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J. Biol. Chem., Vol. 275, Issue 49, 38645-38653, December 8, 2000
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From the Department of Biochemistry, Robert Wood Johnson Medical
School, Piscataway, New Jersey 08854-5635
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
Thr247 residue in EnvZ. Using purified C-terminal domains
of wild-type and mutant EnvZ proteins, we demonstrate that
Thr247 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 Thr247 to
Arg in domain A abolishes its phosphatase activity. Furthermore, using
an in vivo
The Critical Role of the Conserved Thr247 Residue in
the Functioning of the Osmosensor EnvZ, a Histidine Kinase/Phosphatase,
in Escherichia coli*
,, and
-galactosidase activity assay of Taz1-1
(hybrid of the aspartate receptor Tar and EnvZ) constructs of the
Thr247 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 -galactosidase.
*
This work was supported by Grant GM19043 from the National
Institutes of Health and in part by a grant from SmithKline Beecham.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to this work.
§
To whom correspondence should be addressed: Dept. of Biochemistry,
Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ
08854-5635. Tel.: 732-235-4115; Fax: 732-235-4559; E-mail: inouye@rwja.umdnj.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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