Structural Insight into the Mechanism of Oxygen Activation and Substrate Selectivity of Flavin-Dependent N-Hydroxylating Monooxygenases

Abstract

SidA from the human pathogen Aspergillus fumigatus catalyzes the generation of N(5)-hydroxyornithine in the biosynthesis of siderophores, a reaction essential for virulence. The crystal structures of SidA in complex with ornithine and lysine reveal the geometry of the interactions among flavin, NADP(+), and the substrate amine group that underlie the hydroxylation reaction. The structural elucidation of the enzyme in complex with arginine provides insight into the role of electrostatics and hydrogen bonding in the mechanism of oxygen activation in this family of enzymes.

Role of Ser-257 in the Sliding Mechanism of NADP(H) in the Reaction Catalyzed by the Aspergillus fumigatus Flavin-dependent Ornithine N5-Monooxygenase SidA

“…The procedures for expression and purification of the recombinant proteins were essentially as previously described (6,16).…”

“…Orn, ornithine. formed as described previously (5,6,17) to measure the amount of hydroxylated ornithine produced. The standard buffer for the iodine oxidation assay was 100 mM sodium phosphate buffer (pH 7.5).…”

“…Molecular Dynamics (MD) Simulations-All simulations were performed using the GROMACS package (version 4.5.5) (19) with GROMOS96 53a6 force field parameters (20). The coordinates of oxidized wild-type SidA bound to NADP ϩ were taken from Protein Data Base entry 4B63 (6), and the oxidized FAD topology parameters were taken from our previous work (21). The protonation states of ionizable residues were assigned based on pK a values calculated using the Hϩϩ program (22).…”

“…The structure clearly shows that the binding of NADP ϩ is not optimal for hydride transfer but, instead, for its role in the stabilization of the C4a-hydroperoxyflavin ( Fig. 1) (6). The position of NADP ϩ suggests that it undergoes conformational changes after the hydride transfer step.…”

“…These enzymes have been studied for their application in the chemical industry and as potential drug targets (1,3,4). Our group has been interested in the structure and mechanism of action of Aspergillus fumigatus SidA (siderophore A), an N-hydroxylating monooxygenase involved in siderophore biosynthesis (5,6). SidA catalyzes the hydroxylation of ornithine at the N 5 -position (Scheme 1).…”