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Journal of Bacteriology, April 2000, p. 2277-2284, Vol. 182, No. 8
Department of Biochemistry, Virginia
Polytechnic Institute and State University, Blacksburg, Virginia
24061
Received 22 September 1999/Accepted 22 January 2000
Rhodaneses catalyze the transfer of the sulfane sulfur from
thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the
gene, and hence the amino acid sequence, of a 12-kDa rhodanese from
Escherichia coli. Well-characterized rhodaneses are
comprised of two structurally similar ca. 15-kDa domains. Hence, it is
thought that duplication of an ancestral rhodanese gene gave rise to
the genes that encode the two-domain rhodaneses. The glpE
gene, a member of the sn-glycerol 3-phosphate
(glp) regulon of E. coli, encodes the 12-kDa
rhodanese. As for other characterized rhodaneses, kinetic
analysis revealed that catalysis by purified GlpE occurs by way of an
enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. The
Kms for SSO32 Genes of known function belonging to
the glp regulon of Escherichia coli encode
proteins that are responsible for the metabolism of
sn-glycerol 3-phosphate (glycerol-P) and its precursors,
glycerol and glycerophosphodiesters (38). The genes
comprising this regulon belong to five operons. Transcription of all
but the glpEGR operon is negatively regulated by the
glp repressor GlpR, a member of the DeoR family of
transcriptional regulators (34, 65, 69-72). Operon
glpACB, encoding the subunits of the anaerobic glycerol-P dehydrogenase, is located near min 51 of the E. coli genome
(38). Divergently transcribed from glpACB is
glpTQ. The genes glpT and glpQ encode
glycerol-P permease and periplasmic
glycerophosphodiesterase, respectively (38). The
glpFKX operon, at min 89, encodes glycerol diffusion
facilitator, glycerol kinase, and a fructose 1,6-bisphophatase (38; J. L. Donahue, J. L. Bownas, W. G. Niehaus, Jr., and T. J. Larson, unpublished data).
The genes glpE and glpG, together with the gene
encoding the transcriptional repressor, glpR, form a complex
operon at min 77 that is divergently transcribed from glpD (71). The gene glpD encodes the
aerobic glycerol-P dehydrogenase (38).
Prior to this study, the functions of the GlpE protein and
the cytoplasmic membrane-associated GlpG protein were unknown
(71, 72). The function of GlpG remains unknown. However, as
reported by Tatusov et al. (58) and more recently by Hofmann
et al. (23), GlpE exhibits sequence similarity to a
superfamily of transfer proteins including the sulfurtransferases
and the tyrosine and dual-specificity phosphatases. In this work we
show that GlpE is a thiosulfate:cyanide sulfurtransferase (EC
2.8.1.1), an enzyme traditionally given the name rhodanese. Rhodaneses
catalyze the transfer of the sulfane sulfur from thiosulfate to
cyanide, forming thiocyanate and sulfite:
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of a 12-Kilodalton Rhodanese Encoded by
glpE of Escherichia coli and Its Interaction
with Thioredoxin
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
and
CN were 78 and 17 mM, respectively. The apparent
molecular mass of GlpE under nondenaturing conditions was 22.5 kDa,
indicating that GlpE functions as a dimer. GlpE exhibited a
kcat of 230 s1. Thioredoxin 1 from E. coli, a small multifunctional dithiol protein,
served as a sulfur acceptor substrate for GlpE with an apparent
Km of 34 µM when thiosulfate was near its
Km, suggesting that thioredoxin 1 or related
dithiol proteins could be physiological substrates for
sulfurtransferases. The overall degree of amino acid sequence identity
between GlpE and the active-site domain of mammalian rhodaneses is
limited (~17%). This work is significant because it begins to reveal
the variation in amino acid sequences present in the
sulfurtransferases. GlpE is the first among the 41 proteins in
COG0607 (rhodanese-related sulfurtransferases) of the database Clusters
of Orthologous Groups of proteins
(http://www.ncbi.nlm.nih.gov/COG/) for which sulfurtransferase activity
has been confirmed.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Although sulfurtransferases are present in many types of organisms
from all three domains of life (57), their physiological roles are still in question. Proposed roles include cyanide
detoxification (61), sulfur metabolism (15, 66),
and mobilization of sulfur for iron-sulfur cluster biosynthesis or
repair (9, 10, 51, 52).
At least two distinct rhodaneses and the related enzyme mercaptopyruvate sulfurtransferase have been described in E. coli, but the genes for these enzymes have not been mapped nor have the sequences for the proteins been determined (3, 60, 62). In this work, we define for the first time the gene, and hence the amino acid sequence, of a rhodanese from E. coli. In the database Clusters of Orthologous Groups of proteins (57), there are 41 proteins in COG0607 (rhodanese-related sulfurtransferases). To our knowledge, GlpE is the first of these proteins for which sulfurtransferase activity has been confirmed.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials. Unless listed below, the reagents used were purchased from Sigma Chemical Company or Fisher Scientific. Synthetic oligonucleotides were prepared with an Applied Biosystems DNA synthesizer (model 381A) using reagents supplied by Cruachem. New England Biolabs supplied restriction endonucleases and reagents for PCR and cloning. Ammonium thiosulfate and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Aldrich. Ferric nitrate and the sodium salt of mercaptopyruvate were purchased from ICN Pharmaceuticals, Inc. Promega supplied thioredoxin 1 from E. coli. Thioredoxin reductase purified from E. coli was a generous gift from C. H. Williams, Jr. (68).
