The critical role of the conserved Thr247 residue in the functioning of the osmosensor EnvZ, a histidine Kinase/Phosphatase, in Escherichia coli.
The histidine kinase/phosphatase EnvZ helps Escherichia coli adapt to osmotic shock by controlling the phosphorylation state of the transcription factor OmpR, which regulates the levels of the outer membrane porin proteins OmpF and OmpC. We examined the effects of mutating the highly conserved Thr(247) residue in EnvZ. Using purified C-terminal domains of wild-type and mutant EnvZ proteins, we demonstrate that Thr(247) plays a vital role in EnvZ function, variously affecting its autokinase and phosphotransferase activities, but mostly its function as a phosphatase. The cytoplasmic domain of EnvZ (EnvZc) is composed of three segments: the linker domain (residues 180-222), domain A (residues 223-289), and domain B (residues 290-450). It has been shown that the isolated domain A itself can dephosphorylate phosphorylated OmpR. Here we show that mutating Thr(247) to Arg in domain A abolishes its phosphatase activity. Furthermore, using an in vivo beta-galactosidase activity assay of Taz1-1 (hybrid of the aspartate receptor Tar and EnvZ) constructs of the Thr(247) mutants in RU1012 cells expressing ompC-lacZ, we demonstrate that the external signal primarily down-regulates the phosphatase activity of EnvZ. Of the nine EnvZc(T247X) mutants (X = Ser, Ala, Cys, Lys, Asn, Glu, Gln, Tyr, or Arg) analyzed, only Ser functionally substituted for Thr at this position, whereas all the others displayed constitutive expression of beta-galactosidase.
Dutta
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R
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635, USA.
Yoshida
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T
Inouye
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M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D013912:Threonine; D011494:Protein Kinases; D000071677:Histidine Kinase; C057843:envZ protein, E coli; D010749:Phosphoprotein Phosphatases; D001616:beta-Galactosidase