Biochemical Characterization of the CDP-d-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F
Biochemical Characterization of the CDP-d-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F
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DOI：
10.1128/jb.06487-11
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发表时间：
2012-02
影响因子：
3.2
通讯作者：
Quan Wang;Yanli Xu;A. Perepelov;Y. Knirel;P. Reeves;A. Shashkov;Xi Guo;Peng Ding;Lu Feng
中科院分区：
文献类型：
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作者：
Quan Wang;Yanli Xu;A. Perepelov;Y. Knirel;P. Reeves;A. Shashkov;Xi Guo;Peng Ding;Lu Feng
摘要肺炎链球菌是人类的一种主要病原体，与世界上许多疾病有关。荚膜多糖(CPSS)是其主要毒力因子。D-阿拉伯糖醇的生物合成途径，存在于几个肺炎链球菌的CPSS中，但从未被鉴定过。在这项研究中，abpA(以前称为abp1)和abpB(以前称为abp2)基因被克隆，这两个基因先前被报道负责核苷二磷酸(NDP)-d-阿拉伯醇(d-阿拉伯醇的核苷酸激活形式)的合成。对酶产物进行过表达、纯化，并分析其各自的活性。用毛细管电泳法检测到ABPA和AbpB催化反应产生的新产物，用电喷雾电离质谱仪和核磁共振波谱对产物的结构进行了鉴定。结果表明，abpA是编码CDP-5-磷酸木糖的胞苷转移酶基因，负责将CTP转移到D-XLU-5-P形成CDP-d-木糖；abpB是CDP-d-木糖还原酶编码基因，负责CDP-d-木糖转化为CDP-d-阿拉伯醇。测定了底物d-XLU-5-P和CTP的ABPA和底物CDP-d-木糖及其辅因子NADH或NADPH的ABPA的动力学参数，并分析了温度、pH和阳离子对这两种酶的影响。本研究证实了abpa和abpB基因及其产物参与了CDP-d-阿拉伯糖醇的生物合成途径。
ABSTRACT Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of d-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-d-arabinitol (the nucleotide-activated form of d-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a d-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to d-xylulose-5-phosphate (d-Xlu-5-P) to form CDP-d-xylulose, and abpB was characterized to be a CDP-d-xylulose reductase-encoding gene, responsible for the conversion of CDP-d-xylulose to CDP-d-arabinitol as the final product. The kinetic parameters of AbpA for the substrates d-Xlu-5-P and CTP and those of AbpB for the substrate CDP-d-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-d-arabinitol.