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首页> 外文期刊>Biochemistry >Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86 : B7
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Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86 : B7

机译：大肠杆菌O86：B7的UDP-GlcNAc / Glc 4-表异构酶的生化特性

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摘要

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.
机译：革兰氏阴性细菌中脂多糖的O抗原在细菌与宿主的相互作用中起着重要作用。大肠杆菌O86：B7 O单元包含五个糖残基：一个岩藻糖（Fuc）和两个N-乙酰半乳糖胺（GalNAc）和半乳糖（Gal）。先前已对整个O抗原基因簇进行了测序：orf1被分配了gne基因用于UDP-GalNAc的生物合成。为了证实该注释，进行了Gne的过表达，纯化和生化表征。通过使用毛细管电泳，我们显示Gne可以几乎相同地催化UDP-GlcNAc / GalNAc和UDP-Glc / Gal的互变。 UDP-Glc，UDP-Gal，UDP-GlcNAc和UDP-GalNAc的Gne Km值分别为370、295、323和373 AM。将大肠杆菌O86：B7的Gne动力学参数与其他表征的UDP-GlcNAc / Glc 4-表位酶的动力学参数进行比较，结果表明它对四种底物具有宽松的特异性，这是第一种在O抗原中具有这种活性的酶。生物合成。此外，计算得出的UDP-GalNAc和UDP-Gal的k（cat）/ Km值大约是UDP-GlcNAc和UDPGlc的k（cat）/ Km值，这表明Gne对于UDP-GalNAc的差向异构化稍微更有效。 UDP-Gal。一个突变（S306Y）导致非乙酰化底物的差向异构酶活性损失约5倍，但完全废除了N-乙酰化底物的活性，表明残基S-306在确定底物特异性中起重要作用。

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