Bacterial strains andplasmids.
The bacterial strains and
plasmids used or constructed are listed in Table
1. BL21(DE3) harboring pGZ105 was used to
overexpress GlpE for purification. Plasmids pGZ154 and pGZ132, in which
glpE is controlled by the tetracycline-inducible
PN25 promoter, were used to overexpress GlpE in DH5
Z1.
To construct these plasmids, the glpE gene was amplified
from pGZ105 by PCR with the primers 5'-acgAAttcccGctagCaat-3' and
5'-tcactagtttgacagcttatc-3', where uppercase letters indicate
mismatches used for the creation of restriction sites. After
cleavage with EcoRI and XbaI, the amplified product was cloned into the same sites of either pZA3
PN25-luc or pZE2 PN25-luc
(42) to create pGZ154 and pGZ132, respectively. Plasmid
pATCBOA2+6 (34) was used for constitutive
expression of the periplasmic marker alkaline phosphatase.
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Growth media and conditions.
Cultures were grown in
Luria-Bertani broth (43) at 37°C. Antibiotics were
included at 100 µg/ml for ampicillin, 50 µg/ml for kanamycin, 25 µg/ml for chloramphenicol, and 20 µg/ml for spectinomycin where
appropriate. Overexpression of GlpE in cells carrying pGZ105 was
induced in mid-log phase by the addition of 0.5 mM
isopropylthio-
-D-galactopyranoside (IPTG) and in cells carrying pGZ154 or pGZ132 by 0.1 µg of tetracycline per ml. Cultures were grown for 2 to 4 h after induction and then harvested in late
log phase as described below.
Cell fractionation procedures.
DH5Z1 harboring
pATCBOA2+6 (glpT-phoA transcriptional fusion)
and pGZ154 was grown, and expression of GlpE was induced by using
tetracycline as described above. Cells were harvested, and two
fractionation procedures were used for preparation of cell extracts.
(i) Periplasmic fraction. Spheroplasts were prepared by incubation of cells with lysozyme and EDTA essentially as described by Kaback (27). Cells were washed twice with 0.5 volume of 10 mM Tris-HCl (pH 8) and then resuspended in 30 mM Tris-HCl (pH 8)-20% sucrose-10 mM EDTA at an absorbance at 600 nm of approximately 3. Lysozyme was added (0.5 mg/ml) using a freshly prepared stock solution of 25 mg/ml in 10 mM Tris-HCl (pH 8). After incubation at room temperature for 30 min with gentle agitation, spheroplasts were removed by centrifugation for 5 min at 10,000 × g. The supernatant was decanted and saved as the periplasmic fraction. Cell pellets were resuspended in 10 mM Tris-HCl (pH 8) and lysed by sonication (cytoplasmic fraction).
(ii) Freeze-thaw fraction.
The second fractionation
procedure combined freeze-thaw treatment (26) and incubation
with EDTA to disrupt the outer membrane. Cells were harvested by
centrifugation (6,000 × g for 5 min at 4°C) and
washed with 0.5 volume of 25 mM Tris-acetate (pH 8.6)-10 mM ammonium
thiosulfate precooled to 4°C. The cell pellet was stored at 
70°C.
The frozen cells were thawed on ice, resuspended in 1/50 the original
volume of buffer A (50 mM Tris-HCl [pH 7.2], 3 mM EDTA), and
incubated on ice for 30 min. Cells were collected by centrifugation,
and the supernatant fraction was saved. Incubation in buffer A was
repeated twice, and the three supernatant fractions were combined
(freeze-thaw extract). After isolation of the freeze-thaw extract, the
remaining cells were resuspended in 10 mM Tris-HCl (pH 8) and lysed by
sonication (cytoplasmic fraction).
Protein purification.
BL21(DE3) harboring pGZ105 was grown
in 500 ml of Luria-Bertani broth, and expression of GlpE was induced by
the addition of IPTG as described above. Freeze-thaw extraction was
performed as described above. The freeze-thaw extract was loaded onto a prepacked Waters quaternary methylamine (Q)-polymethacrylate
anion-exchange column (10 by 100 mm; Protein-Pak Q15HR 1000Å)
equilibrated at room temperature with buffer A. The flow rate was
maintained at 1.5 ml/min. After being washed with 180 ml of buffer A,
the column was developed with a 45-ml gradient from 0 to 105 mM NaCl in
buffer A, followed by a 112.5-ml gradient from 105 to 255 mM NaCl in buffer A. Fractions containing rhodanese activity, eluted between 150 and 190 mM NaCl, were pooled. Rhodanese was concentrated by reapplying
the pooled fractions diluted twofold with buffer A to the
anion-exchange column. The enzyme was eluted with buffer A containing
750 mM NaCl. Fractions containing rhodanese activity were pooled,
brought to 15% glycerol, and stored in aliquots at 70°C. Protein
concentrations were determined by the method of Bradford
(11) with bovine serum albumin as the standard, using materials purchased from Pierce Chemical Company and the
manufacturer's microassay method.
