6x0k | pdb_00006x0k
Structure of dithionite-reduced SidA ornithine hydroxylase with the FAD "in" and complexed with L-ornithine
Structural highlights
Function[SIDA_ASPFU] Catalyzes the conversion of L-ornithine to N(5)-hydroxyornithine, the first step in the biosynthesis of all hydroxamate-containing siderophores, such as the secreted triacetylfusarinine C (TAFC) involved in iron uptake and the intracellular iron storage compound desferriferricrocin (DFFC). Highly specific for its substrate, only hydrolyzing l-ornithine. Has preference for NADPH over NADH, NADPH playing a role in stabilization of the C4a-hydroperoxyflavin intermediate. Essential for virulence.[1] [2] [3] [4] Publication Abstract from PubMedThe 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 "out" active site conformation characterized by large rotations of the FAD isoalloxazine around the C1'-C2' and N10-C1' bonds, coupled to a 10 A 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 "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. Docking was used to explore the active site of the "out" 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. Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics.,Campbell AC, Stiers KM, Martin Del Campo JS, Mehra-Chaudhary R, Sobrado P, Tanner JJ J Biol Chem. 2020 Jul 28. pii: RA120.014750. doi: 10.1074/jbc.RA120.014750. PMID:32723870[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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