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首页> 外文期刊>Journal of bacteriology >Characterization of GlaKP, a UDP-Galacturonic Acid C4-Epimerase from Klebsiella pneumoniae with Extended Substrate Specificity
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Characterization of GlaKP, a UDP-Galacturonic Acid C4-Epimerase from Klebsiella pneumoniae with Extended Substrate Specificity

机译：具有扩展底物特异性的肺炎克雷伯菌UDP-半乳糖醛酸C4-表异构酶GlaKP的表征

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In Escherichia coli and Salmonella enterica, the core oligosaccharide backbone of the lipopolysaccharide is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. In contrast, Klebsiella pneumoniae lacks phosphoryl groups in its core oligosaccharide but instead contains galacturonic acid residues that are proposed to serve a similar function in outer membrane stability. GlaKP is a UDP-galacturonic acid C4-epimerase that provides UDP-galacturonic acid for core synthesis, and the enzyme was biochemically characterized because of its potentially important role in outer membrane stability. High-performance anion-exchange chromatography was used to demonstrate the UDP-galacturonic acid C4-epimerase activity of GlaKP, and capillary electrophoresis was used for activity assays. The reaction equilibrium favors UDP-galacturonic acid over UDP-glucuronic acid in a ratio of 1.4:1, with the Km for UDP-glucuronic acid of 13.0 μM. GlaKP exists as a dimer in its native form. NAD+/NADH is tightly bound by the enzyme and addition of supplementary NAD+ is not required for activity of the purified enzyme. Divalent cations have an unexpected inhibitory effect on enzyme activity. GlaKP was found to have a broad substrate specificity in vitro; it is capable of interconverting UDP-glucose/UDP-galactose and UDP-N-acetylglucosamine/UDP-N-acetylgalactosamine, albeit at much lower activity. The epimerase GalE interconverts UDP-glucose/UDP-galactose. Multicopy plasmid-encoded glaKP partially complemented a galE mutation in S. enterica and in K. pneumoniae; however, chromosomal glaKP could not substitute for galE in a K. pneumoniae galE mutant in vivo.
机译：在大肠杆菌和沙门氏菌中，脂多糖的核心寡糖主链被磷酸基团修饰。这些残基提供的负电荷对于维持外膜的屏障功能很重要。相比之下，肺炎克雷伯菌在其核心寡糖中缺少磷酸基，但含有半乳糖醛酸残基，被提议在外膜稳定性中起类似的作用。 Gla KP 是一种UDP-半乳糖醛酸C4-表异构酶，可为核心合成提供UDP-半乳糖醛酸，该酶具有生化特性，因为它在外膜稳定性中具有潜在的重要作用。高效阴离子交换色谱法显示了Gla KP 的UDP-半乳糖醛酸C4-表异构酶活性，毛细管电泳用于活性测定。反应平衡相对于UDP-葡萄糖醛酸的比例为UDP-半乳糖醛酸1.4：1，其中UDP-葡萄糖醛酸的 K m 为13.0μM。 Gla KP 以其天然形式的二聚体存在。 NAD + / NADH与酶紧密结合，纯化酶的活性不需要添加补充NAD + 。二价阳离子对酶活性具有意想不到的抑制作用。发现Gla KP 在体外具有广泛的底物特异性。它能够互转换UDP-葡萄糖/ UDP-半乳糖和UDP- N -乙酰氨基葡萄糖/ UDP- N -乙酰半乳糖胺，尽管活性低得多。差向异构酶GalE相互转换UDP-葡萄糖/ UDP-半乳糖。多拷贝质粒编码的 gla KP 部分补充了 S中的 galE 突变。 enterica 和 K。肺炎;但是，染色体 gla KP 不能替代 K中的 galE 。肺炎球菌galE 体内突变体。

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