Reverse phosphotransfer from OmpR to EnvZ in a kinase-/phosphatase+ mutant of EnvZ (EnvZ.N347D), a bifunctional signal transducer of Escherichia coli.
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 (His243) 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 His243 residue.
Dutta
R
R
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Inouye
M
M
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D009097:Multienzyme Complexes; D016296:Mutagenesis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D014157:Transcription Factors; D010770:Phosphotransferases; D011494:Protein Kinases; C057843:envZ protein, E coli; D010749:Phosphoprotein Phosphatases