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

项目总结 微生物产生结构不同的分子，其中许多分子已被成功地重新用于 药剂。这些分子是由依赖于酰基的多酶组装而成的 载体蛋白(ACP)，用于修饰化学中间体并将其传递给一组酶伙伴。 天然酶组件的战略重新设计提供了一条令人兴奋的可能路线来生产新的 抗生素，但任何重新设计方法的成功都取决于对导致 化学产生的ACP-酶相互作用。这项研究的目标是在分子水平上 了解ACP如何与其分子货物和酶伙伴相互作用。 在之前的资助期间，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之间的快速和瞬时相互作用 和两个酶对：酮合成酶(KS)和脱水酶(DH)。这些研究产生了7篇论文，其中40篇 哈弗福德学院本科生获得合著者资格。我们现在寻求利用这些主要的 ACP序列与分子货物同一性之间复杂相互作用的研究进展 指导一种叫做“链隔离”的现象，这种现象被认为在指导 生物催化。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 用于后续分子级结构表征的交联型结构。 这些研究结果将指导未来具有潜力的新型小分子的生物合成。 药学活动。这项工作将在独立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)，从而暴露出~60 让本科生在化学-生物接口上进行深入的研究。

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.