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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03833003/
• 关键词: tetracycline efflux protein; antiporter; drug efflux; antibiotic resistance; site-directed mutagenesis; antiport; membrane protein; gene engineering; テトラサイクリン; 薬剤排出ポンプ; アンチポ-ト; テトラサイクリン耐性; 大腸菌; 膜輸送

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+> - Tetracycline螯合复合物和1980年代末质子的1：1抗焦蛋白。 TETA蛋白，并在其运输功能中鉴定了功能上必不可少的残基。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。在亲水区域的九个保守基本残基中，一个残基ASP70对于传输函数至关重要，而一个残基ASP31对于将蛋白质掺入膜中至关重要。2）在跨膜区域中具有功能重要的残基在跨膜α-螺旋中的垂直条纹上，表明存在类似通道的结构。3）有两个亲水环包含高度保守的残基，它们很可能是底物转运的门。 ，我们声称是四环素/H^+反管的门控通道模型。

项目成果

YAMAGUCHI,A.,et al.: "Serine residues responsible for tetracycline transport are on a vertical stripe including あsp-84....." FEBS Letters. 307. 229-232 (1992)

YAMAGUCHI,A.,et al.：“负责四环素转运的丝氨酸残基位于包括 asp-84 的垂直条纹上……”FEBS Letters 307. 229-232 (1992)。

Yamaguchi, A., Y. Someya, and T. Sawai: "Metal-tetracycline/H^+ antiporter of Escherichia coli encoded by a transposon Tn10: The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3." J. Biol. Chem.267. 19

Yamaguchi, A.、Y. Someya 和 T. Sawai：“由转座子 Tn10 编码的大肠杆菌金属四环素/H^ 逆向转运蛋白：保守序列基序 GXXXXRXGRR 在螺旋 2 和螺旋之间推定的细胞质环中的作用

山口 明人(分担): "続医薬品の開発第4巻薬物の生体膜輸送と組織標的化I" 廣川書店, 327 (1991)

Akito Yamaguchi（撰稿人）：“持续药物开发第 4 卷：药物生物膜转运和组织靶向 I”广川书店，327（1991）

Yamaguchi, A., K. Adachi, T. Akasaka, N. Ono, and T. Sawai: "Metal-tetracycline/H^+ antiporter of Escherichia coli encoded by a transposon Tn10 Histidine 257 plays an essential role in H^+ translocation." J. Biol. Chem.266. 6045-6051 (1991)

Yamaguchi, A.、K. Adachi、T. Akasaka、N. Ono 和 T. Sawai：“由转座子 Tn10 组氨酸 257 编码的大肠杆菌金属四环素/H ^ 反向转运蛋白在 H ^ 易位中发挥重要作用。”

Yamaguchi, A., et al.: "pH-dependent accumulation of tetracycline in Escheri-chia coli." Antimicrob. Agents Chemother.35. 53-56 (1991)

Yamaguchi, A. 等人：“四环素在大肠杆菌中的 pH 依赖性积累。”