Janausch Ingo G, Garcia-Moreno Inma, Unden Gottfried
Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, Becherweg 15, 55099 Mainz, Germany.
J Biol Chem. 2002 Oct 18;277(42):39809-14. doi: 10.1074/jbc.M204482200. Epub 2002 Aug 6.
The two-component regulatory system DcuSR of Escherichia coli controls the expression of genes of C(4)-dicarboxylate metabolism in response to extracellular C(4)- dicarboxylates such as fumarate or succinate. DcuS is a membrane-integral sensor kinase, and the sensory and kinase domains are located on opposite sides of the cytoplasmic membrane. The intact DcuS protein (His(6)-DcuS) was overproduced and isolated in detergent containing buffer. His(6)-DcuS was reconstituted into liposomes made from E. coli phospholipids. Reconstituted His(6)-DcuS catalyzed, in contrast to the detergent-solubilized sensor, autophosphorylation by [gamma-(33)P]ATP with an approximate K(D) of 0.16 mm for ATP. Up to 7% of the reconstituted DcuS was phosphorylated. Phosphorylation was stimulated up to 5.9-fold by C(4)-dicarboxylates, but not by other carboxylates. The phosphoryl group of DcuS was rapidly transferred to the response regulator DcuR. Upon phosphorylation, DcuR bound specifically to dcuB promoter DNA. The reconstituted DcuSR system therefore represents a defined in vitro system, which is capable of the complete transmembrane signal transduction by the DcuSR two-component system from the stimulus (fumarate) to the DNA, including signal transfer across the phospholipid membrane.
大肠杆菌的双组分调控系统DcuSR可响应细胞外C4-二羧酸（如富马酸或琥珀酸）来控制C4-二羧酸代谢相关基因的表达。DcuS是一种膜整合型传感激酶，其传感结构域和激酶结构域位于细胞质膜的两侧。完整的DcuS蛋白（His(6)-DcuS）在含去污剂的缓冲液中过量表达并分离出来。His(6)-DcuS被重组到由大肠杆菌磷脂制成的脂质体中。与去污剂溶解的传感器相比，重组的His(6)-DcuS能催化[γ-(33)P]ATP进行自身磷酸化，对ATP的近似解离常数K(D)约为0.16 mM。高达7%的重组DcuS被磷酸化。C4-二羧酸可将磷酸化刺激提高至5.9倍，但其他羧酸盐则无此作用。DcuS的磷酸基团迅速转移至应答调节蛋白DcuR。磷酸化后，DcuR特异性结合dcuB启动子DNA。因此，重组的DcuSR系统代表了一个明确的体外系统，它能够通过DcuSR双组分系统实现从刺激（富马酸）到DNA的完整跨膜信号转导，包括信号跨磷脂膜的传递。
