Characterization of SLC25A38 as a Mitochondrial Pyridoxal 5′-Phosphate Transporter

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is an essential cofactor for mitochondrial enzymes involved in amino acid metabolism and heme biosynthesis. Despite its central role in cellular homeostasis, the mechanisms regulating PLP import into mitochondria remain poorly understood. The human mitochondrial carrier SLC25A38, previously annotated as a glycine transporter, has been proposed to mediate PLP transport. Indeed, silencing of SLC25A38 selectively reduces mitochondrial PLP levels (Pena et al., 2025). Moreover, several point mutations are associated with pyridoxine-refractory sideroblastic anemia (SIDBA2). We present a computational study to structurally characterize SLC25A38 as a mitochondrial PLP transporter. Structural model was generated using AlphaFold3. Binding pocket was identified using PrankWeb and validated by comparison with homologous known proteins. Ligand binding was predicted through in silico molecular docking of PLP and glycine using GNINA The apo and holo complexes were embedded in a phospholipid bilayer representative of the inner mitochondrial membrane and subjected to 1 μs molecular dynamics simulations in triplicate. Across all replicates, glycine rapidly dissociated from the transporter within 10 ns, indicating low binding stability. In contrast, PLP showed stable binding, supported by MM/PBSA analysis with an average binding energy of -68.63 ± 0.34 kcal/mol. Per-residue energy decomposition identified R96, R187, and K242 as key contributors to PLP stabilization (from -14 to -16 kcal/mol), whereas these residues showed unfavorable contributions in glycine simulations (~1 kcal/mol). Our results support the hypothesis that SLC25A38 may function as a mitochondrial PLP transporter and provide molecular insights into its proposed role.