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

• 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
• 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' -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

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.

项目摘要 微生物产生结构多样的分子，其中许多已被成功地重新利用， 药剂这些分子是由多酶组装而成的， 载体蛋白（ACP）修饰化学中间体并将其转移到一组酶合作伙伴。 天然酶组装的战略重新设计提出了一个令人兴奋的可能途径，以产生新的 抗生素，但任何重新设计方法的成功都取决于对导致 化学生产ACP-酶相互作用。这项研究的目的是获得一个分子水平的 了解ACP如何与其分子货物和酶合作伙伴相互作用。 在上一个资助期，我们的实验室开发了新的分光光度法，使我们能够 揭示ACP与其分子货物之间以及ACP之间的快速和瞬时相互作用 和两种酶的伴侣：酮合酶（KS）和脱氢酶（DH）。这些研究产生了7篇论文，其中40篇 哈佛大学本科生获得合著权。我们现在寻求利用这些主要的 了解ACP序列和分子货物身份之间复杂相互作用的进展， 引导被称为“链螯合”的现象，这被认为在引导中起着关键作用。 生物催化我们还将研究链螯合如何影响ACP-KS结合亲和力，并获得ACP-KS 交联结构用于随后的分子水平结构表征。 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 药物活性。这项工作将在独立的本科生研究的背景下执行 项目和基于课程的本科生研究经验（CURES），从而暴露约60 本科生在化学生物学接口的高级研究。