Cloning, overexpression, and characterization of glutaredoxin 2, an atypical glutaredoxin from Escherichia coli.

Glutaredoxin 2 (Grx2) from Escherichia coli catalyzes GSH-disulfide oxidoreductions via two redox-active cysteine residues, but in contrast to glutaredoxin 1 (Grx1) and glutaredoxin 3 (Grx3), is not a hydrogen donor for ribonucleotide reductase. To characterize Grx2, a chromosomal fragment containing the E. coli Grx2 gene (grxB) was cloned and sequenced. grxB (645 base pairs) is located between the rimJ and pyrC genes while an open reading frame immediately upstream grxB encodes a novel transmembrane protein of 402 amino acids potentially belonging to class II of substrate export transporters. The deduced amino acid sequence for Grx2 comprises 215 residues with a molecular mass of 24.3 kDa. There is almost no similarity between the amino acid sequence of Grx2 and Grx1 or Grx3 (both 9-kDa proteins) with the exception of the active site which is identical in all three glutaredoxins (C9PYC12 for Grx2). Only limited similarities were noted to glutathione S-transferases (Grx2 amino acids 16-72), and protein disulfide isomerases from different organisms (Grx2 amino acids 70-180). Grx2 was overexpressed and purified to homogeneity and its activity was compared with those of Grx1 and Grx3 using GSH, NADPH, and glutathione reductase in the reduction of 0.7 mM beta-hydroxyethyl disulfide. The three glutaredoxins had similar apparent Km values for GSH (2-3 mM) but Grx2 had the highest apparent kcat (554 s-1). Expression of two truncated forms of Grx2 (1-114 and 1-133) which have predicted secondary structures similar to Grx1 (betaalphabetaalphabetabetaalpha) gave rise to inclusion bodies. The mutant proteins were resolubilized and purified but lacked GSH-disulfide oxidoreductase activity. The latter should therefore require the participation of amino acid residues from the COOH-terminal half of the molecule and is probably not confined to a Grx1-like NH2-terminal subdomain. Grx2 being radically different from the presently known glutaredoxins in terms of molecular weight, amino acid sequence, catalytic activity, and lack of a consensus GSH-binding site is the first member of a novel class of glutaredoxins.

where X-S-S-X is HED and X-SH is ␤-mercaptoethanol.
In a crude extract of wild-type E. coli B, Grx1 constitutes only about 2% of the total GSH-disulfide oxidoreductase activity (HED assay) (5,6). Later work resulted in the isolation of two additional glutaredoxins which accounted for the major general GSH-disulfide oxidoreductase activity (6). From their order of elution on Sephadex G-50 the new glutaredoxins were called glutaredoxin 2 (Grx2) and glutaredoxin 3 (Grx3). NH 2terminal sequencing of Grx2 and Grx3 demonstrated that both enzymes contained the typical glutaredoxin motif (Cys-Pro-Tyr-Cys), although Grx2 had an atypical size (M r 27,000) for glutaredoxin. Grx3 (9 kDa) with 82 residues showed 33% sequence identity and a similar secondary structure and tertiary fold (7) as the well characterized Grx1 (8,9). Thus both Grx1 and Grx3 have the thioredoxin fold (10,11) and are members of the thioredoxin (Trx) superfamily of disulfide oxidoreductases. In contrast to Grx3, which has about 5% of the catalytic activity of Grx1 as a disulfide reductant of ribonucleotide reductase, Grx2 lacks such activity (6). The relative abundance of Grx2 in E. coli (6) makes further investigations of the structure and function of this unknown protein necessary to understand the SH-metabolism in the cell. In this study we have cloned and sequenced the gene coding for Grx2 (grxB) and another gene located immediately upstream grxB coding for a potential transmembrane protein. Grx2 showed large differences to previously known glutaredoxins.

MATERIALS AND METHODS
␤-Hydroxyethyl disulfide (HED) was from Aldrich. NADPH and glutathione reductase (yeast) were from Sigma. Diaflo YM10 membranes * This work was supported by grants from the Wenner-Gren foundation, the Swedish Cancer Society (961 and 1806), the Swedish Medical Research Council (13X-3529, 13X-10832, and 13X-11213), the Karolinska Institute, and the Knut and Alice Wallenberg Foundation. 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  were from Amicon. E. coli Grx1 and Grx3, Trx, and thioredoxin reductase were purified to homogeneity as described (12). Oligonucleotides were synthesized by Pharmacia Biotech Ltd. Restriction enzymes, T4 DNA ligase, and Taq polymerase were from Promega.
