The YhcH/YjgK/YiaL (DUF386) family, widely conserved across bacterial species, is involved in essential cellular processes yet remains poorly characterized. YjgK from Salmonella enterica serovar Typhimurium has drawn attention because of its potential role in biofilm formation associated with metal homeostasis, which may be critical for bacterial survival. In this study, we report the crystal structure of YjgK at 1.76 Å resolution, revealing a dimeric arrangement where each monomer consists of a jelly roll-type β-sandwich fold. This fold forms a funnel-shaped cavity, suggesting potential ligand binding. YjgK contains two zinc ions per dimer, which were identified through structural analysis and confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The zinc ions are coordinated by conserved residues (Glu62, His64, Asp69, and His128) to form a tetrahedral geometry. Structural comparisons with homologous proteins revealed significant similarities in their overall fold but distinct differences in their metal ion specificity, with YhcH binding copper and HP1029 binding zinc. Salmonella lacking YjgK increased biofilm formation, while YjgK overexpression hardly influenced biofilm formation. Our findings suggest that the zinc-binding capability of YjgK may play a key role in metal ion homeostasis, contributing to the ability of Salmonella to form biofilm in response to metal-limited environments, such as those encountered during infection. The conservation of DUF386 fold across species, along with variations in metal ion coordination, indicates functional diversification within this family.

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg²⁺ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg²⁺ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg²⁺ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg²⁺ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.