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Originally published In Press as doi:10.1074/jbc.M414326200 on January 10, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11829-11839, March 25, 2005
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Glutathione Synthesis in Streptococcus agalactiae

ONE PROTEIN ACCOUNTS FOR {gamma}-GLUTAMYLCYSTEINE SYNTHETASE AND GLUTATHIONE SYNTHETASE ACTIVITIES*

Blythe E. Janowiak and Owen W. Griffith{ddagger}

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received for publication, December 20, 2004 , and in revised form, January 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Glutamylcysteine synthetase ({gamma}-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the {gamma}-GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae {gamma}-GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L-{gamma}-glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L-{gamma}-glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as {gamma}-GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of {gamma}-GCS-GS (total Mr 85,000) show 32% identity and 43% similarity with E. coli {gamma}-GCS (Mr 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of {gamma}-GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione (L-{gamma}-glutamyl-L-cysteinylglycine, GSH) is the main low molecular weight thiol in many Gram-negative bacteria and in virtually all eukaryotes except those few that lack mitochondria (e.g. Entamoeba histolytica and Giardia duodenalis) (1-3). In all GSH-containing species examined to date, the tripeptide is synthesized by the sequential action of {gamma}-glutamylcysteine synthetase ({gamma}-GCS1; Reaction 1; also known as glutamate cysteine ligase) and GSH synthetase (GS; Reaction 2). The two enzymes are coded by separate genes, gshA and gshB, respectively (gsh1 and gsh2, respectively, in eukaryotes). Glutathione synthesis is not known to occur in the archaebacteria and is rare among Gram-positive bacteria, being identified to date only in some species of Streptococcus, Enterococcus, Lactobacillus, and Clostridium (1, 3). Although some species of Streptococcus (e.g. Streptococcus mutans) are thought to take up GSH from their medium, R. Fahey and co-workers (1, 3, 4) reported that Streptococcus agalactiae contains GSH even when grown on GSH-depleted media.


In preliminary studies, we confirmed that carefully washed S. agalactiae cells contain GSH and showed further that crude homogenates of this species are able to carry out the ATP-dependent synthesis of {gamma}-glutamyl-{alpha}-amino[14C]butyrate from L-glutamate and L-{alpha}-amino[14C]butyrate, a standard assay for {gamma}-GCS activity. Efforts to purify {gamma}-GCS from S. agalactiae were partially successful but were ultimately limited by low enzyme abundance and the instability of the isolated protein. As an alternative, we searched for genomic sequences in S. agalactiae that were consistent with amino acid sequences in the isolated enzyme or that had homology to previously reported prokaryotic or eukaryotic {gamma}-GCS or GS genes. A single sequence coding for an 85-kDa protein was identified. The N-terminal 56-kDa region of that protein is 43% similar to Escherichia coli {gamma}-GCS. No sequences homologous to GS were identified.

The putative S. agalactiae {gamma}-GCS gene was cloned into a Qiagen pQE30 His6 tag expression vector, and the protein was expressed in E. coli and purified to near homogeneity (~98% pure). Proteins unrelated to the 85-kDa putative streptococcal {gamma}-GCS were essentially undetectable. Surprisingly, the purified streptococcal {gamma}-GCS also exhibited GS activity with a specific activity similar to that of {gamma}-GCS. The isolated protein is the first enzyme of GSH synthesis to be identified in a Gram-positive organism and is the first bifunctional {gamma}-glutamylcysteine synthetase-GSH synthetase ({gamma}-GCS-GS) to be reported in any species. A similar gene occurs in several other, mostly Gram-positive bacteria including Listeria, Clostridium, Enterococcus and Pasteurella species.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Biochemical reagents were from Sigma unless indicated otherwise. Di-L-{gamma}-glutamyl-L-cystine was synthesized from GSSG as described (5). L-{alpha}-Amino[14C]butyrate (L-{alpha}-[14C]Aba) (6) and L-buthionine-S-sulfoximine (L-S-BSO) (7) were synthesized as described previously. An expression strain of E. coli, SG13009, was obtained from Qiagen. A sequenced strain of S. agalactiae, 2603 V/R S. agalactiae, was obtained from ATCC (ATCC number BAA-611). E. coli plasmids were obtained commercially: pCR2.1 (BD Biosciences), pREP4 and pQE30 (Qiagen), and pRARE (Promega). S. agalactiae were grown with or without agitation at 37 °C in Todd Hewitt broth (Invitrogen) supplemented with 2% yeast extract (THY medium). E. coli were grown with agitation at 37 °C in 2x YT media (per L: 16 g tryptone, 10 g yeast extract, 5 g NaCl).

Methods
Purification of Endogenous S. agalactiae {gamma}-GCS-GS—Endogenous S. agalactiae {gamma}-GCS activity was isolated and partially purified from 12-liter S. agalactiae cultures by modifying protocols routinely used to purify E. coli (8), rat (9), and human (10) {gamma}-GCS. In brief, S. agalactiae were grown about 8 h (A600 ~ 1.2), and then were harvested by centrifugation (~50-g wet cell mass) and frozen at -80 °C to facilitate cell breakage. The cells were then thawed, resusupended in isolation buffer (50 mM Tris-HCl, pH 7.4, 5 mM L-Glu, 5 mM MgCl2, and 1 mM dithiothreitol), and broken by passage through a French pressure cell. The crude homogenate was clarified by centrifugation, and the supernatant was applied to a 2.5 x 20-cm column of Whatman DE-52 anion exchange resin equilibrated with the isolation buffer. After washing with isolation buffer until A280 ~ 0, S. agalactiae {gamma}-GCS-GS was eluted with a linear gradient established between 400 ml of isolation buffer and 400 ml of isolation buffer containing 0.3 M NaCl. Fractions containing {gamma}-GCS activity, as determined by ADP formation (see below), were pooled, made 5 mM in MnCl2, and applied to a 1 x 8-cm column of ATP affinity resin (C8-linked, 9-atom spacer; Sigma catalog no. A2767) that was equilibrated with isolation buffer that contained 5 mM MnCl2 instead of 5 mM MgCl2. The column was washed successively with ~50 ml of equilibration buffer and ~25 ml of the original Mg2+-containing isolation buffer. S. agalactiae {gamma}-GCS-GS was then eluted with 25 ml of the same buffer supplemented with 1 mM ATP. Fractions that contained {gamma}-GCS activity were pooled and dialyzed against 8 liter of 20 mM HEPES buffer, pH 7.8, containing 1 mM EDTA.

Construction of Plasmids for Expression of S. agalactiae {gamma}-GCS-GS—Genomic DNA was isolated from S. agalactiae as described previously (11), and the putative {gamma}-GCS-GS gene (SAG1821) was isolated by PCR using a nested primer approach. Thus, a fragment containing the {gamma}-GCS-GS gene and ~100 bp of flanking sequences was first amplified using 5'-GATTAATAAGATTGGACTCAAAAG-3' and 5'-ATTATGAGAATTTTTGGAATAGCG-3' as primers. That PCR product was then inserted into a TOPO cloning vector, pCR2.1. Accuracy of the resulting plasmid was confirmed by DNA sequencing, and it was then used as a template in a second PCR step in which primers 5'-CGCGAGATCTCATGATTATCG-3' and 5'-CGCGCTGCAGCCTAGCCTAAGGAAC-3' were used to introduce unique BglII and PstI restriction sites (underlined) at the 5'- and 3'-ends, respectively. The amplified fragment was cut and introduced into the pQE-30 expression vector immediately downstream of the His6 tag site. The insert and flanking regions were sequenced to confirm that the vector insert matched that reported for SAG1821 (12) and coded for the native protein with a N-terminal His6 tag.

Expression and Purification of S. agalactiae {gamma}-GCS—To facilitate expression, S. agalactiae {gamma}-GCS-GS was expressed in SG13009 E. coli cells that were transformed with either pREP4 or pRARE in addition to the {gamma}-GCS-GS-bearing pQE-30 plasmid. The pREP4 plasmid, containing the lacI repressor, was used to prevent {beta}-D-thiogalactopyranoside-independent expression. The pRARE plasmid contains the lacI repressor and also codes for ten tRNAs that recognize six codons known to be rare in E. coli (AUA, AGG, AGA, CUA, CCC, and GGA). The pQE-30, pREP4, and pRARE plasmids code for ampicillin, kanamycin, and chloramphenicol resistance, respectively.

Successful expression required fresh transformation of competent SG13009 [pREP4] or SG13009 [pRARE] E. coli with the {gamma}-GCS-GS-bearing pQE-30 plasmid. A single transformant colony was then used to inoculate a 50-ml starter culture in 2x YT medium supplemented with 100 µg/ml ampicillin and either 50 µg/ml kanamycin or 34 µg/ml chloramphenicol. After growing overnight, 10-ml aliquots of that culture were used to inoculate each of four 1-liter flasks of 2x YT medium supplemented with the appropriate antibiotics. Growth was at 37 °C, and expression of S. agalactiae {gamma}-GCS-GS was induced with 1 mM {beta}-D-thiogalactopyranoside when A600 was ~0.6. Following an additional 15 h of growth at 25 °C, the cells were harvested by centrifugation, and a portion of the cells (~25 g, ~50% of culture yield) was resuspended in ~50 ml of 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM L-glutamate, 5 mM MgCl2, and 5 mM {beta}-mercaptoethanol. Cells were broken by passage through a French pressure cell. Unused cells could be stored at -80 °C for subsequent enzyme isolations.

The crude homogenate was centrifuged at 143,000 x g for 75 min, and the resulting supernatant was applied to a column (2 x 8 cm) of Ni2+-NTA resin (Qiagen) equilibrated with 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM L-glutamate, 5 mM MgCl2, and 5 mM {beta}-mercaptoethanol. After washing with equilibration buffer, {gamma}-GCS-GS was eluted using the same buffer supplemented with 200 mM imidazole. Fractions were monitored for protein (A280), and those eluted with imidazole-containing buffer were assayed for {gamma}-GCS activity and GS activity using the ADP formation assays described below. Fractions showing significant amounts of both activities were pooled and dialyzed overnight at 4 °C against 8 liters of 20 mM HEPES buffer, pH 7.8, containing 1 mM EDTA. Purified enzyme was stored in dialysis buffer at 4 °C or in small aliquots at either -20 or -80 °C in dialysis buffer containing 25% glycerol. Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard (13).

Determination of Quaternary Structure of S. agalactiae {gamma}-GCS-GS—The Mr of S. agalactiae {gamma}-GCS-GS was estimated by fast protein liquid chromatography using a Superdex 200 gel filtration column (10 x 300 mm; Amersham Biosciences) equilibrated and eluted with 20 mM HEPES buffer, pH 7.8, containing 1 mM EDTA. The proteins and other molecules used to produce the standard curve were as follows: thyroglobin (670 kDa), bovine {gamma}-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.35 kDa) ("Gel Filtration Standards"; Bio-Rad). Blue dextran was included to identify the void volume. Standard and sample load volumes were 250 µl, and the flow rate was 0.5 ml/min. Fractions (1 ml) were monitored spectrophotometrically at 280 nm and at 620 nm (blue dextran). For {gamma}-GCS-GS samples, both the {gamma}-GCS and GS activities were assayed and found to co-elute.

Assay of ADP Formation by {gamma}-GCS or GS—To assay {gamma}-GCS activity, amino acid-dependent ADP formation was monitored using a pyruvate kinase- and lactate dehydrogenase-coupled assay modified from a previously described procedure (10). In brief, standard reaction mixtures (final volume 1.0 ml) contained 150 mM Tris-HCl buffer, pH 8.4, 0.2 mM EDTA, 100 mM KCl, 10 mM ATP, 40 mM MgCl2, 100 mM L-glutamate, 10 mM L-cysteine (or 25 mM L-{alpha}-aminobutyrate), 10 mM phospho(enol)pyruvate, 0.42 mM NADH, 12.5 mM ammonium sulfate, 20 IU pyruvate kinase, and 30 IU lactate dehydrogenase. Reaction mixtures were equilibrated to 37 °C, and the reaction was initiated by the addition of S. agalactiae {gamma}-GCS-GS. Background rates were determined in the absence of L-cysteine or L-{alpha}-Aba and subtracted. The rate of ADP formation was assumed to equal the rate of NADH oxidation as monitored at 340 nm ({epsilon} = 6.2 mM-1). One unit of {gamma}-GCS activity is the amount of enzyme forming 1 µmol of product/h.

Assay mixtures for determination of GS were similar to those used for {gamma}-GCS, except L-glutamate and L-cysteine (or L-{alpha}-Aba) were replaced by 25 mM L-{gamma}-glutamyl-L-cysteine (generated in situ by the addition of dithiothreitol to stock solutions of di-L-{gamma}-glutamyl-L-cystine) and 25 mM glycine. Background rates were determined in the absence of L-{gamma}-glutamyl-L-cysteine. One unit of GS is the amount of enzyme forming 1 µmol of product/h.

All kinetic studies characterizing the Km values of {gamma}-GCS and GS substrates were made using the ADP formation assays.

Assay of Dipeptide Synthesis by {gamma}-GCS and GSH Synthesis by GS—To determine dipeptide formation by the {gamma}-GCS activity of S. agalactiae {gamma}-GCS-GS, the incorporation of L-{alpha}-[14C]Aba into L-{gamma}-glutamyl-L-{alpha}-[14C]Aba or the incorporation of L-[35S]cysteine into L-{gamma}-glutamyl-L-[35S]cysteine was quantitated. Reaction mixtures contained, in a final volume of 1 ml, 150 mM Tris-HCl buffer, pH 8.4, 100 mM KCl, 0.3 mM EDTA, 10 mM ATP, 20 mM phosphoenolpyruvate, 10 mM L-glutamate, 10 mM L-{alpha}-[14C]Aba (or 2.5 mM L-[35S]cysteine), 9 units pyruvate kinase, and 1 mM aminooxyacetic acid (to inhibit contaminating transaminases). Reactions were initiated by the addition of crude homogenate or purified {gamma}-GCS-GS, and portions (100 µl) were removed at specified time points and quenched by addition to ice-cold 20 mM acetic acid (1 ml). A 1-ml portion of those solutions was applied to a small column (0.5 x 8 cm) of Dowex 1 (acetate form), and the resin was then washed with 5 column volumes (10 ml) of 20 mM acetic acid to remove unreacted radioactive starting material. Dipeptide product was then eluted with 1.5 M ammonium acetate (4 ml) and quantitated by liquid scintillation counting.

A similar method, involving the incorporation of [14C]glycine into L-{gamma}-glutamyl-L-cysteinyl[14C]glycine ([14C]GSH), was used to determine GSH formation by the GS activity (14). Reaction mixtures were identical to those described for {gamma}-GCS above, except 20 µM [14C]glycine was added and either 10 mM L-cysteine replaced radiolabeled L-cysteine or L-{alpha}-Aba (combined {gamma}-GCS and GS assay) or 25 mM L-{gamma}-glutamyl-L-cysteine (generated in situ as described above) replaced both L-glutamate and radiolabeled L-cysteine or L-{alpha}-Aba.

Synthesis of GSH by {gamma}-GCS-GS was confirmed by assaying for GSH directly. Reaction mixtures contained, in a final volume of 1 ml, 150 mM Tris-HCl buffer, pH 8.4, 0.2 mM EDTA, 100 mM KCl, 10 mM ATP, 40 mM MgCl2, 100 mM L-glutamate, 10 mM L-cysteine, 25 mM glycine, 10 mM phospho(enol)pyruvate, and 20 IU pyruvate kinase. Reactions were initiated by the addition of S. agalactiae {gamma}-GCS-GS. At set time points, 1- or 10-µl aliquots were removed from the reaction mixture and assayed immediately for GSH as described below.

Assay of GSH—Total GSH (GSH + 2x GSSG) was assayed as described previously (15) using a GSSG reductase-dependent enzymatic recycling method based on a procedure originally described by Tietze (16). Quantitation was made using a standard curve constructed with 0-5 nmol of GSH. Reaction mixtures contained, in a final volume of 1 ml, 125 mM sodium phosphate buffer, pH 7.5, 6.3 mM EDTA, 0.21 mM NADPH, 0.6 mM 5,5'-dithiobis-(2-nitrobenzoic acid), and 0.9 IU of yeast GSSG reductase. Reactions were initiated by adding aliquots of a {gamma}-GCS-GS reaction mixture (as described above) or of crude extract (as described below), and the rate of 5,5'-dithiobis-(2-nitrobenzoic acid) reduction, which is linearly proportional to total GSH added, was monitored at 412 nm.

To determine the total GSH content of bacteria, 50-ml cultures were grown in appropriate media (THY for S. agalactiae, 2x YT for E. coli used as controls) for ~18 h, and cells were harvested by centrifugation and washed twice by suspension in 1 ml of phosphate-buffered saline followed by recentrifugation. The cell pellet was then suspended in 500 µl of 20 mg/ml lysozyme in phosphate-buffered saline, and cells were broken by sonication (three 30-s pulses) on ice. Crude homogenates were clarified by centrifugation, and 20 µl of 50% 5'-sulfosalicyclic acid was added to a 200-µl aliquot of the supernatant to precipitate protein. Precipitated proteins were removed by centrifugation, and the supernatant was assayed for total GSH as described.

To confirm that S. agalactiae were able to synthesize GSH rather than simply take it up from the medium, S. agalactiae extracts were assayed for GSH levels after S. agalactiae was grown in chemically defined medium that lacked GSH (17). Other than changing the growth medium, these studies were done identically to those described above.

Inhibition of {gamma}-GCS-GS by L-S-BSO—To characterize initial binding of L-S-BSO (i.e. reversible, competitive inhibition), a Ki was determined using the standard {gamma}-GCS assay for ADP formation with L-glutamate concentrations ranging from 17 to 100 mM and L-S-BSO concentrations ranging from 0 to 10 mM. To characterize irreversible inhibition by L-S-BSO (i.e. mechanism-based inactivation), kinact, KD, and the t1/2 for inactivation were determined. In these studies, S. agalactiae {gamma}-GCS-GS was preincubated in 500-µl reaction mixtures containing 210 mM Tris-HCl buffer, pH 8.2, 0.4 mM EDTA, 140 mM KCl, 10 mM ATP, 35 mM MgCl2, and varying amounts of L-S-BSO (0.62-3.1 mM) at 37 °C. At set time points ranging from 1 to 30 min, 5-µl aliquots were removed and assayed for residual activity using the standard {gamma}-GCS assay for ADP formation. Inhibition progress curves were plotted for each concentration of L-S-BSO (log % remaining activity versus time), and the apparent kinact values were calculated from the slopes of those lines. Reciprocals of the apparent kinact values were replotted versus 1/[L-S-BSO] to establish 1/kinact (y intercept) and -1/KD (x intercept). L-Methionine-SR-sulfoximine (L-SR-MSO) was tested similarly.

Inhibition by GSH—GSH was tested as a competitive inhibitor for both the {gamma}-GCS and GS activities of S. agalactiae {gamma}-GCS-GS using the ADP formation assays. Substrate concentrations of glutamate and {gamma}-glutamylcysteine were altered to be at approximately Km levels ({gamma}-GCS: L-glutamate, 25 mM; L-cysteine, 10 mM; GS: L-{gamma}-glutamyl-L-cysteine, 5 mM; glycine, 25 mM), and GSH was added at concentrations ranging from 0 to 100 mM. GSSG was similarly tested for possible inhibition of the {gamma}-GCS activity.

Inhibition by Cystamine—Cystamine (0-100 mM) was tested as a possible inhibitor or inactivator of either the {gamma}-GCS and GS activities of S. agalactiae {gamma}-GCS-GS using a procedure similar to that used with GSH.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Evidence that S. agalactiae Cells Synthesize GSH and Contain {gamma}-GCS Activity—In preliminary studies, we confirmed that S. agalactiae, grown and washed as described under "Methods," contain substantial levels of GSH as determined by a GSSG reductase-based enzymatic recycling assay (15). Using three independent cultures, levels of total GSH (GSH + 2x GSSG) in S. agalactiae and, for comparison, E. coli were 304 ± 11 and 19 ± 3 nmol GSH/mg of protein, respectively. As reported for some other facultative anaerobes (3), GSH levels in S. agalactiae were found to be sensitive to growth conditions, being 3-fold lower in unagitated (i.e. less aerobic) cultures.

To verify that S. agalactiae are able to synthesize rather than simply take up GSH, S. agalactiae were also grown in a chemically defined medium lacking GSH (see "Methods"), and crude homogenates were assayed for total GSH. Total GSH was 327 ± 12 nmol/mg protein for three independent, agitated cultures.

{gamma}-Glutamylcysteine synthetase activity was determined in crude homogenates of S. agalactiae and E. coli using a dipeptide formation assay in which L-glutamate and L-{alpha}-[14C]Aba are converted to radiolabeled L-{gamma}-glutamyl-L-{alpha}-Aba (L-{alpha}-Aba is stable and frequently used as a surrogate for L-cysteine in established E. coli (8) and mammalian (9, 10) {gamma}-GCS assays). As shown in Fig. 1, crude homogenates of S. agalactiae form L-{gamma}-glutamyl-L-{alpha}-Aba, albeit at rates substantially less that seen with a native E. coli strain (JM105).



