Kinetic Mechanism of L-α-Glycerophosphate Oxidase from Mycoplasma pneumoniae Public Deposited

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  • 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 the reaction mechanism of His₆-MpGlpO follows a ping-pong model. Double-mixing stopped-flow experiments show that after flavin-mediate substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values of the individual rate constants and k [subscript]cat (4.2 s⁻¹ at 4 °C) determined, in addition to the finding that H₂O₂ can bind to the oxidized enzyme suggest that H₂O₂ release is the rate-limiting step for the overall reaction. Results indicate that His₆-MpGlpO contains mixed populations of fast and slow reacting species. Only the fast reacting species predominantly participates in turnovers. Different from other GlpO enzymes previously reported, His₆-MpGlpO can catalyze the reverse reaction of reduced enzyme and DHAP. This result can be explained by the standard reduction potential value of His₆-MpGlpO (-167 ± 1 mV), which is lower than those of GlpO from other species. We found that DL-glyceraldehyde 3-phosphate (GAP) can be used as a substrate in the His₆-MpGlpO reaction, although it exhibited a ~100-fold lower k[subscript]cat value in comparison to the reaction of Glp. These results also imply the involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His₆-MpGlpO reported here should be useful for future studies of drug development against Mycoplasma pneumoniae infection.
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  • Maenpuen, S., Watthaisong, P., Supon, P., Sucharitakul, J., Parsonage, D., Karplus, P. A., ... & Chaiyen, P. (2015). Kinetic mechanism of L–α–glycerophosphate oxidase from Mycoplasma pneumoniae. FEBS Journal, 282(16), 3043-3059. doi:10.1111/febs.13247
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