Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1–4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.

中文翻译：

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脑膜炎奈瑟菌 X 组 N-乙酰氨基葡萄糖-1-磷酸转移酶与其供体底物的相互作用

脑膜炎奈瑟菌X 组是撒哈拉以南非洲地区细菌性脑膜炎的一个新兴原因。X 组的荚膜多糖是N-乙酰氨基葡糖 α(1-4) 磷酸盐的均聚物，是预防与该脑膜炎球菌血清群相关的疾病的疫苗靶标。我们之前已经证明，聚合物的形成是由磷酸转移酶催化的，该磷酸转移酶将N-乙酰氨基葡萄糖-1-磷酸从 UDP- N-乙酰氨基葡萄糖转移到N的 4-羟基上。-乙酰氨基葡萄糖在生长链的非还原端。在这项研究中，我们使用 UDP-GlcNAc 的底物类似物来定义对催化至关重要的酶/供体底物相互作用。我们对磷酸转移酶反应的动力学分析与底物添加和产物释放的顺序机制一致。Wagner 等人设计的新型尿嘧啶修饰类似物的使用。使我们能够评估 CsxA 催化的反应是否与供体依赖性构象变化一致。正如这种糖基转移酶模型所预期的那样，具有大量尿嘧啶修饰的 UDP-GlcNAc 类似物不是底物，而是抑制剂。具有较小碘尿嘧啶取代的类似物是底物和较弱的抑制剂。此外，我们对对N进行修改的类似物的调查糖核苷酸供体的​​-乙酰氨基葡糖残基突出了糖残基C2和C4取代基的重要性。C4 上的羟基和 C2 上的酰基结构对于聚合反应过程中的特异性和底物相互作用非常重要。虽然大多数 C2 修饰的类似物是抑制剂，但乙酰氨基类似物也是底物，表明羰基的重要性。