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：
10669273
负责人：
Adrian Tristan Keatinge-Clay
金额：
$ 31.91万
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 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10669273
关键词：
Acyl Carrier Protein Acyltransferase Anabolism Anthelmintics Antibiotics Antineoplastic Agents Architecture Azithromycin Binding Biological Models Buffers Collaborations Complex Cryoelectron Microscopy Data Development Docking Electron Microscope Electron Microscopy Engineering Ensure Enzymes Evolution Freezing Gatekeeping Goals Human Hydro-Lyases In Vitro Ivermectin Knock-out Learning Mediating Medicine Modeling Molecular Molecular Conformation Motion Mutagenesis Mutate Mutation N-terminal Natural Products Nature Negative Staining Oxidoreductase Physical condensation Physiological Process Production Productivity Reaction Renaissance Research Resolution Role SET Domain Structure Techniques Tertiary Protein Structure Testing Update Visualization design dimer enoyl reductase enzyme model expectation improved in vivo inorganic phosphate insight interest migration particle polyketide synthase polyketides polypeptide self assembly structural biology thioester tool transacylation

项目摘要

A renaissance in the field of modular polyketide synthases has begun. New tools and paradigms are enabling deeper insights into the architectures and activities of these enzymatic assembly lines and are facilitating our long-term goal of applying the synthetic power of modular polyketide synthases to the development and production of new medicines. Using the updated definition of the “module”, our lab has engineered diverse tri- /tetra-/pentaketide synthases that are functional both in vivo and in vitro. While these short assembly lines are uncommon in nature, they are ideal for our structural and functional studies. In Specific Aim 1 we propose to plunge-freeze these assembly lines as they are synthesizing their polyketide products and investigate them by cryo-electron microscopy. Since this approach has enabled us to capture high-resolution, dynamic information of the priming ketosynthase and acyltransferase of a model triketide synthase, we will apply it to synthases that contain other regions of interest. One objective is to learn how the ketoreductase, dehydratase, and enoylreductase processing enzymes are oriented relative to one another and the neighboring ketosynthase+acyltransferase didomains to understand how acyl carrier protein domains move between these enzymes during the extension and processing of polyketide intermediates. Thus, we will investigate at least thirteen engineered tri-/tetraketide synthases and two natural synthases functionally validated in our lab that contain different sets of these processing enzymes. In Specific Aim 2 we propose to elucidate interactions between processed polyketide intermediates and the ketosynthases that gatekeep for them. We have strong hypotheses for how sets of substrate tunnel residues interact with intermediates closest to the reactive thioester to ensure they are properly modified by upstream processing enzymes. Thus, we will appropriately mutate the gatekeeping residues of ketosynthases in less active model synthases as well as model synthases with inactivated upstream processing enzymes and determine whether their productivities improve as predicted. Since our data indicate that polyketide intermediates rigidify the ketosynthase dimer and dimeric ketosynthase+acyltransferase didomains can be readily identified in cryo-electron microscopy studies, we will also perform electron microscopy on stalled synthases to solve structures of polyketide-bound ketosynthase+acyltransferase dimers. In Specific Aim 3 we propose to determine key domain-domain interfaces. We have evidence that interfaces between processing enzymes and downstream KSs drive the ordered self- assembly of synthase polypeptides more than the small interface observed between the C- and N-terminal docking domains and seek structures of representative complexes. We also aim to determine how acyl carrier protein domains dock with ketosynthases during the transacylation reaction. If we are successful in these projects, it will greatly inform the rational engineering of modular polyketide synthases that synthesize new molecules and, ultimately, new medicines.

模块化聚酮合酶领域的复兴已经开始，新的工具和范例正在兴起。更深入地了解这些酶装配线的架构和活动，并正在促进我们长期目标是将模块化聚酮合酶的合成能力应用于开发和使用“模块”的更新定义，我们的实验室设计了多种三重药物。 /四-/五肽合酶在体内和体外均具有功能，而这些短组装线是有效的。它们在自然界中并不常见，是我们在具体目标 1 中建议的结构和功能研究的理想选择。在合成聚酮化合物产品时对这些装配线进行急速冷冻，并通过以下方式进行研究：由于这种方法使我们能够捕获高分辨率的动态信息。模型三酮化合物合酶的引发酮合酶和酰基转移酶的研究，我们将其应用于以下合酶：包含其他感兴趣的区域。一个目标是了解酮还原酶、脱水酶和烯酰还原酶加工酶是相对于彼此和邻近的酶定向的酮合酶+酰基转移酶双结构域，以了解酰基载体蛋白结构域如何在这些结构域之间移动因此，我们至少将研究聚酮化合物中间体的延伸和加工过程中的酶。我们的实验室对 13 种工程三酮/四酮化合物合酶和两种天然合酶进行了功能验证，在具体目标 2 中，我们建议阐明这些加工酶的不同组。在加工的聚酮化合物中间体和为其把关的酮合酶之间，我们拥有强大的优势。关于底物隧道残基组如何与最接近反应性硫酯的中间体相互作用的假设以确保它们被上游加工酶正确修饰，因此，我们将适当地突变它们。活性较低的模型合酶以及具有活性的模型合酶中酮合酶的守门残基灭活上游加工酶并确定其生产率是否如预期提高。由于我们的数据表明聚酮化合物中间体使酮合酶二聚体和二聚体刚性化酮合酶+酰基转移酶双结构域可以在冷冻电子显微镜研究中轻松识别，我们将还对停滞的合酶进行电子显微镜观察，以解析聚酮化合物结合的结构在具体目标 3 中，我们建议确定关键的域-域接口。我们有证据表明加工酶和下游 KS 之间的界面驱动有序的自合酶多肽的组装超过了 C 端和 N 端之间观察到的小界面我们还旨在确定酰基载体的对接结构域并寻找代表性复合物的结构。如果我们在这些方面取得成功，蛋白质结构域就会在转酰基反应过程中与酮合成酶对接。项目，它将极大地指导合成新的模块化聚酮合酶的合理工程分子，最终是新药物。