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Originally published In Press as doi:10.1074/jbc.M501052200 on March 1, 2005

J. Biol. Chem., Vol. 280, Issue 18, 17786-17791, May 6, 2005
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Functional Analysis of Active Site Residues of the Fosfomycin Resistance Enzyme FosA from Pseudomonas aeruginosa*

Zanna Beharry and Timothy Palzkill{ddagger}

From the Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas 77030

Received for publication, January 28, 2005 , and in revised form, February 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Mn2+ 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 Ser94, 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 Km 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The broad-spectrum antibiotic (1R,2S)-epoxypropylphosphonic acid (fosfomycin) is often used in the treatment of urinary tract infections (1), and in combination with other antibiotics, it has shown bactericidal activity against several antibiotic-resistant strains (2–4). Bacterial resistance to fosfomycin may be attributed to either mutations in the uptake and transport mechanisms or the presence of a fosfomycin-modifying enzyme whose product has no antibacterial activity (5–13). Four different fosfomycin-modifying enzymes have been described including FosA, FosB, FosC, and FosX. These enzymes catalyze the formation of a glutathione-fosfomycin (FosA), L-cysteine-fosfomycin (FosB), ATP-fosfomycin (FosC), or water-fosfomycin (FosX) adduct (6, 14, 15).

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). FosA is an unusual GSH transferase (GST)1 because of its high specificity for fosfomycin and the presence of a metal cofactor (5, 14). Furthermore, there is no significant sequence similarity between FosA and the cytosolic (members of the alpha, pi, mu, sigma, theta, zeta, or kappa classes) GSTs. However, the three-dimensional structure of FosA from Pseudomonas aeruginosa exhibits similarity to members of the vicinal oxygen chelate family such as glyoxalase I and the extradiol dioxygenases (19), which are characterized by the ability to bind a metal cofactor with available sites for substrate coordination (20–22).



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  		SCHEME 1
 

The crystal structure of substrate-bound FosA showed that, like other members of the vicinal oxygen chelate family, fosfomycin binds at the active site Mn2+ with distances of 2.0 Å between Mn2+ and one of the phosphonate oxygens and 2.4 Å between Mn2+ and the oxirane oxygen of fosfomycin (Scheme 1) (19). The crystal structure also revealed a number of amino acids (Thr9, Lys90, Ser94, Tyr100, and Arg119) that interact with the bound fosfomycin at the active site (Fig. 1). Thr9 has been proposed to be involved in opening of the epoxide via protonation of the oxirane oxygen (19). Lys90, Ser94, Tyr100, and Arg119 are all within hydrogen bonding distance to the phosphonate oxygens of fosfomycin. In addition, Ser94 bridges the substrate to a potassium ion that is required for maximal activity (Fig. 1) (17, 19).



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  		FIG. 1.
Amino acids within hydrogen-bonding distance to fosfomycin in the FosA dimer generated using ViewerLite (Accelrys, Inc.) and coordinates from 1LQP in the Protein Data Bank (19). All of the residues shown are in the same monomer (orange) with the exception of Thr9, which is from the adjacent monomer (cyan). Mn2+ and K+ are represented as a black and pink sphere, respectively.

 
The key step in the catalytic mechanism of GSTs is the activation of GSH and stabilization of GS– for nucleophilic attack. The "classical" GSTs utilize the hydroxyl group of a serine, threonine (theta class), tyrosine (alpha, mu, pi, and sigma classes), the side chain of an arginine (alpha class), or the thiol group of a cysteine (beta class) to interact with and thereby stabilize the thiolate anion (23, 24). The interactions between FosA and GSH have yet to be determined; however, carbon-1 of fosfomycin, to which GSH is added, is situated at the end of a short channel near the surface of the protein (Fig. 2) (19). Therefore, it is probable that the binding and activation of GSH occur in this channel. Furthermore, the residues highlighted in this channel (with the exception of Trp46) contain a hydroxyl or thiol side chain that could be involved in activating and/or stabilizing GSH/GS–.



