(Received for publication, February 25, 1997, and in revised form, May 12, 1997)
From the Medical Nobel Institute for Biochemistry, Department of
Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden and the Department of Genetics and
Microbiology, Faculty of Sciences, Autonomous University of Barcelona,
Bellaterra, 08193 Barcelona, Spain
Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductases. Either thioredoxin or glutaredoxin is a required electron donor for class I and II enzymes. Glutaredoxins are reduced by glutathione, thioredoxins by thioredoxin reductase. Recently, a glutaredoxin-like protein, NrdH, was isolated as the functional electron donor for a NrdEF ribonucleotide reductase, a class Ib enzyme, from Lactococcus lactis. The absence of glutathione in this bacterium raised the question of the identity of the intracellular reductant for NrdH. Homologues of NrdH are present in the genomes of Escherichia coli and Salmonella typhimurium, upstream of the genes for the poorly transcribed nrdEF, separated from it by an open reading frame (nrdI) coding for a protein of unknown function. Overexpression of E. coli NrdH protein shows that it is a functional hydrogen donor with higher specificity for the class Ib (NrdEF) than for the class Ia (NrdAB) ribonucleotide reductase. Furthermore, this glutaredoxin-like enzyme is reduced by thioredoxin reductase and not by glutathione. We suggest that several uncharacterized glutaredoxin-like proteins present in the genomes of organisms lacking GSH, including archae, will also react with thioredoxin reductase and be related to the ancestors from which the GSH-dependent glutaredoxins have evolved by the acquisition of a GSH-binding site. We also show that NrdI, encoded by all nrdEF operons, has a stimulatory effect on ribonucleotide reduction.
Ribonucleotides are reduced to deoxyribonucleotides by ribonucleotide reductases, which are radical containing enzymes that may be divided into three main classes (1, 2).
The electrons for this reaction are supplied by small redox-active proteins such as thioredoxin (Trx)1 or glutaredoxin (Grx) in the case of class I and II ribonucleotide reductase (3, 4), whereas formate fulfills this function for the anaerobic class III enzyme (5). Thioredoxin and glutaredoxin both contain two redox-active cysteine thiols in their reduced form, which by dithiol-disulfide interchange reduce an acceptor disulfide in the active center of ribonucleotide reductase. The active site sequences of thioredoxins and glutaredoxins are conserved among species, being Cys-Gly-Pro-Cys for thioredoxin and Cys-Pro-Tyr-Cys for glutaredoxin (3, 4). The disulfide in oxidized thioredoxin is regenerated to a dithiol by thioredoxin reductase (TR) and NADPH, whereas oxidized glutaredoxin is reduced by 2 mol of GSH with the formation of GSSG, which is reduced by glutathione reductase (GR) and NADPH.
Escherichia coli contains three different glutaredoxins
(called Grx1, -2, and -3 (6)), which, like all glutaredoxins from other
species, show high activity as general GSH-disulfide oxidoreductases in
a coupled system with GSH, NADPH, and glutathione reductase (7).
Three-dimensional structures for thioredoxins (8) and glutaredoxins (9,
10) show that they have essentially completely unrelated amino acid
sequences but a similar overall fold (often referred to as the
thioredoxin fold), consisting of a central four-stranded -sheet
flanked by three helices in the order
(the
alignment in Fig. 1 includes the location of secondary structure elements in glutaredoxins). Within the thioredoxin superfamily of
proteins (for review, see Ref. 11) two distinct subtypes are the
thioredoxin proteins and the glutaredoxin proteins, the latter having a
conserved GSH-binding site (boxed in Fig. 1), which has been
experimentally determined by NMR (12) for E. coli Grx1.
