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首页> 美国卫生研究院文献>Biochemical Journal >Identification and biochemical characterization of two novel UDP-23-diacetamido-23-dideoxy-α-D-glucuronic acid 2-epimerases from respiratory pathogens
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Identification and biochemical characterization of two novel UDP-23-diacetamido-23-dideoxy-α-D-glucuronic acid 2-epimerases from respiratory pathogens

机译：呼吸道病原体中两种新型UDP-23-diacetamido-23-dideoxy-α-D-glucouronicacid 2-epimerases的鉴定和生化特性

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摘要

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, β-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-α-D-GlcNAc (UDP-N-acetyl-α-D-glucosamine) is converted via four steps into UDP-α-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-α-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-mannuronic acid). UDP-α-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-α-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-α-D-GlcNAc, UDP-α-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-α-D-glucuronic acid) or UDP-α-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucose) as substrates. His6–WbpI or His6–WlbD reactions with UDP-α-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-α- class="small-caps">D-GlcNAc3NAcA, converting it into UDP-α- class="small-caps">D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-α- class="small-caps">D-glucuronic acid 2-epimerases.
机译：铜绿假单胞菌血清群O5的脂多糖以及百日咳博德特氏菌的Band-A三糖的杂多聚O抗原包含二-N-乙酰化的甘露糖醛酸衍生物β-D-ManNAc3NAcA（2,3-diacetamido-2,3 -二脱氧-β-D-甘露糖醛酸）。提出该糖的前体的生物合成需要五个步骤，通过该步骤，UDP-α-D-GlcNAc（UDP-N-乙酰基-α-D-葡糖胺）通过四个步骤被转化为UDP-α-D-GlcNAc3NAcA （UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucouronicacid），然后用WbpI（铜绿假单胞菌）或其直系同源物WlbD（百日咳博德特氏菌）将这种中间体化合物异构化，形成UDP-α-D-ManNAc3NAcA（UDP-2,3-diacetamido-2,3-dideoxy-α-D-mannuronicacid）。 UDP-α-D-GlcNAc3NAcA，拟议的WbpI和WlbD底物，是通过化学合成获得的。过表达His6-WbpI和His6-WlbD，然后使用FPLC通过亲和色谱纯化。毛细管电泳用于分析每种酶的反应，结果表明两种酶均使用UDP-α-D-GlcNAc3NAcA作为底物，并在磷酸钠缓冲液（pH 6.0）中最佳反应。两种酶均未使用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 （3-3-二乙酰氨基-2，3-二脱氧-α-D-葡萄糖）作为底物。毛细管电泳显示，与UDP-α-D-GlcNAc3NAcA发生的His6-WbpI或His6-WlbD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，允许酶-底物反应直接在NMR管中进行，并通过定期获取质子光谱来监测底物向产物的转化。从一维和二维NMR实验收集的数据表明，His6-WbpI催化UDP-α- class =“ D-caps”> D -GlcNAc3NAcA的2表位化，并将其转化为UDP-α - class =“ small-caps”> D -ManNAc3NAcA。这些结果共同提供了证据，表明WbpI和WlbD是UDP-2,3-diacetamido-2,3-dideoxy-α- class =“ small-caps”> D -葡萄糖醛酸2-表位酶。

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