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Learning the Steps to Metalloenzyme Choreography

学习金属酶编排的步骤

基本信息

批准号：
10411948
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/10411948
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 innovation insight metalloenzyme mutant nanosecond oxidation programs protein expression protein purification rational design 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.

抽象。大自然是最终的合成化学家，而金属酶则是最佳的催化剂。实现其金属酶具有前所未有的功能多样性，可调节金属的结构和电子性质。蛋白质的环境中进行反应，从而使困难的转化，往往是对合成化学家来说是个挑战。虽然在结构和力学研究方面取得了巨大进展，尽管酶的作用机理不明确，但这些反应的动力学仍然知之甚少。该提案使用创新的方法来表征协调的原子和电子变化，促进催化在两个含铁金属酶的医学相关类别。实验将根据合理设计对已知的机械行为！可视化溶液和溶液中的其他瞬时催化中间体，水晶。第一个具体的目标集中在底物结合和铁酰血红素形成的模式，免疫抑制人类酶吲哚胺2，3-双加氧酶。该项目将在K99期间完成资金期间，将涉及晶体学表征的反应物复合物和酶促生成的铁基物质。将使用底物/辅因子模拟物稳定中间体，位点- 定向突变体，或利用反应的pH依赖性的冷冻捕获方法。期间独立阶段，第二个目标将使用类似的方法，适用于研究相对未定性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见解因为迄今为止还没有人发表过。第三个也是最后一个目标是发展和应用激光泵浦/X射线探针装置，用于同时收集X射线晶体学和光谱学真实的时间数据，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，并获得世界领先的生物无机专家化学.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。具有接受过物理学的正规教育，我的短期目标是获得必要的湿实验室技能，对我自己的样品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， UV-vis，EPR和金属酶的复杂晶体学表征。在辅导阶段，我将参加一个正式的课程，在异源表达和纯化蛋白质，以及一些其他的，研讨会/会议，旨在增加我对这些技术的接触，并学习管理技能成为一名成功的PI。我在K99期间获得的专业知识将是必要的，以研究更复杂的系统，并开发激光泵浦/X射线探针时间分辨方法，用于研究金属酶在独立阶段。从长远来看，我计划在蛋白质的界面上领导一个多学科的研究项目生物化学和X射线科学。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structural insights into inhibition of the drug target dihydroorotate dehydrogenase by bacterial hydroxyalkylquinolines.

DOI：
10.1039/d1cb00255d
发表时间：
2022-04-06
期刊：
RSC chemical biology
影响因子：
4.1
作者：
Horwitz SM;Blue TC;Ambarian JA;Hoshino S;Seyedsayamdost MR;Davis KM
通讯作者：
Davis KM

Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides.

DOI：
10.1021/jacs.9b07338
发表时间：
2019-09
期刊：
Journal of the American Chemical Society
影响因子：
15
作者：
Micah T. Nelp;Vincent Zheng;K. Davis;K. Stiefel;J. Groves
通讯作者：
Micah T. Nelp;Vincent Zheng;K. Davis;K. Stiefel;J. Groves

Steric enforcement of cis-epoxide formation in the radical C-O-coupling reaction by which (S)-2-hydroxypropylphosphonate epoxidase (HppE) produces Fosfomycin.

DOI：
10.1021/jacs.9b10974
发表时间：
2019-11
期刊：
Journal of the American Chemical Society
影响因子：
15
作者：
Shengbin Zhou;Juan Pan;Katherine M Davis;Irene Schaperdoth;Bo Wang;A. Boal;C. Krebs;J. Bollinger-J.-Bolli
通讯作者：
Shengbin Zhou;Juan Pan;Katherine M Davis;Irene Schaperdoth;Bo Wang;A. Boal;C. Krebs;J. Bollinger-J.-Bolli

Computational Approaches: An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics.