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J. Biol. Chem., Vol. 281, Issue 7, 4380-4394, February 17, 2006

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Characterization of the Bifunctional -Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida^*

From the Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium

Received for publication, August 29, 2005 , and in revised form, December 6, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Glutamate-cysteine ligase (-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. -ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxyl-to-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steady-state levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal -proteobacterial -ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an 85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The -ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (K_i = 13.6 mM). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Glutathione (GSH; L--glutamyl-L-cysteinylglycine) is the predominant low molecular weight peptide thiol present in many Gram-negative bacteria and in virtually all eukaryotes, except those that lack mitochondria (1, 2). Glutathione is made in a highly conserved two-step ATP-dependent biosynthesis pathway by two unrelated peptide bond-forming enzymes (3). In the first and rate-limiting reaction, -glutamate-cysteine ligase (-ECL)² (EC 6.3.2.2 [EC] ) condenses the -carboxylate of L-glutamic acid with L-cysteine to form the dipeptide -glutamylcysteine (-EC), according to

REACTION 1

where Me²⁺ can be magnesium or manganese (4, 5). In the second step, -EC is condensed with glycine in a reaction catalyzed by glutathione synthetase (GS; EC 6.3.2.3 [EC] ), according to

REACTION 2

where Me²⁺ again can be magnesium or manganese.

The activity of eukaryotic -ECL is precisely controlled by nonallosteric glutathione feedback inhibition, the limited availability of cellular L-Cys, and the transcriptional and posttranslational regulation of the expression and activity of the enzyme under various physiological conditions (6). Bacterial glutathione homeostasis is less well characterized, although glutathione feedback inhibition appears to be of major importance, as Escherichia coli expresses its -ECL constitutively (7) and because the E. coli -ECL K_i for glutathione is about 3 mM (8), a value consistent with feedback regulation at physiological glutathione levels.

Recently, the structure of the monomeric -ECL of E. coli has become available (9), which confirms its predicted structural homology with glutamine synthetases (5). By comparing the crystal structures of unliganded ligase and ligase complexed with a sulfoximine-based transition-state analog inhibitor, the cysteine binding site was identified to be formed inductively at the transition state (9). The basis for this induced fit mechanism is a conformational change of a switch loop consisting of residues 240–249. Moreover, this loop forms the starting point of a 58-amino acid stretch that covers the active site and that has been termed the central variable arm, as it varies widely among -ECL family members. Analogously, crystal structures of the unrelated dimeric GS from yeast, unliganded and complexed with its substrate -EC, the ATP analog AMP-PNP, and two magnesium ions, revealed substantial conformational changes in which the free enzyme, which adopts an open conformation, grasps the substrate and ATP analog by forming new secondary structure elements (10). Taken together, the structural studies indicate that glutathione formation requires certain conformational rearrangements in the two unrelated ligases of the pathway, which, however, do not play a role in cooperative or allosteric mechanisms. The reported negative homotropic cooperativity of rat GS toward -EC appears to be the exception to this rule (11).

GS ligases can be grouped into bacterial and eukaryotic protein families, for which pairwise sequence comparisons indicate no significant relationship (12). Crystal structures of the E. coli (13, 14), human (15), and yeast (10) GS ligases, however, adopt a similar fold, which extends across two domains, collectively referred to as the ATP-grasp fold (16). Other members of the ATP-grasp enzyme superfamily include, among others, biotin carboxylase -chain (17), succinate-CoA ligase (18), carbamoylphosphate synthetase (19), cyanophycin synthetase (CphA (20)), and D-Ala-D-Ala ligase (DdlB (21)), from which it can be appreciated that ATP-grasp enzymes carry out ATP-dependent carboxylate to amine/thiol ligase reactions in a number of unrelated biosynthetic pathways, accepting a wide variety of donor and acceptor substrates. In the course of evolution, ATP-grasp folds have therefore been independently recruited to provide the catalytic scaffold for specialized non-ribosomally encoded peptide bond-forming reactions.

Glutahione is considered to play a key role in protecting cells against oxidative toxicity, yet its production among prokaryotes appears to be largely confined to the Gram-negative cyanobacteria and proteobacteria (22). However, a number of Gram-positive bacteria, such as streptococci, enterococci, and clostridiales, collectively classified as firmicutes, have been found to accumulate glutathione (1), whereas other aerobic Gram-positive genera may produce alternative cysteine derivatives to functionally substitute for glutathione, such as streptomycetes, which accumulate the cysteine/sugar condensate mycothiol (23). Recently, Janowiak and Griffith (24) reported that glutathione synthesis occurs atypically in Streptococcus agalactiae, as they identified the functional gene responsible for glutathione accumulation as coding for a bifunctional protein catalyzing both-ECL and GS reactions. Shortly after the Janowiak and Griffith report (24), Gopal et al. (25) ascribed glutathione synthesis in Listeria monocytogenes to a similar bifunctional protein, termed GshF, which, by genetic inactivation, was shown to be essential for aerobic growth and virulence. The GshF fusions consist of a -ECL-like N-terminal domain fused to an ATP-grasp domain. Interestingly, inspection of the genome data base of lactococci, streptococci, and enterococci revealed that not all glutathione-accumulating strains possess a gshF-like sequence in their genomes (such as Lactococcus lactis and Streptococcus thermophilus) and, conversely, that Streptococcus mutans, which possesses a gshF-like gene, takes up rather than synthesizes glutathione (1, 24).

