A look at the phylogenetically widespread class of flavin-dependent monooxygenases shows that little is known of those involved in biosynthetic pathways. Two homologous flavin-dependent enzymes described in this work, ornithine monooxygenase (OMO) from Aspergillus fumigatus and lysine monooxygenase (LMO) from Klebsiella pneumoniae, initiate hydroxamate siderophore biosynthesis in their respective pathogens. Understanding how the enzyme class functions could lead to design of rational inhibitors to be used for the eradication of hydroxamate siderophore-producing pathogens.  At the inception of this project, the OMO and intact LMO proteins had not yet been expressed or characterized from any organism. The chemical and kinetic mechanisms, as well as 3D-structure, for any representative siderophore-associated monooxygenase, were unknown. OMO was especially amenable to spectroscopic characterization and therefore the focus of the majority of this work. OMO was first characterized in the steady-state, as it would exist in the cell. Transient steps were then examined, allowing derivation of a more detailed mechanism and gave the first insights into how the biosynthetic enzymes might be distinct among all those that use flavin. The substrate L-ornithine plays a unique regulatory role in the oxidative half reaction, which has never been described for a flavin-dependent monooxygenase. This led to the pursuit of a study on the oxygen-dependent reaction as a function of substrate analogs and as a function of pH. A type of protein regulation yet again unforeseen in this class of enzymes is demonstrated, which may help to explain how biology regulates, temporally and spatially, the production of the important metabolites that they produce. The structure of a homolog of OMO shows that a strictly conserved histidine in N-hydroxylating monooxygenases resides on the side of the flavin opposite to the site of substrate binding. His91 mutagenesis in OMO shows that this residue plays a role in the formation of a stable flavin-peroxide species, fundamental to substrate hydroxylation.  Altogether these findings define the behavior of a new subclass of flavin-dependent monooxygenases that are involved in important biosynthetic processes. The results may help explain how diverse and biologically vital biosynthetic pathways are regulated, in diverse organisms from bacteria to plants.