Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 284, Issue 16, Pages 10353-10360Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M900521200
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Funding
- Spanish Ministry of Science and Education [CICYT BIO-2006-05668, BFU 2005-0487-C02-02, CSD 200700010]
- Junta de Andalucia [CIV344, CIV1912]
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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 PtodX to the basal level. Calorimetric studies support that TodT binds to the C-terminal kinase module with a K-D of similar to 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.
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