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  		FIG. 2.
Amino acids selected for mutagenesis within the putative GSH-binding site generated using ViewerLite and coordinates from 1LQP in the Protein Data Bank (19). Manganese is represented as a black sphere. Carbon-1 of fosfomycin to which GSH is added is highlighted in green.

 
The low toxicity and broad-spectrum bactericidal activity of fosfomycin have resulted in its increased clinical use, which in turn has resulted in fosfomycin-resistant strains (7, 8). Currently, there are no inhibitors to combat FosA-mediated fosfomycin resistance. To develop effective FosA inhibitors, it will be essential to understand the catalytic mechanism and, consequently, the amino acid determinants of structure and activity. In this report, we have utilized codon randomization and selection for the ability of the resulting clones to confer fosfomycin resistance to Escherichia coli to identify amino acids in the active site of FosA that are critical for function. In addition, site-directed mutagenesis was performed and the activity of the purified proteins was determined. Amino acids were chosen for mutagenesis based on their interactions at the substrate-binding site or in the proposed GSH-binding channel. Site-directed mutagenesis of most of the substrate-binding residues resulted in completely inactive proteins, consistent with the randomization and selection results. None of the proposed GSH-binding variants significantly affected the apparent Km for fosfomycin; however, GSH binding was affected as determined by an increase in apparent Km.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and General Procedures—Fosfomycin, reduced glutathione, and 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) were purchased from Sigma. Other reagents were of the highest commercially available grade. Oligonucleotides were synthesized by Integrated DNA Technologies, Inc. Molecular biology procedures not described in detail below followed those in Sambrook et al. (25). Nucleotide sequencing was performed using an ABI3100 automated sequencer (Applied Biosystems). Protein purity was judged by SDS-PAGE with Coomassie Blue staining (26).

Cloning of fosA—The fosA gene (408 bp) was PCR-amplified with Taq polymerase (Sigma) and the primers FosNde 5'-ACGATACATATGCTTACCGGTCTCAAT-3' and FosXho 5'-ACGATACTCGAGCTAGTCGGCGAAACG-3' (with added NdeI and XhoI restriction sites underlined) or FosSac 5'-ACGATAGAGCTCATGCTTACCGGTCTCAAT-3' and FosXba 5'-ACGATATCTAGACTAGTCGGCGAAACG-3' (with added SacI and XbaI sites underlined) from P. aeruginosa genomic DNA and ligated into the multiple cloning site of pET-24a(+) (NdeI, XhoI) (Novagen) to yield plasmid pFOS2 or pTP123 (SacI, XbaI) (27) to yield plasmid pFOS1. The nucleotide sequence of pFOS1 and pFOS2 was confirmed to be the same as GenBankTM accession number NC002516.

Codon Randomization—Codon randomization was performed using overlap extension PCR (28). 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. The ligation products were transformed into TOP10F' cells (Invitrogen), and the colonies were pooled and stored at –80 °C as glycerol stocks.

Selection of Functional Clones—Functional FosA clones were selected on LB-chloramphenicol (12.5 µg/ml) agar plates containing 240 or 50 µg/ml fosfomycin. The higher concentration of fosfomycin was chosen because it is the maximum concentration where E. coli harboring the pFOS1 plasmid is able to grow, whereas the lower concentration was selected in order to identify variants with partial activity. A randomized library was grown from the corresponding glycerol stock overnight at 37 °C in LB-chloramphenicol media. The culture was diluted 10-, 100-, and 1000-fold and plated on LB-chloramphenicol-fosfomycin plates and incubated overnight at 37 °C. The fosA gene was PCR-amplified from an average of ten colonies, and the entire gene was sequenced.

Construction of FosA Variants—Individual site-directed mutagenesis of residues 9, 39, 46, 48, 90, 94, 100, and 119 to alanine was carried out using the QuikChange Multi site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Plasmid pFOS2 was used as the template with the primers listed in Table I. Replacement with alanine in the resulting plasmids was verified by nucleotide sequencing of the entire fosA gene.


