Purification of a glutathione S-transferase that mediates fosfomycin resistance in bacteria

Abstract

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 a tertiary structure and which lacked marked hydrophobic regions. Visualization of the reaction was achieved by precolunm 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 Km values were 9.4 and 11 mM for fosfomycin and glutathione, respectively. Phosphoenolpyruvate did not interfere with fosfomycin modification. The enzyme was stable at 40C for at least 6 months but progressively lost its activity upon being heated for 60 min at temperatures over 300C.Resistance to fosfornycin, an antibiotic that inhibits the synthesis of peptidoglycan by blocking the formation of N-acetylmuramic acid (11), is exerted by three mechanisms; two of them are located on the chromosome and the third is of plasmid origin. Chromosomal mutants either lack a functional uptake system for L-a-glycerophosphate or glucose-6-phosphate (10, 25) or have a phosphoenolpyruvate:UDP-N-acetylglucosamine enolpyruvyl transferase (pyruvil transferase), which discriminates between phosphoenolpyruvic acid and fosfomycin (26). Plasmid-encoded resistance to fosfomycin (17) is exerted by a gene that is located in a transposon (8,19) and that encodes a 16-kilodalton protein that is located in the cytoplasm; it also encodes constitutive synthesis (7,27). The gene is present in all strains of both environmental and clinical origin that have been isolated in our region of Spain. The presence of this gene was determined on the basis of conjugative transmissibility of fosfomycin resistance (1, 27).The gene product catalyzes the formation of an adduct between fosfomycin and glutathione through the opening of the epoxide ring of the antibiotic and the bonding of its C-1 to the sulfydryl group of the tripeptide cysteine (2). This results in inactivation of the antibiotic. The enzyme is thus a glutathione S-transferase that resembles those found in eucaryotes because of its specificity for glutathione as a substrate and its detoxifying function (14, 16), but it seems to differ from those found in eucaryotes because eucaryotic enzymes do not support fosfomycin modification (2).Here we report the isolation and characterization of fosfomycin:glutathione S-transferase and give preliminary results on the existence of a second enzyme which is responsible for the reaction between glutathione and 1-chloro-2,4-dinitrobenzene, which was previously reported as being carried out by the fosfomycin resistance protein in extracts of fosfomycin-resistant cells (2). MATERIALS AND METHODSBacterial strains. Escherichia coli K-12 JM83 ...

Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane

“…In the past, GST had been characterized in eukaryotes as a detoxifying enzyme. However, several recent reports concerning bacterial GSTs have appeared (1,13,24). It has been shown that GST plays a role in the assimilation of dichloromethane in Methylobacterium spp.…”

Fosfomycin

“…FosA (glutathione S-transferase), the first to be described (in 1988), is a metalloenzyme transferred through plasmids in Enterobacteriaceae. It catalyzes the reaction between glutathione and fosfomycin to an inactive adduct (102,(105)(106)(107). New subtypes, with similar structure, of the gene have been described (fosA2, fosA3, fosA4, and fosA5) (108)(109)(110).…”

Biological Costs and Mechanisms of Fosfomycin Resistance in Escherichia coli

“…Defects in one or both of the transport systems (caused by mutations in the uhpT and glpT structural genes or the regulators) can confer fosfomycin resistance (17,18,31). In addition, plasmid-encoded fosfomycin resistance conferred by an enzyme that inactivates the antibiotic has been described (3)(4)(5).…”