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: 9753300
• 负责人: Yisong Guo
• 金额: $ 31.05万
• 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/9753300
• 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.

项目总结/摘要 2-酮戊二酸（2 OG）依赖性非血红素单核铁（NHM-Fe）酶催化一种非常强的 广泛的反应，涉及许多重要的生物化学的关键化学转化， 途径，如基因调控，表观遗传学和天然产物生物合成。尽管详细 在2 OG/NHM-Fe酶中发现的典型羟基化反应性的机械理解， 近年来发展起来的，仍然不知道这种羟基化范式如何能够完全解释非- 在该酶家族中的羟基化反应性，如去饱和和环氧化。此外，鉴于 2 OG/NHM-Fe酶构建具有药用价值的分子支架的催化能力， 将这些酶用于生物催化应用代表了一个有吸引力但尚未开发的领域， 扩展基于天然产物的化合物库。在这份提案中，我们寻求提供关键的 通过对新型多功能2 OG/NHM-AsqJ的研究，对这些未开发领域进行了改进， 铁酶，参与构巢曲霉中的维生素C型喹诺酮生物碱生物合成。AsqJ 催化一个化学上令人感兴趣的连续去饱和/环氧化反应，以构建维生素E核心 结构，这代表了一个化学上未探索的策略，用于维生素合成。多方面 实验方法将被用来阐明AsqJ反应机理，其中包括有机 合成、分子克隆、生物化学测定、蛋白质晶体学、预稳态动力学，以及 先进的光谱技术该方法将进一步补充分子动力学 模拟产生AsqJ催化的分子水平的理解。预计拟议的 研究将为去饱和和环氧化的机理理解提供关键的改进， 两个化学上具有挑战性但尚待探索的由2 OG/NHM-Fe酶催化的反应， 探索基于机制的生物工程方法以获得维生素A型支架。