Probing and Engineering of Iterative Polyketide Synthase

迭代聚酮合成酶的探索与工程

• 批准号: 9897417
• 负责人: Shiou-Chuan Tsai
• 金额: $ 28.15万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-10 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9897417
• 关键词: Affect; Anabolism; Antibiotics; Antineoplastic Agents; Aromatase; Bacteria; Biomedical Engineering; Chemicals; Complex; Crystallization; Cyclization; Data; Disease Resistance; Doxorubicin; Drug resistance; Engineering; Enzymes; Event; Goals; Individual; Ketones; Knowledge; Lead; Length; Malonates; Modernization; Molecular; Multienzyme Complexes; Mutation; Natural Products; Organic Synthesis; Outcome; Pattern; Pharmacologic Substance; Proteins; Public Health; Resolution; Specificity; Structure; Tetracyclines; Time; Variant; Vision; analog; base; blockchain; carbonyl group; combat; design; enzyme activity; novel therapeutics; oxetane; polyketide synthase; protein protein interaction; structural biology

PROJECT SUMMARY The objective of this proposal is to probe the regio-specificity of iterative type II polyketide synthase (PKS) from bacteria, an enzyme complex comprised of 5 – 10 distinct domains that produce pharmaceutically important natural products. Polyketide diversity is achieved via a controlled variation of starter unit, chain length, and reduction/cyclization patterns. The focus of this proposal is to use chemically synthesized polyketide mimics to probe the regio-specificities of iterative PKS from bacteria (also called “type II PKS”). Specifically, we wish to probe for chain length specificity of the ketosynthase (KS), stereo- and regio-specificities of ketoreductase (KR), and cyclization specificity of aromatase/cyclase (ARO/CYC). Understanding and controlling the regio-specificity of KS, KR and ARO/CYC can potentially lead to new polyketide analogs with synthetic building blocks and new ketoreduction/cyclization patterns. However, past attempt to solve cocrystal structures and to understand the regio-specificity of PKS had been severely hampered by the chemical instability of the poly-beta-ketone substrates. Oxetane has been developed as an isosteric mimic of carbonyl groups. Here, for the first time, we propose to use oxetane as an isosteric substitute for the carbonyl groups of poly-beta-ketone substrates for PKS. We will pursue the following specific aims using a powerful combination of modern organic synthesis and structural biology: AIM 1. Design and synthesis of oxetane-containing mimics of PKS intermediates (Vanderwal), AIM 2. Determine Key Substrate-Protein Interactions in Priming and Elongating Ketosynthase (Tsai), AIM 3. Determine Key Substrate-Protein Interactions in Ketoreductase (KR) and Aromatase/Cyclase (ARO/CYC) Using the Oxetane Probes (Tsai), and AIM 4. Determine Key Protein-Protein Interactions on the Timing of Chain Elongation, Ketoreduction and Cyclization (Tsai). The outcomes will have high scientific impact, because it can potentially change people’s vision about using chemical probes to approach PKS mechanism (Aim 1), elucidate PKS regio-specificities (Aims 2-3), and provide the first type II PKS complex structure that elucidates how protein-protein interactions affect product specificity (Aim 4). It therefore also has potential high overall biomedical impact, because outcomes can be widely applied to PKS bioengineering, leading to new polyketides with different chain length, reduction and cyclization patterns that can subsequently be screened for new therapeutics and bioactivities.

项目摘要 这个建议的目的是探讨区域特异性的迭代类型II 来自细菌的聚酮合酶（PKS），一种由5 - 10个不同的聚酮合酶组成的酶复合物， 生产重要的药物天然产物的领域。聚酮化合物多样性是 通过控制起始单元、链长和还原/环化来实现 模式.该提案的重点是使用化学合成的聚酮化合物模拟物， 探测来自细菌的迭代PKS（也称为"II型PKS"）的区域特异性。 具体地，我们希望探测酮合酶（KS）的链长特异性，立体和立体异构。 酮还原酶（KR）的区域特异性和芳香化酶/环化酶的环化特异性 (ARO/CYC）。了解和控制KS、KR和ARO/CYC的区域特异性， 潜在地导致具有合成结构单元的新的聚酮类似物和新的 酮还原/环化模式。然而，过去试图解决共晶结构和 我知道PKS的区域特异性受到化学不稳定性的严重阻碍 聚β-酮底物的量。氧杂环丁烷已被开发为 羰基。在这里，我们首次提出使用氧杂环丁烷作为 PKS的聚β-酮底物的羰基。我们将继续努力 使用现代有机合成和结构生物学的强大组合的特定目标： AIM 1. PKS中间体的含氧杂环丁烷模拟物的设计和合成（Vanderwal）， AIM 2.确定引发和延长酮合酶中的关键底物-蛋白质相互作用 （Tsai），AIM 3.确定酮还原酶（KR）中的关键底物-蛋白质相互作用， 使用氧杂环丁烷探针（Tsai）的芳香化酶/环化酶（ARO/CYC）和AIM 4。确定关键 蛋白质-蛋白质相互作用对链延长、酮基还原和环化时间的影响 （蔡）。这些结果将具有很高的科学影响力，因为它可能会改变 利用化学探针探讨PKS机理的设想（目的1），阐明 PKS区域特异性（目的2 - 3），并提供第一种II型PKS复合物结构， 阐明了蛋白质-蛋白质相互作用如何影响产物特异性（目的4）。因此， 具有潜在的高整体生物医学影响，因为结果可以广泛应用于 PKS生物工程，导致具有不同链长、还原和 环化模式，随后可以筛选新的治疗和生物活性。