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Updated: Jan 18, 2026

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Published on: March 18, 2012
Functional analysis of R144 in the flavin oxidation mechanism of a flavin-dependent N-monooxygenase
Reeder Robinson1, Korliss Britt1, Pablo Sobrado2
1Department of Biochemistry, Virginia Tech, Blacksburg, Virginia, USA.
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.
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