Characterization of the CDP-D-mannitol biosynthetic pathway in Streptococcus pneumoniae 35A.
Characterization of the CDP-D-mannitol biosynthetic pathway in Streptococcus pneumoniae 35A.
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DOI：
10.1093/glycob/cws113
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发表时间：
2012-12
期刊：
影响因子：
4.3
通讯作者：
Quan Wang;Yanli Xu;A. Perepelov;Y. Knirel;P. Reeves;A. Shashkov;Peng Ding;Xi Guo;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;Peng Ding;Xi Guo;Lu Feng
肺炎链球菌是世界范围内与疾病相关的一种主要人类病原体。衣壳多糖(CPSS)被认为是一个主要的毒力因素，也是疫苗的靶标。发现D-甘露醇存在于几个肺炎链球菌血清型的CPS中。位于CPS基因簇中的两个基因mnp1和mnp2被认为与NDP-d-甘露醇(d-甘露醇的核苷酸活化形式)的合成有关。然而，这一途径从未被实验方法确定，我们的目标是在本研究中对其进行表征。为了实现这一点，我们克隆了mnp1和mnp2两个基因，并对基因产物进行了高效表达、纯化和体外活性分析。用毛细管电泳法检测了Mnp1和Mnp2催化的反应产物，并用电喷雾电离质谱仪和核磁共振波谱对产物进行了验证。我们发现，Mnp1负责将cMP从CTP转移到D-果糖-6-磷酸(Fu-6-P)形成CDP-d-果糖，而Mnp2则催化CDP-d-果糖转化为CDP-d-甘露醇。因此，mnp1(重命名为mnpA)为果糖-6-P胞苷转移酶编码基因，mnp2(重命名为mnpB)为CDP-d-果糖还原酶编码基因。Mnp1在底物(Fu-6-P和CTP)和Mnp2在底物(CDP-d-果糖)和辅因子NADH或NADPH的动力学符合Michaelis-Menten模型。分析了温度、pH和阳离子对这两种酶的影响。这是首次对CDP-d-甘露醇的生物合成途径进行生化鉴定。
Streptococcus pneumoniae is a major human pathogen associated with diseases worldwide. The capsular polysaccharides (CPSs) are considered a major virulence factor and are targets for a vaccine. d-Mannitol was found to be present in the CPS of several S. pneumoniae serotypes. Two genes, mnp1 and mnp2, which are located in the CPS gene cluster, were proposed to be responsible for the synthesis of NDP-d-mannitol (the nucleotide activated form of d-mannitol). However, the pathway has never been identified by experimental methods and we aimed to characterize it in the present study. To achieve this, the two genes, mnp1 and mnp2, were cloned and the gene products were overexpressed, purified, and analyzed in vitro for their respective enzymatic activities. Products of reactions catalyzed by Mnp1 and Mnp2 were detected by capillary electrophoresis and validated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. We show that Mnp1 is responsible for the transfer of CMP from CTP to d-fructose-6-phosphate (Fru-6-P) to form CDP-d-fructose, whereas Mnp2 catalyzed the conversion of CDP-d-fructose to CDP-d-mannitol. Therefore, Mnp1 (renamed as mnpA) was identified as Fru-6-P cytidylyltransferase-encoding gene, and mnp2 (renamed as mnpB) as a CDP-d-fructose reductase-encoding gene. The kinetics of Mnp1 for the substrate (Fru-6-P and CTP) and of Mnp2 for the substrate (CDP-d-fructose) and the cofactor NADH or NADPH fitted the Michaelis-Menten model. The effects of temperature, pH and cations on the two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-d-mannitol has been identified biochemically.