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  		FIG. 1.
Incorporation of L-{alpha}-[14C]Aba into L-{gamma}-Glu-L-{alpha}-[14C]Aba by crude protein extracts of S. agalactiae and E. coli as a function of time. {blacksquare}, S. agalactiae; , JM105 E. coli; {blacktriangleup}, gshA- JM105 E. coli. JM105 E. coli and gshA- JM105 E. coli4 were used as positive and negative controls, respectively.

 
Although the endogenous activity is relatively low, we were able to isolate and partially purify a protein with {gamma}-GCS activity from homogenates of S. agalactiae. In typical preparations, homogenates from ~50 g of cell paste were clarified by centrifugation and sequentially chromatographed on DEAE-cellulose and ATP affinity resins to provide up to ~20 µg of protein having estimated {gamma}-GCS specific activities of 1400 ± 800 units/mg (overall activity yields typically 1-9%). For the best preparation, the isolated protein exhibited two major bands (85 and 55 kDa) on SDS-PAGE (Fig. 2). Efforts to improve the purification protocol by adding steps useful with E. coli or mammalian {gamma}-GCS (e.g. ammonium sulfate fractionation or gel filtration chromatography) met with no success. Furthermore, the isolated enzyme was found to lose {gamma}-GCS activity quickly (>50% in 24 h) when stored at 4, -20, or -80 °C, with or without glycerol.



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  		FIG. 2.
Coomassie Blue-stained SDS-polyacrylamide gel of purification fractions for endogenous S. agalactiae {gamma}-GCS-GS. Lane 1, molecular weight markers (Bio-Rad); lane 2, crude S. agalactiae homogenate; lane 3, DEAE-cellulose column load; lane 4, DEAE-cellulose column pool; lane 5, ATP column load; lane 6, ATP column pool; lane 7, molecular weight markers. The circled band at ~85 kDa was identified as consistent with the SAG1821 S. agalactiae gene by MALDI-TOF analysis with a confidence interval of 2.50 (99%) as determined by Pro-found (Version 4.10.5; The Rockefeller University).

 
Identification and Cloning of the S. agalactiae {gamma}-GCS Gene and Expression and Purification of the Protein—Both major protein bands from the gel in Fig. 2 were subjected to in-gel trypsin digestion and MALDI-TOF analysis of the resulting fragments. The 55-kDa band could not be identified, but the 85-kDa band corresponded to a 1923-bp (641-amino acid) sequence in a 2797-bp contig (gbsa09061:s_agalactiae-strainA909) in the then partially completed S. agalactiae genome.2 While our studies were in progress, that sequence was determined to be associated with a 2250-bp ORF (750 amino acids) in the completed genome (12) and was referred to as SAG1821. In the annotation of the genome, SAG1821 was tentatively identified as coding a putative glutamate-cysteine ligase/amino acid ligase (12). Thus, the SAG1821 sequence codes an 85-kDa protein (750 amino acids) in which the N-terminal 518 amino acids (~56 kDa) show 32% identity (43% similarity) with E. coli {gamma}-GCS (~58 kDa) and the C-terminal 360 amino acids in the sequence (~40 kDa) show 24% identity (38% similarity) to E. coli D-Ala, D-Ala ligase.

As described under "Methods," the SAG1821 sequence was cloned into an E. coli expression vector, pQE30, which codes for an N-terminal His6 tag. The SAG1821-containing plasmid was transformed into SG13009 E. coli bearing either the pREP4 or pRARE plasmid. Transformed cells were grown, induced, harvested by centrifugation, and broken using a French pressure cell. Crude homogenates were clarified by high speed centrifugation, and {gamma}-GCS activity was purified by chromatography on Ni2+-NTA affinity resin (Table I). Unexpectedly, the purified enzyme was found to also catalyze the GS reaction. Since the GS specific activity is higher than reported for any previously purified GS (14, 19, 20), it is apparent that the 85-kDa protein rather than a trace impurity must account for the observed GS activity. Typical purifications using either auxiliary plasmid are summarized in Table I; the {gamma}-GCS/GS specific activity ratio was consistently 0.77 ± 0.03 in several preparations. As shown, the inclusion of pRARE seems to increase expression of S. agalactiae {gamma}-GCS-GS about 2-fold over that seen with pREP4.


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  		TABLE I
Purification of S. agalactiae His6-{gamma}-GCS-GS

 
Analyses by SDS-PAGE show the final preparations of S. agalactiae {gamma}-GCS-GS are highly pure with respect to the expected 85-kDa protein (Fig. 3). In-gel trypsin digestion and MALDI-TOF analysis of the fragments establish that the 85-kDa band corresponds to full-length S. agalactiae {gamma}-GCS-GS (with N-terminal His6 tag), whereas the two most visible trace impurity bands (70 and 55 kDa) correspond to His6-tagged N-terminal fragments of S. agalactiae {gamma}-GCS-GS. Scanning of the stained gel indicates that the 85-kDa band accounts for ~98% of the protein in the final preparations. In contrast to the native enzyme, the {gamma}-GCS (and GS) activity of the isolated His6-tagged enzyme is stable when stored as described under "Methods." Whether the improved stability is due to the His tag per se, to the higher protein concentrations obtained by expression, or to other differences in the isolation procedure is unknown.



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  		FIG. 3.
Coomassie Blue-stained SDS-polyacrylamide gel of purification fractions for S. agalactiae His6-{gamma}-GCS-GS expressed in SG13009[pRARE] E. coli. Lane 1, molecular weight markers (Bio-Rad); lane 2, Ni2+-NTA column load; lane 3, 1-µg Ni2+-NTA column pool; lane 4, 2-µg Ni2+-NTA column pool; lane 5, 4-µg Ni2+-NTA column pool. The marked band at ~85 kDa was confirmed by MALDI-TOF analysis to correspond to S. agalactiae {gamma}-GCS-GS with a confidence interval of 2.50 (99%) as determined by Pro-Found (Version 4.10.5; The Rockefeller University).

 
Determination of the Quaternary Structure of S. agalactiae {gamma}-GCS-GS—Purified S. agalactiae {gamma}-GCS-GS (2.5 mg) was chromatographed on a Superdex 200 gel filtration column, and its Mr was estimated by comparison of its elution position to that of five standards. The results indicated an apparent molecular mass of ~70 kDa, suggesting that S. agalactiae {gamma}-GCS-GS is a monomer (not shown).

Characterization of the {gamma}-GCS Activity of S. agalactiae {gamma}-GCS-GS—Dipeptide synthesis assays (see "Methods"), established that purified {gamma}-GCS-GS catalyzes both the ATP-dependent synthesis of {gamma}-glutamylcysteine from L-glutamate and L-cysteine and the synthesis of {gamma}-glutamyl-{alpha}-aminobutyrate from L-glutamate and L-{alpha}-Aba (not shown). Using the ADP formation assay, the {gamma}-GCS specific activity of S. agalactiae {gamma}-GCS-GS, determined under Vmax conditions (1290 ± 63 units/mg) (Table II) was shown to be in the range of human {gamma}-GCS (1500 units/mg) (10) and about one-half that of E. coli {gamma}-GCS (3353 ± 200 units/mg) (21).


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  		TABLE II
Comparison of Km values of {gamma}-GCS substrates between S. agalactiae {gamma}-GCS-GS and E. coli, human, and A. thaliana {gamma}-GCS enzymes

 
Table II also shows the Km values for the {gamma}-GCS substrates and lists for comparison the Km values for {gamma}-GCS substrates determined for the E. coli, human, and Arabidopsis thaliana enzymes. As shown, Km values for ATP and L-cysteine are similar to those reported for E. coli. In contrast, the Km values for L-glutamate and L-{alpha}-Aba are ~10- and ~2-fold higher, respectively, in S. agalactiae {gamma}-GCS-GS. The high Km of L-glutamate suggested that a L-glutamate analog might be the true substrate. However, activity with the higher and lower glutamate homologs, DL-{alpha}-aminoadipate and L-aspartate, was only ~10% and <1%, respectively, of that seen with L-glutamate (not shown). Several additional analogs have been tested without identifying a substrate more effective than L-glutamate.3 In addition, we confirmed that the observed high Km for glutamate is not due to the His-tagged construct, since a non-His-tagged construct expressed in gshA- JM105 E. coli4 and purified to a specific activity of ~1000 units/mg of protein exhibited a glutamate Km of 25 mM (not shown).

Characterization of the GS Activity of S. agalactiae {gamma}-GCS-GS—As demonstrated by radiolabeled glycine incorporation assays (see "Methods"), purified {gamma}-GCS-GS catalyzes the ATP-dependent synthesis of [14C]GSH from either {gamma}-glutamylcysteine and [14C]glycine or from L-glutamate, L-cysteine, and [14C]glycine (not shown). Synthesis of GSH by S. agalactiae {gamma}-GCS-GS was confirmed using the GSSG reductase-based enzymatic recycling assay described under "Methods" (Fig. 4). As shown, GSH synthesis proceeds linearly after an initial lag that presumably is due to the need to accumulate sufficient L-{gamma}-glutamyl-L-cysteine for efficient GS reaction. The attenuation of GSH formation after ~20 min is due to ATP depletion. In the absence of glycine, synthesis of {gamma}-glutamylcysteine rather than GSH occurs. Thus, under conditions similar to those used in the Fig. 4 studies, incubation of purified {gamma}-GCS-GS with ATP, L-glutamate, and L-[35S]cysteine yields L-{gamma}-glutamyl-L-[35S]cysteine as determined using small columns of Dowex 1 (see "Methods").



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  		FIG. 4.
Formation of GSH from its constituent amino acids, L-glutamate, L-cysteine, and glycine by S. agalactiae {gamma}-GCS-GS. S. agalactiae {gamma}-GCS-GS was incubated with 100 mM L-glutamate, 10 mM L-cysteine, and 25 mM glycine as described under "Methods." , complete reaction mixture; {square}, a similar reaction mixture but without glycine. Both conditions are plotted as means ± S.D. for three independent experiments.

 
S. agalactiae {gamma}-GCS-GS exhibits a GS specific activity, determined under Vmax conditions, of 2383 ± 66 units/mg (Table III), which is 2-fold higher than that of S. pombe GS (1206 ± 18 units/mg) (23), about 4-fold higher than E. coli GS (650 units/mg) (19) and about 7-fold higher than human GS (361 ± 84 units/mg) (20). Table III also shows the Km values for the GS substrates and lists for comparison Km values for the E. coli, human, and rat GS enzymes. As shown, the Km values for L-{gamma}-glutamyl-L-cysteine, L-{gamma}-glutamyl-L-{alpha}-Aba, and glycine are 2-600-fold higher in S. agalactiae than in the other species characterized. The Km value of ATP is substantially lower than that seen with E. coli but 2-fold higher than reported for human GS.


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  		TABLE III
Comparison of Km values of GS substrates between S. agalactiae {gamma}-GCS-GS and E. coli, human, and rat GS enzymes

 
As noted, the C-terminal portion of S. agalactiae {gamma}-GCS-GS has sequence homology to E. coli D-Ala, D-Ala ligase. The purified enzyme has, however, no D-Ala, D-Ala ligase activity as determined by the absence of ADP formation when the GS substrates were replaced by 1-100 mM D-Ala (not shown).

Inhibition of {gamma}-GCS-GS by GSH and Cystamine—Glutathione is a nonallosteric feedback inhibitor for all {gamma}-GCS enzymes studied to date (10, 19, 21). The ability of GSH to inhibit either the {gamma}-GCS or GS activity of S. agalactiae {gamma}-GCS-GS was investigated using GSH concentrations up to 100 mM (ADP formation assays). No significant inhibition (<5%) was seen.

Cystamine is a potent inactivator of mammalian but not E. coli {gamma}-GCS (21, 24, 25). Inhibition of the {gamma}-GCS and GS activity of S. agalactiae {gamma}-GCS-GS by cystamine were tested using concentrations up to 100 mM. No significant inhibition (<5%) was seen.

Inhibition by L-S-BSO and L-SR-MSO—Steady-state kinetic studies were carried out to determine whether L-S-BSO, the active diastereomer of BSO (7), inhibits the {gamma}-GCS activity of S. agalactiae {gamma}-GCS. The studies showed that initial L-S-BSO binding is competitive with L-glutamate and exhibits a Ki of 4.9 ± 0.2 mM. To examine the possibility that L-S-BSO is also an ATP-dependent mechanism-based inactivator of S. agalactiae {gamma}-GCS-GS, as it is with other {gamma}-GCS enzymes (7, 21, 26), the streptococcal enzyme was preincubated with L-S-BSO in the presence of MgATP, and aliquots were assayed at specific time points for residual activity. Fig. 5A shows that S. agalactiae {gamma}-GCS-GS can be fully inactivated by L-S-BSO under the conditions tested, albeit rather slowly compared with other {gamma}-GCS activities (see "Discussion"). A replot of the results from Fig. 5A allowed the initial binding equilibrium constant (KD) and the rate constant for inactivation (kinact) to be determined (Fig. 5B). As shown, KD is ~2.8 ± 0.3 mM, and kinact is 0.13 ± 0.01 min-1 (t1/2 = 5.5 ± 0.5 min).



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  		FIG. 5.
Inhibition of {gamma}-GCS activity from S. agalactiae {gamma}-GCS-GS by L-S-BSO. A, S. agalactiae {gamma}-GCS-GS (200 µg) was preincubated with L-S-BSO in 500-ml reaction mixtures containing 210 mM Tris-HCl, pH 8.4, 0.4 mM EDTA, 140 mM KCl, 10 mM ATP, 40 mM MgCl2, and varying amounts of L-S-BSO: 0 mM (), 0.62 mM ({circ}), 1.23 mM ({blacktriangledown}), 1.86 mM ({triangledown}), 2.47 mM ({blacksquare}), and 3.08 mM ({square}). At the times indicated, 10-µl portions of the reaction mixtures were assayed for residual {gamma}-GCS activity using the ADP formation assay described under "Methods." B, a replot of the results in A using a logarithmic scale in order to calculate apparent kinact values from slopes of the inactivation progress curves. The inset shows a replot of those values to establish 1/kinact as the y intercept and -1/KD as the x intercept. The values shown are means ± S.D. for three independent experiments.

