Biochemical Characterization of dTDP-d-Qui4N and dTDP-d-Qui4NAc Biosynthetic Pathways in Shigella dysenteriae Type 7 and Escherichia coli O7
Biochemical Characterization of dTDP-d-Qui4N and dTDP-d-Qui4NAc Biosynthetic Pathways in Shigella dysenteriae Type 7 and Escherichia coli O7
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DOI：
10.1128/jb.00777-07
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发表时间：
2007-09
影响因子：
3.2
通讯作者：
Ying Wang;Yanli Xu;A. Perepelov;Yu Qi;Y. Knirel;Lei Wang;Lu Feng
中科院分区：
文献类型：
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作者：
Ying Wang;Yanli Xu;A. Perepelov;Yu Qi;Y. Knirel;Lei Wang;Lu Feng
由于存在不同类型的糖和糖键而导致的o抗原变异对受宿主免疫系统威胁的细菌的生存至关重要。7型痢疾志贺氏菌和O7型大肠杆菌的O抗原分别含有4-（n -乙酰甘酰基）氨基-4,6-二脱氧-d-葡萄糖（d-Qui4NGlyAc）和4-乙酰氨基-4,6-二脱氧-d-葡萄糖（d-Qui4NAc），这是在所研究的多糖中不常见的糖。在这项研究中，我们表征了dTDP-d-Qui4N和dTDP-d-Qui4NAc （O抗原中d-Qui4NGlyAc和d-Qui4NAc的核苷酸激活前体）的生物合成途径。克隆了参与两种糖合成的预测基因，并将基因产物过表达并纯化为his标记的融合蛋白。利用纯化后的蛋白进行体外酶促反应，并对反应产物进行毛细管电泳、电喷雾电离质谱和核磁共振谱分析。结果表明，在7型痢疾杆菌和O7型大肠杆菌中，由α-d-葡萄糖-1-磷酸经过葡萄糖-1-磷酸胸苷基转移酶（RmlA）、dtdp -d-葡萄糖4,6-脱水酶（RmlB）和dtdp -4-酮-6-脱氧-d-葡萄糖氨基转移酶（VioA）三个反应步骤合成了dTDP-d-Qui4N。另外一种乙酰转移酶（VioB）在大肠杆菌O7中催化dTDP-d-Qui4N转化为dTDP-d-Qui4NAc。本文描述了VioA和VioB的动力学参数和其他一些性质，并讨论了来自痢疾杆菌7型（VioAD7）和大肠杆菌O7 （VioAO7）的VioA蛋白的差异。据我们所知，这是第一次对VioA和VioB的功能进行生物化学表征。该研究为生产dTDP-d-Qui4N和dTDP-d-Qui4NAc提供了有价值的酶源，它们在制药工业的药物开发中具有潜在的用途。
ABSTRACT O-antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria threatened by host immune systems. The O antigens of Shigella dysenteriae type 7 and Escherichia coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-d-glucose (d-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-d-glucose (d-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-d-Qui4N and dTDP-d-Qui4NAc (the nucleotide-activated precursors of d-Qui4NGlyAc and d-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. It is shown that in S. dysenteriae type 7 and E. coli O7, dTDP-d-Qui4N is synthesized from α-d-glucose-1-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-d-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (VioA). An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-d-Qui4N into dTDP-d-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 (VioAD7) and E. coli O7 (VioAO7) discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-d-Qui4N and dTDP-d-Qui4NAc, which are potentially useful in the pharmaceutical industry for drug development.