Abstract
The metalloglutathione transferase FosA catalyzes the conjugation of glutathione to carbon-1 of the antibiotic fosfomycin, rendering it ineffective as an antibacterial drug. Codon randomization and selection for the ability of resulting clones to confer fosfomycin resistance to Escherichia coli were used to identify residues critical for FosA function. Of the 24 codons chosen for randomization, 16 were found to be essential because only the wild type amino acid was selected. These included ligands to the Mn(2+) and the K(+), residues that furnish hydrogen bonds to fosfomycin, and residues located in a putative glutathione/fosfomycin-binding site. The remaining eight positions randomized were tolerant to substitutions. Site-directed mutagenesis of some of the essential and tolerant amino acids to alanine was performed, and the activity of the purified proteins was determined. Mutation of the residues that are within hydrogen bonding distance to the oxirane or phosphonate oxygens of fosfomycin resulted in variants with very low or no activity. Mutation of Ser(94), which bridges one of the phosphonate oxygens with a potassium ion, resulted in insoluble protein. The Y39A mutation in the putative glutathione-binding site resulted in a 4-fold increase in the apparent K(m) for glutathione. Only two of the amino acids in the substrate-binding site are conserved in the related fosfomycin resistance proteins FosB and FosX, whereas no amino acids in the putative glutathione-binding site are conserved.
Highlights
The best-characterized fosfomycin-modifying enzyme is FosA, which has been shown to be a manganese-containing metalloglutathione transferase that conjugates GSH to carbon-1 of fosfomycin (Scheme 1) (14, 16 –18)
Two of the amino acids in the substrate-binding site are conserved in the related fosfomycin resistance proteins FosB and FosX, whereas no amino acids in the putative glutathione-binding site are conserved
The crystal structure of substrate-bound FosA showed that, like other members of the vicinal oxygen chelate family, fosfomycin binds at the active site Mn2ϩ with distances of 2.0 Å between Mn2ϩ and one of the phosphonate oxygens and 2.4 Å between Mn2ϩ and the oxirane oxygen of fosfomycin (Scheme 1) [19]
Summary
Reagents and General Procedures—Fosfomycin, reduced glutathione, and 5,5Ј-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent) were purchased from Sigma. PFOS1 was used as the template with the external primers FosSac and FosXba and internal complementary primers containing NNN at the codon position to be randomized for amplification of fosA gene fragments using Taq polymerase (Sigma) according to the manufacturer’s instructions. PCR products were purified, and overlapping fragments were combined and the full-length randomized fosA gene was amplified using the FosSac and FosXba primers and ligated into pTP123. A single colony of E. coli BL21 Gold (DE3) harboring pFOS2 (or variant FosA expression plasmid, Table I) was used to inoculate 50 ml of LB-kanamycin and grown aerobically overnight at 37 °C with shaking. This entire culture was used to inoculate
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