Iron is an essential nutrient for most living organisms; however, it is present in low concentration due to its low solubility in water. Some microbes have developed an elaborate system to acquire iron from the environment by producing iron-chelating molecules called siderophores. N-hydroxylating monooxygenases are a group of uncharacterized flavin-dependent monooxygenases involved in the hydroxylation of nitrogen atoms on the side chains of lysine and ornithine in the pathway leading to the biosynthesis of siderophores. This project will employ mutagenesis and kinetic studies, mass spectral analyses, and x-ray crystallography to determine the structure and mechanism of two members of the N-hydroxylating monooxygenase family: N5-ornithine and N6-lysine hydroxylases. The ultimate goal is to understand the mechanisms of amino acid hydroxylation and selectivity and the role of protein motion in catalysis. This information will contribute to our understanding of the mechanism of oxygen activation by flavoenzymes and how nature harnesses oxygenated flavin intermediates for specific chemical outcomes.
The research in this application will serve as a platform for teaching and training high school, undergraduate, and graduate students, enabling them to develop skills and knowledge in molecular biology, mechanistic enzymology, biochemistry, and protein chemistry. Minority students will be integrated into this research through the mentoring activities of the PI in Virginia Tech's Post-Baccalaureate Research and Education and the Initiative to Maximize Student Diversity Programs. Undergraduate and graduate students will gain training in structural biochemistry by participating in an international internship program in the protein crystallography group at the University of Pavia, Italy. High school students visiting the laboratory will be exposed to hypothesis-based research by determining the physiological role of flavin monooxygenase mutants of A. thaliana in conjunction with the Partnership for Research and Education in Plants at Virginia Tech.
