Westman Erin L, McNally David J, Rejzek Martin, Miller Wayne L, Kannathasan Vellupillai Sri, Preston Andrew, Maskell Duncan J, Field Robert A, Brisson Jean-Robert, Lam Joseph S
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1.
Biochem J. 2007 Jul 1;405(1):123-30. doi: 10.1042/BJ20070017.
The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, beta-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-beta-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-alpha-D-GlcNAc (UDP-N-acetyl-alpha-D-glucosamine) is converted via four steps into UDP-alpha-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-alpha-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-mannuronic acid). UDP-alpha-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6-WbpI and His6-WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-alpha-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-alpha-D-GlcNAc, UDP-alpha-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-alpha-D-glucuronic acid) or UDP-alpha-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucose) as substrates. His6-WbpI or His6-WlbD reactions with UDP-alpha-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme-substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6-WbpI catalysed the 2-epimerization of UDP-alpha-D-GlcNAc3NAcA, converting it into UDP-alpha-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases.
铜绿假单胞菌血清群O5脂多糖的杂聚O抗原以及百日咳博德特氏菌的A带三糖都含有二 - N - 乙酰化甘露糖胺糖醛酸衍生物，β - D - ManNAc3NAcA（2,3 - 二乙酰氨基 - 2,3 - 二脱氧 - β - D - 甘露糖醛酸）。推测该糖前体的生物合成需要五个步骤，通过这五个步骤，UDP - α - D - GlcNAc（UDP - N - 乙酰 - α - D - 葡糖胺）经四个步骤转化为UDP - α - D - GlcNAc3NAcA（UDP - 2,3 - 二乙酰氨基 - 2,3 - 二脱氧 - α - D - 葡糖醛酸），然后该中间化合物由WbpI（铜绿假单胞菌）或其同源物WlbD（百日咳博德特氏菌）进行差向异构化，形成UDP - α - D - ManNAc3NAcA（UDP - 2,3 - 二乙酰氨基 - 2,3 - 二脱氧 - α - D - 甘露糖醛酸）。通过化学合成获得了推测为WbpI和WlbD底物的UDP - α - D - GlcNAc3NAcA。His6 - WbpI和His6 - WlbD被过量表达，然后使用快速蛋白质液相色谱（FPLC）通过亲和色谱法进行纯化。使用毛细管电泳分析与每种酶的反应，结果表明这两种酶都使用UDP - α - D - GlcNAc3NAcA作为底物，并且在磷酸钠缓冲液（pH 6.0）中反应最佳。这两种酶都不利用UDP - α - D - GlcNAc、UDP - α - D - GlcNAcA（UDP - 2 - 乙酰氨基 - 2,3 - 二脱氧 - α - D - 葡糖醛酸）或UDP - α - D - GlcNAc3NAc（UDP - 2,3 - 二乙酰氨基 - 2,3 - 二脱氧 - α - D - 葡萄糖）作为底物。如毛细管电泳所示，His6 - WbpI或His6 - WlbD与UDP - α - D - GlcNAc3NAcA的反应产生了一个具有相同保留时间的新峰。为了明确表征反应产物，使酶 - 底物反应直接在核磁共振管中进行，并通过定期采集质子光谱随时间监测底物向产物的转化。从一维和二维核磁共振实验收集的数据表明，His6 - WbpI催化UDP - α - D - GlcNAc3NAcA的2 - 差向异构化，将其转化为UDP - α - D - ManNAc3NAcA。总体而言，这些结果提供了证据表明WbpI和WlbD是UDP - 2,3 - 二乙酰氨基 - 2,3 - 二脱氧 - α - D - 葡糖醛酸2 - 差向异构酶。
