From the Laboratory of Protein Biochemistry and
Protein Engineering, Gent University, 9000 Gent, Belgium, the
§ Department Substances Naturelles, Haute Ecole Luria de
Brouckère, Institut Meurice, 1070 Bruxelles, Belgique, the
¶ Department of Biochemistry and Molecular Biophysics, University
of Arizona, Tucson, Arizona 85721, and the
Department of
Chemistry, University of California, San Diego, La Jolla, California
92093
Received for publication, March 6, 2001, and in revised form, March 22, 2001
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ABSTRACT |
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Among the
Chromatiaceae, the glutathione derivative
Several different oxidoreductase processes shared by eukaryotes
and certain prokaryotes such as the detoxification of environmental chemicals (1, 2), formaldehyde dissimilation (3, 4), and the reduction
of ribonucleotides (5) depend to a certain extent on the glutathione
reductase
(GR1)-dependent
glutathione (GSH) redox cycle. Moreover, in eukaryotes, some reactive
oxygen intermediates are detoxified directly by the action of
glutathione peroxidases (6) and, to a lesser extent, of glutathione
S-transferases (7). Members of another enzyme family of
GSH-dependent thiol-disulfide oxidoreductases, designated
thioltransferases or glutaredoxins (Grx), are believed to act as one of
the primary defenders against mixed disulfides formed after oxidative
damage to proteins (8). Besides these direct and indirect protection
systems against the products of aerobic metabolism relying on GSH
cycling, GSH itself also serves an indirect antioxidant function by
protecting the amino acid cysteine against auto-oxidation (9).
Only three groups of prokaryotes, the Gram-negative cyanobacteria and
purple bacteria and some Gram-positive streptococci and enterococci
(10, 11), produce GSH together with the recycling GR (12). Thus far, no
significant GSH-dependent peroxidase activity has been
reported for a GSH producing prokaryote. On the other hand, substantial
thioltransferase activity is encountered indicating that in terms of
oxygen shielding, GSH metabolism in prokaryotes does not serve a direct
detoxification system for reactive oxygen intermediates but only
maintains disulfides in the reduced state. Prokaryotic aerobes and
pathogens require an array of antioxidant defense mechanisms to protect
themselves against the reactive oxygen intermediates produced by the
incomplete reduction of oxygen during respiration or by the
antimicrobial response of the host phagocytes (13, 14). Detoxification
of the freely diffusible hydrogen peroxide
(H2O2), which in turn can be reduced further via the Fenton reaction to extremely reactive hydroxyl radicals, is
completed by the action of catalases, heme- and manganese-containing peroxidases, and several members of the large multifunctional AhpC/TSA
protein family, recently classified as peroxiredoxins (Prx). To date,
it seems that all bacterial Prx enzymes obtain the necessary reducing
equivalents from the thioredoxin reducing system itself or from a
thioredoxin-like reducing system, because it was demonstrated recently
that the flavoprotein component (AhpF) of the Salmonella
typhimurium alkyl hydroperoxide reductase (AhpCF) system and
bacterial thioredoxin reductase have very similar mechanistic properties (15). Apparently, GSH is never involved in bacterial Prx-reduction.
Chromatium species are anaerobic sulfur-oxidizing
phototrophs that produce glutathione amide (GASH) (16), a GSH
derivative modified at the terminal glycine. An original anaerobic
function for this GASH metabolism was proposed by Pott and Dahl (17) and implies a possible involvement in the transfer of sulfide across
the periplasmic membrane. When grown photoautotrophically on sulfide,
GASH is present in its persulfide form (16), supporting the hypothesis
that the periplasmically formed persulfide becomes transported to the
cytoplasm, where the GASH-bound sulfide is released by the action of a
heterodisulfide reductase. Chromatium species extracts do
show glutathione amide disulfide (GASSAG) reductase activity (16), and
the involvement of GAR as the heterodisulfide reductase in the
hypothesized sulfide transfer mechanism has to be considered because
GSH persulfide reduction was established already for the bovine GR
(18).
Here we present the isolation and successful expression in
Escherichia coli of the Chromatium gracile garB
gene, which has permitted the characterization of the C. gracile GAR enzyme. Further, we provide evidence for the existence
of a novel Prx-containing peroxidase system, probably widespread among
Gram-negative pathogens, that is fueled by the
GAR-dependent redox cycling of the
Chromatium-specific low molecular weight thiol GASH.
Materials
Restriction enzymes were from New England Biolabs (Beverly, MA).
T4 DNA ligase, the DIG DNA labeling kit, and the DIG luminescent detection kit were obtained from Roche Diagnostics. Taq DNA
polymerase was from Amersham Pharmacia Biotech. Plasmid DNA was
prepared on a 30-ml scale using the Qiagen (Crawley, UK) plasmid
purification kit. The pGEM-T and pUC18 plasmids were from Promega
(Madison, WI), and the pET11a plasmid was from Novagen (Madison, WI).
