Functional analysis of R144 in the flavin oxidation mechanism of a flavin‐dependent N‐monooxygenase

Abstract 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 (FADOOH) 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 kcat and a ~300‐fold decrease in kcat/KM. The pH dependence, solvent kinetic isotope effects, and rapid‐reaction kinetics reveal that R144 influences protonation events crucial for the decay of the FADOH intermediate. Structural and kinetic evidence supports a model in which R144 participates in a hydrogen‐bonding network that facilitates flavin oxidation.