A dedicated phosphopantetheinyl transferase for the fredericamycin polyketide synthase from Streptomyces griseus
A dedicated phosphopantetheinyl transferase for the fredericamycin polyketide synthase from Streptomyces griseus
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DOI：
10.1074/jbc.m604895200
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发表时间：
2006-10-06
影响因子：
4.8
通讯作者：
Shen, Ben
中科院分区：
文献类型：
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作者：
Huang, Yong;Wendt-Pienkowski, Evelyn;Shen, Ben
聚酮合成酶只有通过4‘-磷酸天冬氨酸基团与高度保守的丝氨酸残基的共价连接而被翻译后修饰，并且该反应是由磷酸天冬氨酸转移酶(PPTase)催化的，聚酮合成酶才能发挥功能。对灰色链霉菌(Streptomyces Griseus)33kb的弗雷德霉素生物合成基因簇进行克隆和序列分析，发现该基因簇含有fdmW，其推导的基因产物与已知的PPTase具有显著的序列同源性。FdmW的体外生化鉴定证实它是一种PPTase。FdmW的失活导致fdmW产量下降93%，并通过在反式恢复的fdmW中表达fdmW到与野生型菌株相当的水平来补充fdmW：：aac(3)IV突变体。尽管FdmW可以磷酸化各种ACP，但它更喜欢其同源底物FdmH ACP，其K-m为5.8µM，k(CAT)/K-m为8.1亩M(-1)min(-1)，而不是异源ACP，如K-m为1.0×10(2)muM，k(CAT)/K-m为0.6muM(-1)min(-1)的TcmM ACP。这些发现表明，FdmW是fdm生物合成的特异性基因。因此，FdmW代表了第一个从芳香族聚酮生物合成基因簇中鉴定的全息ACP合成酶类型的PPTase。
Polyketide synthases cannot be functional unless their apoacyl carrier proteins (apo-ACPs) are post-translationally modified by covalent attachment of the 4'-phosphopantetheine group to the highly conserved serine residue, and this reaction is catalyzed by phosphopantetheinyl transferases (PPTases). Cloning and sequence analysis of the 33-kb fredericamycin (FDM) biosynthetic gene cluster from Streptomyces griseus revealed fdmW, whose deduced gene product showed significant sequence homology to known PPTases. Biochemical characterization of FdmW in vitro confirmed that it is a PPTase. Inactivation of fdmW resulted in similar to 93% reduction of FDM production, and complementation of the fdmW::aac (3) IV mutant by expressing fdmW in trans restored FDM production to a level comparable with that of the wild-type strain. Although FdmW can phosphopantetheinylate various ACPs, it prefers its cognate substrate, the FdmH ACP, with a K-m of 5.8 mu M and a k(cat)/K-m of 8.1 mu M(-1.)min(-1), to heterologous ACPs, such as the TcmM ACP with a K-m of 1.0 x 10(2) mu M and a k(cat)/K-m of 0.6 mu M(-1.)min(-1). These findings suggest that FdmW is specific for FDM biosynthesis. FdmW therefore represents the first holo-ACP synthase-type PPTase identified from an aromatic polyketide biosynthetic gene cluster.