Metal ions are present in more than 30% of proteins in living organisms and play essential roles in enzyme catalysis by stabilizing substrates, reaction intermediates, and transition states. Notably, in several enzyme families, metal ions have been observed to occupy multiple spatial positions depending on the catalytic stage. However, the molecular principles governing metal ion relocation within the enzyme interior remain poorly understood. Pyruvate aldolase from Achromobacter xylosoxidans (AxADL) catalyzes the Mg²⁺-dependent condensation of formaldehyde and pyruvate to produce 2-keto-4-hydroxybutyrate (2-KHB), with optimal activity at pH 9.0 and 50 °C. Crystal structure analysis reveals that AxADL adopts a canonical triosephosphate isomerase (TIM)-barrel fold and forms a hexamer via C-terminal α-helix exchange between two trimers. Each protomer contains a deep, solvent-accessible active-site pocket lined with highly conserved residues critical for substrate recognition and catalysis. Structural comparison of the Mg²⁺-bound and Mg²⁺/pyruvate-bound complexes reveals two distinct magnesium-binding sites separated by approximately 2.5 Å. Upon substrate binding, the magnesium ion reorganizes the active-site architecture, repositioning itself to directly coordinate the substrate and to create an environment favorable for enolate intermediate formation. Together with ongoing computational analyses and complementary structural and biochemical studies, these findings provide a foundation for elucidating the mechanistic principles underlying dynamic metal ion positioning in enzymes.