Structural and Mechanistic Studies on N-Hydroxylating Monooxygenases Involved in Siderophore Biosynthesis

N-Hydroxylating monooxygenases (NMOs) are flavin dependent enzymes that primarily catalyze the hydroxylation of L-ornithine or L-lysine. This is the first, committed step to siderophore biosynthesis. Pathogenic microbes including Aspergillus fumigatus and Mycobacterium tuberculosis secrete these low molecular weight compounds in order to uptake FeIII from their hosts for their metabolic needs when establishing infection. Therefore, members of this family of enzymes represent novel drug targets for the development of antibiotics. Here, we present the detailed functional and structural analysis of the L-ornithine monooxygenase SidA from Aspergillus fumigatus and the L-lysine monooxygenases MbsG from Mycobacterium smegmatis and NbtG from Nocardia farcinica. The detailed chemical mechanism for flavin oxidation in SidA was elucidated for formation of the C4a-hydroperoxyflavin, deprotonation of L-ornithine, and for the chemical steps of hydrogen peroxide elimination and water elimination. This was performed through a combination of kinetic isotope effect, pH, and density functional theory studies. Also, important residues involved in substrate binding and catalysis were characterized using site-directed mutagenesis for both SidA and NbtG. These include residues involved in coenzyme selectivity, substrate binding, and residues important in C4a-hydroperoxyflavin stabilization and flavin oxidation. The kinetic mechanisms of the L-lysine monooxygenases MbsG and NbtG were characterized which show unique differences with SidA. These include differences in coenzyme selectivity, and C4a-hydroperoxyflavin stabilization. Lastly, the three-dimensional structure of NbtG was solved using X-ray crystallography which is the first structure of a lysine monooxygenase. The structure shows the NADPH-binding domain is rotated ~30° relative to the FAD-binding domain which occludes NADP+ binding in NbtG. Unlike SidA, NbtG does not stabilize a C4a-hydroperoxyflavin and this occlusion observed in the structure might explain this difference. This highlights both the structural and mechanistic diversities among NMOs and the data presented here provides valuable information for the future development of specific inhibitors of NMOs.