The sensor kinase TodS operates by a multiple step phosphorelay mechanism involving two autokinase domains

J Biol Chem. 2009 Apr 17;284(16):10353-60. doi: 10.1074/jbc.M900521200. Epub 2009 Feb 24.


Expression of the Pseudomonas putida tod operon, which encodes enzymes for toluene metabolism, takes place from the P(todX) promoter and is mediated by the TodS/TodT two component system. The sensor kinase TodS has a complex domain arrangement containing two functional modules, each harboring a sensor- and an autokinase domain and separated by a receiver domain. Based on site-directed mutagenesis of phosphoaccepting His-190, Asp-500, and His-760 and in vitro transphosphorylation experiments with recombinant TodS fragments, we show that TodS uses a multiple step phosphorelay mechanism to activate TodT. Toluene binding stimulates exclusively phosphorylation of His-190, which is followed by phosphotransfer to Asp-500 and subsequently to His-760 prior to phosphorylation of TodT Asp-57. Mutation of His-190, Asp-500, and H760A prevented up-regulation of toluene-mediated stimulation of TodT transphosphorylation in vitro and reduced in vivo expression of P(todX) to the basal level. Calorimetric studies support that TodT binds to the C-terminal kinase module with a K(D) of approximately 200 nm and 1:1 stoichiometry. This is the first report of a multiple step phosphorelay mechanism of a sensor kinase that involves two autokinase domains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Histidine / metabolism
  • Mutagenesis, Site-Directed
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Phosphorylation
  • Protein Kinases / chemistry
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Pseudomonas putida / enzymology*
  • Pseudomonas putida / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Toluene / metabolism
  • Trans-Activators / chemistry
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Up-Regulation


  • Bacterial Proteins
  • Peptide Fragments
  • Recombinant Proteins
  • TodT protein, Pseudomonas putida
  • Trans-Activators
  • Toluene
  • Histidine
  • Protein Kinases
  • TodS protein, Pseudomonas putida