Enzymes Involved in Production of Bacterial Isonitrile-containing Natural Products

参与细菌含异腈天然产物生产的酶

• 批准号: 1158169
• 负责人: Andrew Gulick
• 金额: $ 87.09万
• 依托单位: Hauptman-Woodward Medical Research Institute Inc
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158169&HistoricalAwards=false
• 关键词: Enzymes; Involved; Production; Bacterial; Isonitrile

Intellectual Merit. Isonitriles are organic molecules that contain a carbon-nitrogen triple bond. These molecules, which are involved in radical chemistry and are important reagents for multi-component chemical reactions, are both difficult to synthesize chemically and possess an extremely foul odor. These properties have likely limited the study and use of these interesting compounds. Surprisingly, there are many examples of biological isonitrile compounds, found in both terrestrial and marine environments. These compounds are commonly produced by bacterial pathogens, including species that infect fish, plants, insects, and mammals. The mechanistic basis for biosynthesis of isonitrile natural products has not been studied. This project will investigate the enzymology of PvcA, the isonitrile-synthesizing enzyme from a Pseudomonas aeruginosa pathway that is responsible for the synthesis of a novel natural product. This enzyme converts the amino group of tyrosine to an isonitrile, using the C2 carbon of ribulose-5-phosphate as the source of the carbon atom in the isonitrile. This research project will examine the structural and functional mechanisms of the PvcA enzymes from three homologous bacterial pathways. A catalytic mechanism for the PvcA reaction will be tested through the collaborative enzymatic and structural approaches using the crystal structure of the PvcA enzyme from Pseudomonas aeruginosa as an experimental framework. These isonitrile clusters additionally contain PvcB, a non-heme oxygenase that catalyzes pathway-specific two- or four-electron oxidations. The structural and chemical basis for differences in reactions catalyzed by PvcB will also be examined. These experiments will provide detailed mechanistic insights into the structure and function of these novel enzymes, expanding our knowledge of the synthetic capabilities of diverse microorganisms.Broader ImpactThis research project will explore the enzymatic synthesis of isonitriles, an area of research that is virtually unexplored and will therefore provide an exciting research opportunity to graduate and undergraduate trainees. Specifically, this project will support the training of two graduate students. It will additionally support multiple undergraduate students through established, highly successful summer and academic year research programs. Undergraduate students will fully participate in lab activities, will present their work and write lab reports, and will be integrated into the lab so that they understand the contribution made to the larger research study. Students will be trained to identify and address specific hypothesis-driven questions and will receive broad interdisciplinary training in the important areas of chemical, biochemical, and structural biology.This project is jointly supported by the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.

智力优势。异腈是含有碳-氮三键的有机分子。这些分子参与自由基化学，是多组分化学反应的重要试剂，既难以化学合成，又具有极难闻的气味。这些性质可能限制了这些有趣化合物的研究和使用。令人惊讶的是，在陆地和海洋环境中发现了许多生物异腈化合物的例子。这些化合物通常由细菌病原体产生，包括感染鱼类，植物，昆虫和哺乳动物的物种。异腈天然产物生物合成的机理基础尚未研究。本项目将研究PvcA的酶学，PvcA是一种来自铜绿假单胞菌途径的异腈合成酶，负责合成一种新的天然产物。这种酶将酪氨酸的氨基转化为异腈，使用核酮糖-5-磷酸的C2碳作为异腈中碳原子的来源。该研究项目将研究来自三种同源细菌途径的PvcA酶的结构和功能机制。PvcA反应的催化机制将通过协同的酶和结构的方法进行测试，使用铜绿假单胞菌的PvcA酶的晶体结构作为实验框架。这些异腈簇还含有PvcB，一种非血红素加氧酶，催化途径特异性的两个或四个电子氧化。还将研究PvcB催化反应差异的结构和化学基础。这些实验将提供详细的机制洞察到这些新的酶的结构和功能，扩大我们的知识的不同微生物的合成能力。更广泛的影响这个研究项目将探讨异腈的酶促合成，这是一个几乎未探索的研究领域，因此将提供一个令人兴奋的研究机会，研究生和本科生学员。具体而言，该项目将支持培训两名研究生。它还将通过既定的，非常成功的夏季和学年研究计划支持多名本科生。本科生将充分参与实验室活动，展示他们的工作并撰写实验室报告，并将融入实验室，以便他们了解对更大研究的贡献。学生将接受培训，以确定和解决特定的假设驱动的问题，并将在化学，生物化学和结构生物学的重要领域接受广泛的跨学科培训。该项目由分子和细胞生物科学部的生物分子动力学，结构和功能集群以及化学部的生命过程化学计划共同支持。