Structural and Biochemical Characterization of the Flavin-Dependent Siderophore-Interacting Protein from Acinetobacter baumannii

Hannah Valentino; David A Korasick; Tabbetha J Bohac; Justin A Shapiro; Timothy A Wencewicz; John J Tanner; Pablo Sobrado

doi:10.1021/acsomega.1c03047

Structural and Biochemical Characterization of the Flavin-Dependent Siderophore-Interacting Protein from Acinetobacter baumannii

ACS Omega. 2021 Jul 6;6(28):18537-18547. doi: 10.1021/acsomega.1c03047. eCollection 2021 Jul 20.

Authors

Hannah Valentino¹, David A Korasick², Tabbetha J Bohac³, Justin A Shapiro³, Timothy A Wencewicz³, John J Tanner^{2

4}, Pablo Sobrado¹

Affiliations

¹ Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States.
² Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, United States.
³ Department of Chemistry, Washington University in Saint Louis, St. Louis, Missouri 63130, United States.
⁴ Department of Chemistry, University of Missouri, Columbia, Missouri 65211, United States.

Abstract

Acinetobacter baumannii 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 (k _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 (k _obs, 0.02 s^-1) and oxazole pre-Acb-Fe (ox-pre-Acb-Fe) (k _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 Shewanella SIPs. The biochemical and structural data presented here validate the role of BauF in A. baumannii iron assimilation and provide information important for drug design.