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）：现在在结构上表征了聚酮化合酶合酶（PKS）模块的所有折叠组件，但是多模块化PKS组装线（D8 MDA）的典型三维难题（D8 MDA）仍未得到解决。我们对合成酶分量如何在结构和酶上互相接口的有限理解是我们所知的主要差距。为了实现我们的长期目标，即通过工程多模块化PKS来加速天然产物向新的抗生素和抗癌剂开发，以合成有希望的聚酮药物的组合库，必须阐明此信息。我们正处于确定这些最大已知酶的体系结构和活动中，因此我们当前的目标是通过确定其每个域域界面（即单个零件如何适合）来解决多模块化PKS组装线路拼图。从原子分辨率的结构中据报道，以及我们实验室中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化，生物物理和生物信息信息学数据一致的模块和双模型模型。多年来，科学家一直在寻求PKS模块的晶体结构，但我们的模型表明，每个模块都具有柔性的“腰部区域”，例如相关的哺乳动物脂肪酸合酶，而PKS组装线中的主要相互作用实际上是跨模块化边界的。因此，我们将以模型为指导，以获取域如何组装域的物理数据，并测试我们的中心假设，即PKS成分的结构被改变，并通过完整的合酶中形成的域相互作用来增强酶促活性。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（特定目标1）。其他生物物理技术，例如小角度X射线散射（SAX）和不依赖晶体形成的沉积速度分析性超速离心是ALS非常强大的工具，尤其是现在已经确定了每个合成酶成分的原子分辨率结构。因此，即使未获得所需复合物的晶体，也将通过生物物理技术和位置定向诱变的组合来识别域界面（特定目标2）。我们还将通过我的实验室中开发的创新方法来探测PKS模块的结构，该方法利用生物催化和化学生物学工具将PKS模块的产品标记为荧光标记。组件酶如何应对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模块的许多期望的结构和功能细节（特定AIM 3）。我们提出的研究非常重要，因为多模块化PKS会产生许多重要的人类药物，例如抗菌红霉素，抗真菌性两性霉素和抗癌剂epothilone，以及通过对这些分子工厂如何运行的抗癌剂epothilone，我们将能够在新的抗生素和抗生素药物的快速发展中使用它们。