RUI: Collaborative Research: Enzymology of Bacterial Nicotinic Acid Catabolism

这个项目的目标是加强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-杂环芳烃(NHAC)是一类普遍存在的环境污染物，具有潜在的健康风险。恶臭假单胞菌和烟杆菌都是常见的土壤细菌，它们含有通过不同机制分解烟酸的酶，烟酸是一种模型NHAC。在这个项目中，伍斯特学院的马克·J·斯奈德教授和纽约州立大学科特兰学院的凯瑟琳·A·希克斯教授，以及他们的本科生，将合作确定这些细菌酶用来降解烟酸的生化机制。还将在其机构发展以项目为基础的实验室，以加强关于环境问题的生物化学课程。此外，该项目还将通过以中学女孩为对象的社区夏令营(BWISER)教育和激励下一代科学家。为期一周的研究经验将集中在学习现代化学技术，以研究NHAC的降解和细菌在生物修复过程中的重要性。这项研究将利用结构-功能方法建立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，烟曲霉菌的基因组已经被测序，并发现了一组被认为是编码分解代谢酶的基因，这些酶使用一种新的途径降解烟酸。该项目将通过使用CRISPR技术确定敲除特定基因的效果来确认基因的拟议功能。途径中间体将通过LC-MS/MS和1H核磁共振波谱进行鉴定和表征。利用机理研究和蛋白质X射线结晶学相结合的方法，还将对参与激活烟酸的吡啶环进行降解的酶进行表征。这些步骤涉及独特的黄素依赖的单加氧酶，这些单加氧酶扩大了这个超家族成员催化的反应。单加氧酶的工作将集中于阐明底物专一性的结构决定因素，并设计这些酶来激活一系列NHAC进行降解。总之，这个项目有可能建立在我们对细菌进化来降解NHAC的生化策略的理解的基础上。这项工作还将为本科生提供现代生化技术方面的培训，回答有关环境污染的复杂问题所需的技能，并为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优势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.