Mulrooney Erin F, Poon Karen K H, McNally David J, Brisson Jean-Robert, Lam Joseph S
Department of Microbiology, University of Guelph, Ontario, Canada.
J Biol Chem. 2005 May 20;280(20):19535-42. doi: 10.1074/jbc.M500612200. Epub 2005 Mar 18.
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-2-deoxy-alpha-D-glucose or UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) 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.
UDP-N-乙酰-L-岩藻糖胺是铜绿假单胞菌血清型O11脂多糖和金黄色葡萄球菌5型荚膜中L-岩藻糖胺的前体。我们之前已经证明了三种酶WbjB、WbjC和WbjD参与UDP-2-乙酰氨基-2,6-二脱氧-L-半乳糖或UDP-N-乙酰-L-岩藻糖胺（UDP-L-FucNAc）的生物合成。从WbjB-WbjC与初始底物UDP-2-乙酰氨基-2-脱氧-α-D-葡萄糖或UDP-N-乙酰-D-葡萄糖胺（UDP-GlcNAc）的偶联反应中纯化出一种中间化合物，通过核磁共振光谱确定其结构为UDP-2-乙酰氨基-2,6-二脱氧-L-塔罗糖（UDP-L-PneNAc）。然后WbjD可以将这种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的生物合成途径；然而，这些蛋白质中每一种的确切酶活性尚未确定。在这里，我们描述了一种基于快速蛋白质液相色谱（FPLC）的阴离子交换方法，该方法能够分离和纯化由于WbjD引起的C-2差向异构化产物。此外，在核磁共振光谱中应用低温冷却探头对于确定微量物质的结构具有最高的灵敏度，从而能够鉴定该途径的最终产物。我们的结果表明，WbjB具有双功能，在与底物UDP-D-GlcNAc的反应中首先催化C-4、C-6脱水，其次催化C-5差向异构化，产生两种中间体。WbjC也具有双功能，催化第二种中间体的C-3差向异构化，然后在C-4处还原。基于FPLC的方法能够很好地分离WbjD反应的最终产物与其差向异构体/底物UDP-L-PneNAc，并在核磁共振中使用低温冷却探头明确显示最终产物是UDP-L-FucNAc。
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