The present study originated from our interest into the role and metabolism of glutathione in Gram-negative Pasteurellaceae, which, as a consequence of their adaptation to a parasitic life style, are characterized by a reduction in genome information compared with their free-living ancestors (26). In an earlier report, we have shown that Haemophilus influenzae does not accumulate glutathione by biosynthesis but efficiently imports the tripeptide from the growth medium (27). In contrast to other Gram-negatives such as E. coli, the imported glutathione was found to be crucial for H. influenzae, as it scavenges its interior from a wide variety of peroxide chemicals (28, 29). Here, we report on the study of glutathione accumulation by Pasteurella multocida, which forms part of the normal flora in the nasopharynx of many domestic and wild animals. Most human P multocida infections are soft tissue infections caused by animal bites (30). We show that P. multocida accumulates glutathione both by import and biosynthesis. Inspection of the genome data base of P. multocida revealed the presence of a gshF homologous sequence, which was probed as a candidate for glutathione biosynthesis. We show that the P. multocida gshF gene codes for a bifunctional ligase that is functional inside E. coli and carries out complete synthesis of glutathione. Finally, purified recombinant GshF was subjected to steady-state kinetic studies to provide insights into the significance of the bifunctionality of GshF proteins.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Biochemical Reagents—All biochemical reagents used in this study were from Sigma-Aldrich unless indicated otherwise.

Strains and Media—E. coli strain K12 (wild type) was obtained from the E. coli Genetic Stock Center (New Haven, CT). E. coli strains BL21(DE3) (Novagen, Madison, WI) and TOP10 (Invitrogen) were used for the overexpression of recombinant P. multocida GshF and as host for cloning, respectively. The E. coli strains were routinely grown in 37 °C Luria-Bertani (LB) medium, supplemented with either, or a combination of, 100 µg/ml carbenicillin, 50 µg/ml kanamycin or 12.5 µg/ml chloramphenicol, as appropriate, on an orbital shaker rotating at 200 rpm. The P. multocida and S. agalactiae clinical isolates used in this study were kind gifts of Dr. Mario Vaneechoutte (Dept. of Clinical Chemistry, Microbiology, and Immunology, University Hospital, Ghent, Belgium). P. multocida were routinely grown aerobically in BHI (brain-heart infusion broth), which was prepared from a dehydrate (BD Biosciences). To study glutathione accumulation by P. multocida, cells were grown in chemically defined MIc medium, prepared as described by Herriott et al. (31). This medium contains 100 µM cystine as the source of organic sulfur. S. agalactiae was grown without agitation at 37 °C in Todd Hewitt broth (Invitrogen) supplemented with 2% yeast extract (THY media). To prepare agar plates, 1.8% agar was added to the BHI or MIc liquid growth media before autoclaving.

DNA Manipulations—All nucleic acid-modifying enzymes were obtained from New England Biolabs (Beverly, MA) unless indicated otherwise. Plasmid isolations, ligations, and transformations were performed as described (32). Plasmid DNA for ligation experiments was prepared on a 30-ml scale using the Qiagen (Crawley, UK) plasmid purification kit. The sequences of final construct inserts were routinely confirmed by DNA sequencing at GEXbyweb (GENOME express, Meylan, France). P. multocida and S. agalactiae genomic DNA was prepared using the Qiagen genomic tip-100/G protocol according to the manufacturer's instructions.

To verify the in vivo function of the -ECL·ATP-grasp fusion protein (referred to as GshF in this study), a 2,628-bp subgenomic fragment spanning the promoter and terminator regions of the P. multocida gshF gene (see Fig. 1) was amplified via PCR using P. multocida genomic DNA, Taq GoldStar® DNA polymerase (Eurogentec Ltd., Southampton, UK), and the following primer pair: forward primer 5'-GTGATTCAATTATTAGTGC-3' and reverse primer 5'-GTGTTCTTCGTCAATTGC-3'. The PCR fragments were ligated into the pGEM-T vector (Promega, Madison, WI), thereby constructing pGEM-gshF_SG.

To overexpress P. multocida GshF in an E. coli background, we amplified the gshF gene (open reading frame Pm1048) by PCR using P. multocida genomic DNA as a template, the forward primer 5'-TTCCATATGAAAATTCAACATATC-3', and the reverse primer 5'-TTACTCCAGTTCAGGAA-3', hereby introducing an NdeI site (underlined) at the 5' end. The 2,280-bp amplified product was cloned into the pGEM-T vector, and the resulting plasmid was digested with PstI. The linearized plasmid was made blunt-ended with Vent_R® DNA polymerase and was subsequently digested with NdeI. Similarly, the expression plasmid pET-11a (Novagen, Madison, WI) was digested with BamHI, blunt-ended, and digested with NdeI. Then vector and insert were ligated to each other, thereby constructing expression plasmid pET-GshF_Pm.

To overexpress S. agalactiae GshF in an E. coli background, we amplified the gshF gene (open reading frame SAG1821 and 71 bp of terminator region) by PCR using S. agalactiae genomic DNA as a template, the forward primer 5'-CAGCATATGATTATCGATCGACTGTTAC-3', and the reverse primer 5'-GCGAAGCTTCTAGTGATGACAAGGGAT-3', hereby introducing an NdeI site (underlined) at the 5' end and a HindIII (underlined) site at the 3' end. The 2,339-bp amplified product was cloned into the pGEM-T vector, and the resulting plasmid was double digested with NdeI/HindIII. Similarly, the expression plasmid pET-GshF_Pm was NdeI/HindIII double digested, and vector and insert were ligated to each other, thereby constructing expression plasmid pET-GshF_Sa.

