Molecular Analysis of Modular Polyketide Synthases

描述（由申请人提供）：细菌I型聚酮化合物合酶（PKS）由一组有趣的复杂多功能蛋白质组成，其沿着相关酶通过模块化多步骤过程产生结构复杂且临床上重要的天然产物。在过去的十年中，已经发现了许多这种类型的系统，为产生新的天然产物的工程PKS铺平了道路。获得不同微生物系统的可负担的高通量基因组测序正在以不断增加的速度揭示新的PKS、非核糖体肽合成酶（NRPS）和混合PKS-NRPS系统。此外，预测这些代谢系统结构结果的生物信息学工具正在提供快速获得新天然产物的途径。尽管越来越多地获得新的信息，获得详细的生化PKS-NRPS系统的理解是必要的测试功能的预测，并要求严格的实验方法的应用。了解这些细节不仅将扩大我们对PKS-NRPS分子机器的基本知识，而且还提供了操纵它们以扩大化学多样性的新策略。这样的系统是有吸引力的，因为它们有潜力创造新的化学型，在药物发现和开发中具有有价值的应用。尽管取得了显着的进展，理解的分子机制，催化活性，动力学特性，底物特异性和蛋白质-蛋白质识别在天然和混合PKS仍然有限。该竞争性更新申请拟采用多功能且充分表征的委内瑞拉链霉菌匹克罗霉素PKS，以及一系列其他途径，其详细分析已在上一个支持周期中启动。这些系统各自具有迷人的生物化学属性，这将扩大我们对导致天然和混合PKS模块内和之间的功能活性的特异性和结构特征的理解。我们的目标和方法将侧重于评估聚酮化合物链的起始，延伸，2-分支和终止，导致显着的化学多样性的聚酮化合物天然产物的分子细节。这种详细的生化分析，以及结构生物学的整合，以探测底物特异性和合成化学，以开发化学酶的方法，将允许追求我们的长期目标工程PKS系统，有效地产生新的结构，具有显着的潜力作为治疗剂。具体目标包括：模块聚酮合酶的分子分析。设计并采用合成底物和Pik、DEBS和Tyl末端模块，以探索链加载、延伸和加工中的选择性和耐受性。二.分子识别作为模块化PKS中蛋白质-蛋白质相互作用的基础。探索分子参数的对接选择性设计和构建有效的途径，使用天然的，异源的对接结构域组合。 三.模块化系统中终止的分子基础分析。探索大环内酯形成的决定因素与吡克霉素、红霉素、泰乐菌素、互变霉素、Curacin和carmabin的PKS中终止硫酯酶的水解。 四.合成2-分支产物的模块化PKS中新催化结构域和分子相互作用的分析。继续分析苔藓抑素生物合成系统（Bry），包括HMG合酶和2-分支导致修饰的吡喃酮环系统。探索Bry中酰基-ACP同源酶相互作用的基础，包括ACPD：：HMGS、ACPD：：KS和KS：：HMGS）。

DESCRIPTION (provided by applicant): A bacterial type I polyketide synthase (PKS) is comprised of an intriguing set of complex multifunctional proteins that along with allied enzymes generate structurally complex and clinically important natural products via a modular multi-step process. Numerous systems of this type have been discovered over the past decade, paving the way to engineered PKSs that generate novel natural products. Access to affordable high throughput genome sequencing of diverse microbial systems is revealing new PKS, non- ribosomal peptide synthetase (NRPS) and mixed PKS-NRPS systems at an ever-increasing rate. Moreover, bioinformatic tools to predict the structural outcome of these metabolic systems are providing rapid access to new natural products. Despite increasing access to new information, obtaining a detailed biochemical understanding of PKS-NRPS systems is necessary to test functional predictions and demands the application of rigorous experimental approaches. Understanding these details will not only expand our basic knowledge of PKS-NRPS molecular machines, but also provide new strategies to manipulate them to expand chemical diversity. Such systems are attractive due to their potential to create new chemotypes with valuable applications in drug discovery and development. Despite remarkable progress, an understanding of the molecular mechanisms, catalytic activities, kinetic properties, substrate specificity and protein-protein recognition in both natural and hybrid PKSs remains limited. This competing renewal application proposes to employ the versatile and well-characterized Streptomyces venezuelae pikromycin PKS, as well as a series of additional pathways whose detailed analysis has been initiated during the previous cycle of support. These systems each bear fascinating biochemical attributes that will expand our understanding of the specificity and structural features that lead to functional activity within and between native and hybrid PKS modules. Our objectives and approach will focus on assessing the molecular details of polyketide chain initiation, elongation, 2-branching and termination that lead to the remarkable chemical diversity of polyketide natural products. This detailed biochemical analysis, and the integration of structural biology to probe substrate specificity and synthetic chemistry to develop chemoenzymatic approaches will allow pursuit of our long term objective of engineering PKS systems that efficiently generate novel structures with significant potential as therapeutic agents. Specific aims include: I. Molecular Analysis of Modular Polyketide Synthases. Design and employ synthetic substrates and Pik, DEBS, and Tyl terminal modules to explore selectivity and tolerance in chain loading, elongation and processing. II. Molecular recognition as the basis for protein-protein interactions in modular PKSs. Explore molecular parameters of docking selectivity by designing and constructing effective pathways using native, and heterologous docking domain combinations. III. Analysis of the molecular basis for termination in modular systems. Explore the determinants of macrolactone formation vs. hydrolysis by the terminating thioesterases in the PKSs for pikromycin, erythromycin, tylosin, tautomycetin, curacin, and carmabin. IV. Analysis of new catalytic domains and molecular interactions in modular PKSs that synthesize 2-branched products. Pursue analysis of the bryostatin biosynthetic system (Bry) including HMG synthase and 2-branching leading to the modified pyrone ring system. Explore the basis for acyl-ACP cognate enzyme interactions in Bry including ACPD::HMGS, ACPD::KS, and KS::HMGS).