Structural and genetic characterization of the Escherichia coli O180 O antigen and identification of a UDP-GlcNAc 6-dehydrogenase.
Structural and genetic characterization of the Escherichia coli O180 O antigen and identification of a UDP-GlcNAc 6-dehydrogenase.
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DOI：
10.1093/glycob/cws098
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发表时间：
2012-10
期刊：
影响因子：
4.3
通讯作者：
Quan Wang;A. Perepelov;L. Beutin;S. Senchenkova;Yanli Xu;A. Shashkov;Peng Ding;Y. Knirel;Lu Feng
中科院分区：
文献类型：
--
作者：
Quan Wang;A. Perepelov;L. Beutin;S. Senchenkova;Yanli Xu;A. Shashkov;Peng Ding;Y. Knirel;Lu Feng
O抗原是革兰氏阴性菌表面脂多糖的重要组成部分，其变异为血清分型方案提供了主要依据。大肠杆菌O-抗原型O 180于2004年首次被命名，O 180菌株被发现含有毒力因子并引起腹泻。不同的O-抗原形式几乎完全是由于O-抗原基因簇的遗传变异。本研究对E. coli O 180 O抗原。从大肠杆菌中鉴定出一个四糖重复单元，其结构为：→4）-β-D-ManpNAc 3 NAcA-（1 → 2）-α-L-Rhap（I）-（1 → 3）-β-L-Rhap（II）-（1 → 4）-α-D-GlcpNAc-（1→）。coli O 180 O抗原，包括迄今为止在大肠杆菌中未鉴定的残基D-ManpNAc 3 NAcA（2，3-二乙酰氨基-2，3-二脱氧-D-甘露吡喃糖醛酸）。杆菌O-抗原基因簇中的基因基于它们与来自可用数据库的那些基因的相似性被分配功能，并且鉴定了参与UDP-D-ManpNAc 3 NAcA（D-ManpNAc 3 NAcA的核苷酸活化形式）合成的五个基因。克隆了编码参与UDP-D-ManpNAc 3 NAcA生物合成途径初始步骤的酶的gnaA基因，并表达、纯化酶产物并测定其活性。GnaA的特征在于使用毛细管电泳和电喷雾电离质谱，并确定为UDP-GlcNAc 6-脱氢酶。测定了GnaA的动力学参数和理化参数。
The O antigen is an essential component of the lipopolysaccharides on the surface of Gram-negative bacteria and its variation provides a major basis for serotyping schemes. The Escherichia coli O-antigen form O180 was first designated in 2004, and O180 strains were found to contain virulence factors and cause diarrhea. Different O-antigen forms are almost entirely due to genetic variations in the O-antigen gene clusters. In this study, the chemical structure and gene cluster of E. coli O180 O antigen were investigated. A tetrasaccharide repeating unit with the following structure: →4)-β-D-ManpNAc3NAcA-(1 → 2)-α-L-Rhap(I)-(1 → 3)-β-L-Rhap(II)-(1 → 4)-α-D-GlcpNAc-(1→was identified in the E. coli O180 O antigen, including the residue D-ManpNAc3NAcA (2,3-diacetamido-2,3-dideoxy-D-mannopyranuronic acid) that had not been hitherto identified in E. coli. Genes in the O-antigen gene cluster were assigned functions based on their similarities with those from available databases, and five genes involved in the synthesis of UDP-D-ManpNAc3NAcA (the nucleotide-activated form of D-ManpNAc3NAcA) were identified. The gnaA gene, encoding the enzyme involved in the initial step of the UDP-D-ManpNAc3NAcA biosynthetic pathway, was cloned and the enzyme product was expressed, purified and assayed for its activity. GnaA was characterized using capillary electrophoresis and electrospray ionization mass spectrometry and identified as a UDP-GlcNAc 6-dehydrogenase. The kinetic and physicochemical parameters of GnaA also were determined.