RUI: Collaborative Research: Enzymology of Bacterial Nicotinic Acid Catabolism

该项目的目标是提高我们对土壤细菌如何进化出降解重要小分子污染物的能力的理解。N-杂环芳香族化合物（NHACs）是一类广泛存在的环境污染物，具有潜在的健康风险。恶臭假单胞菌和烟酸芽孢杆菌都是常见的土壤细菌，它们含有通过不同机制参与分解烟酸（一种模式NHAC）的酶。在这个项目中，伍斯特学院的马克·J·斯奈德教授和凯瑟琳·A.位于科特兰的纽约学院的州立大学的希克斯，沿着他们的本科研究生，将合作确定这些细菌酶用于降解烟酸的生化机制。还将在这些机构建立以项目为基础的实验室，以加强有关环境问题的生物化学课程。此外，该项目还将通过以社区为基础的夏令营（BWISER）与中学生一起教育和激励下一代科学家。这个为期一周的研究经验将侧重于学习现代化学技术，研究NHACs的降解和细菌在生物修复过程中的重要性。 本研究将从结构-功能的角度探讨酶催化降解烟酸的分子机制。烟酸是了解NHAC代谢的模型化合物。最近，B.已经对烟酸进行了测序，并发现了一组经鉴定编码分解代谢酶的基因，所述分解代谢酶使用新的途径降解烟酸。该项目将通过确定使用CRISPR技术敲除特定基因的效果来确认基因的拟议功能。将通过LC-MS/MS和1H NMR光谱法鉴别和表征途径中间体。使用机械研究和蛋白质X射线晶体学的组合，参与激活烟酸的吡啶环降解的酶也将被表征。这些步骤涉及独特的黄素依赖性单加氧酶，其扩大了由该超家族成员催化的反应。单加氧酶的工作将集中在阐明底物特异性的结构决定因素和工程这些酶激活一系列的NHAC降解。总之，该项目有可能建立在我们对细菌进化为降解NHAC的生化策略的理解基础上。这项工作还将为本科生提供现代生物化学技术的培训，回答有关环境污染的复杂问题所需的技能，以及为STEM领域的职业生涯做准备。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

The goal of this project is to enhance our understanding of how soil-dwelling bacteria have evolved the ability to degrade important small molecule pollutants. N-Heterocyclic aromatic compounds (NHACs) are a class of small molecules that are pervasive environmental pollutants and pose potential health risks. Both Pseudomonas putida and Bacillus niacini are common soil bacteria that contain enzymes involved in breaking down nicotinic acid, a model NHAC, by different mechanisms. In this project, Professor Mark J. Snider at the College of Wooster and Professor Katherine A. Hicks at the State University of New York College at Cortland, along with their undergraduate research students, will collaborate to determine the biochemical mechanisms that these bacterial enzymes use to degrade nicotinic acid. Project-based laboratories will also be developed at their institutions to strengthen the biochemical curricula on environmental issues. In addition, this project will also educate and motivate the next generation of scientists through the community-based summer camp (BWISER) with middle school-aged girls. This week-long research experience will focus on learning modern chemical techniques for studying the degradation of NHACs and the importance of bacteria in bioremediation processes. This research will establish the molecular mechanisms underlying the enzyme-catalyzed degradation of nicotinic acid using a structure-function approach. Nicotinic acid is a model compound for understanding the metabolism of NHACs. Recently, the genome of B. niacini has been sequenced and a cluster of genes putatively identified to code for the catabolic enzymes that degrade nicotinic acid using a novel pathway have been discovered. This project will confirm the proposed functions of the genes by determining the effects of knocking out specific genes using CRISPR technology. Pathway intermediates will be identified and characterized by LC-MS/MS and 1H NMR spectroscopy. Using a combination of mechanistic studies and protein X-ray crystallography, the enzymes involved in activating the pyridine ring of nicotinic acid for degradation will also be characterized. These steps involve unique flavin-dependent monooxygenases that expand the reactions catalyzed by members of this superfamily. Work on the monooxygenases will concentrate on elucidating the structural determinants of substrate specificity and engineering these enzymes for activating a range of NHACs for degradation. Together this project has the potential to build on our understanding of the biochemical strategies bacteria have evolved to degrade NHACs. This work will also provide undergraduate students with training in modern biochemical techniques, skills necessary to answer complex questions about environmental contamination, and preparation for STEM field careers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.