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  		TABLE I
Primers used to construct FosA variants

 
Expression and Purification of Wild Type and Variant FosA Enzymes—The following procedures were used to purify wild type and all of the FosA variants. 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 1 liter of LB-kanamycin, and growth with shaking continued until A600 ~0.6. Protein expression was induced by the addition of isopropyl {alpha}-D-thiogalactopyranoside (1 mM), and after ~4 h of incubation at 37 °C with shaking (A600 ~2), cells were harvested by centrifugation (5,000 x g, 5 min, 4 °C). Cells were lysed using the reagent BugBuster (Novagen) in 50 mM Tris, pH 7.5 (buffer A), according to the manufacturer's instructions, and cell debris was removed by centrifugation (13,000 x g, 20 min, 4 °C), resulting in a pink-brown supernatant. The supernatant was loaded onto a HiTrap Q anion-exchange column (Amersham Biosciences) equilibrated in buffer A and eluted using a 0–1 M NaCl gradient. Fractions containing FosA (determined by SDS-PAGE or enzymatic activity assayed as described below) were concentrated to ~1 ml (Centriprep YM10 membrane, Amicon) and applied to a HiPrep 16/60 Sephacryl S200 size-exclusion column (Amersham Biosciences) equilibrated in buffer A containing 250 mM NaCl (buffer B). The column was eluted at a flow rate of 1 ml/min. Fractions containing FosA (determined by SDS-PAGE or enzymatic activity assayed as described below) were pooled and concentrated to ~0.5 ml and stored at –80 °C in aliquots. Approximately 10–15 mg of purified FosA (quantitated by the method of Bradford (29) using bovine serum albumin as the standard) or variant FosA was obtained per liter of E. coli culture.

FosA Activity Assay—Fosfomycin-dependent glutathione conjugation was detected spectrophotometrically using Ellman's reagent. Unless otherwise stated, assays were carried out in 50 mM HEPES, pH 8, containing 100 mM KCl (buffer C), 0.05 mM MnCl2, 10 mM fosfomycin, 15 mM GSH, and 0.8 µM monomer FosA in a final assay volume of 100 µl at 25 °C. A control reaction under identical conditions but without FosA was also performed. The reactions were allowed to incubate for 5 min followed by the addition of methanol (300 µl) to quench enzyme activity. The concentration of GSH was detected colorimetrically at 405 nm using an EL800 Universal Microplate Reader (Bio-Tek Instruments, Inc.). Assays were carried out in buffer C with Ellman's reagent (0.6 mM) and 5 µl of the methanol-quenched reaction. A standard curve was prepared using GSH. The amount of GSH conjugated was determined by subtracting the "+ FosA" value from the "control reaction" (no FosA) value. The specific activity values are reported as micromolar GSH conjugated/min/micromolar FosA monomer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Codon Randomization and Selection—Random mutagenesis by overlap extension PCR was used to generate libraries containing all of the possible amino acid substitutions at 24 positions in the fosA gene to identify active site residues essential for FosA function. The 24 codons were selected for randomization based on their proximity to the Mn2+, fosfomycin, or a putative GSH-binding site in the active site of FosA. Functional FosA variants conferring fosfomycin resistance to E. coli were selected from randomized libraries that were grown in the presence of 240 µg/ml fosfomycin (the maximum concentration of fosfomycin that E. coli harboring the pFOS1 FosA expression plasmid can survive). In addition, libraries for which only the wild type amino acid was found were also subjected to a less stringent selection on 50 µg/ml fosfomycin to identify variants with partial activity. An average of 10 clones was selected for sequencing to determine the amino acid substitutions that are allowed at each of the 24 positions chosen for randomization. The sequencing results for clones selected on 240 µg/ml fosfomycin are shown in Fig. 3 (top). Positions that do not tolerate substitutions are considered to be critical for FosA structure and function. Accordingly, 16 of the 24 positions randomized were found to be essential for the structure and function of FosA (Fig. 3, boldface), which includes the residues that furnish ligands to the Mn2+ (His7, His64, Glu110) and the K+ (Ser94, Ser98, Glu95), Arg119 and Tyr100 that are within hydrogen-bonding distance to fosfomycin, and eight residues located in a putative fosfomycin/GSH-binding channel. In all of the functional Glu95 clones selected on fosfomycin, a K90R mutation was found. The sequencing of ten clones from the nonselected Glu95 library showed a variety of substitutions (Trp2, Phe, Leu3, Ala, Asp, Pro, Glu) at position 95, but none contained an additional mutation at position 90 indicating that the substitution was not due to a systematic mutation in the library. The minimum inhibitory concentration of fosfomycin for the Glu95 K90R mutant was similar to that of wild type, indicating that the K90R mutation was not selected due to increased resistance levels.



