Chocklett, Samuel Wyatt2017-04-042017-04-042009-12-09etd-12162009-143628http://hdl.handle.net/10919/76915Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus during infection. This siderophore includes N5-hydroxylated L-ornithine in the peptide backbone that serve as iron chelators. Af SidA is the L-ornithine N5-hydroxylase, which performs the first enzymatic step in the biosynthesis of ferrichrome. In this study, Af SidA was recombinantly expressed and purified as a soluble tetramer with a bound FAD cofactor. The enzyme demonstrated typical Michaelis-Menten kinetics in a product formation assay with respect to L-ornithine, but similar experiments as a function NADH and NADPH indicated inhibition at high coenzyme concentrations. Af SidA is highly specific for its substrate; however, it is promiscuous with respect to its coenzyme requirement. A multi-functional role of NADPH is observed since NADP+ is a competitive inhibitor with respect to NADPH and steady-state kinetic experiments indicate that Af SidA forms a ternary complex with NADP+ and L-ornithine for catalysis. Furthermore, in the absence of substrate, Af SidA forms a stable C4a-(hydro)peroxyflavin intermediate that is stable on the second time scale. Af SidA is also inhibited by several halides and the arginine-reactive reagent, phenylglyoxal. Biochemical comparison of Af SidA to other flavin-containing monooxygenases reveal that Af SidA likely proceeds by a sequential-ordered mechanism.en-USIn CopyrightAf SidAhydroxylationAspergillus fumigatusflavinsiderophoreThesishttp://scholar.lib.vt.edu/theses/available/etd-12162009-143628/