Identification of the site of phosphorylation on the osmosensor, EnvZ, of Escherichia coli.
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.
Roberts
D L
DL
Department of Chemistry, University of Wisconsin-Milwaukee 53201.
Bennett
D W
DW
Forst
S A
SA
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D014362:Trypsinogen / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D010750:Phosphoproteins; D006639:Histidine; D014362:Trypsinogen; C057843:envZ protein, E coli