DNA sequencing was performed using an ABI PRISM 377 sequence detection system (Applied Biosystems, Foster City, CA). An AKTA-design FPLC system (Amersham Pharmacia Biotech) was used for all chromatographic protein purification steps (all other chromatographic equipment was
also purchased from Amersham Pharmacia Biotech). All other biochemical
reagents were purchased from Sigma-Aldrich. A Uvikon 943 double beam
UV-visible spectrophotometer (Kontron Instruments, Watford, UK) was
used for the spectroscopic measurements.
Strains and Media
C. gracile (DSM 1712) was grown on Pfenning's
medium (19), supplemented with 1% NaCl, by anaerobic photosynthesis at
a temperature of 30 °C. E. coli strains were grown on LB
medium (Life Technologies, Inc.) supplemented with 100 µg/ml
carbeniciline when necessary. Strain XL-1 blue (New England Biolabs,
Herefordshire, UK) was used as a recipient to detect
N-terminal Amino Acid Sequence Determination
GAR was partially purified according to the method of Chung and
Hurlbert (20). The partially purified enzyme sample was loaded onto an
SDS-polyacrylamide gel, and after electrophoresis, the proteins were
transferred onto a ProBlott membrane (Applied Biosystems) as described
by Maniatis et al. (21). The membrane was stained with
Coomassie Blue, and the protein band corresponding to the GAR enzyme
was subjected to N-terminal sequence determination using a 477A pulsed
liquid sequenator (Applied Biosystems). Sequencing reagents were from
the same firm. Forty-nine residues were identified covering a 51-amino
acid N-terminal stretch,
TQHFDLIAIGGGSGGLAVAEKAAAFGKRVALIESKALGGTXVNVGXVPKKV. The same
procedure was applied to verify the first seven amino acid residues of
the partially purified recombinant Prx/Grx.
SDS-PAGE
Protein samples were subjected to reducing SDS-polyacrylamide
gel electrophoresis (22) and stained with Coomassie Blue or Silver
(23). The total protein concentration was determined by the method of
Bradford (24) using the Bio-Rad 500-0006 kit with bovine serum albumin
as a standard.
DNA Techniques
C. gracile genomic DNA was isolated according to the
cetyl-trimethyl-ammonium-bromide method described by Ausubel et
al. (25). A 150-base pair fragment was obtained from C. gracile genomic DNA via PCR amplification using degenerate primers
based on the N-terminal amino acid sequence of endogenous GAR (forward
primer, 5'-ACICARCAYTTYGAY-3'; reverse primer, 5'-CYTTYTTIGGIACRCA-3'). This PCR fragment was ligated into the pGEM-T vector, and the resulting
construct (pGEM-GAR) was verified by sequence analysis. The pGEM-GAR
construct was used as a template for PCR-based synthesis of a
digoxigenin-labeled probe using the DIG DNA labeling kit. Therefore, a
perfect match primer pair was designed (forward primer, 5'-ACCCAGCATTTCGACCTG-3'; reverse primer, 5'-CCTTCTTGGGCACGCAGC-3'). C. gracile subgenomic DNA fragments, generated by subjecting
the genomic DNA to the action of various restriction enzymes, were screened with the digoxigenin-labeled probe in Southern hybridization experiments. A single signal corresponding to a fragment of ~3.6 kilobases was obtained when the genomic DNA was initially cut with
BamHI. A pUC18-subgenomic library of C. gracile
DNA consisting of BamHI fragments between ~3.4 and 3.9 kilobases was screened by colony hybridization with the
digoxigenin-labeled oligonucleotide. The detection of transformants was
performed with the nonradioactive DIG luminescent detection kit
according to the manufacturer's instructions. Nucleotide sequence
determination of one positive pUC18 derivative was initiated using the
pUC18 universal primers M13F and M13R, and new primers were synthesized
at ~450 nucleotide intervals based on the results of previous sequencing.
