Functional Hybrid Natural Product Synthases by Tracking Acyl Carrier Protein Binding and Conformational Dynamics

通过跟踪酰基载体蛋白结合和构象动力学进行功能性杂化天然产物合成

基本信息

批准号：
10045624
负责人：
Louise Karine Charkoudian
金额：
$ 29.8万
依托单位：
HAVERFORD COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10045624
关键词：
4&apos -phosphopantetheine Actinobacteria class Acyl Carrier Protein Affect Affinity Amino Acid Sequence Amino Acids Anabolism Antibiotics Authorship Behavior Binding Binding Proteins Biological Assay Biology Calorimetry Chemicals Chemistry Collaborations Complex Coupled Coupling Crystallography Engineering Environment Enzyme Interaction Enzymes Escherichia coli Event Fatty Acids Foundations Funding Future Genes Goals Hybrids Hydro-Lyases Lead Length Methodology Methods Modeling Molecular Molecular Conformation Motion Natural Products Outcome Paper Pathway interactions Pharmacologic Substance Play Protein Conformation Proteins Publications Publishing Rattus Research Research Personnel Research Project Grants Role Route Site Specificity Structure Study Subject Training United States National Institutes of Health Work arm base college crosslink design experience experimental study holo-(acyl-carrier-protein) synthase innovation insight microorganism mutant next generation novel polyketide synthase protein protein interaction protein structure function sedimentation velocity small molecule success temporal measurement tool undergraduate research undergraduate student vibration

项目摘要

PROJECT SUMMARY Microorganisms produce structurally diverse molecules, many of which have been successfully repurposed as pharmaceutical agents. These molecules are manufactured by multi-enzyme assemblies, which rely on acyl carrier proteins (ACPs) to modify and transfer chemical intermediates to a team of enzymatic partners. Strategic redesign of natural enzyme assemblies presents an exciting possible route to produce new antibiotics, but the success of any redesign approach hinges on a thorough understanding of what leads to chemically productive ACP-enzyme interactions. The goal of this study is to gain a molecular-level understanding of how ACPs interact with their molecular cargo and enzymatic partners. In the previous funding period, our lab developed new spectrophotometric methodologies that enabled us to unveil the fast and transient interactions between ACPs and their molecular cargo, as well as between ACPs and two enzymatic partners: a ketosynthase (KS) and dehydratase (DH). These studies led to 7 papers with 40 Haverford College undergraduate students earning co-authorship. We now seek to leverage these major advancements to understand the complex interplay between ACP sequence and molecular cargo identity in directing the phenomenon called “chain sequestration,” which is thought to play a critical role in directing biocatalysis. We will also study how chain sequestration effects ACP-KS binding affinity and obtain ACP-KS crosslinked structures for subsequent molecular-level structural characterization. Results from these studies will guide the future biosynthesis of novel small molecules with potential pharmaceutical activity. The work will be executed in the context of independent undergraduate research projects and course-based undergraduate research experiences (CUREs), thereby exposing ~60 undergraduate students to advanced research at the chemistry-biology interface.

项目概要微生物产生结构多样的分子，其中许多已成功地重新利用为这些分子是由多酶组装体制造的，依赖于酰基。载体蛋白（ACP）可修饰化学中间体并将其转移至一组酶促伙伴。天然酶组装体的战略性重新设计为生产新的酶提供了一条令人兴奋的可能途径抗生素，但任何重新设计方法的成功都取决于对导致抗生素的原因的透彻理解本研究的目标是获得分子水平的化学生产力 ACP-酶相互作用。了解 ACP 如何与其分子货物和酶促伙伴相互作用。在上一个资助期间，我们的实验室开发了新的分光光度方法，使我们能够揭示 ACP 与其分子货物之间以及 ACP 之间快速且短暂的相互作用以及两种酶伙伴：酮合酶 (KS) 和脱水酶 (DH) 这些研究发表了 7 篇论文，共 40 篇。哈弗福德学院的本科生获得合着权，我们现在寻求利用这些专业。理解 ACP 序列和分子货物身份之间复杂相互作用的进展引导一种称为“链隔离”的现象，人们认为这种现象在引导我们还将研究链隔离如何影响 ACP-KS 结合亲和力并获得 ACP-KS。用于后续分子水平结构表征的交联结构。这些研究的结果将指导未来具有潜力的新型小分子的生物合成这项工作将在独立的本科生研究的背景下进行。项目和基于课程的本科生研究经验 (CURE)，从而暴露 ~60 本科生在化学-生物学界面进行高级研究。