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：
9084003
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/9084003
关键词：
Active Sites Alcohols Amino Acid Substitution Anabolism Anesthetics Antibiotics Arginine Bacteria Binding Biochemical Pathway Carbon Cell Nucleus Cells 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 Geometry Glutamates Glutarates Guanidines Halogens Hemorrhage 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 Residual state Role Scopolamine Site Soil Structure Testing Transcriptional Regulation Vanadyl Variant Work abstracting adduct alpha ketoglutarate base catalyst cofactor combinatorial computer studies dehydrogenation desaturase extradiol dioxygenase fischerindole frontier halogenation hydroxyl group in vivo innovation insight iron nitrosyl microbial novel therapeutics plant fungi prevent research study 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-（Oxo）谷氨酸依赖性（Fe/2og）二加氧酶羟基酯未活化的脂肪族碳中心在反应中对中央生活过程（例如，新陈代谢及其调节，转录，表观遗传学继承），与多种疾病有关。植物，真菌和细菌已经多样化 Fe/2og-氧化酶平台，用于迷惑的氧化转化阵列包括卤素，环化，脱氢和脂肪族立体化作用碳中心。随着生物合成机械产生大量重要的天然产品药物充满了这种Fe/2og氧酶，可以重新编程的能力通过合理或定向进化的方法，它们可以使微生物/递归方法新型药物化合物的生产。在这个项目中，我们将使用体内和用于设计Fe/2og氧化酶的改变结果变体的体外选择方法抗生素和麻醉药物的途径。我们经过广泛验证的动力学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结构方法结合使用，以合理化用精确的结构和机械术语重新编程。因此，该项目将提供两种用于生产新的潜在药物和机械见解的酶都可以实现这些和其他Fe/2og氧酶的更多合理重编程。