Synthesis of GASH and GASSAG
GSH was synthesized on the Advanced ChemTech 90 peptide
synthetizer (Louisville, KY) using the
N-(9-fluorenyl)methoxycarbonyl (Fmoc) strategy (26)
on a polyamide resin developed for the synthesis of peptide amides (0, 52 mM/g) (Rink Resin, Advanced ChemTech)
(27). Fmoc-L-amino acids (3 eq = 1, 56 mM)
were introduced into the chain as pre-activated pentafluorophenyl
esters (OPfp) in the presence of a 3-fold excess of
1-hydroxybenzotriazole (1 eq). The introduced residues were
Fmoc-Gly-OPfp, Fmoc-Cys(Trt)-OPfp, and
N- Overexpression of GAR and Prx/Grx
The garB gene was PCR-amplified, allowing the
incorporation of NdeI (forward primer,
5'-GGGAATTCCATATGACCCAGCATTTCG-3') and BamHI (reverse
primer, 5'-CACGGATCCTCAGGCCGC-3') sites at the 5' and 3' termini of the
gene, respectively. The PCR amplification of the garA gene
also resulted in an NdeI/BamHI-bordered gene (forward primer, 5'-GGAATTCCATATGTTGCAAGATCG-3'; reverse primer, 5'-CACGGATCCTTAGGCGCTGGCGCGCTCC-3'). The amplified fragments were digested with NdeI and BamHI and were
subsequently cloned into the NdeI/BamHI-digested
expression plasmid pET11a. The resulting constructs (pET-GAR and
pET-Prx/Grx), first verified by nucleotide sequencing, were then
introduced into competent BL21(DE3) E. coli. For the
preparation of the enzyme, one colony was used to inoculate 50 ml of LB
medium containing 100 µg/ml carbeniciline, and the culture was
incubated for 10 h. The culture was used to inoculate 4 liters of
LB medium containing the antibiotic at a ratio of 10 ml/liter. When the
A600 value of the culture reached the value 0.7-1.0, isopropyl- Protein Purification
GAR--
The crude extract from a GAR-producing culture was
clarified by centrifugation at 15,000 × g for 30 min
at 4 °C and fractionated with solid
(NH4)2SO4. The 20-60% saturation
precipitate was dissolved in 20 ml of buffer A (50 mM
sodium phosphate buffer, pH 7.0, containing l mM EDTA and 1 M (NH4)2SO4). The
enzyme solution (2 ml/run) was applied onto a butyl-Sepharose packed HR
16/10 column (Amersham Pharmacia Biotech) equilibrated with buffer A. After loading, the resin was washed with 7 column volumes (CV) of the
same buffer. The elution of GAR occurred with a decreasing step
gradient of (NH4)2SO4 (step 1, 1000-450 mM in 8 CV; step 2, 450-375 mM in 10 CV; step 3, 375-0 mM in 1 CV) at the rate of 2 ml/min. The enzyme eluted during step 3 and the GAR-containing fractions of 10 runs were pooled, concentrated using 15-ml Vivaspin filters (molecular mass cutoff of 5 kDa), and dialyzed against 20 mM Tris-HCl, pH 7.0. The concentrate was further purified
on the ResourceQ anion exchanger (Amersham Pharmacia Biotech)
equilibrated with 20 mM Tris-HCl, pH 7.0. The yellow enzyme
was eluted with a 30-CV linear gradient from 0 to 1 M NaCl
in 20 mM Tris-HCl, pH 7.0, at a rate of 4 ml/min. The
protein eluted at ~0.49 M NaCl. Finally, the pooled
fractions were concentrated and loaded onto a Hiload 16/60 Sephadex 75 gel-sizing column (Amersham Pharmacia Biotech) previously equilibrated
with 10 mM Tris-HCl, pH 7.0. Elution occurred up-flow with
10 mM Tris-HCl, pH 7.0, at a flow rate of 2 ml/min. SDS-PAGE analysis of the pooled yellow-colored fractions
indicated that GAR was purified to homogeneity. The gel-sizing protocol was also applied for native molecular mass determination. Molecular mass standards were derived from a gel filtration calibration kit
(Amersham Pharmacia Biotech) containing ribonuclease A (13.7 kDa),
chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin
(67 kDa), aldolase (158 kDa), and Dextran 2000 (2000 kDa).
The absorption coefficient for the oxidized GAR prosthetic flavin was
determined as described by Macheroux (28), except the cofactor was
released from thermally denatured GAR. The concentration of homogeneous
GAR was determined spectroscopically at 461 nm by using an absorption
coefficient of 11.0 mM Prx/Grx--
The cell debris of a Prx/Grx-containing crude
extract was precipitated by centrifugation at 15,000 × g for 30 min at 4 °C, and the supernatant was
fractionated with solid (NH4)2SO4.
The 30-70% saturation precipitate was dissolved in 20 ml of buffer B
(50 mM sodium phosphate buffer, pH 7.4, containing 1 mM EDTA and 1.5 M
(NH4)2SO4. The enzyme solution (2 ml/run) was then loaded onto an octyl-Sepharose packed HR 16/10 column
(Amersham Pharmacia Biotech) equilibrated with buffer B. After loading,
the resin was washed with the same buffer for 10 CV. The protein was
eluted with a decreasing step gradient of
(NH4)2SO4 (step 1, 1500-450 mM in 8 CV; step 2, 450-375 mM in 15 CV; step
3, 375-0 mM in 1 CV) at a rate of 2 ml/min. Fractions
containing Prx/Grx, as assessed by activity measurements and SDS-PAGE
analysis, were pooled during step 3, concentrated, and dialyzed against
buffer C (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA).