E. coli and Salmonella typhimurium contain the genetic information for two different ribonucleotide reductases belonging to class I, NrdAB (class Ia) and NrdEF (class Ib), with a limited sequence similarity (13) and differences in their allosteric regulation (14). The expression of NrdEF is repressed and insufficient to allow growth of NrdAB-defective cells under aerobic conditions, unless the expression of NrdEF is increased by the presence of additional copies of the nrdEF genes either on the chromosome or on a plasmid (13, 15). Characterization of the proteins encoded by the S. typhimurium nrdEF genes showed a fully functional ribonucleotide reductase that could use E. coli Grx1 (but not thioredoxin) as a hydrogen donor with an apparent Km of 5 µM (16) (cf. 0.15 µM for NrdAB; Ref. 17).
Recently, it was shown by enzyme fractionation that the functional
ribonucleotide reductase in the Gram-positive bacterium Lactococcus lactis grown under microaerophylic conditions is
an NrdEF type of enzyme and that its hydrogen donor protein (NrdH, 72 amino acids) with the active site sequence Cys-Met-Gln-Cys shows
appreciable homology to glutaredoxins (18). Coding sequences for
proteins homologous to NrdH are present upstream of the
nrdEF genes in E. coli and S. typhimurium (81 amino acids), separated by an open reading frame
(Orf2; 136 amino acids). Since we have found that this latter protein
seems to be involved in reduction of ribonucleotides, the protein will
henceforth be referred to as NrdI. An alignment of NrdH and NrdI
proteins from different species are presented in Figs. 1 and 2. The
four genes, nrdH-nrdI-nrdE-nrdF, constitute a unique
transcriptional unit (15). On the assumption that the gene organization
was the same in L. lactis, it was possible to clone the
analogous operon for the L. lactis NrdEF enzyme including the gene (nrdH) for the hydrogen donor protein and
nrdI (18). Since L. lactis has no GSH and the
NrdH proteins also appeared to lack the typical GSH-binding site
identified in Grx1 (12), we wondered how NrdH is reduced. To solve this
question we have cloned and expressed the NrdH protein from E. coli, an organism with a high content of GSH. Characterization of
the NrdH protein showed that it lacked activity with GSH but was a
substrate for thioredoxin reductase. Additionally, and with the aim of
completing the understanding of the function of all the genes that
constitute the conserved nrdEF operon, we have overexpressed
the NrdI protein from S. typhimurium and investigated its
influence on the activity of the NrdEF reductase.
Plasmids pET-24a and pET-15b were from Novagen,
and pGEM-T was from Promega Corp. E. coli DH5
(CLONTECH) was used for general cloning procedures.
BL21(DE3) was from Novagen. Taq DNA polymerase and T4 DNA
ligase were from Pharmacia Biotech Inc. Oligonucleotide primers were
from the Department of Cell and Molecular Biology, Karolinska
Institute. Sephadex G-50 resin was from Pharmacia, and DEAE-cellulose
(DE-52) anion exchanger was from Whatman. Ni2+-NTA agarose
was from Qiagen. E. coli Grx1, Trx, and TR were obtained as
described previously (3, 19, 20), and S. typhimurium NrdEF
proteins (R1E and R2F) were purified as described (16). E. coli NrdAB RR subunits (R1 and R2) were available from this laboratory. The University of Wisconsin Genetics Computer Group (GCG)
package (version 8.0, Open VMS) was used for sequence analysis.
The entire gene for E. coli NrdH was amplified by PCR using plasmid pUA523 (15) as a
template and two primers containing restriction sites for
NdeI and BamHI, respectively (underlined): 5-ATACGACATATGCGCATTACTATTTA-3
and
5
-ACAGGGATCCTCATGCACTGGCCGCG-3
(antisense).
Thirty cycles of amplification were used with annealing at 55 °C.
