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: 10581893
• 负责人: Louise Karine Charkoudian
• 金额: $ 3.31万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10581893
• 关键词: Acyl Carrier Protein; Affinity; Amino Acid Sequence; Anabolism; Antibiotics; Authorship; Binding; Binding Proteins; Biology; Chemicals; Chemistry; Complex; Enzyme Interaction; Enzymes; Funding; Future; Goals; Hybrids; Hydro-Lyases; Methodology; Molecular; Natural Products; Paper; Pharmacologic Substance; Play; Protein Conformation; Research; Research Project Grants; Role; Route; Structure; Work; base; college; crosslink; design; innovation; microorganism; novel; protein structure function; small molecule; success; tool; undergraduate research; undergraduate research experience; undergraduate student

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 微生物产生结构上不同的分子，其中许多已成功地重新定义为 药物。这些分子是由依赖酰基的多酶组件制造的 载体蛋白（ACP）将化学中间体修改并将其转移到酶促伙伴团队中。 自然酶组件的战略重新设计，提出了一条令人兴奋的可能产生新的途径 抗生素，但是任何重新设计方法的成功都取决于对导致的原因的透彻理解 化学生产性的ACP-酶相互作用。这项研究的目的是获得分子级 了解ACP与它们的分子货物和酶促伴侣的相互作用。 在上一个资金期间，我们的实验室开发了新的分光光度法方法，使我们能够 揭示ACP与它们的分子货物之间以及ACP之间的快速和短暂相互作用 和两个酶促伴侣：酮合酶（KS）和脱水酶（DH）。这些研究导致7篇论文，有40篇 Haverford College本科生获得了共同创作。我们现在寻求利用这些专业 了解ACP序列与分子货物身份之间的复杂相互作用的进步 指导称为“链隔离”的现象，该现象被认为在指导中起着至关重要的作用 生物催化。我们还将研究链会话如何影响ACP-KS的结合亲和力并获得ACP-KS 交联结构，用于随后的分子级结构表征。 这些研究的结果将指导具有潜力的新型小分子的未来生物合成 药物活动。这项工作将在独立本科研究的背景下执行 项目和基于课程的本科研究经验（治疗），从而暴露于大约60 本科生在化学生物学界面上进行了高级研究。

项目成果

Tracking carrier protein motions with Raman spectroscopy

• DOI: 10.1038/s41467-019-10184-2
• 发表时间: 2019-05-20
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Epstein, Samuel C.;Huff, Adam R.;Charkoudian, Louise K.
• 通讯作者: Charkoudian, Louise K.

Heterologous Expression, Purification, and Characterization of Type II Polyketide Synthase Acyl Carrier Proteins.

• DOI: 10.1007/978-1-0716-2273-5_13
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)

The Cytochrome P450 OxyA from the Kistamicin Biosynthesis Cyclization Cascade is Highly Sensitive to Oxidative Damage.

• DOI: 10.3389/fchem.2022.868240
• 发表时间: 2022
• 期刊: FRONTIERS IN CHEMISTRY
• 影响因子: 5.5
• 作者: Greule, Anja;Izore, Thierry;Machell, Daniel;Hansen, Mathias H.;Schoppet, Melanie;De Voss, James J.;Charkoudian, Louise K.;Schittenhelm, Ralf B.;Harmer, Jeffrey R.;Cryle, Max J.
• 通讯作者: Cryle, Max J.