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

 描述（由申请人提供）：采用非血红素铁辅因子的酶参与许多重要的细胞过程，包括转录、繁殖和小分子代谢物的产生。这些酶中的一类，铁和2-酮戊二酸（Fe/2 OG）加氧酶，是值得注意的，因为一些成员可以使用相同的活性位点催化多个不同的反应。这些反应性（例如环化，去饱和）中的一些不能通过调整这些酶的已知机制来理解，从而激发对所涉及的化学的更深入理解。莨菪碱-6 β-羟化酶（H6 H）是Fe/2 OG家族的成员，负责将莨菪碱转化为东莨菪碱，东莨菪碱是一种用于治疗运动病和帕金森病等病症的托烷生物碱药物。这种酶首先使莨菪碱羟基化，然后在新生醇上进行后续脱氢以产生环氧环。这第二步在生物学中是非常不寻常的，其机制尚不清楚。在拟议的项目中，将确定H6 H的结构和机制，目的是阐明这种酶如何进行这些不同的反应。X射线晶体学将提供这些反应是如何介导的结构的见解，而停流动力学测量将确定在分子的细节，这两个转换的精确机制。此外，对反应中间体的光谱研究将允许对这些反应中涉及的化学物质进行明确的表征。总之，这项工作将有助于合理化的因素，管理这些酶的不同的反应性，并将作为一个基础，设计新的药用化合物，针对这些酶。

 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.