The sensor kinase TodS operates by a multiple step phosphorelay mechanism involving two autokinase domains.
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.
Busch
Andreas
A
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Prof. Albareda, 1, 18008 Granada, Spain.
Guazzaroni
María-Eugenia
ME
Lacal
Jesús
J
Ramos
Juan Luis
JL
Krell
Tino
T
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014050:Toluene / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C104650:TodT protein, Pseudomonas putida; D015534:Trans-Activators; D014050:Toluene; D006639:Histidine; D011494:Protein Kinases; C104649:TodS protein, Pseudomonas putida