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

 描述(申请人提供)：使用非血红素-铁辅助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。其中一类酶，铁和2-氧戊二酸(Fe/2OG)加氧酶，值得注意的是，一些成员可以催化多个不同的反应使用相同的活性部位。其中一些反应(如环化、脱饱和度)不能通过调整这些酶的已知机制来理解，从而激发了对所涉及化学的更深层次的理解。山梨碱-6羟基酶(H6H)是Fe/2OG家族的成员之一，负责将山梨碱转化为东莨菪碱，东莨菪碱是一种用于治疗运动病和帕金森病等疾病的托烷生物碱药物。这种酶首先使山梨糖胺羟化，然后对初生的酒精进行随后的脱氢反应，生成环氧化环。这第二步在生物学上是非常不寻常的，其机制也不清楚。在拟议的项目中，将确定H6H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射线结晶学将提供对这些反应如何介导的结构洞察，而停流动力学测量将在分子细节上确定这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中涉及的化学物种进行明确的表征。综上所述，这项工作将有助于使控制这些酶的不同反应性的因素合理化，并将作为设计针对这些酶的新药物化合物的基础。

 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.