Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics

Abstract

The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase from <i>Aspergillus fumigatus</i> is a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is an <i>N</i>-monooxygenase that catalyzes the NADPH-dependent hydroxylation of l-ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) or l-ornithine bound (resting state). The resting state structure reveals a new <i>out</i> active site conformation characterized by large rotations of the FAD isoalloxazine around the C1-'C2' and N10-C1' bonds, coupled to a 10-Å movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to the <i>in</i> conformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) and l-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of the <i>out</i> conformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.