Learning the Steps to Metalloenzyme Choreography

学习金属酶编排的步骤

• 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
• 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

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.

抽象的。自然界是最终的合成化学家，而金属酶是催化剂的选择。要实现他们的 前所未有的功能多样性，金属酶调节着细胞的结构和电子性质 进行反应的蛋白质环境，从而实现通常 对合成化学家来说是个挑战。尽管在结构和结构方面的研究已经取得了巨大的进展 机械酶学方面，这些反应的动力学仍然知之甚少。本提案使用 表征原子和电子协同变化的创新方法，这些变化促进了两种 与医学相关的含铁金属酶类。实验将以合理的基础设计 根据已知的机理行为来可视化溶液和水中的其他瞬时催化中间体 克里斯特洛。第一个具体目标集中在底物结合的方式和铁-血红素的形成。 人体免疫抑制酶吲哚胺2，3-双加氧酶。该项目将在K99期间完成 资助期，将涉及反应物络合物和AN的结晶学表征 酶促生成的铁基物种。中间体将使用底物/辅因子模拟物，位点- 定向突变体，或利用反应的pH依赖性的冷冻捕捉法。在.期间 在独立阶段，第二个目标将采用类似的方法来研究相对未定性的 一类钴胺依赖的自由基S-腺苷甲硫氨酸甲基转移酶，参与生物合成 强效抗生素。尽管中间状态模型是可取的，但任何结构都将提供独特的洞察力 添加到这个类的机制中，因为到目前为止还没有发布。第三个也是最终目标，发展和 应用激光泵浦/X射线探头装置同时采集X射线晶体和能谱 实时数据，将同时进行。普林斯顿大学提供了理想的环境， 开始追求这些目标，拥有一流的设施和接触到世界领先的生物无机专家 化学反应。这些资源将得到我在宾夕法尼亚州立大学的共同导师的补充。拥有 接受了正规的物理教育，我的短期目标是获得必要的湿实验室技能， 对我自己的样本进行表征和故障排除。我将接受蛋白质表达和纯化方面的培训，以及 UV-Vis、EPR和复杂的金属酶结晶学表征。在指导阶段， 我将参加一门关于异源表达和纯化蛋白质的正式课程，以及其他一些 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 成为一名成功的个人投资经理所必需的。我在K99期间获得的专业知识将是研究更复杂的课程所必需的 系统，并开发激光泵浦/X射线探针时间分辨方法研究金属酶在 独立阶段。从长远来看，我计划领导一个多学科的蛋白质界面研究项目 生物化学和X射线科学。