The amplified DNA fragment was gel-purified and cloned into vector
pGEM-T according to the manufacturer's protocol, giving rise to
plasmid pUA624. To ascertain that no Taq polymerase-induced mutations were introduced, the cloned fragment was sequenced with fluorescent pUC/M13 universal primers, and the sequence was determined using an automated laser fluorescent DNA sequencer (Pharmacia). pUA624
was digested with NdeI and BamHI, and the
nrdH coding sequence was cloned into the expression vector
pET-24a digested with the same restriction enzymes, downstream from the
inducible T7 promoter and a strong ribosome binding site. Plasmid
(pUA625) from one clone unambiguously confirmed to code for NrdH was
transformed into BL21(DE3) cells, which carry an IPTG-inducible T7 RNA
polymerase gene.
The S. typhimurium nrdI gene was amplified using plasmid pUA335 (13) as a
template and two primers containing restriction sites for
NdeI and BamHI, respectively (underlined):
5-CGACATATGAGCGCGCTCGTCTAC-3
and
5
-CGCGGATCCATGGTTTCCTGC-3
(antisense). The same general procedure described above was used for cloning the nrdI gene
into vectors pGEM-T (pUA626) and pET-15b (pUA627), consecutively. The cloning into NdeI-BamHI-digested pET-15b
introduces a sequence coding for six His codons upstream the
nrdI gene, facilitating purification of the recombinant
protein on Ni2+-NTA-agarose.
E.
coli BL21(DE3) cells containing the pUA625 plasmid were grown in
LB medium at 37 °C in the presence of 50 µg/ml of kanamycin to an
optical density at 600 nm of 0.5 and subsequently induced with 0.4 mM IPTG (final concentration). Four hours after induction, the cells were harvested by centrifugation. The bacterial pellet (4 g)
was dissolved in 6 volumes of 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, and lysed by a combination of lysozyme
(0.2 mg/ml) and sonication. The crude extract obtained after
centrifugation was dialyzed extensively against 20 mM
Tris-HCl, 1 mM EDTA, pH 9.5, and applied to a 100-ml column
of DEAE-cellulose equilibrated with the same buffer. The column was
eluted with 1 liter of 50 mM Tris-HCl, 1 mM
EDTA, pH 8.0. The eluted protein was concentrated by ultrafiltration
using a YM-3 membrane (Amicon) after the addition of 10% glycerol and
0.5 M NaCl (final concentrations). The concentrated protein
was applied to a column (100 × 3 cm2) of Sephadex
G-50 equilibrated with 50 mM Tris-HCl, pH 8.0, 10% glycerol, and 0.5 M NaCl. The eluted protein was
concentrated and stored at 20 °C.
E.
coli BL21(DE3) cells harboring plasmid pUA627 were grown in LB
medium at 30 °C in the presence of 50 µg/ml ampicillin to an
optical density of 0.5 and subsequently induced with 0.4 mM IPTG for 3 h. After centrifugation and extraction by a combination of lysozyme (0.1 mg/ml) and sonication in 5 volumes of binding buffer
(20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole, 0.5 mM CHAPS, 20% glycerol,
0.1% Nonidet P-40, 10 mM -mercaptoethanol), the soluble
crude extract was loaded into a Ni2+-NTA agarose column
according to the manufacturer's protocol. The column was washed with
the same buffer containing 60 mM imidazole, and NrdI was
eluted with 300 mM imidazole. Prior to use, the sample was
dialyzed to remove NaCl, imidazole, and
-mercaptoethanol.
The concentration of the purified NrdH protein was calculated from the absorption at 280 nm using a molar extinction coefficient of 7210 or by Bradford determinations (21) and total amino acid composition. As a complement, the concentration of NrdH was determined from the amount of NADPH consumed upon the addition of TR. The sequence of the first 5 amino acids of the purified NrdH protein was determined by Edman degradation using an ABI 491 Procise instrument.