Bacterial Strains and Plasmids-XL-1 blue and DH5␣ were used for plasmid propagation and cloning purposes. Strain GI698 (13) was a gift Dr. J. McCoy (Genetics Institute, Boston, MA) and was used for overexpression of Grx2.
Measurement of Protein Concentration-Total protein was measured using the Bradford protein assay (14) adapted for use on microtiter plates. For pure Grx2, a molar extinction coefficient of 21,860 M Ϫ1 ⅐cm Ϫ1 at 280 nm was used.
Protein Preparation and Carboxymethylation-A nearly homogenous preparation (more than 90% pure as judged by SDS-PAGE) of E. coli Grx2 (5 nmol in 50 l) purified as described (6), was reduced in 0.5 ml of 50 mM Tris-Cl, pH 8.0, by incubation for 4 h at 4°C in the presence of 1 mM dithiothreitol. Carboxymethylation was performed by the addition of 300 l of 6 M guanidine hydrochloride, 0.4 M Tris-Cl, pH 8.1, 2 mM EDTA, 5 mM neutralized 14 C-iodoacetic acid (Amersham, approximately 2400 cpm/nmol), and incubation for 4 h at 4°C. Reagents and contaminating proteins were removed by reverse-phase chromatography on a C4 (Vydac) HPLC column equilibrated in 0.1% trifluoroacetic acid in water and eluted with a linear gradient of acetonitrile (0 -40% during 60 min, 1 ml/min) containing 0.1% trifluoroacetic acid.
Peptide Generation, Purification, Nomenclature, and Sequence Analysis-HPLC-purified 14 C-carboxymethylated Grx2 was digested for 4 h at 37°C with Lys-C endoprotease (Wako), at an enzyme to protein ratio of 1:10 in 0.1 M ammonium bicarbonate, pH 8.0, containing 1 M guanidine hydrochloride. The resulting peptides (K peptides, Fig. 1) were purified by reverse-phase (C18) HPLC using conditions described above. Peptides (D peptides, Fig. 1) were generated also by cleavage with Asp-N endoprotease (Boehringer-Mannheim) and purified on a C4 HPLC column, see above. Amino acid sequence analysis of all peptides was performed using an Applied Biosystems 470A sequencer and phenylthiohydration detection was by reverse-phase HPLC in a Hewlett-Packard 1090 system (15).
Screening the Kohara Phage Library and Isolation of Phage DNA-An 180-bp PCR product resulting from amplification using the N2 and C1 primers contained sequences corresponding to peptides from Grx2. The PCR product was labeled with 32 P by random priming and used as a probe to screen a nitrocellulose filter containing DNA from the Kohara phage library (16). Phage clone 233 was selected and propagated in E. coli strain Y1090. Phage DNA was purified using the plate lysate method (17), with the modification of resuspending pelleted phage particles in diluent (instead of TE) after DNase I treatment. The modification resulted to higher yields of non-degraded phage DNA.
Construction of Recombinant Plasmids for Sequence Analysis and Overexpression-Phage DNA from clone 233 was digested with BamHI and EcoRI, separated on a 1% agarose gel, transferred to a nitrocellulose membrane, and finally probed using the 180-bp PCR fragment (N2 and C1 primers). A ϳ4.2-kilobase band which hybridized strongly with the probe was cloned into vector pGEM-3Z (Promega) and used for sequence analysis. The nucleotide sequence of the cloned fragment was determined using the Taq DyeDeoxy terminator cycle sequencing on an automated laser fluorescent DNA sequencer (A.L.F., Pharmacia) according to the protocol supplied by the manufacturer. Primers (fluorescent) were made initially based on the 180-bp PCR fragment, and later on the sequences revealed upstream and downstream. Sequencing stopped after the known pyrC and rimJ regions were reached.
