Characterization of C-1027 Enediyne Polyketide Synthase

描述（由申请人提供）：拟定研究的目标是研究迭代I型烯二炔C-1027聚酮合酶（PKS）结构域的功能。保守结构域将通过所提出的活性位点的定点诱变来分析，并且突变体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，需要一个独特的磷酸泛酰巯基乙胺基转移酶（PPT酶），这也被认为是一个结构域内发现的PKS。将通过全面的体内和体外分析（包括拟定活性位点的突变体生成）分析推定ACP和PPT酶结构域的功能。最后，提出烯二炔形成的区域化学是由PKS控制的。这将通过从C- 1027（其含有9-碳烯二炔核心）和加利车霉素（其含有10-碳烯二炔核心）产生杂合PKS来测试。这些结果将提供对烯二炔生物合成的编程规则的深入了解，并将允许产生具有预测化学性质的“非自然”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.