The genome of the Gram-negative bacterial species Roseovarius lutimaris DSM 28463 contains a gene encoding a protein initially predicted to function as a phosphonate transporter across the cell membrane. This protein, termed Phosphonate Transport System Substrate-Binding Protein (Rl PhnD), was the focus of this research project, aiming to elucidate its structural and biochemical characteristics. Structural predictions suggest that Rl PhnD consists of two lobes, the C-lobe and N-lobe, connected by a hinge strand. However, we find that it lacks the conserved residues typically associated with phosphonate binding. Electrostatic surface charge mapping reveals an electronegative surface surrounding the binding pocket of Rl PhnD, which may prevent phosphonate binding. The Rl phnD gene was successfully cloned, overexpressed in E. coli host cells, and purified to homogeneity. In vitro phosphonate binding assays further confirmed that Rl PhnD does not bind phosphonates. Further structural analysis revealed that Rl PhnD shares similarities with transferrin family proteins, including ferric ion binding protein FbpA, suggesting a potential role in metal ion transport rather than phosphonate transport. In vitro metal-binding studies using UV-Vis spectroscopy and nanoDSF demonstrated that Rl PhnD binds metal ions such as Fe³⁺ and other divalent and trivalent ions, with binding facilitated by HCO₃⁻ ions in the buffer. These findings indicate that the previously annotated Phosphonate Transport System Substrate-Binding Protein of Roseovarius lutimaris DSM 28463 is likely not a phosphonate transporter. Instead, it may function as a metal ion transport protein across the cell membrane.