RUI: Collaborative Research: Enzymology of Bacterial Nicotinic Acid Catabolism

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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.