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Mechanisms and Reprogramming of Iron/2-Oxoglutarate Desaturases and Oxacyclases

铁/2-氧戊二酸去饱和酶和氧杂环酶的机制和重编程

基本信息

批准号：
9262989
负责人：
JOSEPH M BOLLINGER
金额：
$ 25.94万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-15 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9262989
关键词：
Active Sites Alcohols Amino Acid Substitution Anabolism Anesthetics Antibiotics Arginine Bacteria Binding Biochemical Pathway Carbon Cell Nucleus Cells Clinical Complex Coupling Crystallography Cyclization Dioxygenases Directed Molecular Evolution Disease Drug Compounding Drug Design Electron Spin Resonance Spectroscopy Engineering Enzymes Epigenetic Process Epoxy Compounds Escherichia coli Event Genetic Recombination Genetic Transcription Geometry Glutamates Glutarates Guanidines Halogens Human Hydrogen Hydrogen Bonding Hydroxylation In Vitro Ions Iron Kinetics Libraries Life Ligands Location Maps Metabolism Metals Methodology Methods Mixed Function Oxygenases Mutagenesis Natural Product Drug Outcome Oxygen Oxygenases Pathway interactions Pharmaceutical Preparations Positioning Attribute Process Production Reaction Regulation Residual state Role Scopolamine Site Soil Structure Testing Vanadyl Variant Work X-Ray Crystallography adduct alpha ketoglutarate catalyst cofactor combinatorial computer studies dehydrogenation desaturase experimental study extradiol dioxygenase fischerindole frontier halogenation hydroxyl group in vivo innovation insight iron nitrosyl microbial novel therapeutics plant fungi prevent screening tool vector

项目摘要

Human iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) dioxygenases hydroxylate unactivated aliphatic carbon centers in reactions that are fundamentally important to central life processes (e.g., metabolism and its regulation, transcription, epigenetic inheritance) and relevant to several diseases. Plant, fungi, and bacteria have diversified the Fe/2OG-oxygenase platform for a bewildering array of oxidative transformations that include halogenations, cyclizations, dehydrogenations and stereoinversions of aliphatic carbon centers. As the biosynthetic machinery generating a large number of important natural-product drugs are replete with such Fe/2OG oxygenases, the ability to reprogram them by either rational or directed-evolution approaches would enable microbial/enzy- matic production of novel drug compounds. In this project, we will use in vivo and in vitro selection methods to engineer outcome-altered variants of Fe/2OG oxygenases on pathways to antibiotic and anesthetic drugs. Our extensively validated kinetic and spectroscopic approaches to structural and functional analysis of these enzymes will be applied in combination with two new, innovative structural methods to rationalize the reprogramming in precise structural and mechanistic terms. The project will thus provide both enzymes for production of new potential drugs and mechanistic insight to enable more rational reprogramming of these and other Fe/2OG oxygenases.

人铁(II)和2-(氧代)戊二酸依赖(Fe/2OG)双加氧酶羟化反应中未活化的脂肪族碳中心对中心生命过程(如新陈代谢及其调控、转录、表观遗传遗传)，并与几种疾病有关。植物、真菌和细菌已经多样化一系列令人眼花缭乱的氧化转化的Fe/20G加氧酶平台包括卤化、环化、脱氢和脂肪族立体转化碳中心。由于生物合成机械产生了大量重要的天然产物药物充满了这样的Fe/2OG加氧酶，即重新编程的能力通过理性或定向进化的方法将使微生物/ENZY- 马季奇制作的新型药物化合物。在这个项目中，我们将使用体内和体内铁/2OG加氧酶突变体的体外筛选方法抗生素和麻醉剂药物的途径。我们经过广泛验证的动能和这些酶的结构和功能分析的光谱方法将是与两种新的、创新的结构方法相结合应用，使以精确的结构和机械术语重新编程。因此，该项目将提供既有用于生产新的潜在药物的酶，也有使对这些和其他Fe/2OG加氧酶进行更合理的重新编程。