Assays with Ribonucleotide ReductaseThe ability of Grx1,
NrdH, and Trx to serve as hydrogen donors for the NrdEF and AB
reductases was determined using the standard ribonucleotide reductase
assay (16, 22). Stoichiometric amounts of R1E/R2F or R1/R2 were
incubated for 20 min at 30 °C with the chosen reductant system in a
final volume of 0.05 ml containing 0.5 mM
[3H]CDP (22 cpm/pmol), 10 mM
MgCl2, 0.1% Nonidet P-40, 50 mM Tris-HCl, pH
8.0, and 0.3 mM dATP (for NrdEF) or 1.5 mM ATP
(for NrdAB). The activity of NrdH was determined in the presence of
either of the three different reducing systems: (i) 4 mM
GSH, 1 mM NADPH, 0.1 µM glutathione reductase
(ii) 1 mM DTT, or (iii) 1 mM NADPH, 0.1 µM thioredoxin reductase (final concentrations). The
activity of Grx1 was determined in the presence of 4 mM
GSH, 1 mM NADPH, 0.1 µM glutathione
reductase, and the activity of Trx was determined in the presence of 1 mM NADPH, 0.1 µM thioredoxin reductase. One unit is defined as the formation of 1 nmol of dCDP/min. For simplicity of comparison, the data for NrdH and Grx1 have been presented as percentages of the extrapolated Vmax obtained
from Lineweaver-Burk plots (except for the data of Grx1 with
NrdAB, where the last data point was set to 100%). Since Trx was
inactive with NrdEF, the activity in Fig. 3C is presented in
units (as specified above).
Assays with Thioredoxin Reductase
The reduction of NrdH, T4-Grx, and Trx by thioredoxin reductase (0.1 µM final concentration) was determined in the presence of 0.5 mM of DTNB, 240 µM NADPH, and 0.1 mg/ml bovine serum albumin in 100 mM Tris-HCl, pH 8.0, 2 mM EDTA in a final volume of 100 µl. The activity was monitored as the increase of absorbance at 405 nm using a Thermomax microplate reader (Molecular Devices).
Determination of the Redox Potential for NrdHThis
determination was performed essentially as described (23, 24) using an
AVIV spectrophotometer model 14DS (AVIV, Lakewood, NJ) at 25 °C.
Absorbance at 340 nm was determined as the average of 150 data points
recorded during 30 s after each step in the experiment. The assays
were performed in a degassed and N2-equilibrated buffer
composed of 100 mM potassium phosphate, 1 mM
EDTA, pH 7.0. In a typical experiment, additions of 6-10
µM NADPH (final concentration) were made to the sample
cuvette, and the absorbance at 340 nm was used to determine the
concentration in the cuvette, followed by the addition of the same
amount of NADPH to the reference cuvette. An equivalent volume of NrdH
protein (final concentration, 6 µM) or buffer (10%
glycerol, 0.5 M NaCl in 50 mM Tris-HCl, pH 8.0) was then added to the sample and reference cuvettes, respectively. Equilibration between NrdH and NADPH was allowed to start by the addition of 10 nM (final concentration) of thioredoxin
reductase to both cuvettes. The equilibrium was then reversed by two
successive additions of NADP+ to final concentrations of
125 and 250 µM. As a control, the redox potential of Trx
(270 mV) was determined with the same procedure.
Reduction of insulin disulfides (50 µM final concentration) was performed in parallel for Grx1, NrdH, T4-Grx, and Trx using a final concentration of 5 µM of each enzyme in the presence of 1 mM DTT in 100 mM potassium phosphate, 1 mM EDTA, pH 6.0. The reaction was followed as the increase of absorbance at 600 nm due to the precipitation of insulin when reduced to A and B chains (25).
The activity of NrdH, T4-Grx, and Trx (final concentration of each enzyme, 5 µM) as reductants of insulin disulfides (100 µM final concentration) was also determined in the presence of 200 µM NADPH, 0.1 µM thioredoxin reductase in 100 mM potassium phosphate, 1 mM EDTA, pH 7.0. The activity was in this case monitored as the decrease in absorbance of NADPH at 340 nm.
