title: Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli.
abstract: EnvZ and OmpR are the sensor and response regulator proteins of a two-component system that controls the porin regulon of Escherichia coli in response to osmolarity. Three enzymatic activities are associated with EnvZ: autokinase, OmpR kinase, and OmpR-phosphate (OmpR-P) phosphatase. Conserved histidine-243 is critical for both autokinase and OmpR kinase activities. To investigate its involvement in OmpR-P phosphatase activity, histidine-243 was mutated to several other amino acids and the phosphatase activity of mutated EnvZ was measured both in vivo and in vitro. In agreement with previous reports, we found that certain substitutions abolished the phosphatase activity of EnvZ. However, a significant level of phosphatase activity remained when histidine-243 was replaced with certain amino acids, such as tyrosine. In addition, the phosphatase activity of a previously identified kinase- phosphatase+ mutant was not abolished by the replacement of histidine-243 with asparagine. These data indicated that although conserved histidine-243 is important for the phosphatase activity, a histidine-243-P intermediate is not required. Our data are consistent with a previous model that proposes a common transition state with histidine-243 (EnvZ) in close contact with aspartate-55 (OmpR) for both OmpR phosphorylation and dephosphorylation. Phosphotransfer occurs from histidine-243-P to aspartate-55 during phosphorylation, but water replaces the phosphorylated histidine side chain leading to hydrolysis during dephosphorylation.
authors.affiliation: Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
authors.forename: W
authors.initials: W
authors.lastname: Hsing
authors.forename: T J
authors.initials: TJ
authors.lastname: Silhavy
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006639:Histidine; D009097:Multienzyme Complexes; D009994:Osmolar Concentration; D010749:Phosphoprotein Phosphatases / Q000235:genetics*; D017354:Point Mutation; D018272:Porins / Q000235:genetics* / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D018272:Porins; D006639:Histidine; C057843:envZ protein, E coli; D010749:Phosphoprotein Phosphatases