Purification and Characterization of the Lipid A Disaccharide Synthase (LpxB) from Escherichia coli, a Peripheral Membrane Protein
Purification and Characterization of the Lipid A Disaccharide Synthase (LpxB) from Escherichia coli, a Peripheral Membrane Protein
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DOI：
10.1021/bi901750f
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发表时间：
2009-12-08
期刊：
影响因子：
2.9
通讯作者：
Raetz, Christian R. H.
中科院分区：
文献类型：
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作者：
Metzger, Louis E.;Raetz, Christian R. H.
大肠杆菌 LpxB 是 GT-B 超家族的反向糖基转移酶，也是 CAZy 数据库家族 19 的成员，催化脂质 A 生物合成的第五步：UDP-2,3-二酰基葡糖胺 + 2, 3-二酰基葡糖胺 1-磷酸 -> 2',3'-二酰基葡糖胺-(β,1'-6)-2,3-二酰基葡糖胺1-磷酸+ UDP。 LpxB 是新抗生素开发的目标，但完全由 LpxB 直向同源物组成的第 19 族成员尚未在机制或结构上得到表征。在这里，我们使用蛋白酶可切割的 His(10) 标记构建体将大肠杆菌和流感嗜血杆菌 LpxB 纯化到接近同质的 10-100 mg 规模。大肠杆菌 LpxB 活性取决于混合胶束测定系统中其底物的总体表面浓度，表明催化作用发生在膜界面处。大肠杆菌 LpxB M-r（类似于 43 kDa）在低盐浓度下用膜沉积，但用高离子强度的缓冲液很大程度上溶解。它用1.6-3.5摩尔磷脂/摩尔LpxB多肽进行纯化。透射电子显微镜揭示了当 LpxB 过度表达时异常细胞内膜的积累。 LpxB 的诱变鉴定出两个保守残基 D89A 和 R201A，没有检测到残留的催化活性。我们的结果为结构研究提供了一个合理的起点。
Escherichia coli LpxB, an inverting glycosyl transferase of the GT-B superfamily and a member of CAZy database family 19, catalyzes the fifth step of lipid A biosynthesis: UDP-2,3-diacylglucosamine + 2, 3-diacylglucosamine 1-phosphate -> 2',3'-diacylglucosamine-(beta,1'-6)-2,3-diacylglucosamine 1-phosphate + UDP. LpxB is a target for the development of new antibiotics, but no member of family 19, which consists entirely of LpxB orthologues, has been characterized mechanistically or structurally. Here, we have purified E. coli and Haemophilus influenzae LpxB to near homogeneity on a 10-100 mg scale using protease-cleavable His(10)-tagged constructs. E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micelle assay system, suggesting that catalysis occurs at the membrane interface. E, coli LpxB M-r similar to 43 kDa) sediments with membranes at low salt concentrations but is largely solubilized with buffers of high ionic strength. It purifies with 1.6-3.5 mol of phospholipid/mol of LpxB polypeptide. Transmission electron microscopy reveals the accumulation of aberrant intracellular membranes when LpxB is overexpressed. Mutagenesis of LpxB identified two conserved residues, D89A and R201A, for which no residual catalytic activity was detected. Our results provide a rational starting point for structural studies.