：
文献类型：
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作者：
Ling Qin;Takeshi Yoshida;Masayori Inouye
大肠杆菌通过组氨酸激酶 EnvZ 及其同源反应调节剂 OmpR 调节其孔蛋白表达。 EnvZ 是一种双功能酶，具有 OmpR 激酶和磷酸化 OmpR (OmpR-P) 磷酸酶活性，从而控制 OmpR-P 的细胞水平。在体外测定系统中，由于 EnvZ 的双重活性，将 OmpR 添加到由 EnvZ 的胞质结构域 (EnvZc) 和 ATP 组成的反应混合物中，会产生几乎不可检测量的 OmpR-P。在这里，我们报告含有孔蛋白基因上游启动子区域（ompF和ompC）的DNA片段可以将OmpR和OmpR-P之间的平衡显着转向OmpR-P。在混合物中发生的四个反应中，只有 EnvZ 磷酸酶活性受到特定 DNA 的严重抑制，这与 Kenney 及其同事之前的报告相反，即 DNA 通过 EnvZ 刺激 OmpR 磷酸化 [Ames, S. K., Frankema, N. & Kenney, L. J. (1999) Proc.国家。阿卡德。科学。美国 96，11792-11797]。 EnvZc的自磷酸化以及从磷酸化的EnvZc到OmpR的磷酸转移不受DNA影响，而OmpR-P的自去磷酸化受到轻微抑制。我们认为，DNA 对 EnvZ 磷酸酶功能的明显抑制作用是由于 OmpR-P 与 DNA 结合而从反应中隔离 OmpR-P 引起的。
Escherichia coli modulates its porin expression through a histidine kinase, EnvZ, and its cognate response regulator, OmpR. EnvZ is a bifunctional enzyme that possesses both OmpR kinase and phosphorylated OmpR (OmpR-P) phosphatase activities and thus controls the cellular level of OmpR-P. In an in vitro-assay system, the addition of OmpR to the reaction mixture consisting of the cytoplasmic domain of EnvZ (EnvZc) and ATP produces a barely detectable amount of OmpR-P because of the dual activities of EnvZ. Here we report that DNA fragments containing the upstream promoter regions of the porin genes (ompF and ompC) can shift the equilibrium between OmpR and OmpR-P dramatically toward OmpR-P. Among the four reactions occurring in the mixture, only the EnvZ phosphatase activity was inhibited severely by the specific DNA, in contrast to the previous report by Kenney and her associates that DNA stimulates OmpR phosphorylation by EnvZ [Ames, S. K., Frankema, N. & Kenney, L. J. (1999) Proc. Natl. Acad. Sci. USA 96, 11792-11797]. The autophosphorylation of EnvZc and the phosphotransfer from phosphorylated EnvZc to OmpR were not affected by DNA, whereas the autodephosphorylation of OmpR-P was inhibited slightly. We propose that the apparent inhibitory effect of DNA on the EnvZ phosphatase function is caused by sequestrating OmpR-P from the reaction as a result of OmpR-P binding to DNA.