Characterization of two β-1,3-glucosyltransferases from Escherichia coli serotypes O56 and O152
Characterization of two β-1,3-glucosyltransferases from Escherichia coli serotypes O56 and O152
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DOI：
10.1128/jb.00160-08
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发表时间：
2008-07-01
影响因子：
3.2
通讯作者：
Feng, Lu
中科院分区：
文献类型：
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作者：
Brockhausen, Inka;Hu, Bo;Feng, Lu
革兰氏阴性菌外膜结合脂多糖（LPS）的O抗原是由重复单元组成的低聚糖，具有不同的结构和抗原性。大肠杆菌的056和0152抗原在重复单元内都含有一个gic - β 1-3-GlcNAc连锁。我们已经克隆并鉴定了两个o抗原基因簇中的基因（wfaP在056和wfgD在0152），它们编码参与该连锁合成的葡萄糖基转移酶。以天然受体底物十一戊烯醇-焦磷酸脂[glcnac - α - po3 - po3 -(CH2)(11)- o -苯基]的合成底物类似物为受体，以UDP-Glc为供体底物，证明wfgD和wfaP都编码葡萄糖基转移酶。用高压液相色谱分离两种糖基转移酶的酶产物，并用核磁共振分析。光谱显示产物中预期的glc - β 1-3-GlcNAc连锁，证实WfaP和WfgD都是UDP-Glc: glcnac -焦磷酸-脂质β -1,3-葡萄糖基转移酶的形式。WfaP和WfgD都有一个DxD序列，该序列被认为通过金属阳离子的配位与核苷酸供体的磷酸基相互作用，并且在C端有一个短的疏水序列，可能有助于将酶与内膜结合。我们发现这些酶具有相似的性质和底物识别能力。它们都需要二价阳离子（Mn2+或Mg2+）才能发挥活性，被洗涤剂失活，具有广泛的最佳pH值，并且需要受体底物中的焦磷酸盐-糖键才能发挥全部活性。缺乏与GlcNAc连接的磷酸或焦磷酸的底物是无活性的。受体底物的脂肪链长度也对活性有影响。
The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The 056 and 0152 antigens of Escherichia coli both contain a Gic-beta 1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in 056 and wfgD in 0152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyropbosphate-lipid [GlcNAc-alpha-PO3-PO3-(CH2)(11)- O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-beta 1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid beta-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn2+ or Mg2+) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.