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

基本信息

批准号：
8426104
负责人：
Levi Michael Stanley
金额：
$ 23.06万
依托单位：
IOWA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8426104
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

项目摘要

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.

项目摘要/摘要在本独立奖项申请途径中描述的研究的目标是提供具有培训和蛋白质工程经验的候选人，以开发人造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 Stanley，Ph。D.目前是NRSA/NIGMS博士后研究员，其直接目标是完成另外一到两年的指导研究以在蛋白质工程领域建立研究技能并将这些新技能应用于结合广泛反应性的人工金属酶的发展过渡金属配合物具有精确控制合成的选择性的能力转型。斯坦利博士的长期职业目标包括建立自己为独立科学家在任职期间的学术地位，并通过产生生物活性化合物和构建块的催化反应的开发。斯坦利博士通过他目前在教授的研究中建立了过渡金属催化的专业知识约翰·哈特维格（John Hartwig）在伊利诺伊大学的实验室。职业发展计划和研究策略将允许候选人将过渡金属催化的知识与当前蛋白质方法相结合工程。该计划的指导阶段将与约翰教授合作进行过渡金属催化领域的领导者哈特维格（Hartwig 蛋白质工程。伊利诺伊大学候选人联合给出者的既定实验室提供理想的环境，以在其各自专业领域的界面进行研究。这在指导阶段的拟议研究将使候选人能够创建一个新颖的平台并开发新的人工金属酶，这些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重点放在这些新人造的应用上金属酶作为区域选择性，化学选择性和立体选择性C-H功能化的催化剂，环丙谁和氮杂性反应。提出的研究计划以四个具体目标概述。第一个目的集中于鉴定金属取代的碳赤霉酶，表现为作为C-H功能化催化剂的活性反应。第二个目的是针对开发生成，表达，纯化的方法并在突变碳赤霉素酶库中取代金属。第三个目标旨在改善定向演化金属取代的碳酸酐酶的活性C-H功能化反应。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，将为候选人提供蛋白质工程和在计划的独立阶段进行了定向演变。第四个目标针对金属取代的碳赤霉酶的分歧演化，以鉴定导致不同的突变体来自共同起点的区域选择性和化学选择性。完成完成的研究目标将在该计划的独立（R00）阶段进行三个和四个。结果这些研究将建立一个新的平台，以生成和改善人工金属酶，并将铺设基础工作，以扩大可用于合成有用的金属酶催化的转化的广度化学家。