Identification of the site of phosphorylation on the osmosensor, EnvZ, of Escherichia coli

J Biol Chem. 1994 Mar 25;269(12):8728-33.


EnvZ is a membrane-bound histidine kinase that functions as an osmotic sensor capable of phosphorylating the regulator protein OmpR in Escherichia coli. To characterize the site of phosphorylation biochemically, we overexpressed a 36-kDa truncated EnvZ protein (Glu-106 to Gly-450) that formed inclusion bodies in the cell. After solubilization, the inclusion body form of EnvZ was cleaved into two major fragments with molecular weights of 25,000 and 10,000. The 25-kDa fragment, EnvZc, was purified and found to exist as a dimer. N-terminal sequence analysis established that cleavage had occurred at Arg-214, indicating that EnvZc contained most of the cytoplasmic domain of EnvZ. After labeling EnvZc with [gamma-32P]ATP, the protein was proteolytically digested, and the resulting peptides were separated by reverse phase chromatography using high performance liquid chromatography. One major radioactive peptide containing greater than 90% of the recovered peptide-associated radioactivity was isolated. Amino acid analysis of this purified peptide indicated that the composition was consistent with a peptide that contained His-243. The amino acid sequence of this peptide was determined to be MAGVSHDLRTP (residues 238-248). These results indicate that His-243 is the major site of phosphorylation on EnvZ and represents the first biochemical characterization of the site of phosphorylation of a membrane histidine kinase of the two-component regulatory family of molecules in bacteria.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Outer Membrane Proteins / chemistry
  • Bacterial Outer Membrane Proteins / metabolism*
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins*
  • Histidine / chemistry
  • Molecular Sequence Data
  • Multienzyme Complexes*
  • Osmolar Concentration
  • Peptide Fragments / chemistry
  • Peptide Mapping
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Trypsinogen / pharmacology


  • Bacterial Outer Membrane Proteins
  • Escherichia coli Proteins
  • Multienzyme Complexes
  • Peptide Fragments
  • Phosphoproteins
  • Histidine
  • Trypsinogen
  • envZ protein, E coli