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The activation of PhoB by acetylphosphate
Author(s) -
McCleary W. R.
Publication year - 1996
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1996.tb02636.x
Subject(s) - biology , autophosphorylation , histidine kinase , response regulator , regulon , biochemistry , dimer , escherichia coli , biophysics , phosphorylation , histidine , enzyme , chemistry , protein kinase a , organic chemistry , gene , mutant
Summary PhoB is a response‐regulator protein from Escherichia coli that controls an adaptive response to limiting phosphate. It is activated by autophosphorylation of a conserved aspartate residue within its regulatory domain. Its primary phospho‐donor is its cognate histidine kinase PhoR; however, it also becomes phos‐phorylated when incubated with acetylphosphate. To further characterize its activation, PhoB was considered to be an acetylphosphatase whose enzymatic mechanism involves a phospho‐enzyme intermediate. The kinetic constants for autophosphorylation were determined using 32 P‐ and fluorescence‐based assays and indicated that PhoB has a K m for acetylphosphate of between 7 and 8 mM. These constants are not consistent with an in vivo role for acetylphosphate in the normal control of the Pho regulon. In addition, when PhoB was phosphorylated by acetylphosphate it eluted from a high‐performance liquid chromatography (HPLC) size‐exclusion column in two peaks. The larger form of PhoB eluted from the column in a similar manner to a chemically cross‐linked dimer of PhoB. The smaller form of PhoB is a monomer. Phosphorylated PhoB bound pho‐ box DNA approximately 10 times tighter than PhoB. These observations show that PhoB forms a dimer when phosphorylated and suggest that the characteristics of activated PhoB result from its dimer ization.