Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida.
Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida.
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DOI：
10.1016/j.fob.2014.03.013
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发表时间：
2014
期刊：
影响因子：
2.6
通讯作者：
de Lorenzo, Victor
中科院分区：
文献类型：
--
作者：
Chavarria, Max;Durante-Rodriguez, Gonzalo;Krell, Tino;Santiago, Cesar;Brezovsky, Jan;Damborsky, Jiri;de Lorenzo, Victor
FBP作为土壤细菌恶臭假单胞菌CRA蛋白的效应子的作用尚不清楚。生化、生物物理和遗传学数据表明，CRA只与代谢激动剂F1P结合。F1P是恶臭假单胞菌CRA蛋白在体内唯一的生理效应因子。CRA的这种调节突起例证了转录因子如何在细菌中多样化。1-磷酸果糖(F1P)是土壤细菌恶臭假单胞菌分解代谢抑制/激活蛋白(CRA)的首选效应物，但其与体内其他代谢中间产物的结合能力尚不清楚。将该微生物(CRAPP)的CRA蛋白与目标DNA序列(PruB启动子)和候选效应因子果糖-1，6-二磷酸(FBP)、葡萄糖-6-磷酸(G6P)和果糖-6-磷酸(F6P)进行迁移率变化分析。1 mM的F1P足以从操作者那里释放大部分CRA蛋白，但需要超过10 mM的FBP或G6P才能释放相同的复合体。然而，等温滴定微量热法未能揭示CRAPP与FBP或G6P之间的任何特定相互作用。为了解决这一悖论，我们在野生型和ΔFruB细胞中测量了PruB-LacZ融合的转录活性，这些细胞生长在改变细胞内F1P和FBP浓度的底物上。结果表明，果糖能刺激PruB的活性，而葡萄糖和琥珀酸不能刺激PruB的活性。这表明CRAPP在体内以一种仅依赖于F1P的方式抑制同源ruBKA操纵子的表达，排除了任何其他生理效应。分子对接和CRA-激动剂相互作用的动态模拟表明，两种代谢物都能结合阻遏物，但CRAPP与F1P和FBP的相对亲和力缺口比大肠杆菌蛋白中的等效距离大三个数量级。这使得恶臭假单胞菌的CRA蛋白唯一的作用是将介质中果糖的存在转化为各种直接和间接的生理反应。
The role of FBP as effector of the Cra protein of soil bacterium Pseudomonas putida is unclear. Biochemical, biophysical and genetic data show that Cra binds only F1P as metabolic agonist. F1P is the only physiological effector of the Cra protein of P. putida in vivo. This regulatory exaptation of Cra exemplifies how transcriptional factors can diversify in bacteria. Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (CraPP) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between CraPP and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that CraPP represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of CraPP for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.
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4.8
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