Mechanistic Determination of the Non-heme Iron Enzyme Hyoscyamine-6-hydroxylase

非血红素铁酶天仙胺-6-羟化酶的机理测定

• 批准号: 9014423
• 负责人: Christopher Pollock
• 金额: $ 5.43万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9014423
• 关键词: Active Sites; Alcohols; Alkaloids; Biological Assay; Biology; Cell physiology; Cells; Chemicals; Chemistry; Complex; Cyclization; DNA; DNA biosynthesis; Deoxyribonucleotides; Deuterium; Development; Drug Design; Enzymes; Epoxy Compounds; Escherichia coli; Family; Foundations; Freezing; Genes; Genetic Transcription; Goals; Health; Heme Iron; Histidine; Histones; Human; Hydrogen Bonding; Hydroxylation; Investigation; Iron; Isotopes; Kinetics; Knowledge; Location; Measurement; Mediating; Metabolism; Mixed Function Oxygenases; Molecular; Motion Sickness; Natural Products; Oxygenases; Parkinson Disease; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Plants; Process; Production; Proteins; Reaction; Reproduction; Ribonucleotides; Role; Scopolamine; Site; Spectrum Analysis; Structure; Tropanes; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; alpha ketoglutarate; base; carboxylate; chemical reaction; chlorite; cofactor; dehydrogenation; demethylation; design; enzyme mechanism; epimerization; halogenation; insight; interest; member; microbial; novel; overexpression; oxidation; pathogen; preference; public health relevance; repaired; research study; small molecule; spectroscopic survey

 DESCRIPTION (provided by applicant): Enzymes that employ non-heme-iron cofactors are involved in many important cellular processes, including transcription, reproduction, and the production of small molecule metabolites. One class of these enzymes, the iron- and 2-oxoglutarate (Fe/2OG) oxygenases, are notable in that some members can catalyze multiple different reactions using the same active site. Several of these reactivities (e.g. cyclizations, desaturations) cannot be understood by adapting the known mechanisms of these enzymes, motivating a deeper understanding of the chemistry involved. The enzyme hyoscyamine-6- hydroxylase (H6H) is a member of the Fe/2OG family and is responsible for the conversion of hyoscyamine to scopolamine, a tropane alkaloid pharmaceutical used in the treatment of conditions such as motion sickness and Parkinson's disease. This enzyme first hydroxylates hyoscyamine and then performs a subsequent dehydrogenation on the nascent alcohol to generate an epoxide ring. This second step is quite unusual in biology and its mechanism is not understood. In the proposed project, structures and mechanisms H6H will be determined with the goal of elucidating how this enzyme conducts these divergent reactions. X-ray crystallography will provide structural insights into how these reaction are mediated, while stopped-flow kinetics measurements will determine-in molecular detail-the precise mechanisms of both transformations. Furthermore, spectroscopic studies on the reaction intermediates will allow for definitive characterization of the chemical species involved in these reactions. Taken together, this work will help to rationalize the factors that govern the divergent reactivity of thse enzymes and will serve as a basis for the design of new medicinal compounds that target these enzymes.

 描述（由适用提供）：员工非血红素辅助因子参与许多重要的细胞过程，包括转录，繁殖和小分子代谢产物的产生。这些酶的一类，即铁和2-氧化甲酸（Fe/2og）氧酶，值得注意的是，有些成员可以使用相同的活性位点催化多个不同的反应。这些反应率中的几种（例如，环化，去饱和）无法通过调整这些酶的已知机制来理解，从而激发了对所涉及的化学反应的更深入的理解。酶羟胺-6-羟化酶（H6H）是Fe/2og家族的成员，负责将羟胺转化为Scopolamine，这是一种用于治疗诸如运动病和帕金森氏病等疾病的型心生物碱药物。该酶首先首先羟基羟基羟胺，然后对新生酒精进行随后的脱氢作用，以产生环氧环。第二步在生物学上是非常不寻常的，而其机制尚不清楚。在拟议的项目中，H6H的结构和机制将以阐明该酶进行这些不同反应的目的确定。 X射线晶体学将提供有关这些反应如何介导的结构见解，而停止流动动力学测量将确定分子细节 - 两种转化的精确机制。此外，对反应中间体的光谱研究将允许对这些反应涉及的化学物种进行明确的表征。综上所述，这项工作将有助于合理化控制这种酶不同反应性的因素，并将作为设计针对这些酶的新药用化合物的基础。