Overexpression of Grx2-grxB was amplified using primers containing NdeI (G2-FNdeI primer: 5Ј-TGGAGGAGTCATATGAAGCTATAC-3Ј) and BamHI (G2-RCBamHI primer: 5Ј-CGCGGCGGGGGATCCTTA-AATCGC-3Ј) sites at the 5Ј and 3Ј termini of the gene, respectively. The amplified fragment was then digested with NdeI and BamHI and was cloned into the pTRXFUS (13) vector replacing the Trx gene and giving rise the Grx2 expression vector named pe1Grx2. Strain GI698 transformed with pe1Grx2 was grown in 1-liter cultures at 30°C. Grx2 expression was induced at an optical density of cells at 600 nm (A 600 ) between 0.4 and 0.5 with a final concentration of 100 g/ml DL-tryptophan. Cells were then grown for up to 16 h in an orbital shaker at 100 rpm and harvested by centrifugation at 6000 ϫ g for 20 min.
Truncated forms of grxB were constructed using the G2-FNdeI primer and primers G2del1RC (for 1-114, 5Ј-GAAATATTTGCGG-GATCCTTATTAAGAAAACTCATC-3Ј) or G2del2RC (for 1-133, 5Ј-AGAGTGGGCCAGGGATCCTTAAAAATTACCCGCGC-3Ј). Both primers contained a BamHI site. The expression strategy for the truncated forms was identical to that used for the wild-type protein with the exception that temperatures of 20 and 25°C also were used in separate experiments for growth and expression.
Purification of Recombinant Grx2-Cells, 6.2 g, were resuspended in 50 ml of 50 mM Tris-Cl, pH 8.0, and placed on ice. Lysozyme (0.5 mg/ml final), phenylmethylsulfonyl fluoride (1 mM final), and DNase I (50 g/ml final) were added sequentially after 5 min each and the mixture was left on ice for 1 h. EDTA was added to a final concentration of 1 mM and the cell lysate was thoroughly sonicated (on ice three times at full power for 1 min). The supernatant was collected after centrifugation at 24,000 ϫ g for 35 min at 4°C and the pellet was washed with 10 ml of 100 mM Tris-Cl, pH 8.0, recentrifuged, and the new supernatant was pooled with the first. The pool was dialyzed overnight against 20 mM Tris base (pH above 10) and was applied to a 200-ml DE52 column (Whatman) equilibrated with the same buffer. Grx2 bound to the column and was eluted by 50 mM Tris-Cl, pH 8.0, in a volume of approximately 2 liters which after concentration through a tangential flow cartridge with a cut-off of 10 kDa (Prep/Scale-TFF cartridge, Millipore) was applied to a column (100 cm ϫ 1.6 cm 2 ) of Sephadex G-50 superfine (Pharmacia) with 100 mM potassium phosphate, pH 7.0, as equilibration and running buffer.
Preparation of Soluble Truncated Forms of Grx2-The 1-114 and 1-133 truncated forms of Grx2 were recovered from the insoluble fraction after cell lysis. The insoluble pellet from a 1-liter culture was washed in 20 ml of 100 mM Tris-Cl, pH 8.0, 1 mM EDTA, recentrifuged, and solubilized with 10 ml of 100 mM Tris-Cl, pH 8.0, 1 mM EDTA, 100 mM dithiothreitol, 8 M urea. The urea extract was diluted 100-fold by dropwise addition to 1 liter of 100 mM Tris-Cl, pH 8.0, 1 mM EDTA at 4°C. The mixture was concentrated to 10 ml on a Diaflo membrane and centrifuged at 24,000 ϫ g for 35 min at 4°C. The supernatant contained the Grx2 1-114 or Grx2 1-133 fragment in soluble form. The purity of these preparations was more than 50% as judged by SDS-PAGE.
Determination of Glutaredoxin Activity (HED assay)-A fresh mixture of 1 mM GSH, 0.2 mM NADPH, 2 mM EDTA, 0.1 mg/ml bovine serum albumin, and 6 g/ml yeast GR was prepared in 100 mM Tris-Cl, 2 mM EDTA, pH 8.0 (3). To 500 l of this mixture in semimicro cuvettes, HED was added to a final concentration of 0.7 mM. After 3 min glutaredoxin was added to the sample cuvettes and the decrease in A 340 was recorded the following 5 min using a Zeiss PMQ3 spectrophotometer. Activity was expressed as micromoles of NADPH oxidized per min using a molar extinction coefficient of 6200 M Ϫ1 ⅐cm Ϫ1 . Steady-state kinetics measurements were performed in a final volume of 100 l using a Molecular Devices Thermomax microplate reader. Values of ⌬A 340 were multiplied by a factor of 4.3 to give the ⌬A 340 of a cuvette with a path length of 1 cm. All activity measurements were made at 25°C.
