Functional analysis of <scp>R144</scp> in the flavin oxidation mechanism of a flavin‐dependent N‐monooxygenase

Flavin-dependent N-monooxygenases (NMOs) are key enzymes in the biosynthesis of hydroxamate-containing siderophores, which are critical virulence factors in pathogenic microorganisms. SidA, an NMO from Aspergillus fumigatus, catalyzes the NADPH- and O₂-dependent hydroxylation of L-ornithine (Orn) via a stable C4a-hydroperoxyflavin (FAD_OOH) intermediate. This study investigates the role of Arg144 in the catalytic cycle of SidA. Site-directed mutagenesis of Arg144 to alanine (R144A) significantly impaired both oxygen consumption and Orn hydroxylation, resulting in an approximately 60-fold reduction in k_cat and a ~300-fold decrease in k_cat/K_M. The pH dependence, solvent kinetic isotope effects, and rapid-reaction kinetics reveal that R144 influences protonation events crucial for the decay of the FAD_OH intermediate. Structural and kinetic evidence supports a model in which R144 participates in a hydrogen-bonding network that facilitates flavin oxidation.