Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM
Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM
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DOI：
10.1016/j.biochi.2012.03.001
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发表时间：
2012-06-01
期刊：
影响因子：
3.9
通讯作者：
Law, Christopher J.
中科院分区：
文献类型：
--
作者：
Holdsworth, Scarlett R.;Law, Christopher J.
多药耐药（MDR）是指细菌同时获得对以前未接触过的结构上不同的化合物的广谱耐药性。MDR主要是通过作为整合膜转运蛋白的蛋白质将药物主动转运出细胞的结果。我们的特点和纯化的推定大肠杆菌MDR转运蛋白，MdTM，一个410个氨基酸残基的蛋白质，属于大型和普遍存在的主要促进超家族。使用生长抑制和全细胞转运测定的MdtM的功能表征揭示了其在E.大肠杆菌细胞对抗菌剂溴化乙锭和氯霉素的耐受性。定点突变研究表明，MdtM天冬氨酸22位残基和高度保守的精氨酸在位置108发挥质子识别的作用。在十二烷基-β-D-吡喃麦芽糖苷洗涤剂溶液中同源过表达MdtM并纯化至均一，并使用尺寸排阻HPLC研究蛋白质在各种洗涤剂溶液中的寡聚状态和稳定性。纯化的MdtM在十二烷基-β-D-吡喃麦芽糖苷溶液中是单体且稳定的，并且在相同的去污剂中以纳摩尔亲和力结合氯霉素。本工作为该类多药物转运蛋白的结构研究奠定了基础。(C)2012年Elsevier Masson SAS。All rights reserved.
Multidrug resistance (MDR) occurs when bacteria simultaneously acquire resistance to a broad spectrum of structurally dissimilar compounds to which they have not previously been exposed. MDR is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. We characterised and purified the putative Escherichia coli MDR transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily. Functional characterisation of MdtM using growth inhibition and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to the antimicrobials ethidium bromide and chloramphenicol. Site-directed mutagenesis studies implied that the MdtM aspartate 22 residue and the highly conserved arginine at position 108 play a role in proton recognition. MdtM was homologously overexpressed and purified to homogeneity in dodecyl-beta-D-maltopyranoside detergent solution and the oligomeric state and stability of the protein in a variety of detergent solutions was investigated using size-exclusion HPLC. Purified MdtM is monomeric and stable in dodecyl-beta-D-maltopyranoside solution and binds chloramphenicol with nanomolar affinity in the same detergent. This work provides a firm foundation for structural studies on this class of multidrug transporter protein. (C) 2012 Elsevier Masson SAS. All rights reserved.