Molecular Analysis of Modular Polyketide Synthases

描述（由申请人提供）：细菌I型聚酮合成酶（PKS）由一组有趣的复杂多功能蛋白组成，与相关酶一起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，为产生新型天然产物的工程PKSs铺平了道路。各种微生物系统的高通量基因组测序正在以越来越快的速度揭示新的PKS，非核糖体肽合成酶（NRPS）和混合PKS-NRPS系统。此外，用于预测这些代谢系统结构结果的生物信息学工具正在为新的天然产物提供快速获取途径。尽管获得新信息的途径越来越多，但获得PKS-NRPS系统的详细生化理解对于测试功能预测是必要的，并且需要应用严格的实验方法。了解这些细节不仅可以扩展我们对PKS-NRPS分子机器的基本知识，还可以提供新的策略来操纵它们以扩大化学多样性。这类系统很有吸引力，因为它们有潜力创造新的化学型，在药物发现和开发中有价值的应用。尽管取得了显著进展，但对天然和杂交PKSs的分子机制、催化活性、动力学性质、底物特异性和蛋白质识别的了解仍然有限。这一竞争性更新申请建议采用多功能和特性良好的委内瑞拉链霉菌派克霉素PKS，以及一系列其他途径，其详细分析已在前一个支持周期中启动。这些系统每个都具有迷人的生化属性，将扩展我们对导致本地和杂交PKS模块内部和之间功能活动的特异性和结构特征的理解。我们的目标和方法将集中于评估导致聚酮天然产物显着的化学多样性的聚酮链起始，延伸，2分支和终止的分子细节。这种详细的生化分析，结合结构生物学来探测底物特异性和合成化学来开发化学酶方法，将使我们能够追求工程PKS系统的长期目标，这些系统可以有效地产生具有重大治疗潜力的新结构。具体目标包括：1 .模块化聚酮合酶的分子分析。设计和使用合成底物和Pik， DEBS和Tyl端子模块来探索链加载，伸长和加工的选择性和容忍度。2。分子识别是模块化PKSs中蛋白-蛋白相互作用的基础。通过设计和构建利用原生和异种对接结构域组合的有效途径，探索对接选择性的分子参数。3。模系统终止的分子基础分析。探索大内酯形成的决定因素与水解的PKSs终止硫酯酶的匹克霉素，红霉素，泰洛星，互变霉素，库拉星和卡马宾。模块化pks合成2支产物的新催化结构域和分子相互作用分析。继续分析苔藓虫素生物合成系统（Bry），包括HMG合成酶和2支化导致的修饰吡酮环系统。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). PUBLIC HEALTH RELEVANCE: The proposed research will focus on elucidating the detailed function of complex biosynthetic machines that create chemically diverse, biologically active natural products. The ability to understand and subsequently engineer these remarkable biochemical systems will create new opportunities to discover and develop effective drugs for the treatment of human diseases, including cancer, infectious diseases, and Alzheimer's.