Brissette R E, Tsung K L, Inouye M
Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Rutgers, Piscataway 08854.
J Bacteriol. 1991 Jan;173(2):601-8. doi: 10.1128/jb.173.2.601-608.1991.
Phosphorylation of OmpR, a transcription activator for ompF and ompC expression, is essential for its function and has been shown to be mediated in vitro by EnvZ, a transmembrane sensory receptor protein. On the basis of the three-dimensional structure of CheY which has an extensive sequence similarity with OmpR, three aspartic residues, D11, D12, and D55, of OmpR are considered to form a triacidic pocket serving as the phosphorylation center. When these aspartic acid residues were replaced with asparagine (D11N) or glutamine (D12Q and D55Q), ompF and ompC expression was almost completely blocked. Two pseudorevertants of the D11N mutation were isolated: one of them is a mutation in EnvZ (G240E), and the other is a mutation in OmpR (S48F). The envZ mutation (G240E) by itself was found to confer a phenotype very similar to that of the well known envZ11 mutation (T247R), suggesting that EnvZ (G240E) is an elevated kinase for OmpR. Consistent with this notion, EnvZ (T247R) was also able to suppress the D11N mutation in OmpR. An in vitro phosphorylation study showed that while the wild-type OmpR was phosphorylated by EnvZ, the D11N OmpR was not. These results suggest that the D11N mutation alters OmpR conformation in such a way that OmpR is very poorly phosphorylated by EnvZ. On the basis of the in vivo and in vitro analysis, the mechanisms by which the G240E mutation in EnvZ and the S48F mutation in OmpR suppress the D11N mutation in OmpR are discussed.
OmpR是ompF和ompC表达的转录激活因子，其磷酸化对其功能至关重要，且已证实在体外由跨膜传感受体蛋白EnvZ介导。基于与OmpR具有广泛序列相似性的CheY的三维结构，OmpR的三个天冬氨酸残基D11、D12和D55被认为形成一个作为磷酸化中心的三酸性口袋。当这些天冬氨酸残基被天冬酰胺（D11N）或谷氨酰胺（D12Q和D55Q）取代时，ompF和ompC的表达几乎完全被阻断。分离出了D11N突变的两个假回复突变体：其中一个是EnvZ中的突变（G240E），另一个是OmpR中的突变（S48F）。发现单独的envZ突变（G240E）赋予的表型与著名的envZ11突变（T247R）非常相似，表明EnvZ（G240E）是OmpR的增强激酶。与此观点一致，EnvZ（T247R）也能够抑制OmpR中的D11N突变。一项体外磷酸化研究表明，野生型OmpR可被EnvZ磷酸化，而D11N OmpR则不能。这些结果表明，D11N突变改变了OmpR的构象，使得EnvZ对OmpR的磷酸化作用非常弱。基于体内和体外分析，讨论了EnvZ中的G240E突变和OmpR中的S48F突变抑制OmpR中D11N突变的机制。
