Biochemical Characterization of Bifunctional 3-Deoxy--d-manno-oct-2-ulosonic Acid (-Kdo) Transferase KpsC from Escherichia coli Involved in Capsule Biosynthesis
Biochemical Characterization of Bifunctional 3-Deoxy--d-manno-oct-2-ulosonic Acid (-Kdo) Transferase KpsC from Escherichia coli Involved in Capsule Biosynthesis
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DOI：
10.1074/jbc.m116.751115
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发表时间：
2016-10-07
影响因子：
4.8
通讯作者：
Whitfield, Chris
中科院分区：
文献类型：
--
作者：
Ovchinnikova, Olga G.;Doyle, Liam;Whitfield, Chris
3-脱氧-d-甘露-辛酸(3-deoxy-d-manno-oct-2-ulosonic，KDO)是细菌脂多糖的重要组成部分，是连接脂肪和碳水化合物的纽带。在所有已知的脂多糖结构中，Kdo残基具有-异构体构型，相应的倒置-Kdo转移酶也得到了很好的表征。最近的研究表明，由ATP结合盒转运蛋白依赖的途径产生的大量革兰氏阴性菌的衣壳多糖也通过保守的Kdo寡糖连接到脂质锚上。在本文报道的研究中，通过核磁共振波谱确定了Kdo连接子的结构，揭示了交替连接的-(24)和-(27)连接的Kdo残基。KPSC含有两个保留-Kdo糖基转移酶结构域，属于GT99家族，负责聚合其糖脂受体上的-Kdo连接子。表达和纯化了KPSC的全长，以及分离的N-末端结构域和含有催化灭活N-末端结构域的突变体蛋白(KPSC D160A)。用合成的受体体外测定这些蛋白的KDO转移酶活性，并用TLC、MS和核磁共振波谱对反应产物进行了表征。N-末端结构域和C-末端结构域分别催化-(24)和-(27)键的形成。基于系统发育分析，我们认为糖基转移酶结构域的连锁特异性在其他细菌的KPSC同源物中是保守的。
3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of bacterial lipopolysaccharides, where it provides the linkage between lipid and carbohydrate moieties. In all known LPS structures, Kdo residues possess -anomeric configurations, and the corresponding inverting -Kdo transferases are well characterized. Recently, it has been shown that a large group of capsular polysaccharides from Gram-negative bacteria, produced by ATP-binding cassette transporter-dependent pathways, are also attached to a lipid anchor through a conserved Kdo oligosaccharide. In the study reported here, the structure of this Kdo linker was determined by NMR spectroscopy, revealing alternating -(24)- and -(27)-linked Kdo residues. KpsC contains two retaining -Kdo glycosyltransferase domains belonging to family GT99 that are responsible for polymerizing the -Kdo linker on its glycolipid acceptor. Full-length Escherichia coli KpsC was expressed and purified, together with the isolated N-terminal domain and a mutant protein (KpsC D160A) containing a catalytically inactivated N-terminal domain. The Kdo transferase activities of these proteins were determined in vitro using synthetic acceptors, and the reaction products were characterized using TLC, mass spectrometry, and NMR spectroscopy. The N- and C-terminal domains were found to catalyze formation of -(24) and -(27) linkages, respectively. Based on phylogenetic analyses, we propose the linkage specificities of the glycosyltransferase domains are conserved in KpsC homologs from other bacterial species.