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: 10197596
• 负责人: Yisong Guo
• 金额: $ 8.37万
• 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/10197596
• 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.

项目摘要/摘要 2-氧基谷氨酸（2OG）依赖性非血红素单核铁（NHM-FE）酶极大地催化 许多重要生物学的关键化学转化涉及的广泛反应范围 途径，例如基因调节，表观遗传学和天然产物生物合成。虽然详细 在2OG/NHM-FE酶中发现的规范羟基反应性的机械理解已有 是近年来开发的，尚不清楚这种羟基化范式如何完全解释非 - 该酶家族中的羟基化反应性，例如去饱和和环氧化。此外，给定 2OG/NHM-FE酶的催化能力构建药品有价值的分子支架， 利用这些酶用于生物催化应用是一个有吸引力但在发达的领域的 扩展基于天然产品的复合库。在此提案中，我们试图提供关键 通过研究ASQJ的研究，对这些发展领域的改进，这是一种新型的多功能2og/nhm-- 与曲霉曲霉中的病毒蛋白型喹啉生物合成有关的Fe酶。 asqj 催化化学有趣的顺序去饱和/环氧反应，以构建病毒蛋白核心 结构，代表了毒素合成的化学未开发的策略。多方面 实验方法将用于阐明ASQJ反应机制，该机制由有机物组成 合成，分子克隆，生化测定，蛋白质晶体学，稳态的态动力学和 高级光谱技术。该方法将进一步补充分子动力学 模拟产生对ASQJ催化的分子水平理解。预计提议 研究将为脱饱和和环氧化的机械理解提供重要的改进， 两种化学具有挑战性但在探索的反应下被2OG/NHM-FE酶催化，进一步 探索基于机制的生物工程方法，以获取毒素型支架。