Determining the Architectures and Activities of Polyketide Synthase Modules

确定聚酮合酶模块的结构和活性

• 批准号: 9263990
• 负责人: Adrian Tristan Keatinge-Clay
• 金额: $ 28.12万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9263990
• 关键词: Amphotericin; Anti-Bacterial Agents; Antibiotics; Antifungal Agents; Antineoplastic Agents; Architecture; Biochemical; Bioinformatics; Biology; Biophysics; Chemicals; Complex; Crystal Formation; Crystallization; Data; Development; Dimensions; Engineering; Enzymes; Epothilones; Erythromycin; Fatty-acid synthase; Goals; Health; Home environment; Human; Immunosuppressive Agents; Individual; Knowledge; Learning; Libraries; Life; Medicine; Mission; Modeling; Molecular; Monitor; Mutagenesis; Mutation; Natural Products; Outcome; Pharmaceutical Preparations; Product Labeling; Public Health; Reporting; Research; Resolution; Roentgen Rays; Scientist; Sirolimus; Site-Directed Mutagenesis; Stretching; Structural Models; Structure; Techniques; Testing; United States National Institutes of Health; X-Ray Crystallography; analytical ultracentrifugation; biophysical analysis; biophysical techniques; burden of illness; combinatorial; flexibility; innovation; operation; peptide synthase; polyketide synthase; predictive modeling; public health relevance; response; sedimentation velocity; structural biology; tool

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.

描述（由申请人提供）：聚酮合酶（PKS）模块的所有折叠组件现已在结构上得到表征，但多模块PKS装配线（d8 MDa）的典型三维难题仍未得到解决。我们对合酶成分如何在结构上和酶学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，即通过设计多模块 PKS 来合成有前景的聚酮化合物药物先导组合库，加速将天然产物开发为新型抗生素和抗癌药物，必须阐明这一信息。我们正处于确定这些已知最大酶的结构和活性的冲刺阶段，因此我们当前的目标是通过确定其每个域-域接口（即各个部分如何组合在一起）来解决多模块 PKS 装配线难题。根据已报告的原子分辨率结构以及我们实验室尚未报告的几种结构信息结构，我们构建了与所有可用的生物化学、生物物理和生物信息学数据一致的模块和双模块模型。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，并且 PKS 装配线内的主要相互作用实际上是跨模块的边界。因此，我们将在模型的指导下获得结构域如何组装的物理数据，并测试我们的中心假设，即通过完整合酶内形成的结构域相互作用，PKS 成分的结构被改变，酶活性得到增强。我们首先寻求通过 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，例如不依赖于晶体形成的小角 X 射线散射 (SAXS) 和沉降速度分析超速离心也是非常强大的工具，特别是现在每个合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，也可以通过生物物理技术和定点诱变相结合来识别结构域界面（具体目标2）。我们还将通过我的实验室开发的创新方法对 PKS 模块的架构进行功能性探索，该方法利用生物催化和化学生物学工具对 PKS 模块的产品进行荧光标记。组分酶如何响应可疑界面处的突变以及关键柔性接头的缩短将有助于揭示 PKS 模块的许多所需的结构和功能细节（具体目标 3）。我们提出的研究具有重要意义，因为多模块 PKS 可以生产许多重要的人类药物，例如抗菌红霉素、抗真菌两性霉素和抗癌剂埃坡霉素，通过加深对这些分子工厂如何运作的了解，我们将能够在新型抗生素和抗癌药物的研发更加迅速。