Aspergillus fumigants SidA Is a Highly Specific Ornithine Hydroxylase with Bound Flavin Cofactor

Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N-5-hydroxylated L-ornithines essential for iron binding. A. fumigatus siderophore A (AfSidA) catalyzes the flavin- and NADPH-dependent hydroxylation of L-ornithine in ferrichrome biosynthesis. AfSidA 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. AfSidA 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, AfSidA 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 AfSidA NADP(+) is a competitive inhibitor with respect to NADPH, demonstrating that AfSidA forms a ternary complex with NADP(+) and L-ornithine during catalysis. The data suggest that AfSidA likely proceeds by a sequential kinetic mechanism.