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。大自然是最终的合成化学家，而金属酶是选择的催化剂。实现他们的 企业多样性，金属酶调节了该结构和电子特性 反应进行的蛋白质环境，从而实现了常常是困难的转化 对合成化学家的挑战。尽管在研究结构和研究中取得了巨大进展 机械酶学，这些反应的动力学仍然很少理解。该建议使用 创新的方法来表征协同的原子和电子变化，这些变化有助于两个 含铁的含铁的金属酶类别。实验将是基于合理设计的 关于已知的机械行为！可视化解决方案和在 结晶。第一个特定目的侧重于底物结合的模式和渡轮 - 渡轮形成 免疫抑制人酶吲哚胺2,3-二氧酶。该项目将在K99期间完成 资金期间，将涉及反应物复合物和A的晶体表征 酶产生的渡轮物种。中间体将使用底物/辅因子模拟物稳定 定向突变体或冻结方法利用反应的pH依赖性。在 独立阶段，第二个目标将采用类似的方法来研究相对未表征 钴拉明依赖性的自由基S-腺苷甲基硫氨基甲基转移酶，参与与生物合成 尽管中间状态模型是理想的，但任何结构都将提供独特的见解 进入该班级的机制，因为没有发布迄今未发表。第三个也是最后一个目标，要发展和 将激光泵/X射线探针设置应用于简单的X射线晶体学和光谱学的集合 实时数据将同时访问。普林斯顿大学提供了理想的环境 启动这些目标，具有出色的设施，并获得了世界领先的生物无机专家 化学。这些资源将由我的宾夕法尼亚州立大学的联合学者完成。有 接受了物理学的正规教育，我的短期目标是获得产生必要的湿实验室技能 表征和故障排除我自己的样本。我将接受蛋白质表达和纯化以及 紫外线，EPR和金属酶的复杂晶体学表征。在修补阶段， 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 需要成为成功的PI。我在K99期间获得的专业知识对于研究更复杂的学习是必要的 系统并开发激光泵/X射线探针时间分辨方法，用于研究金属酶 从长远来看，我计划在蛋白质界面领导多学科研究计划 生物化学和X射线科学。