 
L-Methionine-SR-sulfoximine was also investigated as a potential inhibitor or inactivator of S. agalactiae {gamma}-GCS-GS, but no significant inhibition (<5%) was detected in steady state kinetic studies using up to 80 mM L-SR-MSO. Preincubation of {gamma}-GCS-GS with MgATP and 50 mM L-SR-MSO for 60 min caused only minor and inconsistent inactivation (<10%) versus control incubations lacking L-SR-MSO.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Glutathione synthesis, catalyzed by the sequential action of {gamma}-GCS and GS, is nearly ubiquitous in eukaryotes where the tripeptide serves both directly and through enzyme-mediated reactions as an antioxidant and as a sacrificial nucleophile useful in the detoxification of reactive electrophiles (23, 27, 28). Glutathione synthesis is less common among prokaryotes, but distinct {gamma}-GCS and GS enzymes have been isolated and characterized from E. coli and several other Gram-negative species (8, 19, 29, 30). Although there is substantial evidence that GSH can serve as an antioxidant and sacrificial nucleophile in Gram-negative bacteria, the redundancy of antioxidant defenses and the limited scope of GSH S-transferases in those species suggest that the importance of GSH may not be high (31-33). For example, E. coli in which {gamma}-GCS has been knocked out exhibit no striking phenotype and show only relatively minor increases in sensitivity to a variety of oxidants (34, 35).4

Glutathione synthesis is poorly characterized in Gram-positive prokaryotes. Fahey and co-workers (1) have identified several species of Streptococcus, Enterococcus, and Listeria that contain GSH even when grown on low GSH media, but the actual synthesis of GSH has not been demonstrated, and the pathway and enzymes involved have not been characterized. There are, however, reasons to believe that GSH may have an important role in some Gram-positive bacteria. Thus, Streptococcus sp. are catalase-deficient (36), some Listeria sp. are catalase-deficient (37), and Enterococcus sp. have only weak catalase activity (38), suggesting a reliance on alternative antioxidant defenses including those requiring GSH. Consistent with this view, Caparon and co-workers (39, 40) have shown that knocking out GSH peroxidase in Streptococcus pyogenes increases sensitivity to oxidants and diminishes virulence in mice. The latter observation suggested to us that inhibitors of GSH synthesis, well known for mammalian {gamma}-GCS (26, 27, 41), might be useful for treating streptococcal infections and led us to investigate GSH synthesis in both S. pyogenes and S. agalactiae.

Although we were unable to consistently demonstrate GSH synthesis in S. pyogenes,3 both intact S. agalactiae and homogenates of those cells are clearly able to synthesize GSH as determined by a highly specific enzymatic recycling assay for total GSH and by the glutamate- and cysteine-dependent incorporation of [14C]glycine into an anionic peptide that binds to Dowex 1 and elutes under conditions previously shown to elute GSH (42). Efforts to purify {gamma}-GCS activity from S. agalactiae homogenates were made difficult by the low abundance and instability of the enzyme, but small amounts of partially purified enzyme were obtained. Those preparations established that GSH synthesis in S. agalactiae, as in all other species, proceeds through the initial synthesis of {gamma}-glutamylcysteine, and they provided sufficient amino acid sequence information to directly identify a putative {gamma}-GCS gene in S. agalactiae. Cloning and expression of that gene, SAG1821, led to the remarkable discovery that the isolated protein had both {gamma}-GCS and GS activity. Glutathione synthesis in S. agalactiae thus follows the same pathway previously characterized in eukaryotes and many Gram-negative prokaryotes but utilizes a single bifunctional enzyme that catalyzes both the ATP-dependent formation of {gamma}-glutamylcysteine from glutamate and cysteine and the ATP-dependent addition of glycine to that intermediate to form GSH. Other aspects of GSH metabolism are apparently more conventional in S. agalactiae; the genome, for example, codes for a conventional GSH S-transferase and GSSG reductase (12, 43).

With respect to their interactions with substrates and potential inhibitors, the {gamma}-GCS and GS activities of S. agalactiae {gamma}-GCS-GS show both similarities and differences with previously reported {gamma}-GCS and GS enzymes. The streptococcal {gamma}-GCS activity has significant homology with E. coli {gamma}-GCS, and its Km values for L-cysteine and ATP reflect that similarity. In contrast, {gamma}-GCS-GS has markedly lower affinity for L-{alpha}-Aba and L-glutamate. Since L-{alpha}-Aba is not a physiological substrate, low affinity for that L-cysteine surrogate is not particularly surprising; there is no obvious evolutionary pressure to preserve a cysteine active site with high affinity for L-{alpha}-Aba. Low affinity for L-glutamate, on the other hand, is initially surprising because glutamate is clearly the physiological substrate based on the relative inactivity of glutamate analogs and the observation that S. agalactiae contain genuine GSH as established by both a highly specific enzymatic recycling assay (present work) and earlier studies using high resolution HPLC to detect biological thiols (1). Interestingly, many Gram-positive bacteria, including S. agalactiae, have been reported to maintain exceptionally high intracellular concentrations of L-glutamate (60-100 mM) (44), and it is likely that those concentrations allow GSH synthesis to proceed efficiently despite the high Km for L-glutamate.

The amino acid sequence of the GS domain of {gamma}-GCS-GS is related to D-Ala, D-Ala ligase rather than GS, but previously known GS enzymes and D-Ala, D-Ala ligases all belong to the ATP-grasp superfamily and therefore have similar folds (45). The absence of significant sequence homology between the GS domain of {gamma}-GCS-GS and known GS enzymes means that there is no expectation that Vmax and substrate Km values would be similar, and, in fact, S. agalactiae GS activity exhibits a specific activity that is 2-7-fold higher than reported for known GS enzymes. Taking into account the apparently small molecular mass of the GS domain of {gamma}-GCS-GS (estimated to be 31 kDa) relative to the active subunits of known GS enzymes (E. coli, 52 kDa (19); human, 52 kDa (20); yeast, 56-63 kDa (46, 47)), the catalytic efficiency of the streptococcal GS is 5.4-18-fold greater than seen with other GS enzymes.

With respect to substrate binding, the Km value of the GS domain for ATP is within the range of values seen with known GS enzymes, but the Km values for glycine, L-{gamma}-glutamyl-L-{alpha}-Aba, and L-{gamma}-glutamyl-L-cysteine are significantly higher than seen with other GS enzymes. Again, because L-{gamma}-glutamyl-L-{alpha}-Aba is not a physiological substrate, its high Km is not intrinsically surprising. The relatively high Km for L-{gamma}-glutamyl-L-cysteine is less easily rationalized. We do note that there is no evidence in bacteria for {gamma}-glutamylcyclotransferase, an enzyme that in animals efficiently converts any accumulated {gamma}-glutamylcysteine to 5-oxoproline and cysteine (e.g. it accounts for the 5-oxoprolinuria in GS-deficient patients (48)). Absent a {gamma}-glutamylcyclotransferase, {gamma}-glutamylcysteine is apparently able to accumulate in S. agalactiae until it reaches a concentration high enough to allow the GS reaction to proceed at a good rate despite its high Km for {gamma}-glutamylcysteine. The initial lag in GSH synthesis shown in Fig. 4 is consistent with this view and suggests further that there is no facilitated transfer of {gamma}-glutamylcysteine from the {gamma}-GCS domain to the GS domain. Whether or not accumulation {gamma}-glutamylcysteine also serves some additional purpose in S. agalactiae (e.g. as a substrate for an enzyme other than GS) remains to be determined.