The dialyzed protein solution was applied onto a ResourceQ column
pre-equilibrated with buffer C. The column was washed with 5 CV of
buffer C, and the protein was eluted with a 25-CV linear salt gradient
(0.0-1.0 M NaCl in buffer C) at a flow rate of 3.5 ml/min.
Prx/Grx eluted at 0.22 M NaCl. SDS-PAGE analysis indicated
the presence of the 27.5-kDa Prx/Grx enzyme together with a few
contaminating protein bands.
Mass Determination
Mass spectral analysis of the proteins was performed using
nanospray ionization on a hybrid quadrupole time-of-flight mass spectrometer (Micromass, Whytenshawe, UK). The protein sample was
diluted to ~5 pmol/µl in acetonitrile/water/formic acid (1.1:0.01); 3 µl of the dilutions was loaded in a coated borosilicate needle (Protana, Odense, Denmark). The needle was placed into the quadrupole time-of-flight source, and after breaking the tip, a voltage of 1350 V
was applied. To determine the native molecular weight and the type of
quaternary structure, the solvent was replaced by water. The source
temperature was 30 °C in all cases. The mass spectrometer was
calibrated independently using NaCsI. Mass spectral identification of
the prosthetic flavin of GAR was performed under the same
conditions as described for the subunit mass determination, except the
negative ion mode of analysis was applied.
Enzyme Assay
GAR--
GAR activity was measured by two methods. For all
determinations involving NADH, the GASSAG-dependent NADH
oxidation was monitored at 340 nm ( Prx/Grx--
Prx/Grx-dependent hydroperoxide
reduction was demonstrated in a reconstitution assay by coupling the
hydroperoxide-dependent GASH oxidation to the
GASSAG-dependent NADH oxidation, again monitored at 340 nm.
Unless otherwise stated, the reconstitution assay contained 0.4 units
of GAR, 150 µM NADH, 500 µM GASH, 50 µg/ml partially purified Prx/Grx, and 100 µM
H2O2 or small alkyl hydroperoxides in 0.5 ml of
125 mM potassium phosphate buffer, pH 7.9, and 0.1 mM EDTA. The reaction was started with the addition of the
hydroperoxide. No blank rate of NADH oxidation was observed during the
reconstitution assay devoid of hydroperoxide. In the specificity study,
GASH, GAR, and NADH were replaced by yeast GR (0.4 units), GSH (500 µM), and NADPH (150 µM), respectively.
Nucleotide and Amino Acid Sequence Analysis
The isolated garB gene-containing
BamHI fragment (Fig. 1)
consists of 3519 base pairs and comprises three open reading frames (ORF): garA, garB, and an ORF encoding a
polypeptide that is 67% identical to a hypothelical E. coli
protein. The GAR enzyme-encoding garB gene, 1392 base pairs
long, translates to a polypeptide of 463 amino acids. However, in
accordance with the situation for the E. coli (29) and
Plasmodium falciparum GR enzymes (30) and for the
Trypanosoma congolense trypanothione reductase (31), the
N-terminal amino acid sequence analysis of the endogenous as
well as of the recombinant GAR revealed that the initiator methionine
is post-translationally deleted. Comparison with E. coli GR
(Fig. 2) and Crithidia
fasciculata trypanothione reductase (32) reveals 49 and 34.5%
amino acid sequence identity, respectively, and emphasizes the
conservation of the motifs essential for binding of the prosthetic
group and substrates in the C. gracile enzyme.
-L-glutamyl-L-cysteinylglycine amide, or
glutathione amide, was reported to be present in facultative aerobic as
well as in strictly anaerobic species. The gene
(garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic
organization has been examined. The garB gene is
immediately preceded by an open reading frame encoding a novel 27.5-kDa
chimeric enzyme composed of one N-terminal peroxiredoxin-like domain
followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was
established in vitro to be a glutathione
amide-dependent peroxidase, being the first example of a
prokaryotic low molecular mass thiol-dependent
peroxidase. Amino acid sequence alignment of GAR with the functionally
homologous glutathione and trypanothione reductases emphasizes the
conservation of the catalytically important redox-active disulfide and
of regions involved in binding the FAD prosthetic group and the
substrates glutathione amide disulfide and NADH. By establishing
Michaelis constants of 97 and 13.2 µM for glutathione
amide disulfide and NADH, respectively (in contrast to
Km values of 6.9 mM for glutathione
disulfide and 1.98 mM for NADPH), the exclusive substrate
specificities of GAR have been documented. Specificity for the
amidated disulfide cofactor partly can be explained by the substitution
of Arg-37, shown by x-ray crystallographic data of the human
glutathione reductase to hydrogen-bond one of the glutathione glycyl
carboxylates, by the negatively charged Glu-21. On the other hand, the
preference for the unusual electron donor, to some extent, has to rely
on the substitution of the basic residues Arg-218, His-219, and
Arg-224, which have been shown to interact in the human enzyme with the
NADPH 2'-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest
GAR to be the newest member of the class I flavoprotein disulfide
reductase family of oxidoreductases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-complementation for pGEM-T derivatives on LB plates supplemented
with 80 µM isopropyl-
-D-thiogalactoside and 32 µg/ml
5-bromo-4-chloro-3-indolyl-
-D-galactoside. All
expression plasmids were introduced into competent BL21(DE3) E. coli (Novagen).
