Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: enzymes in the Wbp pathway responsible for O-antigen assembly in Pseudomonas aeruginosa PAO1.
Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: enzymes in the Wbp pathway responsible for O-antigen assembly in Pseudomonas aeruginosa PAO1.
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DOI：
10.1021/bi900186u
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发表时间：
2009-06-16
期刊：
影响因子：
2.9
通讯作者：
Imperali, Barbara
中科院分区：
文献类型：
--
作者：
Larkin, Angelyn;Imperali, Barbara
在机会致病菌铜绿假单胞菌PAO 1（血清型O 5）中发现的脂多糖的B带O-抗原包含一个重复的三糖单元，该单元对于毒力和保护宿主防御系统至关重要。该重复单元中的碳水化合物之一，即罕见的二乙酰化氨基糖醛酸衍生物2，3-二乙酰氨基-2，3-二脱氧-β-d-甘露糖醛酸（ManNAc（3 NAc）A），被认为是由五种酶（WbpA、WbpB、WbpE、WbpD和WbpI）以从UDP-GlcNAc开始的逐步方式产生的。虽然负责这种糖的生物合成的基因是已知的，但只有两个编码的蛋白质（WbpA和WbpI）已被彻底研究。在这份报告中，我们描述了在这一途径，WbpB，WbpE，和WbpD的三个中心酶的克隆，过表达，纯化和生化特性。使用毛细管电泳，RP-HPLC和NMR光谱的组合，我们表明，WbpB和WbpE是一个脱氢酶/氨基转移酶对，通过独特的NAD+再循环途径在偶联反应中将UDP-GlcNAc（3 NH 2）A转化为UDP-GlcNAc（3 NH 2）A。此外，我们证实WbpD催化UDP-GlcNAc（3 NH 2）A的乙酰化，得到UDP-GlcNAc（3 NAc）A。值得注意的是，WbpA、WbpB、WbpE、WbpD和WbpI可以在体外组合以在单个反应容器中产生UDP-ManNAc（3 NAc）A，从而为LPS组装的未来研究提供这种复合糖基供体的供应。这项工作完成了该途径中酶的生化表征，并为潜在的治疗方法提供了新的靶标，以对抗耐药铜绿假单胞菌菌株的感染。
The B-band O-antigen of the lipopolysaccharide found in the opportunistic pathogen Pseudomonas aeruginosa PAO1 (serotype O5) comprises a repeating trisaccharide unit that is critical for virulence and protection from host defense systems. One of the carbohydrates in this repeating unit, the rare diacetylated aminuronic acid derivative 2,3-diacetamido-2,3-dideoxy-β-d-mannuronic acid (ManNAc(3NAc)A), is thought to be produced by five enzymes (WbpA, WbpB, WbpE, WbpD and WbpI) in a stepwise manner starting from UDP-GlcNAc. Although the genes responsible for the biosynthesis of this sugar are known, only two of the five encoded proteins (WbpA and WbpI) have been thoroughly investigated. In this report, we describe the cloning, overexpression, purification and biochemical characterization of the three central enzymes in this pathway, WbpB, WbpE, and WbpD. Using a combination of capillary electrophoresis, RP-HPLC and NMR spectroscopy, we show that WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GlcNAc(3NH2)A in a coupled reaction via a unique NAD+ recycling pathway. In addition, we confirm that WbpD catalyzes the acetylation of UDP-GlcNAc(3NH2)A to give UDP-GlcNAc(3NAc)A. Notably, WbpA, WbpB, WbpE, WbpD and WbpI can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel, thereby providing supplies of this complex glycosyl donor for future studies of LPS assembly. This work completes the biochemical characterization of the enzymes in this pathway and provides novel targets for potential therapeutics to combat infections with drug resistant P. aeruginosa strains.
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