Biosynthesis of UDP-N-acetyl-L-fucosamine, a precursor to the biosynthesis of lipopolysaccharide in Pseudomonas aeruginosa serotype O11
Biosynthesis of UDP-N-acetyl-L-fucosamine, a precursor to the biosynthesis of lipopolysaccharide in Pseudomonas aeruginosa serotype O11
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DOI：
10.1074/jbc.m500612200
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发表时间：
2005-05-20
影响因子：
4.8
通讯作者：
Lam, JS
中科院分区：
文献类型：
--
作者：
Mulrooney, EF;Poon, KKH;Lam, JS
UDP-N-乙酰基-L-岩藻糖胺是铜绿假单胞菌血清型O 11脂多糖和金黄色葡萄球菌5型荚膜中L-岩藻糖胺的前体。我们先前已经证明了三种酶WbjB、WbjC和WbjD参与UDP-2-乙酰氨基-2，6-双脱氧-L-半乳糖或UDP-N-乙酰基-L-岩藻糖胺（UDP-L-FucNAc）的生物合成。纯化来自WbjB-WbjC与初始底物UDP-2-乙酰氨基-2-脱氧-β-D-葡萄糖或UDP-N-乙酰基-D-葡萄糖胺（UDPGlcNAc）的偶联反应的中间体化合物，并且通过NMR光谱确定结构为UDP-2-乙酰氨基-2，6-二脱氧-L-塔罗糖（UDP-L-PneNAc）。然后WbjD可以将该中间体转化为具有相同质量的新产物，这与C-2差向异构化反应一致。这些结果使我们提出了UDP-L-FucNAc生物合成的途径;然而，这些蛋白质中的每一种的确切酶活性尚未确定。在这里，我们描述了一种快速的蛋白质液相色谱（FPLC）为基础的阴离子交换程序，它允许的C-2差向异构化由于WbjD的产品的分离和纯化。此外，低温冷却探针在NMR光谱法中的应用为确定微量材料的结构提供了最大的灵敏度，从而可以识别该途径的最终产物。结果表明，WbjB具有双功能，在与底物UDP-D-GlcNAc的反应中，首先催化C4、C6脱水，然后催化C5差向异构化，生成两个中间体。WbjC也是双官能的，催化第二中间体的C-3差向异构化，然后在C-4处还原。基于FPLC的程序提供了WbjD反应的最终产物与其差向异构体/底物UDP-L-PneNAc的良好分离，并且在NMR中使用低温冷却的探针明确地揭示了最终产物是UDP-L-FucNAc。
UDP-N-acetyl-L-fucosamine is a precursor to L-fucosamine in the lipopolysaccharide of Pseudomonas aeruginosa serotype O11 and the capsule of Staphylococcus aureus type 5. We have demonstrated previously the involvement of three enzymes, WbjB, WbjC, and WbjD, in the biosynthesis of UDP-2-acetamido-2,6-dideoxy-L-galactose or UDP-N-acetyl-L-fucosamine (UDP-L-FucNAc). An intermediate compound from the coupled-reaction of WbjB-WbjC with the initial substrate UDP-2-acetamido-2deoxy-beta-D-glucose or UDP-N-acetyl-D-glucosamine (UDPGlcNAc) was purified, and the structure was determined by NMR spectroscopy to be UDP-2-acetamido-2,6-dideoxy-L-talose (UDP-L-PneNAc). WbjD could then convert this intermediate into a new product with the same mass, consistent with a C-2 epimerization reaction. Those results led us to propose a pathway for the biosynthesis of UDP-L-FucNAc; however, the exact enzymatic activity of each of these proteins has not been defined. Here, we describe a fast protein liquid chromatography (FPLC)-based anion-exchange procedure, which allowed the separation and purification of the products of C-2 epimerization due to WbjD. Also, the application of a cryogenically cooled probe in NMR spectrometry offers the greatest sensitivity for determining the structures of minute quantities of materials, allowing the identification of the final product of the pathway. Our results showed that WbjB is bifunctional, catalyzing firstly C-4, C-6 dehydration and secondly C-5 epimerization in the reaction with the substrate UDP-D-GlcNAc, producing two intermediates. WbjC is also bifunctional, catalyzing C-3 epimerization of the second intermediate followed by reduction at C-4. The FPLC-based procedure provided good resolution of the final product of WbjD reaction from its epimer/substrate UDP-L-PneNAc, and the use of the cryogenically cooled probe in NMR revealed unequivocally that the final product is UDP-L-FucNAc.