-t-butoxycarbonyl-Glu(OPfp)-
-t-butyl.
The efficiency of coupling was always checked by the Kaiser test.
Deprotection of the Fmoc groups was carried out with 20% piperidine in
N,N-dimethylformamide for 15 min. After washings,
the peptide was liberated with a 95:5 (v/v) solution of trifluoroacetic
acid/1,2-ethanedithiol for 90 min under nitrogen. After
filtration, the trifluoroacetic acid was removed under vacuum.
The peptide mixture in water was treated with diethyl ether (1/1) to
eliminate scavengers. GASSAG was prepared according to the method
described by Bartsch et al. (16) and purified by HPLC on a
C18 reverse-phase column.
-D-thiogalactoside was added to a
final concentration of 1 mM. Incubation was continued for
15 h at 37 °C, after which the cells were harvested by
centrifugation (4000 × g) for 15 min, resuspended in
60 ml of sonication buffer (50 mM sodium phosphate buffer,
pH 7.2, containing 1 mM EDTA), and stored at
80 °C
overnight. The cells were broken using a Branson sonicator with four
30-s bursts of 45 watts with 30-s intervals.
1
cm
1/active site.
= 6.22 mM
1 cm
1) in 125 mM
potassium phosphate buffer, pH 7.1, containing 0.1 mM EDTA.
However, in case NADPH was added as the source of reducing equivalents,
monitoring GAR activity was based on the coupling of GASSAG reduction
to the reduction of 5,5-dithio-bis-2-nitrobenzoate, which is measured
at 412 nm (
= 13.6 mM
1
cm
1). Therefore, 500 µM
5,5-dithio-bis-2-nitrobenzoate was added to the enzyme solution
immediately before initiating the reaction. In all cases, the reaction
was started with the simultaneous addition of substrate and reductant
to the quartz cuvette containing the enzyme solution. All reactions
were carried out at 25 °C. The steady-state kinetic data were
analyzed by fitting them to the equation v = Vmax [S]/(Km + [S]).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence of the C. gracile subgenomic BamHI fragment
containing the genes garA and garB,
encoding Prx/Grx and GAR, respectively, together with a third ORF,
encoding an amino acid sequence that is 67% identical to a
hypothetical E. coli protein. The amino acid
sequences shown are deduced from the sequenced nucleotides with the
exception of the boxed residues of the GAR and Prx/Grx
enzyme, which were identified by N-terminal protein sequence analysis.
The first residue of the processed mature GAR protein is indicated by
$. Putative ribosome binding sites and start codons are in
boldface. Asterisks indicate stop codons, and
putative promoter sequences are bold-italic. The
underlined residues of the Prx/Grx deduced polypeptide align
with a distinct class of Prx enzymes, whereas the dashed
underlined residues align with members of the Grx enzyme family
(see "Discussion"). The boxed sequences shown in
capitals are the BamHI sites.
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Fig. 2.
Alignment of the amino acid sequences of
C. gracile GAR, E. coli GR (44),
human erythrocyte GR (43), and E. coli lipoamide
dehydrogenase
-fold region of the
NADH-binding domain (49). Secondary structures are derived from
the crystallographic analysis of the E. coli enzyme (59) and
exemplified via the ESPript 1.9 program. Identical residues at the same
position in all the aligned sequences are in reverse
contrast. Residues interacting with the disulfide substrate in the
human enzyme are marked with an arrow, and the residues that
interact with the C termini of the disulfide substrate are additionally
marked with a star. The characteristic
-fold region
of NAD(P)H-binding domains is underlined, and the residues
involved in the discrimination of the dinucleotide cofactor are marked
with a square.
The garA and garB genes are separated by 185 base pairs and are transcribed in the same direction. A computer search for putative promoter sequences points to the cotranscription of the two ORFs. The enzyme deduced from garA comprises 247 amino acids and seems to be chimeric, having an N-terminal part that is significantly homologous to the Prx family enzymes (33, 34) and a C-terminal domain that is Grx-like (35). Computer alignment of the entire Brassica rapa CPrxII, a Prx peroxidase that has been shown to catalyze the reduction of hydrogen peroxide with the use of electrons from the thioredoxin system (33), with amino acid residues 1-163 of the garA deduced chimeric protein reveals 39% identity and striking homology around the N-terminal cysteine residue, which is believed to be the site of oxidation by peroxides (36). A similar alignment of the E. coli Grx3 (5) with amino acids 171-243 emphasizes the presence of the Grx typical redox-active Cys-Pro-X-Cys motif and reveals 62% homology.
