Probing and Engineering of Iterative Polyketide Synthase

项目总结 这项建议的目的是探讨迭代类型II的区域特异性。 来自细菌的聚酮合成酶(PKS)，是由5-10个不同的 生产具有重要药用价值的天然产品的领域。聚酮多样性是 通过控制起始单元、链长和还原/环化的变化来实现 模式。这项提议的重点是使用化学合成的聚酮模拟物来 探索细菌重复PKS(也称为“II型PKS”)的地区特异性。 具体地说，我们希望探索酮合成酶(KS)、立体声和 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 潜在地导致新的聚酮类似物与合成的构建块和新的 酮还原/环化模式。然而，过去试图解决共晶结构和 了解PKS的区域特异性已受到化学不稳定性的严重阻碍 聚β-酮底物。氧杂环己烷已被开发为一种等量构型的模拟物 羰基。在这里，我们第一次提出用氧杂环己烷作为异构体的替代品 用于PKS的聚β-酮底物的羰基。我们将致力于以下工作 使用现代有机合成和结构生物学的强大组合来实现特定的目标： 目的1.含氧杂环己烷的PKS中间体(Vanderwal)的设计与合成 目的2.确定酮合成酶启动和延长过程中的关键底物-蛋白质相互作用 (Tsai)，目的3.确定酮还原酶(KR)中关键底物与蛋白质的相互作用 使用恶烷探针(TSAI)和AIM的芳香酶/环化酶(ARO/CyC)，4.确定密钥 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 (蔡英文)。研究结果将产生很高的科学影响，因为它可能会发生变化 人们对使用化学探针探讨PKS机制的看法(目标1)，阐明 PKS地区性(目标2-3)，并提供第一种类型II PKS复合体结构， 阐明蛋白质-蛋白质相互作用如何影响产品专一性(目标4)。因此，它还 具有潜在的高整体生物医学影响，因为成果可以广泛应用于 PKS生物工程，导致不同链长、还原和还原的新聚酮 随后可以筛选出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.