Construction of a gshA^-gshB^-E. coli K12 Double Mutant—In E. coli, -ECL and GS are encoded by the genes gshA and gshB, respectively. The gshA^-gshB^-E. coli K12 double mutant was constructed according to the "lambda Red" method (described in Ref. 33). Briefly, PCR fragments of the kanamycin-resistance gene from the pACYC177 plasmid (Fermentas GMBH, St. Leon-Rot, Germany) were amplified with primers that contained a 50-bp 5'-end extension, identical or complementary, respectively, to nucleotides -50 to -1 or 1558 to 1608 of the E. coli K12 1557-bp gshA gene (forward primer, 5'-ACCATTACAGTTATGCTAATTAAAACGATTTTGACAGGCGGGAGGTCAATATGAGCCATATTCAACGGG-3'; reverse primer, 5'-ATTCCAGAGATGAAATTTTGGCCACTCACGAGTGGCCTTTTTCTTTTCTGTTAGAAAAACTCATCGAGCA-3'). K12 E. coli strain, harboring plasmid pKD46, which encodes arabinose-inducible -phage Red recombinase, and grown in medium containing 100 mg/liter ampicillin and 0.04% L-arabinose, was transformed by electroporation with the purified PCR product. Kanamycin-resistant clones, which had integrated the PCR product by homologous recombination in frame to the gshA gene promoter, were selected on LB agar plates supplemented with the appropriate antibiotic, grown at 37 °C, and purified at 42 °C to cure the pKD46 plasmid of the strain. The in-frame replacement of the gshA gene by the PCR product was confirmed by PCR using primers that flank the replaced gshA gene (forward primer, 5'-TGTCTGTTAGCGGGATGG-3'; reverse primer, 5'-CATCCGGGTATGATCGAC-3') (data not shown). The final gshA^-gshB^-E. coli K12 double mutant was then constructed applying an identical methodology, now using the gshA^-E. coli K12 mutant as a host for the electroporation of gshB-interrupting PCR fragments. These PCR fragments contained the chloramphenicol-resistance cassette of the pACYC184 plasmid (Fermentas GMBH) and were amplified with primers that contained a 50-bp 5'-end extension, identical or complementary, respectively, to nucleotides 1–50 or 901–951 of the E. coli 951-bp K12 gshB gene (forward primer, 5'-ATGATCAAGCTCGGCATCGTGATGGACCCCATCGCAAACATCAACATCAACAGCACCTCAAAAACACCAT-3'; reverse primer, 5'-TTACTGCTGCTGTAAACGTGCTTCGATGGCATCCATTAACATTCCGGTGACTACCAGGCGTTTAAGGGCA-3'). The insertion of the PCR product into the gshB gene was confirmed by PCR using primers that flank the interrupted gshB gene (forward primer, 5'-GAGACAACTGCGCTCACC-3'; reverse primer, 5'-GCGCTGTCACTCAGAGTC-3') (data not shown).

Recombinant Expression and Purification of P. multocida and S. agalactiae GshF—An overnight culture of E. coli strain BL21(DE3) bearing pET-GshF_Pm was used to inoculate 5 liters of carbenicillin-supplemented LB medium at a ratio of 10 ml/liter. The expression culture was incubated at 37 °C under vigorous shaking to an optical density at 600 nm (A₆₀₀) of 0.7–0.9, after which isopropyl--D-thiogalactoside was added to a final concentration of 1 mM. After being cultured for another 15 h at 37 °C, the cells were harvested by centrifugation at 4,000 x g for 20 min, at 4 °C. The cell pellets were suspended in 50 mM Tris-HCl, pH 7.4 (5 ml/liter original culture) and sonicated. The suspension was centrifuged at 15,000 x g for 20 min at 4 °C to produce cell-free extract. An identical procedure was applied to obtain recombinant S. agalactiae GshF. To purify either P. multocida or S. agalactiae recombinant GshF, crude protein solution was subjected to medium pressure chromatography using an kta-design fast protein liquid chromatography system (FPLC, Amersham Biosciences). Other chromatographic equipment was also purchased from Amersham Biosciences. The cell-free extract was filtered through a 0.22-µm filter, diluted 3-fold with 50 mM Tris-HCl, pH 7.4, containing 10 mM NaCl (Buffer A), and applied onto a Q-Sepharose FF packed HR 16/10 column pre-equilibrated with Buffer A. The column was then washed with 100 ml of Buffer A followed by elution using an increasing step gradient of 5 mM NaCl increments in Buffer A, each increment generating a 50-ml elution fraction. To the fraction containing the highest amount of GshF, NH₄(SO₄)₂ was added to a final concentration of 1 M, after which precipitated protein was removed by centrifugation (15,000 x g for 20 min at 4 °C). The supernatant was applied onto a butyl-Sepharose FF packed HR 16/10 column that had been equilibrated with Buffer C (Buffer A, with 1 M NH₄(SO₄)₂). The column was washed with Buffer C, and eluate was obtained using a linear gradient of Buffer A at a flow rate of 3 ml/min. GshF started to elute at 75% Buffer A. The fractions containing purest GshF were then applied onto a desalting column (HiPrep^TM 26/10 desalting column; 10 ml/min) pre-equilibrated with buffer A. Eluate was subsequently applied onto 0.5 ml of Source Q anion-exchange resin that had been equilibrated with Buffer A, and GshF was concentrated by a one-step elution from 0 to 300 mM of NaCl. Concentrated GshF (2 ml) was then loaded onto a Superdex G-200 (16/60) gel filtration column (120 ml total volume), pre-equilibrated with 50 mM Tris-HCl, pH 7.4, containing 100 mM NaCl. This buffer was used for down-flow elution at a rate of 1 ml/min. GshF eluted as a single peak and was purified to electrophoretic homogeneity as determined by nonreducing SDS-PAGE. GshF proteins were stored without supplements at -80 °C until use.

