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.

Department(s)

Chemistry

Publication Status

Full Access

Comments

National Science Foundation, Grant CHE‐2003658

Keywords and Phrases

flavin oxidation; flavin-dependent monooxygneases; hydroperoxyflavin; ornithine hydroxylase; siderophore

International Standard Serial Number (ISSN)

1469-896X; 0961-8368

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2025 Wiley, All rights reserved.

Publication Date

01 Oct 2025

PubMed ID

40944420

Download

Full Text Link

Included in

Biochemistry Commons

Share

 
COinS