Determining the Architectures and Activities of Polyketide Synthase Modules

描述（由申请人提供）：聚酮化合物合酶（PKS）模块的所有折叠组分现在已经在结构上表征，但多模块PKS组装线（d8 MDa）的典型三维难题仍然没有解决。我们对合成酶组分如何在结构上和酶促上相互作用的有限理解是我们知识中的主要差距。为了实现我们的长期目标，加快天然产物开发成新的抗生素和抗癌剂的工程多模块PKS合成组合库的有前途的聚酮药物的线索，这方面的信息必须阐明。我们在确定这些最大的已知酶的结构和活性方面处于最后阶段，因此我们目前的目标是通过确定其每个结构域-结构域界面（即各个片段如何组合在一起）来解决多模块PKS组装线难题。从原子分辨率的结构，已经报告，以及几个架构信息结构尚未从我们的实验室报告，我们已经构建了模块和双模块，是符合所有可用的生物化学，生物物理和生物信息学数据的模型。虽然多年来科学家们一直在寻找PKS模块的晶体结构，但我们的模型表明，每个模块都有一个灵活的“腰部区域”，就像相关的哺乳动物脂肪酸合酶一样，PKS组装线内的主要相互作用实际上是跨越模块边界的。因此，我们将引导我们的模型对获得的物理数据域如何组装和测试我们的中心假设，PKS组件的结构被改变和酶活性增强，通过一个完整的合酶内形成的域相互作用。我们首先试图通过X射线晶体学观察最相关的多域复合物（具体目标1）。其他生物物理技术，如小角X射线散射（SAXS）和沉降速度分析超离心，不依赖于晶体的形成也是非常强大的工具，特别是现在每个合成酶组分的原子分辨率结构已经确定。因此，即使没有获得所需复合物的晶体，也将通过生物物理技术和定点诱变的组合来鉴定结构域界面（具体目标2）。我们还将通过在我的实验室开发的创新方法，利用生物催化和化学生物学工具荧光标记PKS模块的产品，功能性地探测PKS模块的架构。组分酶如何对可疑界面处的突变做出反应以及关键柔性接头的缩短将有助于揭示PKS模块的许多所需结构和功能细节（具体目标3）。我们提出的研究是重要的，因为多模块PKSs产生许多重要的人类药物，如抗菌红霉素，抗真菌抗生素，和抗癌剂埃博霉素，并通过增加了解这些分子工厂如何运作，我们将能够利用它们更快地开发新的抗生素和抗癌药物。

DESCRIPTION (provided by applicant): All of the folded components of polyketide synthase (PKS) modules have now been structurally characterized, yet the quintessential three-dimensional puzzle of the multimodular PKS assembly line (d8 MDa) has still not been solved. Our limited understanding of how synthase components structurally and enzymatically interface with one another is the major gap in our knowledge. In order to realize our long-term goal of accelerating the development of natural products into new antibiotics and anticancer agents by engineering multimodular PKSs to synthesize combinatorial libraries of promising polyketide drug leads this information must be elucidated. We are in the home stretch in determining the architectures and activities of these largest known enzymes, thus our current goal is to solve the multimodular PKS assembly line puzzle through determining each of its domain- domain interfaces (i.e. how the individual pieces fit together). From the atomic-resolution structures tha have been reported as well as several architecturally-informative structures not yet reported from our lab, we have constructed models of modules and bimodules that are consistent with all the available biochemical, biophysical, and bioinformatics data. While for many years scientists have sought the crystal structure of a PKS module, our models suggest that each module has a flexible "waist region" like that of the related mammalian fatty acid synthase and that the major interactions within PKS assembly lines are actually across modular boundaries. Thus, we will be guided by our models towards obtaining the physical data of how domains assemble and test our central hypothesis that the structures of PKS components are altered and enzymatic activities are enhanced through domain interactions formed within an intact synthase. We first seek to observe the most relevant multidomain complexes through x-ray crystallography (Specific Aim 1). Other biophysical techniques such as small-angle x-ray scattering (SAXS) and sedimentation velocity analytical ultracentrifugation that do not rely on crystal formation are als very powerful tools, especially now that the atomic-resolution structures of each synthase component have been determined. Thus, even if crystals of desired complexes are not obtained, domain interfaces will be identified through a combination of biophysical techniques and site-directed mutagenesis (Specific Aim 2). We will also functionally probe the architecture of PKS modules through an innovative approach developed in my lab that utilizes biocatalytic and chemical biology tools to fluorescently label products of PKS modules. How component enzymes respond to mutations at suspected interfaces and the shortening of key flexible linkers will help reveal many desired structural and functional details of PKS modules (Specific Aim 3). Our proposed research is significant as multimodular PKSs produce many important human medicines, such as the antibacterial erythromycin, the antifungal amphotericin, and the anticancer agent epothilone, and through an increased understanding of how these molecular factories operate we will be able to utilize them in the more rapid development of new antibiotics and anticancer drugs.