Siderophore A (SidA) catalyzes the first step in the biosynthesis of several hydroxamate containing siderophores in <i>Aspergillus fumigatus</i> and is essential for virulence. The reaction of this flavoenzyme requires NADPH for the hydroxylation of L-ornithine. Here we present structural and mechanistic studies that revealed flavin dynamics as an important step in the mechanism of action of SidA. In the resting state, the flavin ring is present in a new “out” active site conformation featuring a 130° rotation coupled to large (10 Å) movements in a conserved region named the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD into the “in” 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 and structural characterization. Particularly, Met101 was shown to be involved in the mechanism of flavin motion that is required for NADP<sup>+</sup> unload and NADPH reload. Altogether, our results provide insight into how flavin-dependent monooxygenases manage multiple substrates in a complex catalytic cycle.

Mechanism of action of flavin-monooxygenases: Role of flavin dynamics in NADP(H) loading and unloading.

ACS Spring Meeting 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.