Aspergillus fumigatus 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 (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.
Chocklett
Samuel W
SW
Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, USA.
Sobrado
Pablo
P
D001232:Aspergillus fumigatus / Q000201:enzymology*; D002384:Catalysis; D005415:Flavins / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009952:Ornithine / Q000378:metabolism*; D017262:Siderophores / Q000737:chemistry*
D005415:Flavins; D017262:Siderophores; D009249:NADP; D009952:Ornithine; D006899:Mixed Function Oxygenases