ISSN:
1365-2958
Quelle:
Blackwell Publishing Journal Backfiles 1879-2005
Thema:
Biologie
,
Medizin
Notizen:
The two-component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE–CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327–547) showed constitutive autokinase activity and transferred the γ-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB–D56N formed a stable complex with MalE–CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45–176) was overproduced as a soluble, cytoplasmic protein with a C-terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri- and dicarboxylates tested, with high affinity (KD ≈ 5 μM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (ΔH = −76.3 kJ mol−1), whereas the entropy change was opposed (−TΔS = + 46.3 kJ mol−1). The pH dependency of the binding reaction indicated that the dianionic form H-citrate2− is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg–citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1046/j.1365-2958.1999.01536.x
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