Transforming a drug/H+ antiporter into a polyamine importer by a single mutation
Transforming a drug/H+ antiporter into a polyamine importer by a single mutation
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DOI：
10.1073/pnas.1211831109
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发表时间：
2012-10-16
影响因子：
11.1
通讯作者：
Schuldiner, Shimon
中科院分区：
文献类型：
--
作者：
Brill, Shlomo;Falk, Ofir Sade;Schuldiner, Shimon
EmrE是一种来自大肠杆菌的多药反向转运蛋白，它给生物化学家带来了不寻常的惊喜。在这里，我们描述了EmrE，药物/H+反向转运蛋白的多胺进口商的一个单一的突变的转化。微生物中的抗生素耐药性可能由某些染色体位点的突变引起。为了研究这一现象，我们使用定向进化EmrE来评估现有多药转运蛋白中新特异性的发展速度。引人注目的是，当EmrE的随机突变体库被筛选为对两种主要抗菌药物-诺氟沙星、氟喹诺酮和红霉素的抗性时，发现具有单突变的大环内酯类蛋白能够赋予抗性。突变赋予红霉素耐药性的结果从一个完全保守的和必需的色氨酸残基取代甘氨酸，并如预期的那样，这种蛋白质失去了识别和运输的经典EmrE底物的能力。然而，这种蛋白质现在作为一组新的底物：脂肪族多胺的电化学势驱动的进口商发挥作用。这种突变体为理解不同运输方式的功能和进化提供了一个独特的范例。
EmrE, a multidrug antiporter from Escherichia coli, has presented biochemists with unusual surprises. Here we describe the transformation of EmrE, a drug/H+ antiporter to a polyamine importer by a single mutation. Antibiotic resistance in microorganisms may arise by mutations at certain chromosomal loci. To investigate this phenomenon, we used directed evolution of EmrE to assess the rate of development of novel specificities in existing multidrug transporters. Strikingly, when a library of random mutants of EmrE was screened for resistance to two major antibacterial drugs-norfloxacin, a fluoroquinolone, and erythromycin, a macrolide-proteins with single mutations were found capable of conferring resistance. The mutation conferring erythromycin resistance resulted from substitution of a fully conserved and essential tryptophan residue to glycine, and, as expected, this protein lost its ability to recognize and transport the classical EmrE substrates. However, this protein functions now as an electro-chemical potential driven importer of a new set of substrates: aliphatic polyamines. This mutant provides a unique paradigm to understand the function and evolution of distinct modes of transport.