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.

项目总结