The native molecular masses of either P. multocida or S. agalactiae recombinant GshF were determined by gel filtration on a Superdex G-200 (16/60) column with 50 mM Tris-HCl, pH 7.4, containing 100 mM NaCl (1 ml/min). The molecular size standards for gel filtration were -amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). Blue dextran was included to identify the void volume. The concentration of purified protein was determined by the method of Bradford (34) using the Bio-Rad protein assay with bovine serum albumin as the standard.

Agar Disk Diffusion Susceptibility Assays—E. coli (supplemented with the appropriate antibiotics) or P. multocida precultures, grown overnight, were diluted 1:50 to 1:100 in LB or MIc medium, respectively, to an A₆₀₀ of 0.005 and then grown to an A₆₀₀ of 0.75 (mid-exponential phase). Using sterile cotton swabs, cells were confluently inoculated onto LB agar plates (E. coli) or MIc agar plates, either supplemented or not with 100 µM GSSG (P. multocida). Round sterile filters (5.2-mm diameter) were placed in the center of the plates and spotted with 5 µlof 3% hydrogen peroxide (H₂O₂), 0.5 M t-butyl hydroperoxide, 2.78 M methylglyoxal, or 2.0 M diamide. Zones of growth inhibition were measured (in millimeters) after 24 h of incubation at 37 °C (E. coli) in a candle extinction jar (P. multocida). The experiments were performed in triplicate; mean values are reported, with error bars representing the standard error of the mean (S.E.), and were analyzed using the unpaired t test (Graphpad Instat Software, San Diego, CA) with p < 0.05 considered significant.

Determination of Intracellular Glutathione—Overnight grown E. coli (supplemented with the appropriate antibiotics) or P. multocida precultures were diluted 1:50 to 1:100 in LB or MIc medium (either supplemented or not with 100 µM GSSG), respectively, to an A₆₀₀ of 0.005 and then grown to an A₆₀₀ of 0.25 (early exponential phase). Cells were harvested by centrifugation (7,000 x g, 5 min, 4 °C) and were washed once with phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) before being suspended in the same buffer. Cells were subsequently disrupted via sonication, and cell-free extracts were prepared by centrifugation (15,000 x g, 15 min, 25 °C). After determination of total protein, cell-free extracts were incubated at 95 °C for 15 min, and precipitated protein was removed by centrifugation (15,000 x g, 15 min, 25 °C). The sample glutathione concentrations were determined according to the GSSG reductase-based quantification assay described by Tietze (35) using a glutathione standard curve. The results are expressed as nmol of intracellular glutathione/mg of total protein. The experiments were performed in triplicate; mean values are reported with errors representing S.E.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. A genomic map of P. multocida at the Pm1048 (gshF gene) locus and schematic representations of the domain architecture of the GshF ligase and of pairwise comparisons of GshF with its nearest related proteins. Open reading frames are represented as arrows. The subgenomic fragment used in this study to complement gshA-gshB- E. coli is colored blue. The N-terminal -ECL-like and C-terminal ATP-grasp-like domains of GshF are boxed red and yellow, respectively. The question mark illustrates that the junction between the -ECL and ATP-grasp domains cannot be predicted with great certainty on the basis of pairwise sequence comparisons (see "Results" for the arguments). Proteins that are related to either the -ECL-like and ATP-grasp-like domains are represented as black arrows, and protein regions that exhibit pairwise sequence similarities, according to Altschul et al. (37), are indicated by boxes. The sequence identities of these regions with the corresponding GshF domains are depicted above the boxes as % id. The total length of the protein sequences is given in the left column. aa, amino acid(s).

To record GS activity, the standard assay reaction mixtures contained Tris-HCl buffer (200 mM, pH 8.2), -EC (1 mM), Gly (200 mM), MgCl₂ (20 mM), disodium ATP (5 mM), NaCl (100 mM), NADH (0.25 mM), PEP (2 mM), LDH (20 units/ml), PK (7 units/ml), and GshF (added last to start the reaction) in a final volume of 0.50 ml. Background rates were determined in the absence of -EC.

To record the rate of coupled glutathione formation, the standard assay reaction mixtures contained Tris-HCl buffer (200 mM, pH 8.2), sodium L-Glu (50 mM), L-Cys (2 mM), Gly (20 mM), MgCl₂ (20 mM), disodium ATP (5 mM), NaCl (100 mM), and GshF (added last to start the reaction) in a final volume of 1 ml. At 1-min intervals, 50 µl of reaction mixture was transferred to a 950-µl 200 mM phosphate-buffered solution (pH 7.5) containing 1 mM dithionitrobenzoic acid, 500 µM NADPH, and 1 unit of glutathione reductase. As these phosphate concentrations completely inhibit GshF activity (data not shown), mixing instantly blocks the peptide bond forming reactions. The increase in 412 nm absorbance was recorded for 40 s and was, according to Tietze (35), proportional to the amount of total glutathione present in the sample. The amount of glutathione formed was recalculated to micromolar levels. Curve fitting and modeling of kinetic data were performed using the Graphpad PRISM 4.0 software package (GraphPAD Software for Science).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Probing Glutathione Biosynthesis among Pasteurellaceae by Identification and Sequence Analysis of the P. multocida -ECL·ATP-grasp Fusion Protein GshF—In an earlier report, we showed that H. influenzae is not able to synthesize glutathione but instead acquires the thiol-tripeptide by import (27). To determine whether this type of glutathione acquisition is common among other Pasteurellaceae members, we analyzed the translated genome sequences (BLAST search at NCBI (37)) of Haemophilus somnus 129PT, H. somnus 2336, Haemophilus ducreyi 35000HP, Actinobacillus pleuropneumoniae serovar 1 strain 4074, Mannheimia succiniciproducens MBEL55E, and P. multocida subsp. multocida strain Pm70 for homologs of the E. coli K12 glutathione biosynthetic machinery. Although this search produced no significant hits for the GS query, -ECL homologous sequences were apparent from all but the H. ducreyi 35000HP genome. Strikingly, these identified Pasteurellaceae -ECL homologs all combine with an ATP-grasp superfamily sequence to form a fusion protein of 755 amino acids. The P. multocida -ECL·ATP-grasp fusion (Pm1048) was then BLAST-searched against the NCBI microbial genome data base and was found to be homologous to a novel class of bifunctional glutathione biosynthesis proteins, found mostly in low GC Gram-positive bacteria, which catalyze the ATP-dependent formation of glutathione from the constituent amino acids L-Glu, L-Cys, and Gly, as demonstrated recently for the -ECL·ATP-grasp fusions from Streptococcus agalactiae (24) and L. monocytogenes (25). As proposed for the L. monocytogenes -ECL·ATP-grasp fusion (25), we refer to the P. multocida -ECL·ATP-grasp fusion protein as GshF. P. multocida GshF shares 47 and 39% amino acid identity with the GshF proteins of S. agalactiae and L. monocytogenes, respectively.

