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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）戊二酸依赖性（Fe/2 OG）双加氧酶羟基化在反应中未活化的脂肪族碳中心，这对中心生命过程（例如，代谢及其调控，转录，表观遗传遗传），并与多种疾病有关。植物、真菌和细菌已经多样化 Fe/2 OG-加氧酶平台用于一系列令人困惑的氧化转化，包括脂肪族化合物卤化、环化、双取代和立体转化碳中心作为生物合成机器，天然产物药物充满了这样的Fe/2 OG加氧酶，重新编程的能力，通过理性或定向进化的方法，它们将使微生物/酶新型药物化合物的自动生产。在这个项目中，我们将使用在体内和在体外选择方法来工程化Fe/2 OG加氧酶的结果改变的变体，抗生素和麻醉药的途径。我们经过广泛验证的动力学和光谱方法的结构和功能分析这些酶将是结合两种新的、创新的结构方法，在精确的结构和机制方面重新编程。该项目将提供既有用于生产新的潜在药物的酶，这些和其他Fe/2 OG加氧酶的更合理的重编程。