Reverse phosphotransfer from OmpR to EnvZ in a kinase(-)/phosphatase(+) mutant of EnvZ (EnvZ center dot N347D), a bifunctional signal transducer of Escherichia coli
Reverse phosphotransfer from OmpR to EnvZ in a kinase(-)/phosphatase(+) mutant of EnvZ (EnvZ center dot N347D), a bifunctional signal transducer of Escherichia coli
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DOI：
10.1074/jbc.271.3.1424
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发表时间：
1996-01-19
影响因子：
4.8
通讯作者：
Inouye, M
中科院分区：
文献类型：
--
作者：
Dutta, R;Inouye, M
大肠杆菌的EnvZ是一种跨膜组氨酸激酶，属于原核生物中普遍存在的双组分信号转导系统家族，最近在真核生物中发现。响应于介质渗透压的变化，EnvZ调节磷酸化OmpR的水平，其缀合物响应调节ompF和ompC基因的转录因子。EnvZ具有双重相反的酶活性; OmpR-磷酸化酶（激酶）和磷酸-OmpR-去磷酸化酶（磷酸酶）。渗透信号被提出来调节EnvZ的激酶与磷酸酶活性的比率以调节OmpR磷酸化水平。在这项工作中，我们使用了先前鉴定的激酶（-）/磷酸酶（+）EnvZ突变体（EnvZ . N347 D），以证明磷酸-Ompr上的磷酰基转移回EnvZ至野生型蛋白用于自身激酶反应的相同组氨酸残基（His（243））。Phospho-EnvZ .由此形成的N347 D也可以将其磷酰基转移回OmpR。从磷酸化OmpR到EnvZ的磷酸转移反应。ADP抑制N347 D的脱磷酸化反应，Mg ~（2+）促进N347 D的脱磷酸化反应，释放无机磷。这些结果表明OmpR和EnvZ上的磷酰基基团的能级非常相似，并且EnvZ中的磷酸酶反应。N347 D突变体涉及使用相同的His（243）残基逆转从EnvZ到OmpR的磷酸转移反应。
EnvZ of Escherichia coli is a transmembrane histidine kinase belonging to the family of two-component signal transducing systems prevalent in prokaryotes and recently discovered in eukaryotes. In response to changes in medium osmolarity EnvZ regulates the level of phosphorylated OmpR, its conjugate response regulating transcription factor for ompF and ompC genes. EnvZ has dual opposing enzymatic activities; OmpR-phosphorylase (kinase) and phospho-OmpR-dephosphorylase (phosphatase). The osmotic signal is proposed to regulate the ratio of the kinase to the phosphatase activities of EnvZ to modulate the level of OmpR phosphorylation. In this work we used a COOH terminal fragment of a previously identified kinase(-)/phosphatase(+) EnvZ mutant (EnvZ . N347D) to demonstrate that the phosphoryl group on phospho-Ompr is transferred back to EnvZ to the same histidine residue (His(243)) that is utilized for the autokinase reaction by the wild type protein. Phospho-EnvZ . N347D thus formed could also transfer its phosphoryl group back to OmpR. The phosphotransfer reaction from phospho-OmpR to EnvZ . N347D was inhibited by ADP while Mg2+ ions stimulated the dephosphorylation reaction, resulting in release of inorganic phosphate. These results indicate that the energy levels of phosphoryl groups on OmpR and EnvZ are very similar and that the phosphatase reaction in the EnvZ . N347D mutant involves a reversal of the phosphotransfer reaction from EnvZ to OmpR using the identical His(243) residue.