Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli.
Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli.
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DOI：
10.1046/j.1432-1033.2003.03507.x
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发表时间：
2003-04
期刊：
影响因子：
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通讯作者：
Christian Schwoppe;Herbert H. Winkler;H. Neuhaus
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文献类型：
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作者：
Christian Schwoppe;Herbert H. Winkler;H. Neuhaus
UhpC是大肠杆菌中识别外部葡萄糖-6-磷酸（Glc 6P）和诱导转运蛋白UhpT所需的膜结合传感器蛋白。最近，研究表明UhpC也能够转运Glc 6P。在这项研究中，我们调查了这些运输和传感活动是否强制耦合在UhpC。我们在UhpC缺陷型大肠杆菌中表达了His-UhpC蛋白。大肠杆菌菌株，并验证了该构建体不改变Glc 6P传感器系统的基本生物化学性质。精氨酸替代，突变的中央回路，并在UhpC上的运输和传感的盐桥的引入的影响进行了比较。交换R46 C，R266 C和R149 C适度影响UhpC的运输，但强烈降低传感能力。这表明，在UhpC中，对作为转运底物的Glc 6P的亲和力与其对作为诱导物的Glc 6P的亲和力解偶联。11个精氨酸突变体中有4个表现出组成型表型，但具有接近野生型的转运活性，这表明Glc 6P可以通过锁定在诱导构象中的分子转运。螺旋内盐桥的引入增加了UhpC的运输活性，但取消了传感。从中央环的三个保守的残基突变，虽然没有这些显示运输，一个表现出增加的亲和力传感。总之，这些数据表明，运输UhpC是不需要的传感，保守的精氨酸残基是重要的传感，而不是运输，和位于中央亲水环的残基是至关重要的运输和传感。
UhpC is a membrane-bound sensor protein in Escherichia coli required for recognizing external glucose-6-phosphate (Glc6P) and induction of the transport protein UhpT. Recently, it was shown that UhpC is also able to transport Glc6P. In this study we investigated whether these transport and sensing activities are obligatorily coupled in UhpC. We expressed a His-UhpC protein in a UhpC-deficient E. coli strain and verified that this construct does not alter the basic biochemical properties of the Glc6P sensor system. The effects of arginine replacements, mutations of the central loop, and introduction of a salt bridge in UhpC on transport and sensing were compared. The exchanges R46C, R266C and R149C moderately affected transport by UhpC but strongly decreased the sensing ability. This suggested that the affinity for Glc6P as a transported substrate is uncoupled in UhpC from its affinity for Glc6P as an inducer. Four of the 11 arginine mutants showed a constitutive phenotype but had near wild-type transport activity suggesting that Glc6P can be transported by a molecule locked in the inducing conformation. Introduction of an intrahelical salt bridge increased the transport activity of UhpC but abolished sensing. Three conserved residues from the central loop were mutated and although none of these showed transport, one exhibited increased affinity for sensing. Taken together, these data show that transport by UhpC is not required for sensing, that conserved arginine residues are important for sensing and not for transport, and that residues located in the central hydrophilic loop are critical for transport and for sensing.