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  		FIG. 3.
Summary of the sequencing results of clones resistant to fosfomycin. The functional amino acid substitutions identified are labeled above the amino acid position. The superscript numbers represent the number of times that amino acid was observed among the sequenced clones. The positions in boldface do not tolerate substitutions as only the wild type amino acid was selected.

 
The remaining eight positions randomized, Thr9, Tyr62, His112, Lys90, Asp105, Ser50, Arg93, and Asp97, were shown to be tolerant to substitutions, because amino acids other than wild type were found when clones selected on fosfomycin were sequenced (Fig. 1). Thr9 and Lys90, both of which are within hydrogen bonding distance to fosfomycin, clearly showed a preference for wild type or wild type-like amino acids, whereas Asp97 and Asp105 located within the fosfomycin/GSH-binding channel demonstrated a preference for substitutions other than the wild type codon. Ser50, Arg93, and His112, which are all located in the putative fosfomycin/GSH-binding channel, and Tyr62, which is a second coordination sphere ligand to the Mn2+, could be substituted by a variety of different amino acids.

The libraries of the 16 positions that did not tolerate substitutions at 240 µg/ml fosfomycin were selected at a lower concentration (50 µg/ml) to identify variants with partial activity (Fig. 3, bottom). The only positions that allowed substitutions were Cys48 and Tyr128 located in the putative fosfomycin/GSH-binding channel and the Mn2+ ligand Glu110. In each case, however, the wild type amino acid is preferred. The Thr9 random library, which showed a clear preference for the wild type amino acid under the more stringent selection, was also selected at the lower fosfomycin concentration. Because T9G and T9S were selected at the higher fosfomycin concentration, it might be expected that even more variants with partial activity would be found at a lower fosfomycin concentration; however, only the wild type amino acid was selected. This may be due to the fosfomycin concentration being below the Km of Thr9 variants with partial activity. Therefore, partially active variants are catalytically inefficient compared with wild type FosA and cannot compete in the selection process.

Site-directed Mutagenesis and Activities of FosA Variants— Based on the codon randomization results, the following residues were mutated to alanine and the resulting proteins were purified and characterized kinetically: Tyr39; Trp46; Cys48; Ser94; Tyr100; and Arg119. In addition, T9A and K90A were also constructed and purified because the randomization results suggested that the wild type codon is preferred at these positions. Tyr39, Trp46, and Cys48 were chosen for mutagenesis based on their location in a proposed GSH-binding site and the importance of these amino acids in the binding and activation of GSH in other GSTs (Fig. 2). Thr9, Tyr100, Arg119, and Lys90 were selected for mutagenesis because all are within hydrogen-bonding distance to fosfomycin and Ser94 was selected because it is a ligand to the K+.

The turnover number of wild type and variant FosA enzymes under standard assay conditions are listed in Table II. No activity could be detected in assays with K90A, Y100A, or R119A FosA variants. S94A FosA assays with purified enzyme could not be performed because, under the expression and purification conditions employed, this variant was found to be insoluble. The W46A and C48A FosA enzymes exhibited similar specific activities, which were 62 and 74% that of wild type FosA, respectively, whereas the least active variants were the Y39A and T9A enzymes with specific activities of 37 and 3% wild type, respectively. The activities of Y39A, W46A, and C48A variants located in the putative fosfomycin/GSH-binding channel do not correlate with the in vivo selection results that showed these positions to be critical for FosA structure function. This discrepancy may be attributed to the sensitivity of the in vivo selection, which is dependent on the intracellular concentration of substrates and stability of the protein in vivo among other conditions. However, the kinetics of Y39A, W46A, and C48A do lend support for a role of these residues in the binding and/or activation of GSH as described below. Previous mutagenesis studies of the FosA Mn2+ ligands demonstrated that each of the three ligands (His7, His64, Glu110) could independently be substituted with Gln and still retain significant activity (17). However, in this study, substitutions were only allowed at Glu110 and only observed at the lower fosfomycin concentration.