Assay of Thioredoxin Activity-The ability of Grx2 to serve as a substrate of thioredoxin reductase was determined in the reduction of 5,5Ј-dithiobis(2-nitrobenzoic acid). This was followed at 412 nm in 100 mM Tris-Cl, pH 8.0, 2 mM EDTA, 0.1 mg/ml bovine serum albumin, 0.2 mM NADPH, 35 nM E. coli thioredoxin reductase, and 0.5 mM 5,5Јdithiobis(2-nitrobenzoic acid) as described (12). E. coli thioredoxin was used as a positive control.
Preparation of Cellular Fractions from E. coli-Periplasmic fractions were prepared after three cycles of quick freezing and thawing in dry ice-ethanol bath, in phosphate-buffered saline at an A 600 of 40 (18). The supernatant after centrifugation was collected as the periplasmic lysate fraction. To prepare osmotic shock fractions, cells were initially resuspended (A 600 ϭ 40) in ice-cold 20% sucrose, 20 mM Tris-Cl, pH 8.0, with or without 1 mM EDTA for 10 min. They were then resuspended in ice-cold 100 mM Tris-Cl, pH 8.0, 1 mM EDTA and left on ice for 10 min. Cells were finally centrifuged and the supernatant was collected as the osmotic shock fraction (13).
SDS-PAGE-This was performed according to Laemmli (19) using the Bio-Rad mini gel apparatus. Gels were stained with Coomassie Blue.

RESULTS
Cloning of grxB-The sequence for the 18 NH 2 -terminal residues of Grx2 including its active site was determined previously (6). We now digested Grx2 with either Lys-C endoprotease or Asp-N endoprotease and separated the peptides by HPLC. Altogether 26 peptides were analyzed by automated Edman degradation (Fig. 1). Based on the NH 2 -terminal sequence and internal peptide K8, primers were designed (N2 and C1) which following PCR resulted in the generation of an 180-bp PCR product containing part of the sequence of grxB. The 180-bp PCR product was used as a probe to screen the Kohara phage library (16) and a positive clone ( 233) was identified. After cleavage of DNA with EcoRI and BamHI a 4.2-kilobase fragment which hybridized with the PCR probe was cloned and sequenced. The novel sequence revealed in the 4.2-kilobase fragment was named grxB region (1943 bp) (Fig. 1) and contained the sequences of grxB and another open reading frame (ORF) upstream grxB.
The 645-bp ORF for grxB encodes a 215-amino acid residue protein with a predicted molecular mass of 24.3 kDa. The deduced amino acid sequence was in agreement with the available amino acid sequence data from the peptides derived from purified Grx2. The NH 2 -terminal methionine is coded for by GTG. A consensus ribosomal binding site (GGAGG) is located 6 bp upstream of the initiator GTG codon. There are two Ϫ35 TTGACa promoter consensus sequences upstream of grxB there but no obvious Ϫ10 TAtAaT consensus (Fig. 1).
grxB is partially overlapping with the pyrC region data base entry (Fig. 2) and is located 802 bp upstream from the actual E. coli gene for dihydroorotase (pyrC) (23.4 min) (20). The gene encoding the NH 2 -terminal acetylase of ribosomal protein S5 (rimJ) (23.7 min) (21) starts 1216 bp upstream from grxB, but is translated from the complementary DNA strand. In fact both grxB and pyrC regions are translated anticlockwise.
Sequence and Structure of a Novel Putative Membrane Protein-An ORF corresponding to 402 amino acids is located immediately upstream of grxB, before rimJ ( Figs. 1 and 2). A GGAGG consensus ribosomal binding site is located three bases upstream from the first Met residue or 12 bp upstream to an alternative GTG-encoded Met. The TMAP program for the prediction of transmembrane proteins (22) aligned the putative protein to well established transmembrane proteins and a membrane orientation model was constructed (Fig. 3). The grxB region containing grxB and the preceding ORF has been submitted to the EMBL data base with accession number X92076.
