Elucidate Mechanisms of Quinolone Alkaloid Biosynthesis via Iron(II)/2-Oxoglutarate Dependent Enzymes: Diverse, but Controlled Reactivity

通过铁 (II)/2-氧戊二酸依赖性酶阐明喹诺酮生物碱生物合成的机制：多样但受控的反应性

• 批准号: 10458319
• 负责人: Yisong Guo
• 金额: $ 11.17万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458319
• 关键词: Alkaloids; Anabolism; Anti-Bacterial Agents; Antiviral Agents; Area; Aspergillus nidulans; Biochemical; Biological; Biological Assay; Biomedical Engineering; Catalysis; Cations; Chemicals; Comparative Study; Crystallography; Cytochrome P450; Deuterium; Development; Enzymes; Epigenetic Process; Exhibits; Family; Gene Expression Regulation; Goals; HIV; Hydrogen; Hydroxylation; Intercept; Iron; Kinetics; Knowledge; Label; Lead; Libraries; Logic; Malignant Neoplasms; Metabolism; Methods; Molecular; Molecular Cloning; Mononuclear; Natural Products; Nature; Organic Synthesis; Outcome; Oxidants; Oxygen; Pathway interactions; Pharmacologic Substance; Play; Preparation; Proteins; Quinolones; Reaction; Regulation; Research; Role; Scheme; Site; Solvents; Structure; Structure-Activity Relationship; Techniques; Time; Variant; Viral Cancer; X-Ray Crystallography; alpha ketoglutarate; analog; anti-cancer; anticancer activity; antimicrobial; base; design; drug development; drug discovery; electronic structure; functional group; improved; insight; interest; member; non-Native; novel; scaffold; simulation

Project Summary/Abstract 2-Oxoglutarate (2OG) dependent nonheme mononuclear iron (NHM-Fe) enzymes catalyze an exceedingly broad scope of reactions that are involved in key chemical transformations of many important biological pathways, such as gene regulation, epigenetics, and natural product biosynthesis. Although detailed mechanistic understandings of the canonical hydroxylation reactivity found in 2OG/NHM-Fe enzymes have been developed in recent years, it remains unknown how this hydroxylation paradigm can fully explain non- hydroxylation reactivity in this family of enzymes, such as desaturation and epoxidation. Furthermore, given the catalytic abilities of 2OG/NHM-Fe enzymes to construct pharmaceutically valuable molecular scaffolds, exploiting these enzymes for biocatalysis applications represents an attractive but under developed area for expanding natural product based compound libraries. In this proposal, we seek to provide critical improvements on these under developed areas through the studies of AsqJ, a novel multifunctional 2OG/NHM- Fe enzyme that is involved in Viridicatin-type quinolone alkaloid biosynthesis in Aspergillus nidulans. AsqJ catalyzes a chemically interesting sequential desaturation/epoxidation reaction to construct Viridicatin core structure, which represents a chemically unexplored strategy for Viridicatin synthesis. A multi-faceted experimental method will be utilized to elucidate AsqJ reaction mechanisms, which consists of organic synthesis, molecular cloning, biochemical assays, protein crystallography, pre-steady state kinetics, and advanced spectroscopic techniques. This method will be further supplemented with molecular dynamic simulations to generate molecular level understandings of the AsqJ catalysis. It is expected that the proposed research will provide critical improvements to the mechanistic understandings of desaturation and epoxidation, two chemically challenging but under explored reactions catalyzed by 2OG/NHM-Fe enzymes, and further explore mechanism based bioengineering approach to access viridicatin-type scaffolds.

项目概要/摘要 2-氧戊二酸 (2OG) 依赖性非血红素单核铁 (NHM-Fe) 酶催化 涉及许多重要生物的关键化学转化的广泛反应 途径，例如基因调控、表观遗传学和天然产物生物合成。虽然详细 对 2OG/NHM-Fe 酶中典型羟基化反应性的机制理解 近年来发展起来，目前尚不清楚这种羟基化范式如何充分解释非 该酶家族中的羟基化反应，例如去饱和和环氧化。此外，鉴于 2OG/NHM-Fe 酶构建具有药学价值的分子支架的催化能力， 利用这些酶进行生物催化应用是一个有吸引力但尚未开发的领域 扩大基于天然产物的化合物库。在本提案中，我们寻求提供关键的 通过 AsqJ（一种新型多功能 2OG/NHM-）的研究，对这些欠发达地区进行了改进 Fe酶参与构巢曲霉中Viridicatin型喹诺酮生物碱的生物合成。阿斯吉 催化化学上有趣的连续去饱和/环氧化反应以构建 Viridicatin 核心 结构，它代表了化学上尚未探索的 Viridicatin 合成策略。一个多面的 将利用实验方法来阐明 AsqJ 反应机理，该反应机理包括有机 合成、分子克隆、生化测定、蛋白质晶体学、前稳态动力学和 先进的光谱技术。该方法将进一步补充分子动力学 模拟以产生对 AsqJ 催化的分子水平理解。预计拟议的 研究将为对去饱和和环氧化的机理理解提供重要的改进， 2OG/NHM-Fe 酶催化的两个化学上具有挑战性但尚未探索的反应，以及进一步 探索基于机制的生物工程方法来获取绿色素型支架。