Abstract
Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N5-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-dependent hydroxylation of l-ornithine in ferrichrome biosynthesis. Af SidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to l-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. Af SidA is highly specific for its amino acid substrate, only hydroxylating l-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, Af SidA can be reduced by NADPH, and a C4a-(hydro)peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by l-ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP+ in the stabilization of intermediates in the reaction of Af SidA. NADP+ is a competitive inhibitor with respect to NADPH, demonstrating that Af SidA forms a ternary complex with NADP+ and l-ornithine during catalysis. The data suggest that Af SidA likely proceeds by a sequential kinetic mechanism. © 2010 American Chemical Society.
Recommended Citation
S. W. Chocklett and P. Sobrado, "Aspergillus Fumigatus SidA is a Highly Specific Ornithine Hydroxylase with Bound Flavin Cofactor," Biochemistry, vol. 49, no. 31, pp. 6777 - 6783, American Chemical Society, Aug 2010.
The definitive version is available at https://doi.org/10.1021/bi100291n
Department(s)
Chemistry
International Standard Serial Number (ISSN)
1520-4995; 0006-2960
Document Type
Article - Journal
Document Version
Citation
File Type
text
Language(s)
English
Rights
© 2024 American Chemical Society, All rights reserved.
Publication Date
10 Aug 2010
PubMed ID
20614882