In mammals and E. coli, GSH synthesis is regulated in part by feedback inhibition of {gamma}-GCS by GSH (27, 28). We were surprised, therefore, to find that GSH inhibits neither the {gamma}-GCS nor the GS activity of {gamma}-GCS-GS. This observation does, however, provide a possible explanation for the observation that S. agalactiae maintain a much higher intracellular GSH concentration than E. coli (304 ± 11 nmol/mg protein versus 19 ± 3 nmol/mg protein; see "Results") despite the fact that {gamma}-GCS activity is lower in S. agalactiae homogenates. The high levels of GSH in S. agalactiae have been noted previously (43), and it may be that, since the bacteria lack catalase, it is advantageous for them to accumulate GSH. We speculate further that the high Km for L-glutamate may provide an alternative regulation of GSH synthesis if under conditions of limiting nutrients the glutamate concentration decreases to a level that limits {gamma}-GCS activity, preventing depletion of free L-cysteine. Studies addressing this possibility have not to our knowledge been carried out but would be of interest.

Cystamine, L-S-BSO and L-SR-MSO are not physiological regulators of {gamma}-GCS or GS but are well studied pharmacological inhibitors. Cystamine inhibits mammalian and protozoan {gamma}-GCS by forming a mixed disulfide with a cysteine residue in the L-glutamate binding site (24, 25, 49, 50). E. coli {gamma}-GCS lacks the active site cysteine residue and is not inhibited (8). Failure of cystamine to inhibit S. agalactiae {gamma}-GCS activity is consistent with that domain's sequence homology with the E. coli enzyme.

The reaction mechanisms of E. coli and mammalian {gamma}-GCS have been elucidated (27). In brief, after substrates bind, ATP phosphorylates the {gamma}-carboxylate of glutamate, forming enzyme-bound {gamma}-glutamylphosphate and ADP. The {alpha}-amino group of cysteine then attacks the {gamma}-carboxyl of {gamma}-glutamylphosphate, forming {gamma}-glutamylcysteine and Pi. Methionine sulfoximine, which inhibits both glutamine synthetase and {gamma}-GCS, and BSO, which is {gamma}-GCS-selective, are well studied inhibitors that exploit this mechanism (26, 41). Both sulfoximines bind initially in competition with L-glutamate with the sulfoximine S bound at the site normally occupied by the {gamma}-carboxylate of glutamate. Initial binding is typically tight, because the tetrahedral sulfoximine S mimics the tetrahedral transition state formed during the attack of the nitrogen of the second substrate (i.e. NH3 or cysteine) on {gamma}-glutamylphosphate. In addition, once MSO or BSO is bound, ATP phosphorylates the sulfoximine N to form a very tightly but not covalently bound inhibitor (mechanism-based inhibition). In all cases, the L-S-diastereomer of the inhibitor is the only isomer phosphorylated and the only one causing potent inhibition.

Although both E. coli5 and mammalian (51) {gamma}-GCS are inhibited by MSO, we find that L-SR-MSO causes no significant inhibition of S. agalactiae {gamma}-GCS activity even when preincubated with the enzyme in the presence of MgATP and the absence of L-glutamate, conditions that favor binding and phosphorylation of the inhibitor. Inhibition of {gamma}-GCS is typically better with BSO, because the S-butyl moiety partially mimics the side chain of L-{alpha}-Aba or, less effectively, L-cysteine (26, 41). Streptococcal {gamma}-GCS activity is, in fact, inhibited by L-S-BSO, albeit less effectively than E. coli or mammalian {gamma}-GCS. Binding of L-S-BSO to {gamma}-GCS-GS (KD) exhibits 42- and 350-fold lower affinity than seen with E. coli and mammalian {gamma}-GCS, respectively (Table IV), and these ratios are semiquantitatively confirmed by steady-state initial rate studies (Ki values) which show that initial binding affinity of L-S-BSO to {gamma}-GCS-GS is 75- and 2500-fold lower than seen with E. coli and rat {gamma}-GCS, respectively. The value for kinact, a measure of the rate of inhibitor phosphorylation, is 2.3- and 30-fold lower for S. agalactiae {gamma}-GCS-GS than for E. coli and rat {gamma}-GCS, respectively (Table IV). Relatively weak initial binding of L-S-BSO to S. agalactiae {gamma}-GCS may be due, in part, to the very poor affinity of the streptococcal enzyme for L-{alpha}-Aba, which, as noted, BSO mimics. A similar consideration may apply with E. coli {gamma}-GCS, which is less effectively inhibited by L-S-BSO than human {gamma}-GCS and which also uses L-{alpha}-Aba less efficiently. Much higher affinity for E. coli {gamma}-GCS is exhibited by an S-2-carboxybutyl analog of BSO (30, 52), and it will be of interest to determine whether that derivative is also a better inhibitor of {gamma}-GCS-GS.


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  		TABLE IV
Comparison of inhibition by L-S-BSO between S. agalactiae {gamma}-GCS-GS and E. coli, A. thaliana, and rat {gamma}-GCS enzymes

 
As noted, S. agalactiae {gamma}-GCS-GS is the first bifunctional GSH synthesis enzyme to be identified. A schematic of the polypeptide chain is shown in Fig. 6A in which the portions attributed to the {gamma}-GCS domain and GS domain are based on homology with E. coli {gamma}-GCS and D-Ala, D-Ala ligase, respectively. Based on those homologies, the domains overlap by about 158 amino acids, and studies are currently under way to elucidate the boundary more exactly by expression of the isolated domains.3 Both E. coli {gamma}-GCS (54) and D-Ala, D-Ala ligase are monomers (55, 56), indicating the absence of surface features resulting in quarternary structure. Consistent with this absence, gel filtration studies indicate that {gamma}-GCS-GS is also a monomer.



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  		FIG. 6.
Sequence analysis of S. agalactiae {gamma}-GCS-GS. A, schematic of S. agalactiae {gamma}-GCS-GS showing regions having homology with E. coli {gamma}-GCS (blue line) and E. coli D-Ala, D-Ala ligase (green line). B, phylogenetic tree of all currently identified putative {gamma}-GCS-GS enzymes; {gamma}-GCS-GS activity has been confirmed only in the S. agalactiae {gamma}-GCS-GS enzyme (highlighted in yellow). The scale represents 20 nucleotide substitutions (x 100) as determined by the ClustalW method (53). Sequence analysis was carried out using the Megalign program from the Lasergene software suite (DNAStar, Madison, WI), and the phylogenetic tree was constructed using NJPlot (18).

