Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86:B7
Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86:B7
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DOI：
10.1021/bi0612770
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发表时间：
2006-11-21
期刊：
影响因子：
2.9
通讯作者：
Wang, Peng George
中科院分区：
文献类型：
--
作者：
Guo, Hongjie;Li, Lei;Wang, Peng George
革兰氏阴性菌脂多糖o抗原在菌宿主相互作用中起重要作用。大肠杆菌O86: b7o -单位含有五个糖残基：一个焦点（Fuc）和两个n -乙酰半乳糖胺（GalNAc）和半乳糖（Gal）。整个o抗原基因簇先前已测序：orf1被指定为UDP-GalNAc生物合成的基因基因。为了证实这一注释，我们进行了基因的过表达、纯化和生化表征。通过毛细管电泳，我们发现基因对UDP-GlcNAc/GalNAc和UDP-Glc/Gal的催化作用几乎相同。UDP-Glc、UDP-Gal、UDP-GlcNAc和UDP-GalNAc基因的Km值分别为370、295、323和373 AM。从大肠杆菌O86: B7中分离得到的基因动力学参数与其他已知的UDP-GlcNAc/Glc 4-外膜酶的动力学参数比较表明，该基因对这四种底物具有较弱的特异性，是首个在o抗原生物合成中具有这种活性的酶。此外，计算出的UDP-GalNAc和UDP-Gal的k(cat)/ k -m值大约是UDP-GlcNAc和UDPGlc的2-4倍，表明基因对UDP-GalNAc和UDP-Gal的外聚化效率略高。其中一个突变（S306Y）导致对非乙酰化底物的epimase活性降低了约5倍，但对n-乙酰化底物的活性完全丧失，这表明残基S-306在确定底物特异性方面起着重要作用。
The O-antigen of lipopolysaccharide in Gram-negative bacteria plays an important role in bacterium-host interactions. Escherichia coli O86: B7 O-unit contains five sugar residues: one fucose ( Fuc) and two each of N-acetylgalactosamine ( GalNAc) and galactose ( Gal). The entire O-antigen gene cluster was previously sequenced: orf1 was assigned the gne gene for the biosynthesis of UDP-GalNAc. To confirm this annotation, overexpression, purification, and biochemical characterization of Gne were performed. By using capillary electrophoresis, we showed that Gne can catalyze the interconversion of both UDP-GlcNAc/GalNAc and UDP-Glc/Gal almost equally well. The Km values of Gne for UDP-Glc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc are 370, 295, 323, and 373 AM, respectively. The comparison of kinetic parameters of Gne from Escherichia coli O86: B7 to those of other characterized UDP-GlcNAc/Glc 4-epimerases indicated that it has relaxed specificity toward the four substrates, the first characterized enzyme to have this activity in the O-antigen biosynthesis. Moreover, the calculated k(cat)/K-m values for UDP-GalNAc and UDP-Gal are approximately 2-4 times higher than those for UDP-GlcNAc and UDPGlc, suggesting that Gne is slightly more efficient for the epimerization of UDP-GalNAc and UDP-Gal. One mutation ( S306Y) resulted in a loss of epimerase activity for non-acetylated substrates by about 5-fold but totally abolished the activity for N-acetylated substrates, indicating that residue S-306 plays an important role in the determination of substrate specificity.