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.

项目摘要/摘要 依赖于2-羟基戊二酸(2OG)的非血红素单核铁(NHM-Fe)酶过度催化 广泛的反应范围，涉及许多重要生物的关键化学转化 基因调控、表观遗传学和天然产物生物合成等途径。虽然详细 对20G/NHM-Fe酶的典型羟化反应的机理理解有 近年来已经发展起来，但尚不清楚这种羟化范式如何完全解释非 这类酶的羟化反应活性，如脱饱和度和环氧化。此外，鉴于 2OG/NHM-Fe酶构建有药用价值的分子支架的催化能力 开发这些酶用于生物催化应用是一个有吸引力但不发达的领域 扩大基于天然产物的化合物文库。在这项提案中，我们寻求提供关键的 通过对新型多功能2OG/NHM--ASQJ的研究，改善了这些欠发达地区的状况。 参与维达菌素型喹诺酮类生物碱生物合成的铁酶。AsqJ 催化化学上有趣的一系列去饱和/环氧化反应以构建维拉丁核心 结构，这代表了一种化学上未曾探索过的合成维他丁的策略。一个多方面的 将利用实验方法阐明AsqJ反应机理，其中包括有机 合成，分子克隆，生化分析，蛋白质结晶学，稳态前动力学，以及 先进的光谱技术。这种方法将进一步得到分子动力学的补充 模拟以产生对AsqJ催化的分子水平的理解。预计拟议的 研究将对减饱和和环氧化的机理理解提供关键的改进， 由20G/NHM-Fe酶催化的两个具有化学挑战性但未被探索的反应，以及进一步 探索基于机理的生物工程方法来获得波形抑制素型支架。