Staphyloferrin B is a hydroxycarboxylate siderophore that is crucial for the invasion and virulence of Staphylococcus aureus in mammalian hosts where free iron ions are scarce. The assembly of staphyloferrin B involves four enzymatic steps, in which SbnH, a pyridoxal 5′-phosphate (PLP)-dependent decarboxylase, catalyzes the second step. Here, we report the X-ray crystal structures of S. aureus SbnH (Sa SbnH) in complex with PLP, citrate, and the decarboxylation product citryl-diaminoethane (citryl-Dae). The overall structure of Sa SbnH resembles those of the previously reported PLP-dependent amino acid decarboxylases, but the active site of Sa SbnH showed unique structural features. Structural and mutagenesis analysis revealed that the citryl moiety of the substrate citryl- l -2,3-diaminopropionic acid (citryl- l -Dap) inserts into a narrow groove at the dimer interface of Sa SbnH and forms hydrogen bonding interactions with both subunits. In the active site, a conserved lysine residue forms an aldimine linkage with the cofactor PLP, and a phenylalanine residue is essential for accommodating the l -configuration Dap of the substrate. Interestingly, the freestanding citrate molecule was found to bind to Sa SbnH in a conformation inverse to that of the citryl group of citryl-Dae and efficiently inhibit Sa SbnH. As an intermediate in the tricarboxylic acid (TCA) cycle, citrate is highly abundant in bacterial cells until iron depletion; thus, its inhibition of Sa SbnH may serve as an iron-dependent regulatory mechanism in staphyloferrin B biosynthesis. Image 1 • SbnH is a key decarboxylase in the biosynthesis of staphyloferrin B in S taphylococcus aureus. • Cocrystal structures of Sa SbnH with cofactor, product, and citrate were determined. • Residues in substrate recognition were identified, and confirmed by mutagenesis. • Citrate can bind to the active site and efficiently inhibit the activity of Sa SbnH. • The structures enrich our understanding of the catalysis and regulation of SbnH. [ABSTRACT FROM AUTHOR]