Rick Paul D, Barr Kathleen, Sankaran Krishnan, Kajimura Junko, Rush Jeffrey S, Waechter Charles J
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
J Biol Chem. 2003 May 9;278(19):16534-42. doi: 10.1074/jbc.M301750200. Epub 2003 Mar 5.
The assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or "flippases." The bacterial genes proposed to encode these translocases have been collectively designated wzx genes. The wzxE gene of Escherichia coli K-12 has been implicated in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid III), the donor of the trisaccharide repeat unit in the biosynthesis of enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol. Chem. 274, 35129-35138) provided indirect evidence that the wzx(016) gene product of E. coli K-12 encoded a translocase capable of mediating the transbilayer movement of N-acetylglucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and many lipopolysaccharide O antigens. Therefore, genetic and biochemical studies were conducted to determine if the putative Wzx(O16) translocase was capable of mediating the transport of N-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a water-soluble analogue of GlcNAc-P-P-Und. [(3)H]GlcNAc-P-P-Ner was transported into sealed, everted cytoplasmic membrane vesicles of E. coli K-12 as well as a deletion mutant lacking both the wzx(016) and wzxC genes. In contrast, [(3)H]GlcNAc-P-P-Ner was not transported into membrane vesicles prepared from a wzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a pyrophosphoryl-linked saccharide and an unsaturated alpha-isoprene unit in the carrier lipid. These results support the conclusion that the wzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli.
许多细菌细胞表面多糖的组装需要多聚异戊二烯连接的糖中间体跨细胞质膜进行跨双层运动。一般认为，糖脂中间体的横向扩散是由称为转位酶或“翻转酶”的整合膜蛋白介导的。拟编码这些转位酶的细菌基因被统称为wzx基因。大肠杆菌K-12的wzxE基因与Fuc4NAc-ManNAcA-GlcNAc-P-P-十一异戊烯醇（脂质III）的跨双层运动有关，脂质III是肠道细菌共同抗原（ECA）生物合成中三糖重复单元的供体。先前的研究（费尔德曼，M.F.，马罗拉达，C.L.，蒙泰罗，M.A.，佩里，M.B.，帕罗迪，A.J.，和瓦尔瓦诺，M.（1999年）《生物化学杂志》274，35129 - 35138）提供了间接证据，表明大肠杆菌K-12的wzx(016)基因产物编码一种能够介导N-乙酰葡糖胺基焦磷酸十一异戊烯醇（GlcNAc-P-P-Und）跨双层运动的转位酶,GlcNAc-P-P-Und是ECA和许多脂多糖O抗原合成中的早期中间体。因此，进行了遗传和生化研究，以确定假定的Wzx(O1(16))转位酶是否能够介导N-乙酰葡糖胺基焦磷酸神经醇（GlcNAc-P-P-Ner）的运输，GlcNAc-P-P-Ner是GlcNAc-P-P-Und的水溶性类似物。[³H]GlcNAc-P-P-Ner被转运到大肠杆菌K-12的密封外翻细胞质膜囊泡以及一个同时缺失wzx(016)和wzxC基因的缺失突变体中。相比之下，[³H]GlcNAc-P-P-Ner没有被转运到由wzxE基因缺失突变体制备的膜囊泡中，代谢放射性标记实验揭示了脂质III在该突变体中的积累。WzxE转运系统通过识别载体脂质中的焦磷酸连接的糖和不饱和α-异戊二烯单元表现出底物特异性。这些结果支持了wzxE基因编码一种参与大肠杆菌中脂质III跨双层运动的膜蛋白的结论。
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