View larger version (64K): [in this window] [in a new window]   FIGURE 2. Multiple amino acid sequence alignment of residues 1–480 of P. multocida GshF with monofunctional -ECL proteins. EC, Escherichia coli; Vc, Vibrio cholera; Ca, Clostridium acetobutylicum; Pm, Pasteurella multocida. The alignment was generated using T-coffee (68) and was printed using the ESPript 2.1 software package. Conserved residues are depicted in white on a red background. Physicochemically conserved residues are depicted in red. Overall conserved regions are framed in blue. Top, sequence secondary structures correspond to the E. coli -ECL crystal structure (Protein Data Bank accession code 1V4G [PDB] ). Bottom, secondary structures are predicted from the GshF amino acid sequence using the PSIPRED Protein Structure Prediction Server (69). The E. coli -ECL crystal structure (9) comprises two structural domains: a catalytic domain (residues 18–387 and 442–518) and a small domain (residues 1–16 and 388–441; helices -1, -14, and -15, colored brown). These domains are linked through a disulfide bridge (C372–C396), which is absent in the C. acetobutylicum and P. multocida sequences. Two highly variable arms cover the active site (boxed green), the N-terminal variable arm (residues 105–144) and the central variable arm (residues 240–298), both of which exhibit deletions only in the P. multocida sequence. Twelve amino acids, probably involved in substrate binding (depicted in white on a purple background), are found to be highly conserved among the aligned -ECL sequences. The residues depicted in brown at the end of the P multocida GshF sequence form the starting amino acid stretch of the ATP-grasp-like domain shown in Fig. 3.

On the basis of PSI-BLAST sequence similarity analysis, Copley and Dhillon (12) classified -ECL proteins into three groups. The first group consists primarily of sequences from -proteobacteria, the second of sequences from non-plant eukaryotes, and the third primarily of sequences from flowering plants and -proteobacteria. The N-terminal -ECL domain of GshF proteins falls within the first group of -proteobacterial sequences, although sequence similarities are rather low (ranging from 27 to 32% sequence identity) and cover only the central part of the catalytic domain of the group 1 -ECL proteins (see legend for Fig. 2). This figure shows a multiple sequence alignment of the typical group 1 -ECL sequences of E. coli and Vibrio cholera, together with the N-terminal portions of the P. multocida and S. agalactiae GshF fusions and the -ECL sequence of C. acetobutylicum. Interestingly, the nonfused C. acetobutylicum -ECL protein exhibits a higher and more overall similarity to the freely existing -proteobacterial -ECL sequences (36% overall sequence identity) than to the N-terminal -ECL-like portions of GshF proteins. On the basis of the recent crystal structure of E. coli -ECL (9), substrate-binding residues were identified of which all but one (which is physicochemically conserved) are strictly conserved in the -ECL domain of P. multocida GshF. Although the C-terminal 150 amino acid residues of the E. coli -ECL protein do not align significantly using the BLAST algorithm, complete structural conservation appears on the basis of a comparison between the observed and predicted secondary structures of the E. coli -ECL and the N-terminal -ECL domain of the P. multocida GshF, respectively (Fig. 2). On the basis of this comparison, the N-terminal 462 amino acid residues appear to fold into the -ECL domain of the P. multocida GshF fusion, leaving 295 amino acid residues to form the ATP-grasp domain.