The genomes of the Haemophilus species, Yersinia pestis, Pasteurella multocida, Vibrio cholerae, Bordetella pertussis, and Thiobacillus ferrooxidans, also enclose a homologue of the Prx/Grx coding sequence along with a glutathione reductase gene, but only the latter organism has both genes being organized in an operon as is the case in C. gracile.
Characterization of Recombinant GAR
Physical and Spectral Characterization--
Recombinant GAR was
overexpressed and purified to homogeneity (Fig.
3) as described under "Experimental
Procedures." Because E. coli strain BL21(DE3) contains an
active glutathione reductase gene, the possibility of contamination of
the vector-encoded GAR with the chromosomally encoded GR had to be
considered. However, the two disulfide reductases were well resolved by
chromatography, because neither GR activity nor a GR-derived additional
mass spectral peak of 48,641 Da was detected after the analysis of
finally purified GAR.
|
A subunit mass of 49,030 Da obtained using nanospray ionization on a
quadrupole time-of-flight mass spectrometer agrees well with the value
calculated from the deduced amino acid sequence, 49,159 Da, minus 131 Da corresponding to the N-terminal methionine residue being absent in
the native enzyme. A native molecular mass of 98,000 Da was obtained
from gel filtration chromatography and mass spectrometric analysis,
which is consistent with a dimeric quaternary structure, a
characteristic feature of the glutathione reductase family. The
strength of the GAR dimer is significantly increased in the presence of
copper ions, because boiling and SDS treatment failed to disrupt the
GAR dimer conformation after the addition of CuCl2 at
micromolar levels (Fig. 3). We established that the gradual increase in
copper-mediated dimer strength concurs with the gradual decrease of GAR
activity observed when comparable CuCl2 concentrations were
added to the reaction mixture (data not shown). Mn2+,
Ni2+, Zn2+, Hg2+, and
Ca2+ ions were also tested in their capacity to capture the
dimer state, but they all failed to do so. Copper is, after mercury, the most effective metal ion inhibitor of members of the glutathione reductase family (20, 37). The visible absorption spectrum of oxidized
GAR shows maxima at 273, 378, and 461 nm, with a shoulder at 481 nm,
indicating the presence of a flavin cofactor. The spectral ratio
A280/A461 of the
homogeneous enzyme was determined to be 7.2 and is similar to other
glutathione reductases (7.2 and 7.1 for the E. coli (29) and
Enterococcus faecalis (11) enzymes, respectively). To
identify the flavin prosthetic group, the flavin was liberated by
thermal denaturation of the protein at 100 °C for 20 min. The
resulting free flavin showed absorption maxima at 370 and 450 nm, and
the ratio of A461 of the enzyme-bound flavin to
A450 of the free flavin yielded an absorption
coefficient for the GAR enzyme of 461 = 11.0 mM
1 cm
1 (S.D. = 0.1 mM
1 cm
1, n = 4). The liberated flavin was purified by HPLC and identified by
negative ion mode mass spectral analysis to be FAD. Upon anaerobic titration with NADH, the spectrum of the enzyme exhibits decreases in
absorbance at 461 nm and increases at ~530 nm (Fig.
4), demonstrating the formation of a
charge-transfer complex between one of the newly reduced active site
thiols and the oxidized FAD.
|
Kinetic Characterization--
C. gracile GAR exhibits a
high specificity for the substrate GASSAG and the reductant NADH, both
of which display Michaelis-Menten saturation kinetics (data not shown).
The Km for GASSAG with 100 µM NADH was
97 ± 5 µM, and a turnover number of 14,981 ± 80 µmol min1 µmol
1 FAD was calculated,
yielding a kcat/Km of 2.6 106 M
1 s
1. The
apparent Km and kcat values
for NADH with 500 µM GASSAG were 13.2 ± 1.8 µM and 11,210 ± 90 µmol min
1
µmol
1 FAD, respectively, yielding a
kcat/Km of 14.2 106 M
1 s
1. The
observed poor activity of the reductase with either glutathione disulfide or NADPH are, in the case of the former substrate, mostly because of the weak binding to the active site of GAR
(Km = 6.9 ± 0.2 mM,
kcat = 8759 ± 65 µmol min
1
µmol
1 FAD), whereas in the case of the dinucleotide,
binding as well as catalysis are seriously diminished (apparent
Km = 1.98 ± 0.1 mM and apparent
kcat = 805 ± 24 µmol min
1
µmol
1 FAD).
Reconstitution of a GASH-dependent Peroxidase
System--
Even though the amino acid sequence of the C. gracile Prx/Grx chimeric enzyme has no homology with recently
characterized eukaryotic non-selenium glutathione peroxidases (38, 39)
and no GSH-dependent peroxidase activity was observed in
Chromatium vinosum extracts (40), we tested the possibility
that the C. gracile GASH metabolism could serve a direct
antioxidant function by neutralizing peroxides. Many Prx-type enzymes
have indeed been proven to be potent peroxide reducers through the use
of electrons coming from different sources (33, 34, 41, 42), and
evidently no GSH-dependent peroxidase activity could have
been observed in GASH-producing Chromatium species.
Therefore, recombinant Prx/Grx was partially purified (see
"Experimental Procedures"), after which the first seven amino acid
residues were verified by N-terminal sequence analysis. A polypeptide
with a molecular mass of 27,500 Da was visible upon SDS-PAGE analysis
(Fig. 3), matching the value of 27,426 Da calculated from the deduced
amino acid sequence. The hydroperoxide-dependent
Prx/Grx-catalyzed oxidation of GASH was established in
vitro, and as to be expected, the reaction is fueled by NADH via
GASH redox cycling (Fig. 5).
Nevertheless, the GSH reducing system also seems to be an effective
electron donor (Fig. 5, curves 4, 7, and
9). The Prx/Grx enzyme displays a similar activity with
hydrogen peroxide (Fig. 5b) and small alkyl hydroperoxides
(Fig. 5, a and c), indicating broad substrate specificity.
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DISCUSSION |
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GASH, -L-glutamyl-L-cysteinylglycine
amide, is a recently discovered derivative of GSH that has been found
in strictly anaerobic as well as in aerotolerant Chromatium
species (16). Extracts of Chromatium species were found to
contain an enzyme activity capable of reducing oxidized GASH (16). The
work reported here demonstrates that the activity is carried out by a
new member of the class I flavoprotein disulfide reductase family of
oxidoreductases, which includes GR (43, 44), trypanothione reductase
(45), mycothione reductase (46),
-glutamylcysteine reductase (9), mercuric reductase (47, 48), and lipoamide dehydrogenase (49). We could
establish numerous structural and spectral homologies between the
C. gracile GAR and glutathione reductases (50), making it
clear that GAR is a new GR homologue. However, in addition to the
altered substrate specificity of GAR, another striking difference lies
in the fact that GAR displays a strong preference for NADH as the
electron donor. Both specificity alterations are demonstrated by the
summary of the kinetic parameters given in Table
I. The table further illustrates that the
C. gracile GAR and the human GR share comparable kinetic
parameters, taking the preferred coenzymes and substrates into
consideration. This can be demonstrated additionally by comparing the
ratios of the specificity constants for the preferred substrates to
those for the biologically irrelevant substrates. For the C. gracile enzyme, the apparent kcat/Km(NADH)/kcat/Km(NADPH) = 2088 and
kcat/Km(GASSAG)/kcat/Km(glutathione disulfide) = 122, which are similar to the inverse ratios of the human enzyme (2158 and 87.7, respectively).
|
Three-dimensional structure analysis of the GR of human erythrocytes provided a detailed knowledge of the structural features in the active site that impart the specificity for the coenzyme (NADPH) and the substrate (glutathione disulfide) (51). Sequence alignment shows replacements disclosing in part the preference of GAR for NADH and GASSAG (Fig. 2). Seventeen of twenty-one active site substrate interacting residues, direct or solvent-based, are either identical or similar to those of the human enzyme. Three of the four nonconserved residues interact with the C-terminal glycines of the substrate. Of particular interest is Arg-37, which forms a salt bridge with the first substrate glycine carboxylate in the human GR enzyme and is substituted by Glu-21 in GAR. Comparison with the currently characterized GR homologues reveals that the enzyme of the trypanosomatids, which also catalyzes the reduction of a GSH derivative having a C-terminal amidation (N1,N8-bis(glutathionyl)spermidine), displays a negative charge in this region as well. This indicates that the Chromatium enzyme may be the only bacterial enzyme found so far that is specific for GASSAG (cf. for many enzymes in the family, the substrate specificity has not yet been examined).
In most NADH-dependent redox enzymes, the functional domain
primarily responsible for binding the dinucleotide takes the form of a
-unit (52) with a highly conserved
Gly-X-Gly-X-X-Gly sequence motif
between the first
-strand and the succeeding
-helix (53). In
NADPH-binding domains the third glycine residue is often replaced by
alanine (54). Furthermore, the negatively charged 2'-phosphate group of
the AMP moiety of NADPH rests against a cushion provided by positively
charged side chains. In the region corresponding to the
-unit
in the GAR sequence, the complete Gly-X-Gly-X-X-Gly sequence motif is
observed, and in addition, all three basic residues (Arg-218, His-219,
and Arg-224) interacting in the human GR enzyme with the NADPH
2'-phosphate group are replaced (Leu-197, Glu-198, and Phe-203,
respectively). The negative charge caused by Glu-198 is likely
to be the primary cause of the change in reductant specificity. No
other GR has a negative charge in this region, and therefore no other
GR is thought to preferentially utilize NADH.
NADH-dependent enzymes are almost exclusively involved in the oxidative degradations that yield ATP, whereas NADPH-dependent enzymes, with few exceptions, are confined to the reactions of reductive biosynthesis. Therefore, the proposed anaerobic function for the Chromatium species-specific GASH metabolism as a sulfide carrier necessary for cytoplasmic sulfide oxidation (17) looks attractive. By analyzing the genomic organization of the garB gene encoding the crucial enzyme in GASH metabolism, we aimed to broaden our understanding with regard to the proposed sulfide carrier system. We found that the gene is immediately preceded by an ORF (which we termed garA because of the established cooperation between the garA and garB gene products) encoding a 27.5-kDa chimeric enzyme, clearly assembled from two enzyme entities separated by a seven-amino acid stretch containing three proline residues. The N-terminal component is strikingly homologous to Prx enzyme family members found in yeast, plants, and animals already characterized to reduce peroxides in a thioredoxin-dependent manner (33, 34, 55), whereas the second component is homologous to glutaredoxins, which are known to reduce diverse disulfide bonds in a coupled system with GSH, NADPH, and GR. Based on the number of conserved cysteine residues and the properties revealed by immunoblot analysis, the Prx proteins can be subdivided into the subfamilies of the 1Cys-Prx and 2Cys-Prx proteins (56). Phylogenetic analysis, however, indicates that the Prx-like component of the C. gracile Prx/Grx peroxidase together with its homologues constitute a separate Prx type (57). We produced the recombinant 27.5-kDa Prx/Grx chimeric enzyme in E. coli and showed that it possesses peroxidase activity in reconstitution experiments using hydroperoxides, GASH, GAR, and NADH. Differing from the assumption that GSH-dependent hydroperoxide metabolism is catalyzed by a single enzymatic entity (6), we have demonstrated that the C. gracile-specific GASH metabolism (or the physiological irrelevant GSH metabolism) can deliver electrons to a peroxidase, which is most likely assembled from two enzymatic entities, and is capable of reducing H2O2 and small alkyl hydroperoxides at comparable rates.
These data, which show that the C. gracile Prx reducing electrons are Grx-derived (whereas in plants and animals those electrons come from the thioredoxin system), are additional support for the observation that thioredoxin and Grx systems can partially substitute for each other in vivo by reducing the same targets.
In conclusion, looking for a primordial anaerobic function for
glutathione (amide) metabolism in the ancient phototrophic bacterium
C. gracile (41) resulted in the identification of a unique
cascade of oxidoreductase components that catalyzes GASH-mediated hydroperoxide metabolism. The possibility that the distribution of the
Prx/Grx enzyme among Chromatium species determines to a certain extent the oxygen sensitivity of this bacterial family, containing strictly anaerobic as well as aerotolerant representatives, is currently a subject of study. Overall, next to the unique cascade of
oxidoreductase components that uses the electrons provided by the redox
capacity of trypanothione to finally reduce hydroperoxides (33), the
C. gracile GASH-dependent peroxidase system
illustrated here is the second low molecular weight
thiol-dependent alternative for the well documented members
of the eukaryote-specific (seleno-cysteine-containing) GSH peroxidase
family. Because we have found homologues of the Prx/Grx peroxidase in
the translated genomes of several Gram-negative pathogens, which are
supposed to synthesize GSH, Prx/Grx is assumed to be a member of a
novel enzyme family crucial for prokaryotic GSH-dependent
direct protection against reactive oxygen intermediates.
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FOOTNOTES |
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* This work was supported by the Fund for Scientific Research-Flanders Grant 3G003601 and the Bijzonder Onderzoeksfonds of the Gent University Grant 12050198.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Fax: 32-9-264-53-38; E-mail: Jozef.vanbeeumen@rug.ac.be.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M102026200
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ABBREVIATIONS |
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The abbreviations used are: GR, glutathione reductase; GSH, glutathione; Grx, glutaredoxin; Prx, peroxiredoxin; GASH, glutathione amide; GASSAG, glutathione amide disulfide; GAR, glutathione amide reductase; Prx/Grx, chimeric enzyme composed of one Prx and one Grx homologous domain; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Fmoc, N-(9-fluorenyl)methoxycarbonyl; OPfp, pre-activated pentafluorophenyl esters; HPLC, high pressure liquid chromatography; CV, column volumes; ORF, open reading frame.
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