GSH-Disulfide Oxidoreductase AssaysGSH-disulfide
oxidoreductase assays were performed as described (17), measuring the
reduction of -hydroxyethyl disulfide by GSH at the expense of NADPH
as monitored at 340 nm. A standard of purified E. coli Grx1
was used in each experiment as a positive control.
The gene for E. coli NrdH was amplified by PCR using primers with designed restriction sites. The PCR product was cloned into the T7 RNA polymerase-dependent expression vector pET-24a. Using this system, the NrdH protein was expressed to levels of around 30% of total soluble protein, as judged by densitometry scans of SDS-polyacrylamide gels. The Mr of the overexpressed polypeptide (9 kDa) was in accordance with the expected size for NrdH. The protein was purified to homogeneity but showed poor solubility. Thus, to avoid precipitation of the protein when concentrated, the final steps of the purification were done in a buffer containing 10% glycerol and 0.5 M NaCl. Purified NrdH protein was subjected to N-terminal amino acid sequence determination, confirming the homogeneity of the protein and also showing an unprocessed initiator Met residue as expected.
The sequence homology between NrdH proteins and glutaredoxins has been
noted, with E. coli Grx3 being the closest glutaredoxin homologue (18). A prediction of the secondary structure elements of
E. coli NrdH using the PHD program (26) produced a similar pattern of secondary structure elements (with the exception of 4) as
experimentally determined for Grx1 and Grx3 (9, 27). Fig.
1 shows a refined alignment where the predicted
secondary structure elements of E. coli NrdH have been
matched against the known secondary structure elements of E. coli Grx3 (27). The presence of a proline (Pro-52 in E. coli NrdH) in the NrdH proteins in the same relative position as
the conserved cis-proline in glutaredoxins reemphasizes the
structural similarity between NrdH proteins and glutaredoxins. The
conclusion that NrdH proteins lack the conserved GSH-binding site found
in glutaredoxins is still valid in this refined alignment. It is
noteworthy that E. coli NrdH may be aligned with E. coli Grx3 with essentially no introduction of gaps. This could
provide a basis for further studies of E. coli NrdH intended
to introduce a GSH-binding site by changing the relevant amino acids of
NrdH to the corresponding ones found in the GSH-binding site of Grx3,
which has been shown to be essentially identical to the GSH-binding
site of Grx1.2
Since we have found that a sequence homologous to nrdI found between nrdH and nrdE in E. coli and S. typhimurium (15) is present in all known nrdEF operons (e.g. L. lactis (18), Mycoplasma genitalium (28), and Bacillus subtilis (29)), one should suspect a function for this unknown protein in the ribonucleotide reduction reaction. Searching for functional similarities of NrdI in the protein sequence data bases was unsuccessful. Fig. 2 shows the predicted amino acid sequence alignment of all known nrdI products. To investigate the effect of NrdI in the in vitro assay of S. typhimurium NrdEF ribonucleotide reductase, we overexpressed and partially purified the recombinant NrdI protein of this bacterium.
After cloning the PCR-amplified nrdI into the vector pET-15b, the His-tagged NrdI protein was expressed to levels of around 30% of total protein. Only a small fraction of the overexpressed protein was found in the soluble fraction of the crude extract. This soluble NrdI material was purified by one-step chromatography on Ni2+-NTA resin, resulting in material that was about 50% pure. The size of the recombinant protein was higher (17 kDa) than the size of the expected NrdI polypeptide (15 kDa) due to the His tag. Any attempt to increase the solubility of the recombinant NrdI during growth or extraction (e.g. by decreasing the growth temperature or IPTG concentration or by additions of NaCl, glycerol, detergents, or reducing agents such as DTT) was unsuccessful. NrdI could be purified in the presence of guanidinium hydrochloride or urea, but the protein precipitated when the denaturing agent was removed by dialysis.
