GONUTS has been updated to MW1.31 Most things seem to be working but be sure to report problems.

Have any questions? Please email us at ecoliwiki@gmail.com

PMID:10973966

From GONUTS
Jump to: navigation, search
Citation

Dutta, R, Yoshida, T and Inouye, M (2000) The critical role of the conserved Thr247 residue in the functioning of the osmosensor EnvZ, a histidine Kinase/Phosphatase, in Escherichia coli. J. Biol. Chem. 275:38645-53

Abstract

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.

Links

PubMed Online version:10.1074/jbc.M005872200

Keywords

Amino Acid Sequence; Amino Acid Substitution; Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Bacterial Proteins; Conserved Sequence; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Kinetics; Multienzyme Complexes/chemistry; Multienzyme Complexes/genetics; Multienzyme Complexes/metabolism; Mutagenesis, Site-Directed; Osmolar Concentration; Phosphoprotein Phosphatases/chemistry; Phosphoprotein Phosphatases/metabolism; Protein Kinases/chemistry; Protein Kinases/metabolism; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Threonine; Trans-Activators/chemistry; Trans-Activators/metabolism; beta-Galactosidase/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:ENVZ

GO:0046777: protein autophosphorylation

ECO:0000315:

P

EnvZ mutants cannot properly autophosphorylate in Fig 3.

complete
CACAO 4641

ECOLI:ENVZ

GO:0016301: kinase activity

ECO:0000314:

F

Figure 4 and Figure 5. Phosphotransfer from phosphorylated EnvZc and EnvZc(T247X) mutant proteins to OmpR.A. EnvZ-P, phosphorylated EnvZ.


complete
CACAO 4647

ECOLI:ENVZ

GO:0004721: phosphoprotein phosphatase activity

ECO:0000315:

F

figure 7 shows phosphatase activity of mutants relative to wild type

complete
CACAO 4686

ECOLI:ENVZ

involved_in

GO:0046777: protein autophosphorylation

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

See Help:References for how to manage references in GONUTS.