Directed Evolution of Metal-Substituted Carbonic Anhydrases for Catalysis

用于催化的金属取代碳酸酐酶的定向进化

• 批准号: 8598905
• 负责人: Levi Michael Stanley
• 金额: $ 22.9万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598905
• 关键词: Active Sites; Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; Amino Acids; Area; Award; Azides; Catalysis; Chemistry; Collaborations; Data; Development; Development Plans; Dialysis procedure; Doctor of Philosophy; Engineering; Environment; Enzymes; Evolution; Excision; Exhibits; Foundations; Generations; Genes; Goals; Health; Histidine; Human; Hydrogen Bonding; Illinois; Ions; Isoenzymes; Knowledge; Laboratories; Lead; Libraries; Mediating; Medical; Mentors; Metals; Methods; Mutagenesis; Mutation; National Institute of General Medical Sciences; National Research Service Awards; Organism; Pathway interactions; Phase; Positioning Attribute; Postdoctoral Fellow; Procedures; Protein Engineering; Protocols documentation; Reaction; Reporting; Research; Roentgen Rays; Science; Scientist; Site; Sulfonamides; Synthesis Chemistry; System; Training; Transition Elements; Universities; Zinc; abstracting; amidation; analog; base; carbene; carbonate dehydratase; career; career development; catalyst; cycloaddition; design; diazo compound; directed evolution; experience; high throughput screening; human disease; improved; metal complex; metalloenzyme; mutant; nitrene; novel; professor; programs; research and development; scaffold; screening; skills

Project Summary/Abstract The objectives of the research described in this Pathway to Independence Award application are to provide the candidate with training and experience in protein engineering to develop artificial metalloenzymes that catalyze new or improved synthetic reactions to prepare biologically active compounds. The candidate, Levi Stanley, Ph. D., is currently an NRSA/NIGMS postdoctoral fellow with the immediate goal of completing an additional one to two years of mentored research to establish research skills in the field of protein engineering and to apply these new skills to the development of artificial metalloenzymes that combine the broad reactivity of transition metal complexes with the ability of enzymes to exquisitely control the selectivity of synthetic transformations. Dr. Stanley's long-term career goals include establishing himself as an independent scientist in a tenure-track academic position and contributing to the improvement of human health through the development of catalytic reactions that generate bioactive compounds and building blocks to such compounds. Dr. Stanley has established expertise in transition metal catalysis through his current research in Professor John Hartwig's laboratory at the University of Illinois. The career development plan and research strategy will allow the candidate to combine his knowledge of transition metal catalysis with current methods for protein engineering. The mentored phase of the program will be carried out in collaboration with Professor John Hartwig, a leader in the field of transition metal catalysis, and Professor Huimin Zhao, an expert in the field of protein engineering. The established laboratories of the candidate's co-mentors at the University of Illinois provide an ideal environment to conduct research at the interface of their respective areas of expertise. The proposed research in the mentored phase will allow the candidate to create a novel platform for the discovery and development of new artificial metalloenzymes that catalyze reactions that are useful to synthetic chemists. The independent phase of the proposed research will focus on the application of these new artificial metalloenzymes as catalysts in regioselective, chemoselective, and stereoselective C-H functionalization, cyclopropanation, and aziridination reactions. The proposed research program is outlined in four specific aims. The first aim is focused on the identification of metal-substituted carbonic anhydrases that exhibit activity as catalysts for C-H functionalization reactions. The second aim is directed toward developing methods for the generation, expression, purification, and substitution of the metal in mutant carbonic anhydrase libraries. The third aim is designed to improve by directed evolution the activity of metal-substituted carbonic anhydrases that are active for C-H functionalization reactions. The first two aims and the beginning of the third aim will be the primary focus of the mentored (K99) phase and will provide the background necessary for the candidate to conduct protein engineering and directed evolution during the independent phase of the program. The fourth aim is directed toward the divergent evolution of metal-substituted carbonic anhydrases to identify mutants that lead to different regioselectivities and chemoselectivities from a common starting point. Research toward the completion of aims three and four will be conducted during the independent (R00) phase of this program. The results of these studies will establish a new platform to generate and improve artificial metalloenzymes and will lay the groundwork to expand the breadth of transformations catalyzed by metalloenzymes that are useful to synthetic chemists.

项目总结/摘要 本独立之路奖申请中描述的研究目标是提供 具有蛋白质工程培训和经验的候选人，以开发人工金属酶， 催化新的或改进的合成反应以制备生物活性化合物。候选人李维 斯坦利博士，目前是NRSA/NIGMS博士后研究员，其近期目标是完成一项 额外的一到两年的指导研究，以建立蛋白质工程领域的研究技能 并将这些新技术应用于人工金属酶的开发， 过渡金属配合物与酶的能力，精细地控制合成的选择性， 转变斯坦利博士的长期职业目标包括成为一名独立的科学家 在一个终身制的学术职位，并通过促进人类健康的改善， 开发产生生物活性化合物和这些化合物的结构单元的催化反应。 博士Stanley通过他目前在Professor的研究建立了过渡金属催化方面的专业知识 约翰·哈特维希在伊利诺伊大学的实验室。职业发展计划和研究战略将 允许候选人联合收割机将其过渡金属催化的知识与当前蛋白质的方法相结合 工程.该计划的辅导阶段将与约翰教授合作进行 过渡金属催化领域的领军人物Hartwig教授和催化领域的专家Huimin Zhao教授 蛋白质工程候选人的共同导师在伊利诺伊大学建立的实验室 提供一个理想的环境，在各自的专业领域进行研究。的 在指导阶段提出的研究将允许候选人创建一个新的发现平台 和开发新的人工金属酶，催化对合成化学家有用的反应。 拟议研究的独立阶段将侧重于这些新的人工 金属酶在区域选择性、化学选择性和立体选择性C-H官能化中作为催化剂， 环丙烷化和氮丙啶化反应。 拟议的研究计划概述了四个具体目标。第一个目标是集中在 鉴定表现出作为C-H官能化催化剂活性金属取代的碳酸酐酶 反应.第二个目的是开发用于产生、表达、纯化 和突变碳酸酐酶文库中金属的取代。第三个目标是通过以下方式改进 定向进化对C-H官能化有活性的金属取代的碳酸酐酶的活性 反应.前两个目标和第三个目标的开始将是指导的主要重点（K99） 阶段，并将提供必要的背景候选人进行蛋白质工程和 在程序的独立阶段进行定向进化。第四个目标是针对 金属取代碳酸酐酶的趋异进化，以鉴定导致不同的 区域选择性和化学选择性。为完成以下工作进行研究： 目标三和目标四将在该方案的独立（R 00）阶段进行。的结果 这些研究将为人工金属酶的产生和改良建立新的平台， 为扩大金属酶催化的转化的广度奠定了基础， 化学家。