Activity of NrdH and NrdI with the NrdAB and NrdEF Ribonucleotide ReductasesThe activity of NrdH was compared with that of E. coli Grx1 and Trx as reductants of the NrdAB enzyme from E. coli and the NrdEF enzyme from S. typhimurium. NrdH was found to be a functional hydrogen donor for both enzymes in the presence of either 1 mM DTT or thioredoxin reductase/NADPH but not in the presence of GSH/glutathione reductase/NADPH. The Vmax of NrdH (in units/µg of the R1 or R1E subunits) was similar to that of Grx1 and Trx with the NrdAB enzyme and similar to the Vmax of Grx1 with the NrdEF enzyme. As shown in Table I and Fig. 3A, NrdH showed a lower Km value with the S. typhimurium NrdEF enzyme than with the E. coli NrdAB enzyme, whereas the opposite was found to be the case for E. coli Grx1 (Fig. 3B). This tendency for NrdH was even more pronounced when the assays were performed in the presence of 1 mM DTT instead of thioredoxin reductase/NADPH. Thus, the apparent Km value of NrdH as a hydrogen donor for NrdEF was repeatedly found to be lower (0.3-0.6 µM) in the presence of 1 mM DTT than in the presence of NADPH/thioredoxin reductase (Km = 1.2 µM). This finding is hard to reconcile with the finding that NrdH is efficiently reduced by thioredoxin reductase (see below). Nevertheless, the results clearly demonstrate that NrdH is a more specific hydrogen donor for NrdEF than for NrdAB, whereas the opposite is the case for Grx1. E. coli Trx was a hydrogen donor for NrdAB but not for NrdEF (Fig. 3C) as observed previously (16).
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The partially purified NrdI recombinant protein stimulated the NrdEF
ribonucleotide reductase activity when the NrdH protein was used as a
hydrogen donor, not only when NrdH was reduced by thioredoxin
reductase/NADPH (Fig. 4A) but also, although
to a minor extent, when it was reduced by 0.5 mM DTT. On
the other hand, there was no stimulatory effect when NrdEF was reduced
by Grx1 (either using DTT or GSH/glutathione reductase/NADPH; Fig. 4B) or by DTT alone up to 20 mM. A similar
stimulatory effect of NrdI could also be seen for NrdAB in the presence
of Trx and thioredoxin reductase/NADPH, while the effect in the
presence of NrdH/thioredoxin reductase/NADPH was almost nonexistent.
Even in the presence of NrdI, Trx remained inactive with NrdEF.
Activity of NrdH with Thioredoxin Reductase
Having established that NrdH is a substrate for thioredoxin reductase, we next chose to characterize how NrdH performed as a substrate for this enzyme compared with the other known substrates, Trx and T4-Grx. Using the standard DTNB reduction assay in the presence of NADPH/thioredoxin reductase at pH 8.0, we found that the three enzymes had similar Km values in this assay and that the Vmax for Trx was only somewhat higher than for NrdH or T4-Grx (Table II).
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Since NrdI stimulated both the activity of NrdH plus NrdEF and that of Trx plus NrdAB in the presence of NADPH/thioredoxin reductase, we tested whether NrdI would affect the reduction of NrdH or Trx by thioredoxin reductase in the DTNB assay. No effect by NrdI was seen.
Redox Potential of the NrdH EnzymeThe ability of NrdH to be
reduced efficiently by thioredoxin reductase allowed the determination
of its redox potential by assessing the equilibrium constant with
NADP+/NADPH. Calculated from a standard state redox
potential for NADP+/NADPH of 315 mV, a redox potential of
248.5 ± 1.5 mV was obtained for NrdH.
Reduction of
insulin disulfides is a classical assay for thioredoxin (25). As shown
in Fig. 5, NrdH was almost as potent a reductant as Trx
in this system and was much more potent than Grx1 and T4-Grx. The same
relative order of activity among Trx, NrdH, and T4-Grx was also
obtained when thioredoxin reductase/NADPH was used instead of DTT.