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  		TABLE II
Kinetic values of wild type and variant FosA enzymes

 
Table II lists the apparent Km, kcat, and catalytic efficiency values for wild type and the FosA variants for fosfomycin and GSH. The apparent Km values of wild type FosA for fosfomycin and GSH are essentially the same as that previously reported (19). For those variants within hydrogen-bonding distance to fosfomycin, kinetic data could only be obtained with T9A FosA because no activity could be detected with the other variants under the standard assay conditions employed and S94A FosA was insoluble. The T9A mutation showed the greatest effect on the binding of fosfomycin. The apparent Km of this variant for fosfomycin increased 10-fold over wild type along with a 6-fold decrease in kcat, resulting in an overall catalytic efficiency with respect to fosfomycin nearly two orders of magnitude lower than wild type. The dependence of the kinetics on the concentration of GSH was most affected by the mutation of Tyr39 to alanine. The apparent Km for GSH increased by ~4-fold, and the kcat decreased by ~7-fold, resulting in an overall decrease in catalytic efficiency by approximately an order of magnitude compared with wild type.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Fosfomycin has many advantageous pharmacological properties, and its activity alone or in combination with other antibiotics against vancomycin-resistant enterococci, quinolone-resistant E. coli, biofilms, and other antibiotic-resistant strains suggests that its usage will increase (2–4, 30–31). Resistance to fosfomycin by FosA-, FosB-, FosC-, or FosX-catalyzed alteration of the antibiotic, however, threatens its therapeutic value and demonstrates the need for effective FosA inhibitors. Currently, there are no known inhibitors of any of the fosfomycin resistance enzymes. Therefore, a thorough understanding of the structure-activity relationship of these antibiotic resistance enzymes is a necessary component in the discovery of effective therapeutics.

In this report, a combination of codon randomization and functional selection and site-directed mutagenesis was used to identify residues important for FosA function. All of the residues selected in the substrate-binding site were shown to be essential because the variants showed very little or no activity. As seen in the crystal structure of the FosA-fosfomycin complex (19), their function appears to be to properly position the substrate to promote catalysis. However, the complete lack of activity observed with the alanine-substituted variants (with the exception of T9A FosA) may suggest an additional role for these residues perhaps in proper folding and/or stability of the protein dimer.

A sequence alignment of FosA with the cysteine-dependent FosB and the FosX hydrolase (Fig. 4) shows conservation of the metal ligands (His7, His64, Glu110) along with Tyr100 and Arg119 that are within hydrogen-bonding distance to two of the phosphonate oxygens of fosfomycin in FosA. It has been reported that this arginine in FosX (Arg127) is also involved in substrate binding (32), and it most probably plays a similar role in FosB. Lys90 and Ser94, neither of which is conserved in FosB or FosX, were also demonstrated to be important (although Lys90 tolerates conservative substitutions) for FosA catalysis. These residues are also within hydrogen-bonding distance to the phosphonate oxygens of fosfomycin. These interactions may also be found in FosB and FosX but involving different amino acids. Additionally, Ser94 is apparently required for maintaining the structural integrity of FosA because mutation to alanine resulted in insoluble protein. Ser94 bridges one of the phosphonate oxygens of fosfomycin with a potassium ion that is required for maximal activity of FosA (Fig. 1) (17, 19). Interestingly, neither the cysteine conjugation reaction nor the hydration reactions catalyzed by FosB or FosX are enhanced by monovalent cations; however, both enzymes are capable of the addition of GSH to fosfomycin but at significantly lower rates than FosA (15, 32). Furthermore, only two of the five amino acids that coordinate the potassium ion in FosA (19) are conserved in FosB and FosX. One of these residues, Ser98, in FosA is within hydrogen-bonding distance to the Mn2+ ligand Glu110 and therefore may be required for activity. This may suggest that, in the evolution of the fosfomycin resistance enzymes, the structural requirements for a potassium ion-binding site were acquired specifically for optimal GSH-conjugating activity.



