Single bifunctional UDP-GlcNAc/Glc 4-epimerase supports the synthesis of three cell surface glycoconjugates in Campylobacter jejuni
Single bifunctional UDP-GlcNAc/Glc 4-epimerase supports the synthesis of three cell surface glycoconjugates in Campylobacter jejuni
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DOI：
10.1074/jbc.m407767200
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发表时间：
2005-02-11
影响因子：
4.8
通讯作者：
Wakarchuk, WW
中科院分区：
文献类型：
--
作者：
Bernatchez, S;Szymanski, CM;Wakarchuk, WW
空肠弯曲菌NCTC 11168的主要细胞表面碳水化合物（脂寡糖、荚膜和糖蛋白N-连接七糖）含有Gal和/或GalNAc残基。GalE是该细菌中唯一注释的UDP-葡萄糖4-差向异构酶。这些糖中存在GalNAc残基，表明GalE可能是UDP-GlcNAc 4-差向异构酶。在与C.空肠糖基转移酶和糖核苷酸差向异构平衡研究。因此，GalE具有UDP-GlcNAc 4-差向异构酶活性，并更名为Gne。纯化的MalE-Gne融合蛋白对于UDP-GlcNAc和UDP-GalNAc的K-m（app）值为1087和1070 μ m，而对于UDP-Glc和UDP-Gal的K-m（app）值为780和784 μ m。UDP-GalNAc和UDP-Gal的k（cat）和k（cat）/K-m（app）值比UDP-GlcNAc和UDP-Glc高3至4倍。MalE-Gne的动力学参数与其它特征性细菌UDP-GlcNAc 4-差向异构酶的动力学参数的比较表明，Gne是一种双功能UDP-GlcNAc/Glc 4-差向异构酶。通过使用来自铜绿假单胞菌的UDP-GlcNAc 4-差向异构酶WbpP的结构来模拟Gne的UDP糖结合位点。注意到了一些小的差异，这些可能解释了C.空肠在空肠弯曲菌的gne突变体中，毛细管电泳-质谱法显示脂寡糖被至少五种糖截短。此外，糖蛋白N-连接的七糖和胶囊都不再被高分辨率魔角旋转NMR检测到。这些数据表明，Gne是在C.空肠NCTC 11168。
The major cell-surface carbohydrates (lipooligosaccharide, capsule, and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues. GalE is the sole annotated UDP-glucose 4-epimerase in this bacterium. The presence of GalNAc residues in these carbohydrates suggested that GalE might be a UDP-GlcNAc 4-epimerase. GalE was shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-epimerase activity and was renamed Gne. The K-m(app) values of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087 and 1070 gm, whereas those for UDP-Glc and UDP-Gal are 780 and 784 mum. The k(cat) and k(cat)/K-m(app) values were three to four times higher for UDP-GalNAc and UDP-Gal than for UDP-GlcNAc and UDP-Glc. The comparison of the kinetic parameters of MalE-Gne to those of other characterized bacterial UDP-GlcNAc 4-epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-epimerase. The UDP sugar-binding site of Gne was modeled by using the structure of the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa. Small differences were noted, and these may explain the bifunctional character of the C. jejuni Gne. In a gne mutant of C jejuni, the lipooligosaccharide was shown by capillary electrophoresis-mass spectrometry to be truncated by at least five sugars. Furthermore, both the glycoprotein N-linked heptasaccharide and capsule were no longer detectable by high resolution magic angle spinning NMR. These data indicate that Gne is the enzyme providing Gal and GalNAc residues with the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168.