Strikingly, these 295 C-terminal amino acid residues of GshF exhibit 34–36% sequence identity with a region enclosed in the bifunctional cyanophycin synthetase (CphA) (Fig. 1). The 857–901-amino acid CphA proteins, found mostly in cyanobacterial backgrounds, catalyze the ATP-dependent synthesis of the storage polymer multi-L-arginylpoly-L-aspartic acid (cyanophycin), thereby sequentially adding an aspartic acid residue and an arginine residue to a -Asp-Arg primer (38). These two ATP-dependent carboxyl-to-amine ligase reactions take place in the active sites of two distinct domains: a C-terminal domain, starting approximately just before the second half of the sequence, which falls into a superfamily of Rossman fold-containing peptide ligases including certain murein ligases and folyl poly--glutamate ligase; and an N-terminal domain that groups within the ATP-grasp superfamily of ATP-dependent ligases (20). A multiple sequence alignment of the "founding members" of the ATP-grasp superfamily, the D-Ala-D-Ala ligase (DdlB) sequences of E. coli K12 and Salmonella typhimurium LT2 (16), together with the N-terminal portion of the CphA enzymes of Bordetella pertussis and Anabaena variabilis and the C-terminal ATP-grasp domains of the GshF sequences of S. agalactiae and P. multocida, is presented in Fig. 3. The E. coli DdlB crystal structure revealed a three-domain organization of the ligase, of which the central (also called the lid domain) and C-terminal domains, collectively described as the ATP-grasp fold, each containing two antiparallel -strands and a loop, form the active site cleft for ATPase activity (21). The ATP-grasp fold residues of the aligned sequences shown in Fig. 3 exhibit overall similarity, although two comparable and major insertions are apparent in the C-terminal domains of the aligned CphA and GshF sequences compared with the DdlB sequences; these insertions cluster the former two proteins into a phylogenetically distinct branch of ATP-grasp fold-containing proteins (25). Nonetheless, the three loops that close over the catalytic cavity in the liganded E. coli DdlB structure are among the most conserved regions in the alignment, and, moreover, 8 of 10 DdlB MgATP^2--binding residues are physicochemically conserved in both the CphA and GshF aligned sequences.

In summary, sequence analysis and secondary structure prediction demarcate two functional modules in the P. multocida GshF sequence. The N-terminal residues 1–462 appear to fold into a complete group 1 -ECL structure, whereas the C-terminal residues 480–757 adopt the classical two-domain ATP-grasp fold. In this scenario, the two functional modules are connected via an 18 amino acid linker region.

P. multocida Cells Acquire Glutathione Both by Biosynthesis and by Import—Because the P. multocida genome contains genes encoding genuine glutathione reductase and glutaredoxin, as well as the recently discovered PGdx glutathione-dependent peroxidase (39), we expected to find glutathione in crude lysates of washed P. multocida cells. Indeed, P. multocida cells grown overnight on BHI agar plates were found to contain 72 ± 12 nmol of glutathione/mg of protein, as determined by a highly specific glutathione reductase-based enzymatic recycling assay. To verify that P. multocida is able to synthesize rather than simply take up glutathione, as has been demonstrated for its close relative H. influenzae (27), cells were grown up to the early exponential phase of growth in a chemically defined liquid medium, either supplemented or not with 100 µM GSSG, and analyzed for intracellular glutathione. The total glutathione content of GSSG-supplemented cultures (170 ± 20 nmol of glutathione/mg of protein) was found to be 7.4-fold that of cultures grown in the absence of the oxidized tripeptide (23 ± 5 nmol of glutathione/milligram of protein). These results strongly suggest that P. multocida acquires glutathione by both biosynthesis and import.

Imported Glutathione Does Not Provide a Higher Level of Protection against Excessive Oxidative Stress in P. multocida—To rationalize the observed redundancy in glutathione acquisition, we set up disk diffusion assays by which we examined the sensitivities of P. multocida cells to different oxidants in relation to the availability of external glutathione. Four different oxidants were tested, each generating a specific type of oxidative stress; H₂O₂, t-butyl hydroperoxide, diamide, and methylglyoxal generated general peroxide stress, membrane peroxidation, thiol-disulfide stress, and carbonylic stress, respectively. With the exception of methylglyoxal, the recorded growth inhibition zones for the other three oxidants were independent of available external glutathione (Fig. 4). These results suggest that glutathione acquisition by biosynthesis builds up a sufficient pool to fully protect P. multocida against oxidative stress. A recent computer simulation of the glyoxalase pathway in Leishmania infantum showed that the intracellular concentration of methylglyoxal is controlled by the rate of its formation and by the concentration of trypanothione (40), the functional counterpart of glutathione in trypanosomatids. In accordance with this model, the 7.8-fold higher levels of glutathione inside GSSG-supplemented cultures provide a higher level of protection against exogenous supranormal levels of methylglyoxal, suggesting that, in response to this type of electrophilic stress, glutathione biosynthesis inside cells grown in the absence of external glutathione is not (sufficiently) induced to produce the intracellular glutathione pools of cells that accumulate glutathione by both biosynthesis and import.

View larger version (74K): [in this window] [in a new window]   FIGURE 3. Multiple amino acid sequence alignment of residues 481–757 of P. multocida GshF with ATP-grasp fold containing D-Ala-D-Ala-ligases (DdlB) and cynophycin synthetases (CphA). Ec, Escherichia coli; St, Salmonella typhymurium; Av, Anabaena variabilis; Bp, Bordetella pertussis; Sa, Streptococcus agalactiae; Pm, Pasteurella multocida. The alignment was generated using T-coffee (68) and was printed using the ESPript 2.1 software package. Conserved residues are depicted in white on a red background. Physicochemically conserved residues are depicted in red. Overall conserved regions are framed in blue. Difference regions are shown on an orange background. Top, sequence secondary structures correspond to the E. coli DdlB crystal structure (Protein Data Bank accession code 1I0V). Bottom, secondary structures are predicted from the GshF amino acid sequence using the PSIPRED Protein Structure Prediction Server (69). The domain organization of E. coli DdlB (16, 21) is depicted by color-coded bars above its sequence, with black, dark gray, and light gray representing the N-terminal domain, the central B-domain, and the C-terminal domain, respectively. The latter two domains, which characterize the ATP-grasp fold (16), form a cleft that grasps the MgATP2- substrate with the aid of three flexible loops (framed in green), the B-loop (residues 146–152), the -loop (residues 205–221), and the J-loop (residues 273–281) (framed in green), all three of which turn out to be highly conserved among the aligned ATP-grasp-like sequences. Moreover, conserved residues known to be involved in MgATP2- binding in structurally characterized ATP-grasp enzymes are indicated in white on a purple background, of which 8 of 10 are also conserved in the GshF sequences. Following the -loop, GshF as well as CphA sequences exhibit similarly large insertions, which clusters them into a phylogenetically distinct branch of ATP-grasp proteins (25).