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  		FIG. 4.
Amino acid sequence alignment of GSH transferase FosA (6), cysteine transferase FosB (14), and fosfomycin hydrolase FosX (15) generated using the program Multialin (36). Residues known to furnish ligands to Mn2+ are indicated with an asterisk. Residues interacting with fosfomycin are indicated by (^) and those in the putative GSH-binding site are indicated by (#). Inclusion of FosC that phosphorylates fosfomycin did not show conservation of the metal ligands and thus was omitted from this alignment.

 
Thr9, which is within hydrogen-bonding distance to the oxirane oxygen in both FosA and FosX, has been suggested to play a role in catalysis by acting as a proton donor in facilitating the opening of the epoxide (19, 32). Mutation of Thr9 to alanine caused a significant decrease in activity and the affinity of the enzyme for fosfomycin, consistent with Thr9 playing a critical role in catalysis. The analogous residue in the mammalian GSTs that show conjugating activity with epoxide substrates is a tyrosine (24). This tyrosine is essential for activity, and the crystal structures of the substrate- and product-bound forms show that the side-chain hydroxyl is within hydrogen-bonding distance to the oxirane oxygen in the epoxide substrate and in the resulting ring-opened product (33). Therefore, although FosA and the mammalian GSTs show essentially no sequence similarity, both enzymes apparently utilize similar active site substrate interactions to facilitate catalysis. Thr9 is conserved in FosX, and an overlay of the FosX and FosA structures shows that this residue is in a similar position in both active sites (32). However, Thr9 is not conserved in FosB where it is replaced with leucine. There are other mechanistic differences between FosA and FosB such as a preference for Mg2+ over Mn2+ and cysteine as opposed to GSH as the thiol donor (15). Furthermore, the catalytic efficiency of FosA with GSH is an order of magnitude greater than FosB with cysteine (15), which may be the result of this leucine substitution at the Thr9 position presumably in close proximity to the oxirane oxygen.

There is no crystal structure of a GSH·FosA complex currently available. Therefore, a putative GSH-binding site has been proposed based on the position of fosfomycin in the active site. Tyr39, Trp46, and Cys48 are located in this region and were mutated to alanine in order to determine whether they affect GSH-binding and/or catalysis. Tyr39 and Cys48 were selected because of the importance of similar residues in mammalian and bacterial GSTs in GSH binding and activation (24, 34). The Y39A variant exhibited a slight decrease in affinity for GSH and showed a significant decrease (~14-fold) in catalytic efficiency. The other variants exhibited only modest changes in their kinetics. Studies of a mammalian GST showed that mutation of the tyrosine that stabilizes GS– through hydrogen bonding did not affect GSH binding but did decrease the catalytic efficiency by at least 100-fold (35). FosA shares essentially no structural homology with the mammalian GSTs, and so it is difficult to draw any conclusions by analogy. However, the results are consistent with Tyr39 being involved in GSH binding (35).

The random libraries used in this study to identify residues critical for the inactivation of fosfomycin may also prove useful for inhibitor development. As putative FosA inhibitors become available, the random libraries could be used for structure-function studies on inhibitor-binding sites as well as to rapidly assess the potential for the evolution of inhibitor resistance due to amino acid substitutions in the vicinity of the enzyme active site.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant AI32956 (to T. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX 77030. Tel.: 713-798-5609; Fax: 713-798-7375; E-mail: timothyp{at}bcm.tmc.edu.

1 The abbreviation used is: GST, glutathione S-transferase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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