PURIFICATION OF A GLUTATHIONE S-TRANSFERASE THAT MEDIATES FOSFOMYCIN RESISTANCE IN BACTERIA

The enzyme that modifies fosfomycin by formation of an adduct with glutathione was purified 12-fold with a 56% activity yield by passage through DEAE Sephacel and high-performance liquid chromatography molecular exclusion columns. Its functional form was a homodimer of two 16,000-dalton polypeptides, which possibly showed an antiparallel α tertiary structure and which lacked marked hydrophobic regions. Visualization of the reaction was achieved by precolumn derivatization of glutathione and the adduct, separation by high-performance liquid chromatography, and fluorescence detection of both compounds. Temperature and pH optima were 20 to 30°C and 8.25, respectively; Mn2+, Fe2+, and Co2+ enhanced the rate of modification; and K(m) values were 9.4 and 11 mM for fosfomycin and glutathione, respectively. Phosphoenolpyruvate did not interfere with fosfomycin modification. The enzyme was stable at 4°C for at least 6 months but progressively lost its activity upon being heated for 60 min at temperatures over 30°C.