In contrast to glutaredoxins, the NrdH protein lacked detectable activity in the GSH-disulfide oxidoreductase assay, where glutaredoxins show a high activity (data not shown).
The NrdH protein was originally discovered as the hydrogen donor for L. lactis NrdEF, a class Ib enzyme that is the active ribonucleotide reductase under aerobic conditions in this organism (18). The juxtaposition of nrdH and nrdI upstream of nrdEF in a conserved operon suggests a specific involvement of NrdH and NrdI in the ribonucleotide reduction process. Since the NrdEF enzyme from E. coli and S. typhimurium serves as an excellent model system for class Ib enzymes, we have now extended the characterization of the proteins encoded by this operon to include NrdH and NrdI. We have found NrdH to be an efficient hydrogen donor for ribonucleotide reductase, with higher specificity for the NrdEF enzyme than for the NrdAB enzyme. This reaction was stimulated modestly by the addition of NrdI by an as yet unknown mechanism. Furthermore, we show that NrdH is a good substrate for thioredoxin reductase and that it has no detectable activity with NrdAB or NrdEF in the presence of GSH. A summary of the biochemical properties of NrdH in comparison with other redox active proteins from E. coli and phage T4 is presented in Table III.
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Functionally, NrdH thus behaves like the classical thioredoxin (3, 30) of E. coli, sharing some of its biochemical properties including a low redox potential and the ability to reduce insulin disulfides. However, the sequences of the two proteins are not related. Instead, NrdH shows sequence homology with E. coli glutaredoxins (Fig. 1). Glutaredoxins function as dithiol shuttles during ribonucleotide reduction. Their distinction is that they contain a glutathione binding site and that glutathione reduces their active cysteines. With one known exception, they are not reduced by thioredoxin reductase, the exception being the glutaredoxin induced by phage T4, which for this reason originally was classified as a thioredoxin (31). This protein was later renamed T4-Grx (32), since thioredoxin reductase can be fully substituted by GSH (33) and also because of the sequence homology with E. coli Grx1, which became apparent upon the determination of the primary structure of the latter (34). The three-dimensional structure of T4-Grx is also more related (10) to the known structure of E. coli Grx1 (9) than to thioredoxin.
In addition to NrdH, the known substrates for E. coli thioredoxin reductase are Trx and phage T4-Grx. The sequence homology between these three proteins is, however, restricted (21%) between NrdH and phage T4-Grx and essentially insignificant between NrdH (or T4-Grx) and Trx. This prevents us from making any predictions of the residues that are involved in the interaction with thioredoxin reductase. However, given the predicted high structural similarity (Fig. 1) between in particular E. coli Grx3 and NrdH it might be possible to mutate Grx3 with the aim of making it a substrate for thioredoxin reductase. The identity of the residues to be changed, however, is not obvious and will require careful analysis of three-dimensional structures.
Conceivably, the inability of NrdH to use GSH could be an effect of the
redox potential of the protein being too low to allow an efficient
reduction by GSH. We have found that E. coli NrdH has a
redox potential of 248 mV at pH 7.0 compared with
270 mV for
thioredoxin (23). Since this number is quite similar to the
240 mV
determined for T4-Grx (31, 35), which is relatively efficiently reduced
by GSH, the lack of activity of NrdH with GSH can best be explained by
the protein lacking the residues needed to interact with GSH.
Furthermore, NrdH did not show any activity in the general assay for
glutaredoxins using the artificial disulfide
-hydroxyethyl disulfide
as a substrate, supporting our conclusion that the protein does not
catalyze GSH-dependent disulfide reductions.
