Probing and Engineering of Iterative Polyketide Synthase

项目概要 该提案的目的是探讨迭代类型 II 的区域特异性 来自细菌的聚酮合酶 (PKS)，这是一种由 5 – 10 个不同的酶组成的酶复合物 生产药学上重要的天然产物的领域。聚酮化合物多样性为 通过起始单元、链长度和还原/环化的受控变化来实现 模式。该提案的重点是使用化学合成的聚酮化合物模拟物 探测细菌迭代 PKS 的区域特异性（也称为“II 型 PKS”）。 具体来说，我们希望探究酮合酶（KS）的链长特异性、立体和 酮还原酶 (KR) 的区域特异性和芳香酶/环化酶的环化特异性 （ARO/CYC）。了解和控制 KS、KR 和 ARO/CYC 的区域特异性可以 可能会产生具有合成结构单元的新聚酮化合物类似物和新的 酮还原/环化模式。然而，过去解决共晶结构和 了解 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。氧杂环丁烷已被开发为等排模拟物 羰基。在这里，我们首次建议使用氧杂环丁烷作为等排替代品 PKS 聚-β-酮底物的羰基。我们将追求以下目标 利用现代有机合成和结构生物学的强大组合实现特定目标： 目的 1. 设计和合成含氧杂环丁烷的 PKS 中间体模拟物 (Vanderwal)， 目标 2. 确定启动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-蛋白质相互作用和 使用氧杂环丁烷探针 (Tsai) 和 AIM 的芳香酶/环化酶 (ARO/CYC) 4. 确定关键点 蛋白质-蛋白质相互作用对链延长、酮还原和环化时间的影响 （蔡）。结果将产生巨大的科学影响，因为它可能会改变 人们对使用化学探针来研究 PKS 机制（目标 1）的愿景，阐明 PKS 区域特异性（目标 2-3），并提供第一个 II 型 PKS 复合结构， 阐明蛋白质-蛋白质相互作用如何影响产品特异性（目标 4）。因此它也 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、还原和还原的新型聚酮化合物 随后可以筛选新的治疗方法和生物活性的环化模式。

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.