View larger version (55K): [in this window] [in a new window]   FIGURE 4. Disk diffusion susceptibility assays to study the effect of exogenous glutathione on oxidative stress tolerance of P. multocida. Zones of growth inhibition were plotted (in millimeters) as mean values ± S.E. The differences in susceptibility between cells grown in the presence of glutathione and cells grown in the absence of exogenous tripeptide were only considered significant (unpaired t test, p < 0.05) for the methylglyoxal experiment. t-BOOH, t-butyl hydroperoxide.

Characterization of the Individual -ECL and GS Activities of the Bifunctional GshF Protein of P. multocida—By using the steady-state coupled enzyme ATPase assay (36), which couples the rephosphorylation of the ADP product to the decomposition of NADH, the ATP-dependent synthesis of -EC from L-Glu and L-Cys, as well as the ATP-dependent synthesis of glutathione from -EC and Gly, was demonstrated to be catalyzed by the GshF bifunctional protein of P. multocida. Next, the kinetic parameters were determined for both individual activities (summarized in Table 2). For comparison, Table 2 also includes the corresponding values for the S. agalactiae GshF (24) together with those of the E. coli monofunctional counterparts (4, 42). It is interesting to note that no deviations from linearity were apparent in Eadie-Hofstee plots of the initial velocity data, indicating that both peptide bond-forming reactions are catalyzed by the bifunctional GshF without the involvement of cooperative mechanisms.

View this table: [in this window] [in a new window]   TABLE 2 Steady-state kinetic constants of the individually assayed -ECL and GS activities of the bifunctional GshF glutathione synthetase of P. multocida (Pm) Both activities were assayed on the basis of ADP formation as described under "Materials and Methods." The counterpart values for the homologous GshF protein from S. agalactiae (Sa) (24) and for the monofunctional -ECL (4) and GS (42) ligases from E. coli strain B (Ec) are given for comparison.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Native molecular weight determination of the recombinant GshF proteins of P. multocida (black line) and S. agalactiae (green line). See "Materials and Methods" for experimental details. P. multocida and S. agalactiae GshF enzymes eluted as 220- and 191-kDa complexes, which points to a dimeric quaternary structure. Inset, SDS-PAGE analysis of P. multocida GshF, before (track 1, 150 µg of crude cell-free lysate proteins from a GshF overexpression culture) and after (track 2, 5.25 µg of purified GshF) the four-step purification protocol described under "Materials and Methods."

Glutathione Feedback Inhibition of the P. multocida GshF-catalyzed -ECL Reaction—Glutathione is a non-allosteric feedback inhibitor for all monofunctional -ECL enzymes studied to date. In these reports, feedback inhibition mechanisms were consistently analyzed toward L-Glu. Strikingly, the -ECL activity catalyzed by S. agalactiae GshF was reported to be insensitive to feedback inhibition by glutathione (24), a result that was reproduced here, as shown in Fig. 6D. To study the effect of glutathione on the -ECL activity catalyzed by the P. multocida GshF bifunctional enzyme, increasing glutathione concentrations were included in the assay mixtures composed to follow steady-state enzyme activities as a function of L-Glu concentration (Fig. 6). The direct plot (Fig. 6A) shows that low millimolar levels of glutathione significantly reduced activity and that increasing the concentration of L-Glu did not overcome the inhibition. Plotting the reciprocal of velocity against the reciprocal of L-Glu concentration (1/v against 1/S, Fig. 6B) yielded all straight lines that meet in a joint intercept on the abscissa, indicating pure noncompetitive inhibition. However, a secondary plot of the ordinate intercepts against glutathione concentration is nonlinear, and therefore the true noncompetitive inhibition constant K_i cannot be read directly from the x-intercept. Yet, the ordinate intercepts fit to Equation 1,

(Eq. 1)

where Or is ordinate intercept, n is the Hill coefficient, V_max is maximum velocity, and K_i is true noncompetitive inhibition constant), indicating noncompetitive feedback inhibition characterized by a K_i of 13.6 mM, which is sensitive to positive cooperativity characterized by a Hill coefficient of 1.65.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Glutathione feedback inhibition of the -ECL activity of P. multocida GshF toward L-Glu. -ECL activities were recorded on the basis of ADP formation as described under "Materials and Methods." A, direct plot of steady-state initial velocities as a function of L-Glu yields hyperbolic curves in the presence of 0 mM (diamonds), 10 mM (inverted triangles), 20 mM (triangles), and 30 mM (circles) of glutathione. B, double reciprocal plot (1/v0 versus 1/S) of the data from A. C, secondary plot of the ordinate intercepts shown in B as a function of glutathione concentration; the curve was produced by fitting the data to Equation 1 (see "Results" for details) using a nonlinear regression routine. D, effect of glutathione on the partially substrate-saturated -ECL activities of the GshF ligases from P. multocida and S. agalactiae, depicted as percent of remaining activity versus tripeptide concentration.