Where does this leave the NrdH protein? It lacks a glutathione binding
site, and its active site cysteine residues are not reduced by
glutathione. It was actually discovered in an organism that lacks
glutathione. Therefore, it may not functionally be classified as a
glutaredoxin, despite the sequence homology (Fig. 1). In the
phylogenetic tree (Fig. 6), it is apparent that NrdH proteins form a separate group, on the same branch as glutaredoxins but
definitely separated from thioredoxins. Since the two groups are easily
distinguished by the presence of several typical conserved residues (8,
10, 11), we chose to refrain from classifying NrdH as a thioredoxin
despite its thioredoxin-like activity profile. E. coli
contains only one known thioredoxin so far, but yeast has two isoforms
(36) and plants have many isoforms (37). All of these thioredoxins are
at least 105 residues long and do in comparison with glutaredoxins
contain one additional -sheet and one
-helix preceding the
domain, which thioredoxins share with
glutaredoxins. They also contain conserved residues (E. coli
Trx numbering) such as Asp-26, Trp-31, and Pro-40, which have no
equivalents in the NrdH proteins.
We would like to point out that the two glutaredoxin-like proteins, clustered with the NrdH proteins in the phylogenetic tree (Fig. 6), also might be substrates for thioredoxin reductase. As a speculation, we would like to extend this hypothesis to other glutaredoxin-like proteins present in the genomes of many organisms lacking GSH, including archae (38, 39). Glutaredoxins are the simplest members of the thioredoxin superfamily, and the glutaredoxin fold is present in all of the members (here including GSH-peroxidases and glutathione S-transferases (11). A reasonable evolutionary scenario could actually be that NrdH and similar glutaredoxin-like proteins are related to the progenitors of the thioredoxin superfamily, from which the other members evolved by divergent evolution (the glutaredoxins simply by the acquisition of a GSH-binding site).
A genetic study has shown that disruption of the gene for thioredoxin reductase (trxB), but not that of thioredoxin (trxA), is accompanied by increased disulfide bond formation in the cytoplasm of E. coli (40). This finding suggested that there are additional substrates for thioredoxin reductase in E. coli that may channel electrons from this enzyme for disulfide reduction (40, 41). An obvious candidate for this role is NrdH. A function of NrdH in general disulfide reduction in the cytoplasm is, of course, dependent on the levels of expression of this protein, which are currently unknown. Since extensive studies of E. coli mutants lacking thioredoxin failed to identify any additional thioredoxin reductase-coupled protein (42), the expression of NrdH is likely to be low, as is the case for NrdEF. The poor solubility of the protein is, however, a factor to consider for the lack of detection of NrdH protein in extracts of E. coli.
This paper demonstrates that the NrdH gene located upstream of the nrdEF genes in E. coli codes for a protein that has a higher specificity as a hydrogen donor for NrdEF than for NrdAB. By analogy with the situation in L. lactis, it would thus seem that most NrdEF enzymes use NrdH as the functional in vivo hydrogen donor. However, the recently completed sequence determination of the M. genitalium genome (28) shows the presence of an NrdEF type of ribonucleotide reductase but no NrdH-like protein. This could provide an example of an NrdEF enzyme that interacts with, for example, thioredoxin for which M. genitalium contains a coding sequence. Thus, care should be taken not always to associate NrdEF with NrdH, as the situation in L. lactis, E. coli, and S. typhimurium would suggest.
The mechanism of the stimulatory effect of NrdI on the activity of NrdH with NrdEF and on the activity of Trx with NrdAB remains elusive. Since the presence of nrdI in the known nrdEF loci is more conserved than that of nrdH, we believe that the NrdI protein has an important in vivo function for the activity of NrdEF. It is surprising that all of the gene products encoded by the nrdEF operon are fully functional proteins also in E. coli and S. typhimurium, since this operon seems to be poorly transcribed and knock-out mutants of the nrdEF genes have no phenotype (15). The conservation of the operon does, however, suggest an important in vivo function that at present is not understood.
We are indebted to Isidre Gibert for suggestions and contributions to the phylogenetic analysis of protein sequences.