Department of Medicine, Division of Rheumatology, Queen's University, Kingston, Ontario, Canada.
Glycobiology. 2012 Aug;22(8):1092-102. doi: 10.1093/glycob/cws081. Epub 2012 May 3.
The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.
肠出血性 O157 型大肠杆菌是最著名的细菌病原体之一，其 O 抗原重复单元结构的序列为[-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]。大肠杆菌 O157 的 O 抗原基因簇包含负责组装该重复单元的基因，其中包括 wbdN。尽管克隆了许多 O 抗原基因，但很少对参与 O 抗原合成的酶进行生化特性分析。在这项工作中，我们在大肠杆菌 BL21 中表达了 wbdN 基因，并对其进行了 His 标签纯化。使用供体底物 UDP-[14C]Glc 和合成的受体底物 GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph 对 WbdN 活性进行了表征。通过高压液相色谱分离酶产物，质谱分析表明 WbdN 将一个 Glc 残基转移到受体上。产物结构的核磁共振分析表明 Glc 通过 WbdN 以β1-3 键连接到 GalNAc 上。WbdN 包含一个保守的 DxD 基序，需要二价金属离子才能发挥全部活性。WbdN 活性的最佳 pH 值在 7 到 8 之间，对 UDP-Glc 作为供体底物具有高度特异性。缺乏二磷酸基团的 GalNAcα 衍生物作为底物无活性，并且该酶不会将 Glc 转移到 GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph 上。我们的结果表明，WbdN 是一种特异性的 UDP-Glc:GalNAcα-二磷酸脂质β1,3-Glc 转移酶。该酶是开发抑制剂以阻断 O157 抗原合成的靶标。