Expression and Purification of Recombinant Grx2-The gene sequence used for the overexpression of Grx2 was identical to the chromosomal one with the exception that the GTG initiator codon was substituted with ATG to increase expression levels since GTG has been reported as a less efficient start codon compared with ATG (23). Using the overexpression system for Trx fusions (13) with Grx2 replacing Trx, recombinant Grx2 was expressed in a soluble state as high as 50% of total cell protein (Fig. 4). Chromatographies on columns of DE52 cellulose and Sephadex G-50 superfine resulted in a homogeneous protein. A yield of 40% from the purification procedure was obtained. A summary of the purification procedure and yields is shown in Table I. The molecular mass of the recombinant Grx2 was determined as 24.3 kDa by matrix-assisted laser desorption/ionization mass spectrometry. This value is identical to that predicted from the genomic sequence. Peptide sequencing showed that the initiation Met was present in preparations of the recombinant enzyme as was also the case for protein isolated from E. coli cells (6). The specific activity of the recombinant Grx2 (316 units/mg, Table I) is identical to the value published before (6) for the wild type enzyme which was based on a M r of 27,000 rather than the correct value of 24,300.
Grx2 Activity as a GSH-disulfide Oxidoreductase-Previous results (6) suggested that a large part of the GSH-disulfide oxidoreductase activity in E. coli cells measured by the standard HED assay (Equations 1 and 2) is due to Grx2. To compare the steady-state contribution of HED reducing activities of the three E. coli glutaredoxins, we determined their apparent K m and k cat values for GSH using the standard HED assay which employs final concentrations of 1 mM GSH and 0.7 mM HED. Comparison was also made with human glutaredoxin (hGrx) (24,25). Due to the spontaneous reaction between HED and GSH leading to increased NADPH consumption, we only determined rates up to 4.5 mM GSH (Fig. 5). The kinetic data (Fig. 5, Table II) showed that Grx2 had the lowest apparent K m for GSH and the highest turnover.
Expression and Characterization of Truncated Mutants of Grx2-Sequence alignment of Grx2 with Grx1 and Grx3 showed surprisingly very little homology and identities (Fig. 6). The active site is identical in all three proteins and since Grx2 contains two Cys residues, these must be the catalytic ones located in a similar relative position as in Grx1 and Grx3. Sequence analysis using data base programs revealed some limited homologies to glutathione S-transferases (GSTs) and protein disulfide isomerases (PDIs) (Fig. 6). Since Grx2 seemed to contain Grx-like and PDI-like parts (Fig. 6), we constructed and expressed relevant truncated mutant proteins to examine their possible functional autonomy. The first (amino acids 1-114) mutant corresponded to a part of Grx2 which is long enough to include a Grx-like domain (ϳ80 amino acids). The second mutant was extended to amino acid 133 to include the part with homology to bovine PDI. Both mutants were expressed at high levels but in insoluble states (Fig. 4). Resolubilization from 8 M urea gave 50% pure preparations but no activity could be detected with the standard HED assay for concentrations of the truncated forms up to 10 M (assuming a 50% purity, data not shown). In comparison, activity of the least active of the four glutaredoxins, Grx1, is detected readily at concentrations of 0.001 M.
Grx2 as a Reductant of Insulin Disulfides-Trx and Grx1 reduce disulfides in insulin, with Grx1 being a much less efficient enzyme (3). Our results show that Grx2 and Grx3 did not reduce insulin using 1 mM GSH (Fig. 7). This probably reflects an unfavorable enzyme substrate interaction and/or an elevated redox potential for Grx2 and Grx3. The fact that purified Grx2 was obtained in a fully reduced state, as measurements of free SH-groups showed, suggests that Grx2 has a high redox potential and is rather oxidizing.
Grx2 Is Not a Substrate for Thioredoxin Reductase-Recombinant alkaline phosphatase can be expressed as an active protein with all its correct disulfides in E. coli trxB mutants, but this cannot happen in trxA mutants lacking thioredoxin (26). This suggests the presence of an alternative substrate for thioredoxin reductase which reduces and inactivates alkaline phosphatase (26). Grx2 did not reduce 5,5Ј-dithiobis(2-nitrobenzoic acid), demonstrating that it was not a substrate for thioredoxin reductase (data not shown). To examine whether GSH was required for thioredoxin reductase-Grx2 interactions, standard HED assays were carried with thioredoxin reductase substituting for glutathione reductase. Grx2 was not reduced under these conditions as measured at 340 nm showing that it did not react with thioredoxin reductase through GSH.
