title: The sensor kinase TodS operates by a multiple step phosphorelay mechanism involving two autokinase domains.
abstract: 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.
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Prof. Albareda, 1, 18008 Granada, Spain.
authors.forename: Andreas
authors.initials: A
authors.lastname: Busch
authors.forename: María-Eugenia
authors.initials: ME
authors.lastname: Guazzaroni
authors.forename: Jesús
authors.initials: J
authors.lastname: Lacal
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: Ramos
authors.forename: Tino
authors.initials: T
authors.lastname: Krell
mesh_terms: 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
chemical_list: 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