Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa
Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa
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DOI：
10.1016/j.pep.2010.01.007
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发表时间：
2010-06-01
影响因子：
1.6
通讯作者：
Liang, Zhao-Xun
中科院分区：
文献类型：
--
作者：
Murugan, Elavazhagan;Kong, Rong;Liang, Zhao-Xun
酰基载体蛋白磷酸二酯酶 (AcpH) 是唯一已知可从全酰基载体蛋白 (ACP) 中去除 4'-磷酸泛酰胆碱基部分的酶，全酰基载体蛋白 (ACP) 是脂质和其他细胞代谢物生物合成所必需的一大类蛋白质。在这里，我们报道，在优化表达和纯化条件后，来自铜绿假单胞菌的 AcpH (paAcpH) 可以在大肠杆菌中过表达，成为可溶且稳定的蛋白质。这标志着对易于聚集的大肠杆菌AcpH的改进，后者只能通过重新折叠从包涵体获得的多肽来获得。通过可溶性重组蛋白，我们发现 PaAcpH 在八种载体蛋白中对来自脂肪酸合成途径的 ACP 表现出优先的底物特异性。我们进一步表明，PaAcpH 会水解并释放来自多域聚酮化合物合酶的 4'-磷酸泛酰胆碱基团连接的产物，证明该酶完全能够水解酰化的 ACP 底物。 (C) 2010 Elsevier Inc. 保留所有权利。
Acyl carrier protein phosphodiesterases (AcpH) are the only enzymes known to remove the 4'-phosphopantetheinyl moiety from holo acyl carrier proteins (ACP), which are a large family of proteins essential for the biosynthesis of lipid and other cellular metabolites. Here we report that the AcpH (paAcpH) from Pseudomonas aeruginosa can be overexpressed in Escherichia coli as a soluble and stable protein after optimization of the expression and purification conditions. This marks an improvement from the aggregation-prone E. coli AcpH that could only be obtained by refolding the polypeptide obtained from the inclusion body. With the soluble recombinant protein, we found that PaAcpH exhibits preferred substrate specificity towards the ACPs from the fatty acid synthesis pathway among eight carrier proteins. We further showed that PaAcpH hydrolyzes and releases the 4'-phosphopantetheinyl group-linked products from a multidomain polyketide synthase, demonstrating that the enzyme is fully capable of hydrolyzing acylated ACP substrates. (C) 2010 Elsevier Inc. All rights reserved.