Intracellular Localization of Grx2-To examine the intracellular localization of Grx2, periplasmic and cytosolic fractions of non-transformed E. coli cells and transformants were analyzed by SDS-PAGE (Fig. 8). Grx2 was clearly cytosolic. The enzyme was not released (even when overexpressed) by osmotic shock conditions which is different from Trx (1, 13) and Grx1 (1).

DISCUSSION
This paper describes the cloning and sequence determination of a genomic region containing grxB and presents findings on the characterization of recombinant Grx2. Previously two other glutaredoxins from E. coli, both 9-kDa proteins, were shown to have the same active site sequence Cys-Pro-Tyr-Cys and to catalyze GSH-disulfide oxidoreductions via a GSH-binding site (7,27). Grx2 contains the same active site sequence Cys-Pro-Tyr-Cys as the other two glutaredoxins and is highly active in the general GSH-disulfide oxidoreductase assay with the small disulfide HED as a substrate. Grx2, however, has a larger size and lacks activity as a hydrogen donor (protein disulfide reductase) for ribonucleotide reductase (6). Alignment of Grx2 with other proteins showed no further extensive homology to known glutaredoxins or thioredoxins (Fig. 6). Grx2 therefore lacks the conserved sequences of Grx1 and Grx3 defining a GSH-binding site previously experimentally verified by NMR for the mixed disulfide between E. coli Grx1 C14S and GSH (28). Amino acid residues of Grx2 that may be the homologues to amino acids of Grx1 involved in binding to GSH (underlined) are Y11 (Y13 in Grx1), QVP 49 (TVP 60 in Grx1), and YVD 70 (YTD 74 in Grx1).
Data base searches showed that Grx2 was weakly homologous to sequences of two GSH S-transferases (GSTs) (Grx2 amino acids 47-69 and 86 -112), a relative of GST, crystallin SL-11 (28) (Grx2 amino acids 16 -71), and PDIs of different organisms (70 -180) (Fig. 6). The significance of this is unknown. Secondary structure prediction using the method of Rost and Sander (29) suggested that the first third of Grx2 (2-70) contained an alternation of ␤-sheets and ␣-helices (␤␣␤␣␤␤), while the remaining part (84 -212) was predominantly helical (Fig. 6). The ␤␣␤␣␤␤ prediction for the NH 2terminal third of the molecule is similar to the characteristic ␤␣␤␣␤␤␣ secondary structure of the thioredoxin fold found in E. coli Trx (10), Grx1 (8), Grx3 (7), glutathione peroxidase (30), and GSH S-transferases (GST) (31). In the latter a first Trxlike domain (ϳ80 amino acids) is connected (through a short linker of 8 amino acids for GST) (32) to a second domain (ϳ130 amino acids) composed of ␣ helices (31). Although the amino acid sequence of Grx2 was markedly different from other members of the Trx superfamily or GSTs including the known E. coli GST (Fig. 6) (33), the NH 2 -terminal third of the molecule may have the thioredoxin fold. E. coli DsbA, for example, has almost no sequence homology to Trx (34) but contains a Trxlike domain. To examine whether Grx2 contained a fully active Trx/Grx-like domain at its NH 2 terminus, we overexpressed two truncated mutant proteins. The first (1-114) included the putative ␤␣␤␣␤␤␣ fold and the second (1-133) included a part with homology to bovine PDI (Fig. 6). Both overexpressed pro- The alignment of Grx2 with the different PDI was made using the BLAST program software of the National Center for Biotechnology Information. Secondary structure information was obtained using the secondary structure prediction software of Rost and Sander (29). L, prediction for loop; ␣, prediction for ␣-helix; ␤; prediction for ␤-pleated sheet structure. Alignment to GST from Pseudomonas (GST Ps), GST 6.0, and S-crystallin SL11 was performed using the TMAP program (22). Identical amino acids are boxed. tein fragments were inactive in the glutaredoxin assay (HED assay). These data suggested that the GSH-disulfide oxidoreductase activity of Grx2 requires parts of the COOH-terminal half of the molecule and is not confined to a Grx1-like NH 2 -terminal subdomain. In comparison, human PDI contained distinct Trx domains which can be expressed as soluble proteins which catalyze thiol-disulfide exchange reactions (35). Grx2 therefore appears more similar to GSTs in which the GSH-binding site is not confined exclusively to the ␤␣␤␣␤␤␣ Grx-like first domain but also includes residues from the second domain (␣4/␣5 helix-turn-helix segment and COOH