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Siderophores acquire iron from hosts under iron-limiting conditions and play an essential role in the survival of microorganisms. Siderophore-interacting proteins (SIPs) from microbes release iron from the siderophore complex by reducing ferric iron to ferrous iron, but the molecular mechanism of iron reduction remains unclear. To better understand the molecular mechanism of SIPs, we herein report the crystal structure of Aeromonas hydrophila SIP (AhSIP) in complex with flavin adenine dinucleotide (FAD) as a cofactor. AhSIP consists of an N-terminal FAD binding domain and a C-terminal NADH binding domain, which are connected by a linker region. AhSIP showed unique structural differences in the orientation of the cofactor binding lobes when compared with SIP homologs. This study identified a cluster of three basic residues (Lys48, His259 and Arg262) in AhSIP distributed around a potential substrate binding pocket. In addition, AhSIP, containing the NADH binding motif E(L)VL-X 3 -GE, belongs to the group I subfamily. Our results show the diverse cofactor and substrate binding sites of the SIP family. • Crystal structure determination of the Siderophore-interacting protein SIP-FAD complex from A. hydrophila. • AhSIP consist of N-terminal FAD binding domain and C-terminal NADH binding domain. • The potential substrate binding pocket of AhSIP and putative NADH binding sites are proposed. [ABSTRACT FROM AUTHOR]

Bacillus possesses the peptide toxin porulation-elaying rotein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth. [ABSTRACT FROM AUTHOR]