When assaying the in vitro kinetics of the individual GshF activities using the steady-state coupled enzyme ATPase assay, we were surprised to notice that under saturating concentrations of the substrates to form the -EC intermediate, the steady-state rate of ADP formation remained unaffected whether or not 20 mM of Gly was included in the reaction mixture. This phenomenon is also demonstrated in Fig. 8, in which the Michaelis-Menten plots of steady-state velocities against varying L-Glu concentrations are compared for assay mixtures containing L-Glu, L-Cys, and MgATP^2- (-ECL reaction) and assay mixtures containing L-Glu, L-Cys, Gly, and MgATP^2- (-ECL + GS reactions). On the basis of this experiment, it appears as if the GS ligase reaction is increasingly inhibited by the degree of saturation of the -ECL activity and finally, at full saturation of the latter activity, is completely inactivated.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Plot of the initial velocities of glutathione formation versus varying Gly concentrations of the coupled -ECL-GS reaction shows a physiologically relevant affinity (Km of 1.72 mM) for the C-terminal amino acid substrate. The rates of glutathione formation were recorded via a discontinuous assay as described under "Materials and Methods." Inset, the progress curves for the -ECL-GS-coupled reaction saturated with its substrates L-Glu, L-Cys, and MgATP2- versus varying concentrations of Gly. These time courses show an apparent lag period before reaching a steady state.

Steady-state Transient Time Analysis Is Inconsistent with Substrate Channeling—The presence of two consecutive enzymatic activities on GshF raises the possibility that the intermediate, -EC, is channeled between the catalytic sites. Because both consecutive reactions are supposed to be irreversible (cf. irreversible monofunctional counterpart enzymes and no ADP accumulation as a result of the coupled ATPase assay to record GshF activity) and because no deviations from simple Michaelis-Menten kinetics have been observed for either reaction, the progress curves of glutathione formation shown in Fig. 7 may be described (45, 46) by

(Eq. 2)

where [GSH]_t = glutathione concentration produced at time t, v₀ is steady-state velocity, t is time, and is transient time. The latter parameter is actually of great importance in describing a coupled enzyme system, because it gives an indication of the speed with which the coupled enzyme system reaches a steady state. In cases wherein channeling has been proven, the channeling step is very fast (>1000 s^-1), and accordingly, no transient time, along with an immediate steady state, has been observed using steady-state kinetics (47, 48). Fitting the time course of the P. multocida GshF-coupled activity saturated with substrates (50 mM L-Glu, 2.5 mM L-Cys, 20 mM Gly, and 5 mM MgATP^2-)to Equation 2 gives a value of 48.4 s, indicating that substantial -EC intermediate has to accumulate before the attainment of a steady state. These accumulated levels of -EC at the steady state are described by

(Eq. 3)

and were calculated to be 28 µM. When the K_m value of the GS activity of GshF for -EC (90 µM) is not influenced by the preceding coupled -ECS activity, then the steady-state -EC concentration may furthermore be described (45, 46) by

(Eq. 4)

where [-EC]_ss is steady-state -EC concentration; v₀, steady-state velocity; K_m2, the Michaelis constant of the GS activity for -EC; and V₂, maximal velocity of the GS reaction center. Substituting [-EC]_ss = 28 µM, v₀ = 11.1 s^-1, and K_m2 = 90 µM gives a V₂ value of 46.5 s^-1, which is close to the apparent k_cat value for the individual GS activity obtained in this work (Table 2). In summary, the generalized theory of the transient time for sequential noninteracting enzyme reactions obeying Michaelis-Menten kinetics, as first described by Easterby (45), is applicable to the linked consecutive peptide bond-forming reaction centers in GshF, without the need to consider metabolite channeling.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Gram-negative glutathione-containing cyanobacteria and proteobacteria accumulate glutathione via a de novo biosynthesis pathway that consists of two unrelated monofunctioanl ATP-dependent amino acid ligases called -ECL and GS. In this respect, our previous results with H. influenzae were surprising, as this -proteobacterium of the family Pasteurellaceae acquires glutathione solely via a highly specific import system (27). The motivation for the present research was to evaluate whether other Pasteurellaceae members were atypical also with respect to glutathione acquisition. By conducting this survey, novel hybrid sequences, referred to as GshF sequences, containing a typical -proteobacterial -ECL fused to an ATP-grasp-like domain, were discovered to be present in the genomes of strains of H. somnus, A. pleuropneumoniae, M. succiniciproducens, and P. multocida, yet no such sequences were found in the genomes of a number of H. influenzae and H. ducreyi strains. By in vivo complementation studies in a gshA^-gshB^-E. coli double mutant background and through in vitro kinetic studies with a recombinant and purified P. multocida GshF preparation, we have proven that the natural hybrid catalyzes both well known consecutive peptide bond-forming reactions toward the formation of glutathione, strongly suggesting that a large number of Pasteurellaceae strains accumulate glutathione via de novo synthesis, whereas others, such as H. influenzae and H. ducreyi, accumulate the tripeptide via import.

A BLASTP search with 303 microbial genomes at NCBI (37) identified 19 species, of both Gram-negative and Gram-positive signature, containing a GshF homologous sequence. Among Gram-negative bacteria, the distribution of GshF sequences appears to be confined to members of the Pasteurellaceae family, with Desulfotalea psychrophila LSv54, a -proteobacterium, as the single exception. Fourteen of the 19 GshF-containing bacteria are Gram-positive species, which are classified as firmicutes. The GshF ligases of L. monocytogenes and S. agalactiae have recently been shown to be functional multidomain glutathione synthetases (24, 25) and, in case of L. monocytogenes, GshF was found to be essential for aerobic growth and virulence. Interestingly, some of the Gram-positive genera that harbor GshF, such as streptococci and enterococci, have been found to accumulate glutathione by either or both of the two presently recognized ways of glutathione acquisition, import and complete de novo synthesis (1, 24, 49).

View larger version (21K): [in this window] [in a new window]   FIGURE 8. Plots of th