Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway
Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway
复制标题
MraY 转位酶酶活性的脂质需求和脂质 II 合成途径的体外重建
DOI：
10.1074/jbc.m115.664292
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发表时间：
2016-01-29
影响因子：
4.8
通讯作者：
Bernhard, Frank
中科院分区：
文献类型：
--
作者：
Henrich, Erik;Ma, Yi;Bernhard, Frank
针对关键蛋白质靶标的新化合物的筛选必须不断跟上新出现的抗生素耐药性。虽然细菌细胞壁生物合成的周质酶已成为第一批药物靶标，但针对膜整合催化剂的化合物几乎不可用。一个有前途的未来目标是整合膜蛋白MraY催化细菌肽聚糖生物合成的细胞质途径内的第一个膜相关步骤。然而，大多数MraY同源物在细胞表达系统中的表达是具有挑战性的，并且限制了生化分析。我们报告了通过合成无细胞表达方法从各种人类病原体中有效生产MraY同源物及其随后的表征。源自百日咳博德特氏菌、幽门螺杆菌、肺炎衣原体、伯氏疏螺旋体和大肠杆菌以及枯草芽孢杆菌的MraY同系物使用洗涤剂胶束或与限定膜组装的预成型纳米盘共溶解。所有来自革兰氏阴性细菌的MraY酶对洗涤剂敏感，并且需要含有带负电荷的脂质的纳米盘以获得稳定的和功能性折叠的构象。相反，革兰氏阳性B.枯草芽孢杆菌MraY不仅耐受洗涤剂，而且对其脂质环境的特异性也较低。纳米盘复合物能够与无细胞表达的可溶性酶MurA-F和膜相关蛋白MurG组合重建完整的体外脂质I和脂质II形成管道。作为未来筛选平台的原理证明，我们证明了特异性抑制剂磷霉素、feglymycin和衣霉素对体外脂质II生物合成的抑制作用。
Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY.nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.