terminus) (31,32). Grx2 residues that may be the homologues of GST residues known to interact with GSH (underlined) are Y6 (Y6 in GST, Y7 in GST, and Y8 in GST␣) (31), V in QVP 49 (QVP 55 in seven different GST␣s) (31), and perhaps the W in WL 90 (WL 47 in eight different GSTs) (31). Biochemically Grx2 was different from GSTs as it was inactive in the standard 1-chloro-2,4-dinitrobenzene assay for GST activity (36) and did not form the -complex with 1,3,5-trinitrobenzene (37) as GSTs do. 2 Furthermore, Grx2 is a monomeric and not a dimeric enzyme either in its reduced or oxidized form as determined by Superdex G-75 chromatography in either 20 or 100 mM potassium phosphate, pH 7.0. In comparison, all known GSTs form homodimeric complexes in solution (31). The ORF immediately upstream grxB was predicted as a transmembrane protein, using the TMAP program for the prediction and alignment of transmembrane proteins (22). Homologies were observed with other transmembrane transporter proteins and a membrane orientation model was constructed (Fig. 3) after sequence alignment with the metal-tetracycline/H ϩ antiporter 1 which is thought to contain 12 membrane spanning segments (38). In this model the ORF follows the positive inside rule for transmembrane proteins (39) as it contains one Arg in the periplasm compared with 25 Arg, and Lys in the cytosol. Similarities with other transmembrane proteins reside on the sequence GAIADRFGAK between potential ␣2 and ␣3 (GXXXD(R/K)XGR(R/K) is the consensus for transmembrane transporters) (40). Transporters which aligned with the ORF include four tetracycline antiporters (TetA, B, C, and D), the quinolone resistance protein from Staphylococcus aureus (NorA) (41), and the multidrug resistance protein from Bacillus subtilis (Bmr) (42). Since these transporters belong to class II of transmembrane proteins (40), we suggest that the ORF before Grx2 also is a member of this class.
The ORF transporter contains 5 Cys residues (Fig. 3), although none with the thioredoxin CXXC active-site motif. Cysteine residues also were present in other transmembrane proteins that aligned with the ORF (not shown). Of particular interest is whether the ORF transporter (63 bp upstream grxB), Grx2, and the protein encoded by the ORF before pyrC (169 bp downstream grxB) are functionally coupled (in principle the transporter could export a product delivered from Grx2) and belong to the same operon. The latter could be possible because although there are two Ϫ35 TTGACa promoter consensus sequences upstream from grxB there is no obvious Ϫ10 TAtAaT consensus. Also, there is no obvious consensus for the termination of transcription downstream from the ORF before grxB or grxB itself. However, there is a transcriptional terminator downstream from the ORF before pyrC and expression of the pyrC genes is known to be independent from genes upstream (20).
The catalytic activity of recombinant Grx2 and three other classical glutaredoxins was compared in the GSH-disulfide oxidoreductase assay varying the concentrations of GSH with the HED concentration constant (0.7 mM). All the different glutaredoxins had similar apparent K m values for GSH ( Fig. 5 and Table II) but Grx2 showed the highest apparent k cat . Grx2 previously was calculated to represent a major part of the total glutaredoxin activity in an E. coli Grx1 null mutant (6). Since the physiological concentrations of GSH in E. coli is from 3.5 to 6.6 mM (43), Grx2 should have the potential of being a major catalyst in GSH-utilizing reactions. The situation may be more complicated. Grx1 has been reported (6) to amount to only 2% of total E. coli oxidoreductase activity (HED assay) (5) but its levels are highly variable and depend on other factors (for example, the levels of ribonucleotide reductase, Trx and GSH) which regulate the intracellular redox environment (44). If Grx2 levels are regulated is unknown and requires further studies.
The biological function of Grx2 is unknown and will require further studies. If Grx2 has a high redox potential it may be 2 R. Morgenstern, Karolinska Institute, Department of Toxicology, personal communication. involved in GSH-dependent thiol-disulfide exchange reactions making disulfides in proteins. Such reactions and other potential physiological roles linked to the transporter can be addressed by gene inactivation experiments currently in progress.