Determining the Architectures and Activities of Polyketide Synthase Modules

确定聚酮合酶模块的结构和活性

基本信息

批准号：
9918938
负责人：
Adrian Tristan Keatinge-Clay
金额：
$ 30.63万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9918938
关键词：
Active Sites Acyl Carrier Protein Acyltransferase Affect Anti-Bacterial Agents Antifungal Agents Antineoplastic Agents Architecture Bacillus subtilis Bioinformatics Biology Biophysics C-terminal Catalysis Cells Chemicals Complex Cryoelectron Microscopy Crystallography Docking Electron Microscopy Electrons Elements Engineering Enzymatic Biochemistry Enzymes Erythromycin Polyketide Synthase Estrogen receptor positive Evolution Fatty Acids Filament Future Gatekeeping Goals Human Hydro-Lyases Immunosuppressive Agents In Vitro Investigation Learning Length Measures Medicine Methodology Molecular Machines Mutagenesis Mutate Mycobacterium tuberculosis N-terminal Natural Products Negative Staining Polymers Positioning Attribute Process Reaction Research Resolution Specificity Structure System Tertiary Protein Structure Titan To specify Update Work austin base biophysical tools dimer drug discovery instrument physical model polyketide synthase reconstitution tomography

项目摘要

ABSTRACT Our research on polyketide assembly lines is helping bring about a paradigm shift for how sets of component enzymes cooperate to biosynthesize polyketide natural products. The updated definition of a module, with the ketosynthase domain positioned at its downstream end, affects every level of modular polyketide synthase enzymology. Each of these levels must be further explored to achieve our long-term goal of reprogramming polyketide assembly lines to synthesize designer molecules and accelerate the drug discovery process. Our highest-resolution proposal is to study how ketosynthases gatekeep such that only one type of polyketide intermediate is selected by a module to be further elongated by the downstream assembly line (Specific Aim 1). This will be accomplished through methodology we have developed to crystallographically observe polyketides bound in ketosynthase active sites and measure the activity of ketosynthases mutated at suspected gatekeeping residues; engineered triketide synthases will also aid in this effort. From structures determined by our lab and others, we hypothesize that several uncharacterized domain interfaces are present within modules. We seek to structurally elucidate these interfaces within the context of the newly-defined module (Specific Aim 2). Thus, through crystallography and cryo-electron microscopy the structures of multidomain fragments possessing upstream processing enzymes and a downstream ketosynthase will be determined. We will also continue our efforts to characterize transient interfaces that form during the reaction cycle as acyl carrier protein domains present polyketide intermediates to cognate enzymes for catalysis. Our lab has collected several pieces of structural evidence for higher-order architecture. In the bacillaene polyketide synthase, a three-helix element adjacent to the ketosynthase domain seems to zipper homodimeric assembly lines into ~100 MDa assembly sheets observable within Bacillus subtilis cells. We propose to understand the structures of such biosynthetic megacomplexes by reconstituting them in vitro and observing them through electron microscopy (Specific Aim 3). Our lab is already visualizing Pks12 from Mycobacterium tuberculosis both in its “bimodular” and its polymeric assembly line states. We seek to determine how modules stack to construct higher-order architecture in such systems. Through investigations at each of these levels, an overall picture of the architectures and activities of polyketide assembly lines will emerge that will be particularly significant to the future engineering of these medicinally-relevant molecular machines.

抽象的我们对聚酮化合物装配线的研究正在帮助实现组件组的范式转变酶配合生物合成聚酮化合物天然产物模块的更新定义，具有：酮合酶结构域位于其下游末端，影响模块化聚酮合酶的每个水平必须进一步探索这些水平中的每一个，以实现我们重编程的长期目标。聚酮化合物装配线用于合成设计分子并加速药物发现过程。最高分辨率的提案是研究酮合成酶如何守门，使得只有一种类型的聚酮化合物中间体由模块选择，由下游装配线进一步拉长（具体目标 1). 这将通过我们开发的晶体学观察方法来实现聚酮化合物结合在酮合酶活性位点并测量突变的酮合酶的活性可疑的守门残基；工程化的三酮合酶也将有助于这一努力。由我们的实验室和其他人确定，我们捕获到存在几个未表征的域接口我们寻求在新定义的上下文中从结构上阐明这些接口。模块（具体目标 2）因此，通过晶体学和冷冻电子显微镜的结构。具有上游加工酶和下游酮合酶的多结构域片段将是我们还将继续努力表征反应过程中形成的瞬态界面。循环作为酰基载体蛋白结构域将聚酮化合物中间体呈现给同源酶以进行催化。实验室已经收集了一些 bacillaene 中高阶结构的结构证据。聚酮合酶，一种与酮合酶结构域相邻的三螺旋元件，似乎可以拉链同源二聚体我们建议将其组装成约 100 个可在枯草芽孢杆菌细胞内观察到的 MDa 组装片。通过体外重构和观察来了解此类生物合成巨型复合物的结构通过电子显微镜（具体目标 3），我们的实验室已经对来自分枝杆菌的 Pks12 进行了可视化。结核病的“双模块”和聚合装配线状态我们试图确定模块的状态。通过对每个级别的研究，可以在此类系统中构建高阶架构。聚酮装配线的架构和活动的总体情况将会出现对于这些医学相关分子机器的未来工程特别重要。