Department of Microbiology and Immunology, Infectious Diseases Research Group, University of Western Ontario, London, ON, Canada, N6A 5C1.
Biochem J. 2011 Oct 15;439(2):235-48. doi: 10.1042/BJ20110890.
The capsule of Campylobacter jejuni strain 81-176 comprises the unusual 6-deoxy-α-D-altro-heptose, whose biosynthesis and function are not known. In the present study, we characterized enzymes of the capsular cluster, WcbK and WcaG, to determine their role in 6-deoxy-altro-heptose synthesis. These enzymes are similar to the Yersinia pseudotuberculosis GDP-manno-heptose dehydratase/reductase DmhA/DmhB that we characterized previously. Capillary electrophoresis and MS analyses showed that WcbK is a GDP-manno-heptose dehydratase whose product can be reduced by WcaG, and that WcbK/WcaG can use the substrate GDP-mannose, although with lower efficiency than heptose. Comparison of kinetic parameters for WcbK and DmhA indicated that the relaxed substrate specificity of WcbK comes at the expense of catalytic performance on GDP-manno-heptose. Moreover, although WcbK/WcaG and DmhA/DmhB are involved in altro- versus manno-heptose synthesis respectively, the enzymes can be used interchangeably in mixed reactions. NMR spectroscopy analyses indicated conservation of the sugar manno configuration during catalysis by WcbK/WcaG. Therefore additional capsular enzymes may perform the C3 epimerization necessary to generate 6-deoxy-altro-heptose. Finally, a conserved residue (Thr(187) in WcbK) potentially involved in substrate specificity was identified by structural modelling of mannose and heptose dehydratases. Site-directed mutagenesis and kinetic analyses demonstrated its importance for enzymatic activity on heptose and mannose substrates.
空肠弯曲菌 81-176 株的荚膜由不寻常的 6-去氧-α-D--altro-庚糖组成，其生物合成和功能尚不清楚。在本研究中，我们对荚膜簇的酶 WcbK 和 WcaG 进行了表征，以确定它们在 6-去氧-altro-庚糖合成中的作用。这些酶与我们之前表征的假结核耶尔森氏菌 GDP-甘露庚糖脱水酶/还原酶 DmhA/DmhB 相似。毛细管电泳和 MS 分析表明，WcbK 是一种 GDP-甘露庚糖脱水酶，其产物可被 WcaG 还原，并且 WcbK/WcaG 可以使用 GDP-甘露糖作为底物，尽管效率低于庚糖。比较 WcbK 和 DmhA 的动力学参数表明，WcbK 的底物特异性较宽松是以牺牲 GDP-甘露庚糖的催化性能为代价的。此外，尽管 WcbK/WcaG 和 DmhA/DmhB 分别参与了 altro-和 manno-庚糖的合成，但这两种酶可以在混合反应中互换使用。NMR 光谱分析表明，在 WcbK/WcaG 催化过程中，糖 mannose 构型保持不变。因此，可能需要其他荚膜酶来完成生成 6-去氧-altro-庚糖所必需的 C3 差向异构化。最后，通过甘露糖和庚糖脱水酶的结构建模，鉴定出一个保守残基（WcbK 中的 Thr(187)）可能参与了底物特异性。定点突变和动力学分析证明了其对庚糖和甘露糖底物酶活性的重要性。
