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 该提案的目的是探测迭代II型的区域特异性 来自细菌的聚酮化合物合酶（PK），这是5 - 10个不同的酶复合物 产生物理上重要天然产品的领域。聚客体多样性是 通过启动单元，链长和还原/环化的受控变化实现 模式。该提案的重点是使用化学合成的聚酮化合物模仿 探测细菌迭代PK的区域特异性（也称为“ II型PK”）。 具体而言，我们希望探究酮合酶（KS），立体声和立体声和的链长度特异性 酮核酶（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的区域特异性，KR和ARO/CYC可以 有可能导致新的聚酮化合物类似物，具有合成的构建块和新的类似物 酮类化合物/环化模式。但是，过去试图解决共晶结构和 了解PK的区域特异性已被化学不稳定性严重阻碍 聚酮基酮底物的。驱烷已被开发为同义模仿 羰基。在这里，我们首次提议将氧烷用作同义替代品 PKS的聚β-酮基质的羰基。我们将追求以下 使用现代有机合成和结构生物学的有力组合的特定目的： 目标1。含氧烷的模拟物​​的设计和合成PKS中间体（Vanderwal）， AIM 2。确定启动和拉长酮合酶中的关键底物 - 蛋​​白质相互作用 （TSAI），AIM 3。确定酮核酶（KR）和 使用Oxetane探针（TSAI），AIM 4。确定钥匙 蛋白质 - 蛋白质相互作用在链伸长，开蛋白和环化的时机上的相互作用 （Tsai）。结果将产生很大的科学影响，因为它可能会改变 人们对使用化学问题接近PK机制的愿景（AIM 1），阐明 PKS区域特异性（目标2-3），并提供了第一个II型PKS复数结构 阐明蛋白质 - 蛋白质相互作用如何影响产品特异性（AIM 4）。因此也是如此 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，还原和的新聚酮化合物 随后可以筛选新的疗法和生物活性的环化模式。