Molecular Analysis of Modular Polyketide Synthases

描述(申请人提供)：细菌I型聚酮合成酶(PKS)由一组耐人寻味的复杂多功能蛋白质组成，与相关酶一起通过模块化的多步骤过程产生结构复杂和具有临床重要性的天然产物。在过去的十年里，已经发现了许多这种类型的系统，为制造产生新的天然产品的工程PKS铺平了道路。获得负担得起的不同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、非核糖体多肽合成酶(NRPS)和PKS-NRPS混合系统。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的天然产品的途径。尽管越来越多地获得新的信息，但获得对PKS-NRPS系统的详细生化理解对于测试功能预测是必要的，并要求应用严格的实验方法。了解这些细节不仅将扩大我们对PKS-NRPS分子机的基础知识，还将为操纵它们以扩大化学多样性提供新的策略。这类系统很有吸引力，因为它们具有创造新的化学类型的潜力，在药物发现和开发中具有重要的应用价值。尽管取得了显著的进展，但对天然和杂化PKS的分子机制、催化活性、动力学性质、底物专一性和蛋白质-蛋白质识别的了解仍然有限。这一竞争性的更新申请建议使用多功能和特征良好的维尼链霉菌皮克罗霉素PKS，以及一系列额外的途径，其详细分析已在前一个支持周期中启动。这些系统都具有令人着迷的生化属性，这些属性将扩大我们对导致天然和杂交PKS模块内和之间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集中于评估聚酮链的起始、延伸、2-支化和终止的分子细节，这些细节导致聚酮天然产物具有显著的化学多样性。这种详细的生化分析，以及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，将使我们能够追求设计PKS系统的长期目标，从而有效地产生具有巨大治疗潜力的新型结构。具体目标包括：1.模块聚酮合成酶的分子分析。设计和使用合成底物和Pik、Debs和TYL端子模块，以探索链加载、延伸和加工中的选择性和公差。II.分子识别作为模块化PKSS中蛋白质-蛋白质相互作用的基础。通过设计和构建使用本地和异源对接结构域组合的有效途径，探索对接选择性的分子参数。三、模块化体系中终止的分子基础分析。探索吡克罗霉素、红霉素、泰乐菌素、直链霉菌素、咖喱和卡马宾的PKS中终止硫酯酶对大内酯形成和水解的决定因素。IV.合成2-支化产物的模块化PKSS中新的催化结构域和分子相互作用的分析。对Bryostatin生物合成系统(Bry)进行分析，包括HMG合成酶和2-分支，从而得到改进的吡喃酮环系统。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)。 与公共健康相关：拟议的研究将侧重于阐明复杂的生物合成机器的详细功能，这些机器创造出具有化学多样性、生物活性的天然产品。理解并随后设计这些非凡的生化系统的能力将创造新的机会，发现和开发有效的药物来治疗人类疾病，包括癌症、传染病和阿尔茨海默氏症。

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.