Characterization of C-1027 Enediyne Polyketide Synthase

描述(申请人提供)：拟议研究的目标是研究迭代类型Iendiyne C-1027聚酮合成酶(PKS)结构域的功能。保守结构域将通过建议的活性位点的定点突变进行分析，突变体将通过体内分析来表征。据推测，PKS含有一个不寻常的酰基载体蛋白(ACP)结构域，该结构域需要一个独特的磷脂酰基转移酶(PPTase)，该酶也被认为是在PKS中发现的一个结构域。推测的ACP和PPTase结构域的功能将通过全面的体内和体外分析进行分析，其中包括建议的活性位点的突变产生。最后，提出烯二炔形成的区域化学受PKS的控制。这将通过由C-1027和Calicheamicin创建混合PKSS来进行测试，C-1027包含9碳的烯二炔核心，Calicheamicin包含10碳的烯二炔核心。这些结果将为烯二炔生物合成的编程规则提供洞察力，并将允许用预测的化学方法生成“非天然”的天然烯二炔。

DESCRIPTION (provided by applicant): The goal of the proposed research is to study the functionality of the domains of the iterative type I enediyne C-1027 polyketide synthase (PKS). Conserved domains will be analysized by site-directed mutagenesis of the proposed active sites, and the mutants characterized by in vivo analysis. It is hypothesized the PKS contains an unusual acyl carrier protein (ACP) domain that requires a unique phosphopantetheinyl transferase (PPTase), which is proposed to also be a domain found within the PKS. The function of the putative ACP and PPTase domain will be analyzed by a thorough in vivo and in vitro analysis, which includes mutant generation of the proposed active sites. Finally, it is proposed that the regiochemistry of enediyne formation is controlled by the PKS. This will be tested by the creation of hybrid PKSs from C- 1027, which contains a 9-carbon enediyne core, and calicheamicin, which contains a 10-carbon enediyne core. The results will provide insights into the programming rules of enediyne biosynthesisand will allow for the generation of "unnatural" natural enediynes with predicted chemistry.