Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida
Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida
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DOI：
10.1074/jbc.m110.187583
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发表时间：
2011-03-18
影响因子：
4.8
通讯作者：
de Lorenzo, Victor
中科院分区：
文献类型：
--
作者：
Chavarria, Max;Santiago, Cesar;de Lorenzo, Victor
分解代谢产物阻遏物/激活物（Cra）蛋白是通过革兰氏阴性菌的中心代谢途径的碳通量的全局传感器和调节器。要检查的性质的效应器（或效应器），信号这样的通量恶臭假单胞菌的蛋白质，该土壤微生物的Cra因子已被纯化和表征，并确定其三维结构。超离心、凝胶过滤和迁移率变动分析表明，无效应子的Cra是一种二聚体，其结合fruBKA簇启动子区的操纵基因DNA序列。此外，果糖1-磷酸（F1 P）被发现最有效地解离的Cra-DNA复合物。通过等温滴定量热法计算的F1 P-Cra-DNA相互作用的热力学参数显示，该因子与DNA序列5 '-TTAAACGTTTCA-3'紧密缔合（K-D = 26.3 +/- 3.1 nM），并且F1 P以每个Cra单体1.06 +/- 0.06个分子的表观化学计量和209 +/- 20 nM的K-D结合蛋白质。其他可能的效应物，如果糖1，6-二磷酸，在测定条件下未显示出对调节剂的显著亲和力。此外，在2埃分辨率下Cra及其与F1 P的共晶体的结构显示F1 P最佳地适合效应器口袋的几何形状。因此，我们的研究结果挑选出F1 P作为恶臭假单胞菌Cra蛋白的首选代谢效应子。
The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5'-TTAAACGTTTCA-3' (K-D = 26.3 +/- 3.1 nM) and that F1P binds the protein with an apparent stoichiometry of 1.06 +/- 0.06 molecules per Cra monomer and a K-D of 209 +/- 20 nM. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-angstrom resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida.