(Received for publication, November 28, 1994)
From the
A protein has been purified to homogeneity from crude extracts of the hyperthermophilic archaeon Pyrococcus furiosus based on its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol; the protein has a molecular mass of 24.8 kDa and a pI of 4.9, and it is highly heat-stable. The first 29 amino acid residues at the N terminus of the P. furiosus protein were determined by Edman degradation, and its gene was cloned in Escherichia coli. The amino acid sequence derived from the DNA sequence contains the CPYC sequence, which is typical of the active site of glutaredoxin (also called thioltransferase). The C-terminal portion of the P. furiosus protein, containing the conserved sequence, shows sequence similarity with glutaredoxins from different sources. The P. furiosus protein can reduce disulfide bonds in L-cystine in the presence of GSH (the thioltransferase activity) with an optimum pH of 8.0. The expression of the P. furiosus protein, with full activity, in E. coli at a very high level (21% of total soluble protein) is described; the recombinant protein was purified to homogeneity by merely two successive heat treatments and gel filtration chromatography. The features of the P. furiosus protein here described are discussed in light of the current knowledge about the ubiquitous family of protein disulfide oxidoreductases.
The ubiquitous family of protein disulfide oxidoreductases comprises proteins whose cysteine residues in the active site sequence CXXC undergo reversible oxidation-reduction. The protein disulfide isomerase in eucaryotic endoplasmic reticulum, thioredoxin, and glutaredoxin (also known as thioltransferase) are the most thoroughly studied members of the family. Protein disulfide isomerase (a dimer of 57 kDa) has the sequence CGHC in the active site; the protein disulfide isomerase homologue in Escherichia coli, named DsbA, is a monomer of 189 residues with the active sequence CPHC. Protein disulfide isomerase and DsbA catalyze the reduction, oxidation, and reshuffling of protein disulfides depending on the redox potential, thus playing a primary role in the correct structure and, hence, in the proper functioning of protein molecules (reviewed by Freedman(1992), Bardwell and Beckwith(1993), and Loferer and Hennecke(1994)). Thioredoxin, similar to glutaredoxin in molecular mass (about 12 kDa), has at the N terminus the active sequence CGPC, with the only exception represented by the thioredoxin C2 of Corynebacterium nephridii where A replaces G (reviewed by Holmgren(1985) and Eklund et al.(1991)). Glutaredoxin has at the N terminus a redox disulfide bond in the sequence CPYC, with the exceptions of glutaredoxins from T4 (CVYC), pig liver, and rice (CPFC) (reviewed by Holmgren, 1989). Thioredoxin and glutaredoxin have been postulated to be involved in a number of different cellular processes, as cofactors for dithiol-disulfide exchange reactions on small and protein disulfides.
All living beings can be grouped into three evolutionarily distinct
kingdoms: Eucarya, Bacteria, and Archaea (Woese et al., 1990).
All but two genera of hyperthermophiles, the microorganisms that grow
at and above 100 °C, belong to the kingdom of Archaea. Eucarya and
Archaea have a common ancestor, which is not shared by Bacteria; the
first organisms to have diverged from the Eucarya/Archaea lineage were
the hyperthermophiles, considered to be the most ancient of all extant
life. Pyrococcus furiosus, the best studied among the
hyperthermophiles that reduce elemental sulfur (S°) to
HS, is a strictly anaerobic heterotroph; S° is reduced
probably to remove toxic H
, not to gain the energy for
growth that is obtained by an unusual fermentative type metabolism
(Adams, 1994). The enzymes and the proteins so far isolated from P.
furiosus exhibit unique features with respect to their
non-archaeal counterparts (for a review see Adams, 1993).
This paper reports the isolation from P. furiosus of a novel 24.8-kDa protein disulfide oxidoreductase, which exhibits glutaredoxin-like properties. The cloning and the high level expression of the coding gene in E. coli are also described.
DNA electrophoresis on 1% agarose gels in 90 mM Tris borate, 20 mM EDTA (TBE buffer) and plasmid
transformations of E. coli cells were carried out as described
by Sambrook et al.(1989). DNA sequencing was carried out by
the dideoxy chain termination method (Sanger et al., 1977)
with [S]dATP, using the Sequenase version 2.0
sequencing kit (USB) on alkali-denatured double-stranded templates; the
universal M13 forward and reverse primers or specific synthetic
oligonucleotides were used.
The standard assay mixture for the thioltransferase activity consisted of 0.1 M sodium phosphate buffer, pH 8.0, 1 mM EDTA, 10 mM GSH, 2.5 mML-cystine, 0.2 units of glutathione reductase, 0.3 mM NADPH in the absence or in the presence of the P. furiosus protein (final volume, 1 ml); the enzyme activity was monitored at 340 nm and 30 °C. The net enzymatic reaction rate, expressed as micromoles of NADP formed per minute, was obtained by subtraction of the nonenzymatic reaction rate from the total rate. The activity dependence on pH was determined by the standard assay mixture in which 0.1 M sodium phosphate was used from pH 6 to 8 and 0.1 M Tris-HCl from 8 to 9 (0.12 µM protein was used for each assay).
Ten grams of bacteria underwent freeze-thawing twice; were mixed
with 30 g of sand and 100 ml of 20 mM Tris buffer, pH 8.4, 2
mM EDTA, 1 M NaCl; and were homogenized in a Homni
Mixer. The homogenate was centrifuged at 4,000 g for
20 min at 4 °C to remove the sand and ultracentrifuged at 160,000
g for 90 min at 4 °C; the supernatant represented
the crude extract.
Material for N-terminal amino acid sequence analysis was prepared by C4 reverse-phase HPLC.
The recombinant protein
was purified to homogeneity, as judged from SDS-PAGE analysis, by the
following procedure, starting from the crude extract of transformed
cells grown at 1.0 A cell density and induced
for 18 h with IPTG.
Figure 1: SDS-PAGE analysis of the active sample during the purification procedure of the P. furiosus protein. Lane 1, pooled active fractions from DEAE-Sepharose column; lane 2, pooled active fractions from Superdex 75 column; lane 3, pooled active fractions from Bio-Gel P30 column; lane 4, molecular weight standards. Samples (6 µg/lane), treated and run as described under ``Experimental Procedures,'' were stained with Coomassie Blue.
Isoelectric focusing gel electrophoresis of the homogeneous protein revealed one band whose isoelectric point was 4.9.
The protein purified from P. furiosus was endowed with an extreme resistance toward heating: after 3 h of incubation at 90 °C, its effect on the insulin precipitation reaction was comparable with that of the native protein.
The strategy used to establish a representative
genomic bank in pGEM7Zf(+) plasmid (Fig. 2) was highly
efficient for the insertion of one single DNA fragment from P.
furiosus genome per vector molecule (Cannio et al., 1994). The specificity as a probe of the oligonucleotide mixture
and the stringency conditions were previously determined by Southern
blot analysis of the genomic DNA digested with different restriction
enzymes. After two sequential screenings, 5 clones out of 5
10
exhibiting positive hybridization signals were isolated;
one clone, named pDR7, had a 2.3-kb region encompassing the complete
gene sequence. The hybridization of the whole coding sequence as a
probe to the bacterial chromosomal DNA produced results consistent with
the restriction maps of the isolated clone, thus confirming that the
recombinant clone suffered no rearrangements during the cloning
procedures.
Figure 2: Restriction endonuclease map of the genomic fragment in pDR7 plasmid carrying the gene coding for the P. furiosus protein. The arrows indicate the sequencing strategy, which employed synthetic oligonucleotides (PfN, PfC, Pf1, Pf2, Pf3, and Pf4) and universal M13 primers on fragments suitably subcloned (see ``Experimental Procedures'' for details).
Fig. 3shows the nucleotide sequence of the gene and the deduced primary structure of the P. furiosus protein. A 675-bp open reading frame (ORF) starting at ATG (position 106) and ending at TAA (position 783) as well as extended flanking regions were found. Upstream to the initiation codon, the putative archaebacterial TATA box (at positions 76-81) and the ribosome binding site (at positions 98-102) could be identified (Reiter et al., 1988). The 226 amino acid residues derived from the ORF matched up to the calculated size of the native protein whose N-terminal sequence lacks the initial methionine. The residues from Phe-141 to Cys-149 identify the FXXXXCXXC motif, which is a part of the active site of the enzymes that catalyze thiol/disulfide interchange reactions; the sequence CPYC from Cys-146 to Cys-149 is the canonical active disulfide of glutaredoxins. The sequence CQYC from residue 35 to residue 38 is not conserved in the active site of any of the known protein disulfide oxidoreductases.
Figure 3: Nucleotide sequence (numbering at right) of the gene coding for the P. furiosus protein and the deduced amino acid sequence (numbering at left). The underlined nucleotides represent putative archaebacterial TATA box (from 76 to 81) and ribosome binding site (from 98 to 102). The amino acid residues that identify the conserved active site of glutaredoxin (from 146 to 149) are in boldface.
When the primary structure of the P. furiosus protein was compared with GenBank using the Fasta
program, similarity with the active site region of some glutaredoxins
was detected. We aligned the sequence CPYC of the P. furiosus protein with the conserved active site sequences of glutaredoxins
from different sources and maximized similarity by introducing gaps.
The moiety of the P. furiosus protein from residue 122 was
compared with the full primary structures of glutaredoxins (Fig. 4). Five residues, valine 140, cysteines 146 and 149 of
the active site, proline 194, and glycine 202, are common among all
nine proteins, whereas proline 147, aspartic 186, valine 193 and 197,
and glycine 210 are common among all but one of the proteins (numbering
refers to the complete sequence of the P. furiosus protein).
The highest identity (40%) was with the protein isolated from the
methanogenic archaeon Methanobacterium thermoautotrophicum;
the lowest (23%) was with T4 glutaredoxin. Prediction of secondary
structures of the P. furiosus protein, obtained by Chou-Fasman
analysis, revealed that the sequence CPYC, located in a turn, is
preceded by a -sheet (residues 137-144) and followed by a
large
-helix (residues 151-168). Similar structural features
have been identified in the active site region of all glutaredoxins
isolated (McFarlan et al.(1992), and references therein).
Figure 4: Comparison of the amino acid sequences of different glutaredoxins. The alignment was performed as described under ``Results.'' The sequences were from the following sources (in parentheses): M. thermoautotrophicum glutaredoxin (McFarlan et al., 1992), E. coli glutaredoxin (Hoog et al., 1983), yeast glutaredoxin (Gan et al., 1990), bovine glutaredoxin (Papayannopoulos et al., 1989), rabbit glutaredoxin (Hopper et al., 1989), pig glutaredoxin (Gan and Wells, 1987), rice glutaredoxin (Minakuchi et al., 1994), and T4 glutaredoxin (Sjoberg and Holmgren, 1972). Positional numbers refer to the sequence of the P. furiosus protein. The residues identical to the sequence of the P. furiosus protein are in boldface. The boxed residues indicate the conserved active site sequence of glutaredoxin.
Figure 5:
DTT-dependent insulin reduction. The
DTT-dependent reduction of bovine insulin disulfides was carried out as
described under ``Experimental Procedures'' in the absence
() or in the presence of different concentrations of pure P.
furiosus protein:
, 0.2 µM;
, 0.6
µM;
, 1.2
µM.
Glutaredoxin has an inherent GSH-dependent disulfide reductase activity (named the thioltransferase activity) that can be assayed by coupling to saturating concentrations of glutathione reductase, which reduces GSSG in the presence of NADPH (Axelsson et al., 1978). By using L-cystine as disulfide substrate, we demonstrated that the P. furiosus protein exhibits thioltransferase activity. The variations in activity with GSH and L-cystine concentrations showed that the maximal rate was obtained at 10 mM GSH and 2.5 mML-cystine; inhibition of the reaction rate was detected by further increasing the concentrations of the reactants. The thioltransferase activity of the P. furiosus protein showed a linear dependence on the protein amount up to 0.12 µM, inhibition being detected at higher protein concentrations (Fig. 6A); when the activity was assayed over the pH range of 6-9, a sharp optimum was found at pH 8.0 (Fig. 6B). Most glutaredoxins have been reported in the literature to have basic pH optima.
Figure 6:
A, dependence of the thioltransferase
activity on the concentration of pure P. furiosus protein. The
standard assay mixture (see ``Experimental Procedures'')
contained different amounts of protein. B, dependence of the
thioltransferase activity on pH. The activity was assayed in sodium
phosphate buffer () and Tris-HCl buffer (
) at the indicated
pH values, as described under ``Experimental Procedures.'' In A and B, the activity is expressed as the net
enzymatic velocity; each value is the average of two separate
experiments.
E. coli Rb791-competent cells were transformed with the newly constructed
pGlx expression vector and allowed to grow in aliquots up to 0.5, 1,
1.5, 2, and 2.5 A cell densities; the expression
of the P. furiosus protein was induced by the addition of 1
mM IPTG to the culture media, the induction time being 16 h in
each case. The five extracts and the extract from E. coli cells transformed with the expression vector lacking any inserted
sequence (the negative control) were assayed for the thioltransferase
activity using the same protein amount: maximum activity was found in
the extract from transformed cells grown at 1 A
cell density. To further optimize the expression of the protein,
transformed cells grown at 1 A
cell density were
exposed to 1 mM IPTG for increasing time lengths, and the
thioltransferase activity was assayed on each extract as described; the
results showed that the maximum level of expression was reached after
an 18-h induction.
For preparative purposes, the crude extract from
transformed cells grown at 1 A cell density and
induced for 18 h with IPTG underwent two successive thermal
precipitation steps (at 65 and 70 °C); most of the E. coli proteins became insoluble and were removed by centrifugation,
whereas recombinant protein was recovered soluble and active in the
supernatant. Following the thermoprecipitation steps, about
70-80% of the E. coli proteins were removed, yielding an
almost homogeneous 24.8-kDa protein, as judged by SDS-PAGE (Fig. 7); minor contaminants were removed by a gel filtration
chromatography. The recombinant protein and the native one were
indistinguishable in pI, thermal stability, and catalytic activities.
Figure 7: SDS-PAGE analysis of the steps during the purification of expressed P. furiosus protein. Lane 1, E. coli crude extract; lane 2, sample after heat treatments; lane 3, sample from Superdex 75 column; lane 4, molecular mass standards. Samples (6 µg/lane), treated and run as described under ``Experimental Procedures,'' were stained with Coomassie Blue.
In the framework of a project aimed at studying protein disulfide oxidoreductases from Archaea, we purified a heat-stable redox protein of 24.8 kDa and pI 4.9 from the crude extract of the hyperthermophile P. furiosus. The purification conditions described yielded 0.5 mg of pure protein from 10 g of cell paste; this yield did not vary significantly when the purification was carried out in the presence of DTT. The low cellular amount of a protein poses a limit to its structural and functional characterization; therefore, we assessed a high level expression system of the recombinant P. furiosus protein cloned in E. coli by optimization of both the growth time of the transformed cells and the induction time with IPTG. Based on the consideration that a heat-stable protein expressed in a mesophilic host differs from the endogenous proteins for its resistance against heating, the extract of E. coli was enriched for the P. furiosus protein by two simple thermal precipitation steps. The expression system and the purification procedure described allowed us to obtain fully active protein in a very large amount: 18 mg of pure protein starting from 85 mg of E. coli crude extract. The crystallization of the P. furiosus protein is currently being carried out with the purpose of obtaining information about its three-dimensional structure.
The protein from P. furiosus described here is clearly a member of the family of protein disulfide oxidoreductases; the presence of the sequence CPYC and the demonstration of its thioltransferase activity prompt us to call it ``glutaredoxin-like'' protein. However, it is an unusual protein. All glutaredoxins described so far have the conserved active sequence located at their N terminus; the sequence CPYC is situated at the C-terminal portion of the P. furiosus protein, which shows sequence similarity with glutaredoxins from different sources (Fig. 4). The dithiol in the sequence CQYC at the N terminus of the P. furiosus protein (residues 35-38) is not conserved in any of the protein disulfide oxidoreductases described so far. We exclude the possibility that this dithiol could be considered a homologue to the additional dithiol that is present in all mammalian glutaredoxins with the conserved sequence CIGGC and whose function is not clear. Even if the canonical sequence CPYC is the best candidate for being responsible for the catalytic activity of the P. furiosus protein, we cannot rule out a contribution of the additional dithiol. The expression system described is highly suitable for site-directed mutagenesis studies that can provide precise information about which residues contribute to the catalytic activity and to what extent.
The molecular mass of the P. furiosus protein, 24.8 kDa, is unusual for most
glutaredoxins, which have a typical size of 10-12 kDa. E.
coli contains two glutaredoxins of 10 kDa and one glutaredoxin of
27 kDa, named Grx2, whose N-terminal sequence shows the conserved site
CPYC (Aslund et al., 1994). One cannot simply assume that the P. furiosus protein described here is the homologue of Grx2;
nevertheless, a class of large glutaredoxins may exist. The protein of
24 kDa isolated from the aerobic hyperthermophilic archaeon Sulfolobus solfataricus (Guagliardi et al., 1994),
called thioredoxin, is likely to be the homologue of the P.
furiosus protein; the two proteins have similar amino acid
compositions and pI as well as comparable insulin reductase activity
and thioltransferase activity. ()The chloroplasts of the
unicellular green algae Scenedesmus obliquus contain a
thioredoxin of 28 kDa (Langlotz et al., 1986; Langlotz and
Follmann, 1987); the cyanobacterium Anabaena sp.7119 has a
thioredoxin of 25.5 kDa, which shares amino acid content with the S. obliquus protein (Whittaker and Gleason, 1984). These
proteins have been classified as thioredoxins based on their capability
to stimulate several disulfide-containing enzymes. However, the S.
obliquus protein does not show immunoreactivity with the E.
coli thioredoxin, and fluorescence studies suggest that the
tryptophan residues in the Anabaena protein are not in the
vicinity of the active site, as typical of such residues in
thioredoxins. The determination of the primary structures of the large
redox proteins will be of great interest, as will the eventual
elucidation of their functional specialization.
In Bacteria and Eucarya, protein disulfide oxidoreductases are involved in many fundamental cellular processes; protein disulfide isomerase is responsible for the catalytic rearrangements necessary for the correct folding of nascent protein molecules, whereas the thioredoxin system (thioredoxin, NADPH, and thioredoxin reductase) and the glutaredoxin system (glutaredoxin, GSH, NADPH, and glutathione reductase) have been proposed to participate in several different cellular processes through disulfide reductase activity. The information available on protein disulfide oxidoreductases from Archaea is too scanty to define their physiological function(s). The protein described here from P. furiosus and that from S. solfataricus can catalyze the reduction of disulfide bonds in proteins and small compounds; both proteins failed to reactivate scrambled RNase A and to form disulfide bonds in reduced denatured RNase A (data not shown). The protein isolated from the strictly anaerobic methanogen M. thermoautotrophicum serves as a hydrogen donor for the E. coli ribonucleotide reductase and catalyzes the DTT-dependent reduction of insulin disulfides but does not show thioltransferase activity (Schlicht et al., 1985; McFarlan et al. 1992).
The reducing system utilized by the archaeal protein disulfide oxidoreductases is also puzzling. We were unable to detect NADPH-dependent glutathione reductase in crude extracts of P. furiosus and S. solfataricus. To the best of our knowledge, the extracts of different Archaea are devoid of GSH or related peptides, even if the occurrence of other monothiols cannot be ruled out. McFarlan et al.(1992) suggested that the glutaredoxin from M. thermoautotrophicum may utilize a reducing system based on hydrogenase and ferredoxin. Ferredoxins and hydrogenases have been isolated from different Archaea (Adams(1993, 1994), and references therein), and in our opinion, the possibility exists that a ferredoxin-dependent reducing system might work in Archaea. Of course, this hypothesis must await further experimental support.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z38068[GenBank].