Characterization of C-1027 Enediyne Polyketide Synthase

描述（由申请人提供）：拟议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。保守结构域将通过提出的活性位点的定点诱变和体内分析表征的突变体来分析。假设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酸antetheinyl转移酶（PPTase），这也被认为是PKS中发现的结构域。假设的ACP和PPTase结构域的功能将通过彻底的体内和体外分析进行分析，其中包括拟议活性位点的突变体生成。最后，提出了PKS控制了内二炔形成的区域化学。这将通过从含有9个碳烯二炔核心的C- 1027和含有10个碳烯二炔核心的calicheamicin中创建杂交PKSs来进行测试。该结果将提供对烯二炔生物合成的编程规则的见解，并将允许生成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.