FUNCTION OF CONSERVED HISTIDINE-243 IN PHOSPHATASE-ACTIVITY OF ENVZ, THE SENSOR FOR PORIN OSMOREGULATION IN ESCHERICHIA-COLI

EnvZ and OmpR are the sensor and response regulator proteins of a two- component system that controls the porin regulon of Escherichia coli i n response to osmolarity. Three enzymatic activities are associated wi th EnvZ: autokinase, OmpR kinase, and OmpR-phosphate (OmpR-P) phosphat ase. Conserved histidine-243 is critical for both autokinase and OmpR kinase activities. To investigate its involvement in OmpR-P phosphatas e activity, histidine-243 was mutated to several other amino acids and the phosphatase activity of mutated EnvZ was measured both in vivo an d in vitro. In agreement with previous reports, we found that certain substitutions abolished the phosphatase activity of EnvZ. However, a s ignificant level of phosphatase activity remained when histidine-243 w as replaced with certain amino acids, such as tyrosine. In addition, t he phosphatase activity of a previously identified kinase(-) phosphata se(+) mutant was not abolished by the replacement of histidine-243 wit h asparagine. These data indicated that although conserved histidine-2 43 is important for the phosphatase activity, a histidine-243-P interm ediate is not required. Our data are consistent with a previous model that proposes a common transition state with histidine-243 (EnvZ) in c lose contact with aspartate-55 (OmpR) for both OmpR phosphorylation an d dephosphorylation. Phosphotransfer occurs from histidine-243-P to as partate-55 during phosphorylation, but water replaces the phosphorylat ed histidine side chain leading to hydrolysis during dephosphorylation .