L-α-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-α-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H₂O₂). The catalytic properties of the recombinant His₆-GlpO from Mycoplasma pneumoniae (His₆-MpGlpO) were investigated with transient and steady-state kinetics and ligand binding. The results indicate that...