 
To investigate the possible presence of the {gamma}-GCS-GS-dependent GSH synthesis pathway in other organisms, the SAG1821 sequence was blasted against the NCBI completed and partially completed bacterial genome data bases. As shown in Fig. 6B, homologous sequences were identified in 13 additional species: S. mutans, Streptococcus suis, Streptococcus thermophilus, Haemophilus somnus, Mannheimia succiniciproducens, Pasteurella multocida, Enterococcus faecalis, Enterococcus faecium, Listeria innocua, Listeria monocytogenes, Clostridium perfringens, Desulfotalea psychrophila, and Lactobacillus plantarum. These species are not closely related. Although most of the {gamma}-GCS-GS-containing bacteria are Gram-positive organisms and are classified as firmicutes, four are Gram-negative proteobacteria and are members of the {delta}/{epsilon}-proteobacteria (i.e. D. psychrophila) or {gamma}-proteobacteria (i.e. H. somnus, M. succiniciproducens, and P. multocida). Taken together, these results indicate that {gamma}-GCS-GS enjoys a broad, albeit sparse, distribution and is coded by a novel gene, which we designate gshAB in analogy to the previous designation of the {gamma}-GCS and GS bacterial genes as gshA and gshB, respectively.

It should be noted that GSH synthesis has not yet been directly demonstrated for any organism with the gshAB gene except S. agalactiae. S. mutans, which has the gene, has, in fact, been reported to take up rather than synthesize GSH (4), a result that we have confirmed using S. mutans grown on defined chemical media.3 Whether the gshAB gene in S. mutans is inactive or only expressed under specific growth conditions is under investigation. Other organisms having a gshAB gene have not been examined, although Fahey et al. (1) have shown that L. monocytogenes, S. thermophilus, E. faecalis, E. facium, and C. perfringens contain GSH, as determined by HPLC of its bimane derivative.

Recent analyses indicate that prokaryotic and eukaryotic {gamma}-GCS enzymes along with the mechanistically related glutamine synthetases represent a {gamma}-GCS and glutamine synthetase superfamily composed of four {gamma}-GCS families and three glutamine synthetase families (57). By that analysis, the putative {gamma}-GCS-GS enzymes group into the prokaryote III {gamma}-GCS family but are distinct from previously identified members of that group (Fig. 7). A recent analysis of the evolution of GSH biosynthesis genes proposes that {gamma}-GCS enzymes originated from a single gene in cyanobacteria and diverged by lateral gene transfer to other organisms (58). In keeping with this view, we speculate that the {gamma}-GCS-GS group separated from the remaining prokaryote III {gamma}-GCS enzymes by an early lateral gene transfer event, acquired its GS domain, and then diverged further by additional lateral gene transfer among the unrelated species in which it is now present.



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  		FIG. 7.
Phylogenetic tree showing the four families of {gamma}-GCS enzymes that appear to originate from a common ancestor. The proposed bifunctional {gamma}-GCS-GS family (boxed in red, S. agalactiae {gamma}-GCS-GS highlighted in yellow) are related most closely to a prokaryotic family containing mostly {gamma}-proteobacteria. The scale represents 100 nucleotide substitutions (x 100) as determined by the ClustalW method (53). Sequences were obtained from the NCBI data base, and sequence analysis was carried out using the Megalign program from the Lasergene software suite (DNAStar, Madison, WI), and the phylogenetic tree was constructed using NJPlot (18).

 
In contrast to the {gamma}-GCS enzymes, prokaryotic and eukaryotic GS sequences are so divergent that it is uncertain whether they are homologous or products of convergent evolution (58). As noted, the GS domain of {gamma}-GCS-GS is homologous to known D-Ala, D-Ala ligase sequences but is only weakly related to any known GS. Fig. 8 shows that the GS domain of the {gamma}-GCS-GS family can be grouped with the prokaryotic GS sequences but only as a distinct branch that diverges very early. We believe it is likely that the GS domain of {gamma}-GCS-GS was, in fact, acquired by gene duplication of D-Ala, D-Ala ligase and that it evolved into a GS activity after {gamma}-GCS-GS separated from the other prokaryotic III family {gamma}-GCS enzymes as shown in Fig. 7.



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  		FIG. 8.
Phylogenetic tree showing the two distinct families of GS enzymes, separated into (mostly) eukaryotes (top bracket) and prokaryotes (lower bracket). The proposed bifunctional enzyme family, {gamma}-GCS-GS sequences (boxed in red, S. agalactiae {gamma}-GCS-GS highlighted in yellow) cluster together and form a unique family related distantly to the prokaryotic GS enzymes. The scale represents 100 nucleotide substitutions (x 100) as determined by the ClustalW method (53). Sequences were obtained from the NCBI data base, sequence analysis was carried out using the Megalign program from the Lasergene software suite (DNAStar, Madison, WI), and the phylogenetic tree was constructed using NJPlot (18).

 
Finally, we note that the discovery of a unique bifunctional GSH synthesis enzyme in a group that includes potentially pathogenetic bacteria may offer a novel target for antibiotic development. Because both the {gamma}-GCS and the GS domain sequences of {gamma}-GCS-GS are unrelated to the corresponding human sequences, it should be possible to design inhibitors selective for the bacterial activities. The observation that the absence of GSH peroxidase reduces virulence of S. pyogenes (39, 40) encourages the view that pharmacological ablation of GSH, a required GSH peroxidase substrate, would also reduce virulence, presumably by diminishing the bacteria's ability to resist peroxide oxidants generated by an immune response.


   FOOTNOTES
 
* The studies reported were supported in part by an American Heart Association Predoctoral Fellowship Award (to B. E. J). A preliminary account of these studies was presented in March 2004 at the Oxygen Club of California World Congress in Santa Barbara, CA.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 Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226. Tel.: 414-456-8778; Fax: 414-456-6510; E-mail: griffith{at}mcw.edu.

1 The abbreviations used are: {gamma}-GCS, {gamma}-glutamylcysteine synthetase; GS, GSH synthetase; L-S-BSO, L-buthionine-S-sulfoximine; L-SR-MSO, L-methionine-SR-sulfoximine; L-{alpha}-[14C]Aba, L-{alpha}-Amino[14C]butyrate; NTA, nitrilotriacetic acid; HPLC, high pressure liquid chromatography. Back

2 H. Tettelin, V. Masignani, M. J. Cieslewicz, J. A. Eisen, S. Peterson, M. R. Wessels, I. T. Paulsen, K. E. Nelson, I. Margarit, T. D. Read, L. C. Madoff, A. M. Wolf, M. J. Beanan, L. M. Brinkac, S. C. Daugherty, R. T. DeBoy, S. Durkin, J. F. Kolonay, L. A. Umayam, R. Madupu, M. R. Lewis, D. Radune, N. B. Fedorova, D. Scanlan, H. Khouri, S. Mulligan, H. A. Carty, R. T. Cline, J. Gill, M. Scarselli, M. Mora, E. T. Iacobini, C. Brettoni, G. Galli, M. Mariani, F. Vegni, D. Maione, D. Rinaudo, R. Rappuoli, J. L. Telford, D. L. Kasper, G. Grandi, and C. M. Fraser (2002) direct submission to the Institute for Genomic Research (Rockville, MD). Back

3 B. E. Janowiak and O. W. Griffith, unpublished results. Back

4 M. A. Hayward and O. W. Griffith, unpublished results. Back

5 B. S. Kelly and O. W. Griffith, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Michael A. Hayward for technical assistance and thank Drs. Dara Frank (Medical College of Wisconsin), Francis Peterson (Medical College of Wisconsin), and Michael Caparon (Washington University, St. Louis, MO) for helpful advice and discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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