Dual Role of NADP(H) in the Reaction of a Flavin Dependent N-hydroxylating Monooxygenase

Abstract

Aspergillus fumigatus siderophore A (Af SidA) is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine, producing N 5-hydroxyornithine. This is the first step in the biosynthesis of hydroxamate-containing siderophores in A. fumigatus. Af SidA is essential for virulence, validating this enzyme as a drug target. Af SidA can accept reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme. When the enzyme is reduced with NADPH and reacted with molecular oxygen, a C4a-hydroperoxyflavin intermediate is observed. When the enzyme is reduced with NADH, the intermediate is 2-fold less stable. Steady-state kinetic isotope effect values of 3 and 2 were determined for NADPH and NADH, respectively. The difference in the isotope effect values is due to differences in the rate of flavin reduction by these coenzymes. A difference in the binding mode between these coenzymes was observed by monitoring flavin fluorescence. Limited proteolysis studies show that NADP+, and not NAD+, protects Af SidA from proteolysis, suggesting that it induces conformational changes upon binding. Together, these results are consistent with NADPH having a role in flavin reduction and in the modulation of conformational changes, which positions NADP+ to also play a role in stabilization of the C4a-hydroperoxyflavin. © 2012 Elsevier B.V. All rights reserved.

Department(s)

Chemistry

Comments

National Science Foundation, Grant MCB-1021384

Keywords and Phrases

Anti-fungal drug target; Aspergillus fumigatus; C4a-hydroperoxyflavin; Flavin-dependent monooxygenase; N-hydroxylating; SidA

International Standard Serial Number (ISSN)

1878-1454; 1570-9639

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 Elsevier, All rights reserved.

Publication Date

01 Jun 2012

PubMed ID

22465572

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