Valentino, HannahKorasick, David A.Bohac, Tabbetha J.Shapiro, Justin A.Wencewicz, Timothy A.Tanner, John J.Sobrado, Pablo2021-08-042021-08-042021-07-062470-1343PMC8296543 (pmc)http://hdl.handle.net/10919/104576<i>Acinetobacter baumannii</i> is an opportunistic pathogen with a high mortality rate due to multi-drug-resistant strains. The synthesis and uptake of the iron-chelating siderophores acinetobactin (Acb) and preacinetobactin (pre-Acb) have been shown to be essential for virulence. Here, we report the kinetic and structural characterization of BauF, a flavin-dependent siderophore-interacting protein (SIP) required for the reduction of Fe(III) bound to Acb/pre-Acb and release of Fe(II). Stopped-flow spectrophotometric studies of the reductive half-reaction show that BauF forms a stable neutral flavin semiquinone intermediate. Reduction with NAD(P)H is very slow (<i>k</i> _obs, 0.001 s^-1) and commensurate with the rate of reduction by photobleaching, suggesting that NAD(P)H are not the physiological partners of BauF. The reduced BauF was oxidized by Acb-Fe (<i>k</i> _obs, 0.02 s^-1) and oxazole pre-Acb-Fe (ox-pre-Acb-Fe) (<i>k</i> _obs, 0.08 s^-1), a rigid analogue of pre-Acb, at a rate 3-11 times faster than that with molecular oxygen alone. The structure of FAD-bound BauF was solved at 2.85 Å and was found to share a similarity to <i>Shewanella</i> SIPs. The biochemical and structural data presented here validate the role of BauF in <i>A. baumannii</i> iron assimilation and provide information important for drug design.Pages 18537-18547application/pdfenCreative Commons Attribution-NonCommercial-NoDerivatives 4.0 International0904 Chemical Engineering0912 Materials EngineeringArticle - Refereed2021-08-04https://doi.org/10.1021/acsomega.1c03047628Sobrado, Pablo [0000-0003-1494-5382]34308084 (pubmed)2470-1343