Determining the Architectures and Activities of Polyketide Synthase Modules

描述(申请人提供)：聚酮合成酶(PKS)模块的所有折叠组件现在都已被结构表征，但多模块PKS装配线(D8MDA)的典型三维谜题仍未解决。我们对合成酶成分如何在结构上和酶作用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，即通过工程多模块PKSS来合成有前景的聚酮类药物先导化合物的组合文库，加速天然产物开发成为新的抗生素和抗癌药物，这些信息必须得到阐明。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-结构域接口(即单个片段如何结合在一起)来解决多模块PKS装配线难题。从已经报道的原子分辨结构以及我们实验室尚未报道的几个建筑信息结构，我们已经构建了与所有可用的生化、生物物理和生物信息学数据一致的模块和双模模型。虽然多年来科学家一直在寻找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“腰区”，就像相关的哺乳动物脂肪酸合成酶一样，并且PKS装配线内的主要相互作用实际上是跨越模块边界的。因此，我们将在我们的模型的指导下，获得结构域如何组装的物理数据，并验证我们的中心假设，即通过在完整的合成酶中形成的结构域相互作用，改变PKS组分的结构，增强酶活性。我们首先寻求通过X射线结晶学来观察最相关的多域配合物(具体目标1)。其他不依赖于晶体形成的生物物理技术，如小角X射线散射(SAXS)和沉积速度分析超速离心法都是非常强大的工具，特别是现在已经确定了合成酶每个组分的原子分辨结构。因此，即使没有获得所需复合体的晶体，也将通过生物物理技术和定点突变的组合来识别结构域界面(特定目标2)。我们还将通过我的实验室开发的一种创新方法，从功能上探索PKS模块的体系结构，该方法利用生物催化和化学生物学工具来荧光标记PKS模块的产品。组成酶如何对可疑界面上的突变做出反应，以及关键柔性连接子的缩短，将有助于揭示PKS模块许多所需的结构和功能细节(特定目标3)。我们提出的研究具有重要意义，因为多模块PKS生产许多重要的人类药物，如抗菌红霉素、抗真菌两性霉素和抗癌剂埃博西隆，通过对这些分子工厂如何运作的了解，我们将能够利用它们来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.