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

Publication Type:

Journal Article


J Biol Chem (2020)


<p>The SidA ornithine hydroxylase from Aspergillus fumigatus 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 N-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 &quot;out&quot; active site conformation characterized by large rotations of the FAD isoalloxazine around the C1&#39;-C2&#39; and N10-C1&#39; 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 &quot;in&quot; 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 &quot;out&quot; 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.</p>

6X0H, 6X0I, 6X0J, 6X0K