The O antigen is indispensable for the full function and virulence of pathogenic bacteria. During O-repeating unit (RU) biosynthesis, committed glycosyltransferases (GTs) transfer various sugars from an activated sugar donor to the appropriate lipid carrier sequentially. While the nucleotide sequence specific for O antigen of pathogenic bacteria is already known, the exact substrate specificity of most hypothetical GTs have yet be characterized. In the present paper, we report the biochemical characterization of one alpha-glucosyltransferase, WfgE, a member of GT family 4. This enzyme is implicated in the pentasaccharide RU biosynthetic pathway of E. coli O152 O antigen. A chemoenzymatically synthesized acceptor (GlcGlcNAc α-PP-O(CH₂)₁₀CH₃) was used to characterize the WfgE activity. The enzyme product was determined to have a 1,2-linkage using strategy based on collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSⁿ). The lack of a DxD motif and its high activity without divalent metal ions suggests that WfgE belongs to the GT-B fold superfamily. The enzyme is specific for beta-glucose or galactose-terminating acceptor substrates, and in particular UDP-glucose but also UDP-galactose as donor substrates. Our results suggest that WfgE catalyses the addition of the third sugar residue of the E. coli O152 O-RU. The recombinant GST-WfgE was solubilized and further purified to homogeneity via GST affinity chromatography, paving the way for structure-function relationship studies.

中文翻译：

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大肠杆菌O152 O抗原重复单元生物合成第三步中涉及的新葡萄糖基转移酶的表达，纯化和表征。

O抗原对于病原菌的全部功能和毒力必不可少。在O重复单元（RU）生物合成过程中，定型糖基转移酶（GTs）会将各种糖从活化的糖供体依次转移到合适的脂质载体上。尽管已知对病原细菌的O抗原具有特异性的核苷酸序列，但尚未鉴定出大多数假想的GT的确切底物特异性。在本论文中，我们报道了一个α-葡萄糖基转移酶WfgE（GT家族4的成员）的生化特性。该酶与大肠杆菌O152 O抗原的五糖RU生物合成途径有关。化学酶促合成受体（GlcGlcNAcα-PP-O（CH ₂）₁₀ CH ₃）用来表征WfgE活性。使用基于碰撞诱导的离解电喷雾电离离子阱多级串联质谱（CID-ESI-IT-MS ⁿ）的策略，确定酶产物具有1,2-链接。DxD基序的缺乏及其没有二价金属离子的高活性表明WfgE属于GT-B折叠超家族。该酶对β-葡萄糖或半乳糖终止的受体底物具有特异性，特别是作为供体底物的UDP-葡萄糖但也有UDP-半乳糖。我们的结果表明，WfgE催化大肠杆菌O152 O-RU的第三个糖残基的添加。重组的GST-WfgE可以溶解，并通过GST亲和层析进一步纯化至同质，为结构-功能关系研究铺平了道路。