Learning the Steps to Metalloenzyme Choreography

学习金属酶编排的步骤

基本信息

批准号：
10174959
负责人：
Katherine Marie Davis
金额：
$ 24.9万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10174959
关键词：
Allosteric Regulation Anabolism Antibiotics Behavior Binding Bioinorganic Chemistry Biological Biology Catalysis Chemistry Cobalamin Collection Complement Complex Crystallization Cytochrome P450 Data Dependence Development Disease Disputes Education Educational workshop Electron Spin Resonance Spectroscopy Environment Enzymatic Biochemistry Enzymes Exposure to Facility Accesses Freezing Funding Goals Health Heme Human Hydrogen Bonding Interdisciplinary Study Ions Iron Lasers Lead Learning Mentors Metabolism Metals Methodology Methods Methyltransferase Modeling Monitor Mononuclear Multienzyme Complexes Muramidase Nature Nitric Oxide Pathway interactions Pennsylvania Phase Photosensitivity Physics Porphyrins Positioning Attribute Process Production Property Protein Biochemistry Proteins Proteome Publishing Pump Reaction Resources Roentgen Rays Role S-Adenosylmethionine Sampling Scheme Science Site Site-Directed Mutagenesis Spectrum Analysis Structural Biochemistry Structure System Techniques Testing Time Training Transition Elements Tryptophan 2,3 Dioxygenase Universities Variant Visualization Vitamin B 12 absorption analog anti-cancer therapeutic base biochemical tools catalyst cofactor combat cytotoxic design experience experimental study improved inhibitor/antagonist innovation insight metalloenzyme mutant nanosecond oxidation programs protein expression protein purification skills spectroscopic data structural biology symposium

项目摘要

Abstract. Nature is the ultimate synthetic chemist, and metalloenzymes the catalysts of choice. To achieve their unprecedented functional diversity, metalloenzymes modulate the structural and electronic properties of the protein environment in which the reaction proceeds, thus enabling difficult transformations that are often challenging for synthetic chemists. Although enormous progress has been made in the study of structural and mechanistic enzymology, the dynamics of these reactions remain poorly understood. This proposal uses innovative methods to characterize the concerted atomic and electronic variations that facilitate catalysis in two medicinally-relevant classes of iron-containing metalloenzymes. Experiments will be rationally designed based on known mechanistic behavior to!visualize otherwise transient catalytic intermediates both in solution and in crystallo. The first specific aim focuses on the mode of substrate binding and ferryl-heme formation in the immunosuppressive human enzyme indoleamine 2,3-dioxygenase. This project will be completed during the K99 funding period and will involve crystallographic characterization of both the reactant complex and an enzymatically-generated ferryl species. Intermediates will be stabilized using substrate/cofactor mimics, site- directed mutants, or freeze-trapping methods exploiting the pH dependence of the reaction. During the independent phase, the second aim will use a similar approach applied to study the relatively uncharacterized class of cobalamin-dependent radical S-adenosylmethionine methyltransferases, involved in the biosynthesis of potent antibiotics. Although intermediate state models are desirable, any structure would provide unique insight into the mechanism of this class as none have been published to-date. The third and final aim, to develop and apply a laser pump/X-ray probe setup for the simultaneous collection of X-ray crystallographic and spectroscopic real time data, will be pursued concurrently. Princeton University provides the ideal environment in which to initiate pursuit of these goals, with excellent facilities and access to the world’s leading experts in bioinorganic chemistry. These resources will be complemented by my co-mentor at the Pennsylvania State University. Having received a formal education in physics, my short-term goals are to acquire wet lab skills necessary to generate, characterize and troubleshoot my own samples. I will be trained in protein expression and purification as well as UV-vis, EPR and sophisticated crystallographic characterization of metalloenzymes. During the mentored phase, I will attend a formal course in heterologous expression and purification of proteins, as well as a number of other workshops/conferences designed to increase my exposure to these techniques and learn the management skills required to be a successful PI. The expertise I acquire in the K99 period will be necessary to study more complex systems and develop laser pump/X-ray probe time-resolved methods for the study of metalloenzymes during the independent phase. In the long-term, I plan to lead a multidisciplinary research program at the interface of protein biochemistry and X-ray science.

摘要：大自然是最终的合成化学家，而金属酶是实现其目标的首选催化剂。金属酶涵盖功能多样性，调节结构和电子特性反应进行的蛋白质环境，从而实现通常难以实现的困难转化尽管在结构和化学研究方面取得了巨大进展，但这对合成化学家来说仍然是一个挑战。机械酶学，这些反应的动力学仍然知之甚少。创新方法来表征协调的原子和电子变化，促进两种催化作用将根据含铁金属酶的药用相关类别合理设计实验。根据已知的机械行为，可视化溶液中和水中的其他瞬态催化中间体第一个具体目标集中于底物结合模式和铁血红素形成。免疫抑制人类酶吲哚胺2,3-双加氧酶这个项目将在K99期间完成。资助期，并将涉及反应物复合物和酶促生成的 Ferrel 物种将使用底物/辅因子模拟物、位点进行稳定。定向突变体或利用反应过程中 pH 依赖性的冷冻捕获方法。独立阶段，第二个目标将使用类似的方法来研究相对无特征的一类钴胺素依赖性自由基 S-腺苷甲硫氨酸甲基转移酶，参与尽管中间态模型是理想的，但任何结构都可以提供独特的见解。迄今为止尚未发布第三个也是最后一个目标，即开发和实施此类机制。应用激光泵/X 射线探头装置同时收集 X 射线晶体学和光谱普林斯顿大学将为同时追求实时数据提供理想的环境。凭借优良的设施和接触世界领先的生物无机专家的机会，开始追求这些目标我在宾夕法尼亚州立大学的合作导师将补充这些资源。接受过正规的物理学教育，我的短期目标是获得必要的湿实验室技能，以产生，我将接受蛋白质表达和纯化方面的培训，并对我自己的样品进行表征和故障排除。金属酶的紫外可见分光光度计、EPR 和复杂的晶体学表征。我将参加蛋白质异源表达和纯化的正式课程，以及许多其他课程旨在增加我对这些技术的接触并学习管理技能的研讨会/会议要成为一名成功的 PI，我在 K99 时期获得的专业知识将是学习更复杂的知识所必需的。系统并开发激光泵/X射线探针时间分辨方法来研究金属酶从长远来看，我计划领导蛋白质界面的多学科研究项目。生物化学和 X 射线科学。