Studies on the Bacterial Tetracyclin/H^+ Antiport Mechanisms using Site-directed Mutagenesis

利用定点诱变研究细菌四环素/H^反向转运机制

基本信息

批准号：
03833003
负责人：
YAMAGUCHI Akihito
金额：
$ 1.22万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Bacterial tetracycline resistance is based on the active efflux of the drug. We revealed that the tetracycline resistance protein (TetA) is a 1:1 antiporter of Mg^<2+>-tetracycline chelation complex and proton at late 1980's.In this study, we established many site-directed mutants of the transposon Tn10- encoded TetA protein and performed the identification of the functionally essential residues in its transport function. As a result, we revealed some important principles common to the other membrane transport proteins as follows:1) All of the three acidic residues and one basic residue located in the putative transmembrane region of the TetA protein are essential for its transport function. In contrast, among eight conserved acidic residues in the hydrophilic region, only one residue, Asp66, is essential. Out of nine conserved basic residues in the hydrophilic region, one residue, Asp70, is essential for the transport function and one residue, Asp31, is essential for incorporation of the protein into the membrane.2) Functionally important residues in the transmembrane region are arranged on vertical stripes in the transmembrane alpha-helices, indicating the presence of the channel-like structure.3) There are two hydrophilic loops comprising highly conserved residues, which are likely to play as a gate for substrate translocation.On the basis of these observations, we claimed the gating-channel model for tetracycline/H^+ antiport.

细菌四环素耐药性基于该药物的主动外排。我们透露，四环素抗性蛋白（TETA）是Mg^<2+>的1：1抗焦蛋白 - 四环素螯合复合物和质子在1980年代末。在这项研究中，我们确立了许多位置定向的转座子TN10 TN10 TN10-TETA TETA蛋白质的突变体，并在具有功能功能的功能中执行了具有功能功能的识别。结果，我们揭示了其他膜转运蛋白共有的一些重要原理，如下所示：1）所有三个酸性残基和一个位于TETA蛋白推定的跨膜区域中的一个基本残基对于其运输功能至关重要。相反，在亲水区的八个保守酸性残基中，只有一个残基ASP66至关重要。在亲水性区域的九个保守基本残基中，一个残基ASP70对于运输功能至关重要，而一个残基ASP31对于将蛋白质掺入膜中至关重要。2）在跨膜区域中在功能上重要的残基排列在跨膜alpha-helpha-Helece的垂直条纹上，指示液体效果，指示液体效应，是指元素的流长型，是元素的效果。高度保守的残基很可能是底物易位的门。在这些观察结果的基础上，我们声称是四环素/H^+ antiport的门控通道模型。