Fosfomycin Resistance Proteins: A Nexus of Glutathione Transferases and Epoxide Hydrolases in a Metalloenzyme Superfamily
Introduction
Fosfomycin, (1R, 2S)‐epoxypropylphosphonic acid, is a broad‐spectrum antibiotic produced by certain strains of Streptomyces (Hendlin, 1969). The antibiotic functions by covalent modification of the enzyme (UDP‐N‐acetylglucosamine‐3‐enolpyruvyl transferase or MurA) that catalyzes the first committed step in peptidoglycan biosynthesis (Kahan, 1974). Shortly after its introduction into the clinic, plasmid‐mediated resistance to the drug was observed (Llaneza 1985, Mendoza 1980, Villar 1986). The resistance gene was subsequently established to encode a 16‐kDa polypeptide (FosA) that catalyzed the addition of glutathione (GSH) to the antibiotic rendering it inactive (Fig. 1) (Arca 1988, Arca 1990). A detailed biochemical analysis revealed that FosA was a Mn(II)‐dependent metalloenzyme with a requirement for K+ for optimum catalytic activity (Bernat 2001, Bernat 1997, Bernat 1999) Sequence analysis and the three‐dimensional structure of FosA places the protein in the vicinal oxygen chelate (VOC) superfamily of proteins, which includes extradiol dioxygenases, glyoxalase I, and methylmalonyl‐CoA epimerase (Armstrong 2000, Pakhomova 2004, Rife 2002).
Queries of microbial genome sequences indicated the existence of several homologs of FosA. For example, Pseudomonas aeruginosa harbors a gene encoding a FosA homologue (FosAPA) that shares a high degree (60%) of sequence identity with the plasmid‐encoded protein described previously. Structural and mechanistic analysis of this protein established that it catalyzes the Mn(II) and K+‐dependent addition of GSH to fosfomycin and confers resistance to fosfomycin in the biological context of Escherichia coli (Rife 2002, Rigsby 2004).
Several other more distantly related FosA homologs that exhibit 30–40% sequence identity led to the discovery of two additional mechanistically distinct classes of fosfomycin resistance proteins. The genomes of Bacillus subtilis and Staphylococcus aureus and related microorganisms encode proteins that catalyze the addition of L‐cysteine to fosfomycin (Fig. 1) (Cao et al., 2001; Rigsby and Armstrong, unpublished results). These enzymes, called FosB, require Mg2+ for optimum activity and do not show a dependence on K+ or any other monovalent cation. The evolution of this distinct activity was driven in part by the fact that these microorganisms do not make GSH. The FosB enzymes characterized to date seem to be genuine fosfomycin resistance proteins in that they confer measurable resistance to the antibiotic in Escherichia coli even though the concentration of L‐cysteine in this organism is quite low (Rigsby and Armstrong, unpublished results).
A third class of resistance proteins, FosX, has been shown to have fosfomycin hydrolase activity (Fig. 1). Fosfomycin hydrolases encoded in the genomes of Mesorhizobium loti and the pathogen Listeria monocytogenes have been characterized with respect to their structure and mechanism (Fillgrove et al., 2003). The regiochemistry of the addition of water is the same as that observed for FosA and FosB. Like FosA, these two proteins require Mn(II) for catalysis. However, neither requires a monovalent cation for optimal activity. The enzyme from M. loti (FosXML) is not a very good resistance protein because of its low catalytic activity. However, FosXML is unique in that it is catalytically promiscuous having both GSH transferase and fosfomycin hydrolase activities, although it does neither reaction very well. The gene encoding FosXML is part of a putative phn operon in this organism that is presumably involved in phosphonate metabolism. Although the biological role of FosXML is probably not resistance to the antibiotic, it has been proposed to be a possible progenitor of the fosfomycin resistance proteins (Fillgrove et al., 2003). In contrast, FosXLM from Listeria monocytogenes is a very efficient fosfomycin hydrolase that confers robust resistance to the antibiotic in E. coli.
This chapter reports the procedures for expression and purification of FosA, FosB, and FosX proteins from six microbial sources. The quantification of enzyme activities by a fluorescence‐detected high‐performance liquid chromatography (HPLC) assay (FosA and FosB) and a gas chromatography‐mass spectrometry (GC‐MS) (FosX) assay are described. In addition, a rapid semiquantitative assay of FosA and FosX activities by 31P‐NMR is reported. The chapter concludes with the kinetic constants and biological activity for the six resistance proteins.
Section snippets
Plasmid‐Encoded FosA from Transposon TN2921 (FosATN) (Bernat et al., 1997)
The TN2921 coding sequence flanked by NdeI and EcoRI restriction sites was synthesized from multiple oligonucleotides by successive rounds of polymerase chain reaction (PCR) and confirmed by sequencing as described previously (Bernat et al., 1997). The final PCR product was ligated into pET20b(+) expression vector. Protein was overexpressed in E. coli BL21(DE3) cells transformed with this plasmid. Bacteria were grown in LB media containing 100 μg/ml ampicillin at 37° to an OD600 = 1.0 before
Assay of FosA and FosB by Fluorescence‐Detected HPLC (Bernat et al., 1997, 1998; Cao et al., 2001)
Activity assays for FosA and FosB rely on the detection of the free amino group of the peptide or amino acid adduct of the antibiotic after derivatization with the fluorescent reagent 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate (AQC) and separation of the derivatives by reversed‐phase HPLC. For FosA activity assays, typically 50–150 nM enzyme was preincubated with MnCl2 and KCl in 20 mM TMA/HEPES, pH 8.0, before mixing with glutathione (GSH). Reactions were equilibrated to 25° and were
Physical and Catalytic Characteristics
The physical and catalytic characteristics of the fosfomycin resistance proteins described in this article are listed in Table I. All of the proteins are dimers in solution. There is a correlation between the kcat/KMfos and the biological efficacy of the enzyme in conferring resistance to fosfomycin in E. coli.
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