Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain.
Protein Eng Des Sel. 2012 Apr;25(4):159-69. doi: 10.1093/protein/gzs001. Epub 2012 Feb 3.
Two-component systems (TCSs) play key roles in the adaptation of bacteria to environmental changes. In prototype TCSs a single phosphoryl transfer between the sensor kinase and response regulator occurs, whereas phosphorelay TCSs are characterised by a His1-Asp1-His2-Asp2 phosphorylation cascade. The TodS/TodT TCS controls the expression of a toluene degradation pathway and the TodS sensor kinase operates by a three-step internal phosphorelay. Based on TodS we report the construction of a minimal form of TodS, termed as Min-TodS, that contains only three of the seven TodS domains. Min-TodS is composed of the N-terminal PAS sensor domain as well as the C-terminal dimerisation/phosphotransfer domain and catalytic domain of TodS. We have conducted a comparative analysis of the phosphorelay TCS with its prototypal derivative. We demonstrate that Min-TodS binds effector molecules with affinities comparable with those observed for TodS. Min-TodS forms a TCS with TodT and toluene increases the amount of TodT-P. In contrast to TodS, toluene does not stimulate Min-TodS autophosphorylation. The half-life of Min-TodS-P was significantly increased as compared with TodS. Analysis of TodSD500A revealed that the hydrolysis of the acylphosphate of the receiver domain is responsible for the reduced half-life of TodS. The regulation of P(todX) expression by Min-TodS/TodT and TodS/TodT in response to different effectors are compared. The Min-TodS/TodT system was characterized by a higher basal activity but a lower magnitude of response. Data will be discussed in the context that the phosphorelay system appears to be better suited for the control of a degradation pathway for toxic compounds.
双组分系统 (TCS) 在细菌适应环境变化中起着关键作用。在原型 TCS 中，传感器激酶和响应调节剂之间只发生一次磷酸转移，而磷酸接力 TCS 的特征是 His1-Asp1-His2-Asp2 磷酸化级联。TodS/TodT TCS 控制甲苯降解途径的表达，TodS 传感器激酶通过三步内部磷酸接力作用发挥作用。基于 TodS，我们构建了一个最小形式的 TodS，称为 Min-TodS，它只包含 TodS 的七个结构域中的三个。Min-TodS 由 N 端 PAS 传感器结构域以及 C 端二聚化/磷酸转移结构域和 TodS 的催化结构域组成。我们对磷酸接力 TCS 与其原型衍生物进行了比较分析。我们证明 Min-TodS 与 TodS 一样能够结合效应分子，亲和力相当。Min-TodS 与 TodT 形成 TCS，甲苯增加 TodT-P 的量。与 TodS 相反，甲苯不会刺激 Min-TodS 自磷酸化。与 TodS 相比，Min-TodS-P 的半衰期显著增加。对 TodSD500A 的分析表明，受体结构域的酰基磷酸酯的水解是 TodS 半衰期缩短的原因。比较了 Min-TodS/TodT 和 TodS/TodT 对不同效应物的 P(todX) 表达的调节。Min-TodS/TodT 系统的基础活性较高，但响应幅度较低。数据将在以下背景下进行讨论：磷酸接力系统似乎更适合控制有毒化合物的降解途径。
