The ABC exporter IrtAB imports and reduces mycobacterial siderophores

Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores to scavenge iron from host proteins(1). M. tuberculosis produces two classes of siderophores, lipid-bound mycobactin and soluble carboxymycobactin(2, 3). Functional studies revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ABC transporter IrtAB(4), which features an additional cytoplasmic siderophore interaction domain (SID)(5). However, IrtAB’s predicted ABC exporter fold seemingly contradicts its import function. Here, we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the SID to facilitate iron release. Structure determination by X-ray crystallography and cryo-EM confirms IrtAB’s ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB resulting in a partially collapsed inward-facing substrate binding cavity. The SID is positioned in close proximity to the inner membrane leaflet, which allows the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB prefers mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore import machinery in mycobacteria.