CMP-N-acetylneuraminic acid synthetase from Escherichia coli K1 is a bifunctional enzyme -: Identification of minimal catalytic domain for synthetase activity and novel functional domain for platelet-activating factor acetylhydrolase activity
CMP-N-acetylneuraminic acid synthetase from Escherichia coli K1 is a bifunctional enzyme -: Identification of minimal catalytic domain for synthetase activity and novel functional domain for platelet-activating factor acetylhydrolase activity
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DOI：
10.1074/jbc.m400143200
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发表时间：
2004-04-23
影响因子：
4.8
通讯作者：
Jin, C
中科院分区：
文献类型：
--
作者：
Liu, GC;Jin, CS;Jin, C
大肠杆菌cMP-NeuAc合成酶(EC 2.7.7.43)催化CTP和NeuAc合成cMP-NeuAc，这是K1菌株形成胶囊化聚唾液酸酯所必需的。将其氨基酸序列与其他细菌的氨基酸序列进行比对，发现保守的基序位于其N端，而C端似乎是多余的。在此基础上，构建了一系列cMP-NeuAc合成酶编码区3‘端的缺失片段，并在大肠杆菌中进行了表达。结果发现，cMP-NeuAc合成酶所需的催化结构域位于N-末端，由1-229个氨基酸组成。采用基于二级结构同源搜索的三级结构预测策略，将C端半部分识别为牛脑血小板活化因子乙酰水解酶I的α1亚基。生化分析表明，由228-418氨基酸组成的C端半部分具有血小板活化因子乙酰水解酶活性。酶性质和底物专一性与牛脑α1亚基相似。尽管其生理功能尚不清楚，但已有研究表明，大肠杆菌的α1亚单位样结构域可能参与血脑屏障的穿透。
Escherichia coli CMP-NeuAc synthetase (EC 2.7.7.43) catalyzes the synthesis of CMP-NeuAc from CTP and NeuAc, which is essential for the formation of capsule polysialylate for strain K1. Alignment of the amino acid sequence of E. coli CMP-NeuAc synthetase with those from other bacterial species revealed that the conserved motifs were located in its N termini, whereas the C terminus appeared to be redundant. Based on this information, a series of deletions from the 3'-end of the CMP-NeuAc synthetase coding region was constructed and expressed in E. coli. As a result, the catalytic domain required for CMP-NeuAc synthetase was found to be in the N-terminal half consisting of amino acids 1-229. Using the strategy of tertiary structure prediction based on the homologous search of the secondary structure, the C-terminal half was recognized as an alpha1-subunit of bovine brain platelet-activating factor acetylhydrolase isoform I. The biochemical analyses showed that the C-terminal half consisting of amino acids 228-418 exhibited platelet-activating factor acetylhydrolase activity. The enzyme properties and substrate specificity were similar to that of bovine brain alpha1-subunit. Although its physiological function is still unclear, it has been proposed that the alpha1-subunit-like domain of E. coli may be involved in the traversal of the blood-brain barrier.