Structural and Biochemical Characterization of SbnC as a Representative Type B Siderophore Synthetase

Staphyloferrin B is a key siderophore secreted by Staphylococcus aureus to acquire ferric ions from a host during infection, and its biosynthetic pathway has been validated to develop efficient antibacterial agents. Herein, we report the crystal structure of AMP-bound SbnC from S. aureus (SaSbnC) as the first representative structure of type B synthetases in the biosynthesis of α-hydroxycarboxylate siderophores. While type B synthetases specifically use α-ketoglutarate (α-KG) as their carboxylic acid substrate, SaSbnC showed unique structural features in the substrate pocket compared with the type A and C synthetases. Screening of α-KG analogues suggested that the hydrogen-bonding interaction between the α-carbonyl group of α-KG and residue Lys552 is a key determinant for the substrate selectivity of type B synthetases. Interestingly, citrate, the product of the tricarboxylic acid cycle and the substrate of type A synthetases, was found to inhibit the activity of SaSbnC with an IC₅₀ value of 83 μM by mimicking α-KG binding, suggesting a potential regulatory role of the tricarboxylic acid cycle, whose activity is under the control of the intracellular iron concentration, to SaSbnC and other type B synthetases. These results provide critical new information to understand the structure, function, and regulation of type B synthetases in the siderophore-based iron acquisition system employed by a large number of pathogenic microbes.