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首页> 外文期刊>Biochemistry >Purification and Characterization of the Lipid A Disaccharide Synthase (LpxB) from Escherichia coli, a Peripheral Membrane Protein
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Purification and Characterization of the Lipid A Disaccharide Synthase (LpxB) from Escherichia coli, a Peripheral Membrane Protein

机译：纯化和表征的脂质A双糖合酶（LpxB）从大肠杆菌，一种外周膜蛋白。

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摘要

Escherichia coli LpxB, an inverting glycosyl transferase of the GT-B superfamily and a member ofnCAZy database family 19, catalyzes the fifth step of lipid A biosynthesis: UDP-2,3-diacylglucosamine þ 2,n3-diacylglucosamine 1-phosphate f 20n,30n-diacylglucosamine-(β,10n-6)-2,3-diacylglucosamine 1-phosphate þnUDP. LpxB is a target for the development of new antibiotics, but no member of family 19, which consistsnentirely of LpxB orthologues, has been characterized mechanistically or structurally. Here, we have purifiednE. coli and Haemophilus influenzae LpxB to near homogeneity on a 10-100 mg scale using protease-cleavablenHis10-tagged constructs. E. coli LpxB activity is dependent upon the bulk surface concentration of itsnsubstrates in a mixed micelle assay system, suggesting that catalysis occurs at the membrane interface. E. colinLpxB (Mr ∼ 43 kDa) sediments with membranes at low salt concentrations but is largely solubilized withnbuffers of high ionic strength. It purifies with 1.6-3.5 mol of phospholipid/mol of LpxB polypeptide.nTransmission electron microscopy reveals the accumulation of aberrant intracellular membranes when LpxBnis overexpressed. Mutagenesis of LpxB identified two conserved residues, D89A and R201A, for which nonresidual catalytic activity was detected. Our results provide a rational starting point for structural studies.
机译：大肠杆菌LpxB是GT-B超家族的反向糖基转移酶，是nCAZy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þ2，n3-二酰基葡糖胺1-磷酸f 20n， 30n-二酰基氨基葡萄糖-（β，10n-6）-2,3-二酰基氨基葡萄糖1-磷酸þnUDP。 LpxB是开发新抗生素的目标，但是没有机制19的成员，该家族完全由LpxB直向同源物组成，没有被机械地或结构上地表征。在这里，我们已经纯化了。大肠杆菌和流感嗜血杆菌LpxB使用蛋白酶可裂解的His10标签构建物以10-100 mg的比例接近均质。大肠杆菌LpxB活性取决于混合胶束测定系统中其n底物的整体表面浓度，这表明催化作用发生在膜界面。 E. colinLpxB（Mr〜43 kDa）沉积物在低盐浓度下具有膜，但在高离子强度的缓冲液中可大量溶解。它用1.6-3.5摩尔磷脂/摩尔LpxB多肽纯化。n透射电子显微镜显示LpxBnis过表达时异常细胞内膜的积累。 LpxB的诱变确定了两个保守的残基，D89A和R201A，其检测到非残留的催化活性。我们的结果为结构研究提供了一个合理的起点。

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