Estimation of subunit and native molecular masses.
Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed as described by Laemmli (33) on 15%
polyacrylamide gels. A modified sample loading buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.02% bromophenol blue,
and 1.5 M -mercaptoethanol was prepared immediately prior to use.
Use of the less-reducing buffer described by Laemmli (33)
resulted in migration of GlpE as a doublet. Protein was visualized with Fast Stain from Zoion Biotech, Inc.
Assay of sulfurtransferase activity. Two methods were used to measure the sulfurtransferase activity of GlpE. Assays were performed at ambient temperature (approximately 22°C).
(i) First method. During purification of GlpE and for characterization of the enzyme, the assay used was essentially that described previously to quantify thiocyanate (3, 66). Assays contained 100 mM Tris-acetate (pH 8.6), 50 mM ammonium thiosulfate, 50 mM KCN, and enzyme in a final volume of 0.5 ml. Reactions were initiated by the addition of KCN and terminated, after 0.5 to 2 min, by the addition of 0.25 ml of 15% formaldehyde. Color was developed by the addition of 0.75 ml of ferric nitrate reagent[100 g of Fe(NO3)3 · 9H2O and 200 ml of 65% HNO3 per 1,500 ml]. Assays were clarified by centrifugation, and the absorbance at 460 nm was determined (66). One unit of enzyme is defined as the amount that catalyzes the production of 1 µmol of thiocyanate per minute and corresponds to an absorbance change at 460 nm of 2.8 in this system.
(ii) Second method. Assays to measure the ability of GlpE to transfer sulfur from thiosulfate to thioredoxin 1 were performed essentially as described previously for the bovine liver rhodanese (48). Each assay (1 ml, final volume) contained 50 mM potassium phosphate (pH 8.2), 0.1 U of thioredoxin reductase per ml, 50 µM NADPH, and the indicated amount of thioredoxin 1. Cuvettes containing all reagents except NADPH were used as blanks. NADPH was added, and the reaction mixtures were allowed to equilibrate for at least 1 h. Periodic measurements of the absorbance at 340 nm were used to ensure that the mixtures had reached equilibrium. After equilibrium had been reached, either purified GlpE (4 µM monomer, 48 µg/ml), ammonium thiosulfate (80 mM), or both were added.
The basis for this assay is that when thioredoxin 1, which contains the active-site motif WCGPC, accepts a sulfane sulfur from rhodanese, a persulfide is formed at the N-terminal cysteine within the active site. The C-terminal cysteine within the active site then reacts with the persulfide, yielding sulfide and oxidized thioredoxin 1. Thioredoxin reductase acts to reduce the disulfide bond of oxidized thioredoxin 1, with the concomitant oxidation of NADPH (48). The decrease in absorbance at 340 nm was measured to determine the rate of NADPH oxidation.Other enzyme assays. Alkaline phosphatase activity was determined by utilizing the substrate p-nitrophenylphosphate and monitoring the increase in absorbance at 410 nm (34). Glucose 6-phosphate dehydrogenase activity was determined by following the increase in absorbance at 340 nm due to reduction of NADP in the presence of glucose 6-phosphate (21).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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GlpE is a sulfurtransferase.
A PSI-BLAST search (4)
using GlpE as the query revealed that GlpE exhibits sequence similarity
to the family of sulfurtransferases that includes rhodaneses and
mercaptopyruvate sulfurtransferases and to the family of tyrosine and
dual-specificity phosphatases (23, 58). Together, these two
families of proteins constitute a superfamily of transfer proteins
(23, 58). Figure 1 illustrates alignment of GlpE with the conserved regions of several
representative members of this superfamily. A comprehensive
discussion of the superfamily, along with alignments for 37 members,
has been published recently (23).
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Cellular location of GlpE.
Two distinct rhodanese activities
have been described previously for E. coli. One is released
from cells only after sonication (62). The other is
"accessible" to substrates when intact cells are added to assay
mixtures and is released from cells by freeze-thaw treatment (3,
62). Release of a protein by freeze-thaw treatment could indicate
a periplasmic localization. However, some cytoplasmic proteins are
released from cells treated with Tris-EDTA (8). Like the
"accessible" enzyme, we found that GlpE was released from cells by
freeze-thaw treatment (Table 3) and by
most other methods that have been used for isolating periplasmic
proteins, including treatment with chloroform (5) and
osmotic shock (49) (data not shown). GlpE, however, has no
N-terminal targeting sequence that would direct it to the periplasm.
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Purification of GlpE.
The modified freeze-thaw treatment
quantitatively released rhodanese activity from intact cells that
overexpress GlpE, thereby facilitating purification of the enzyme
(Table 3). The freeze-thaw extract was subjected to
anion-exchange chromatography. Three peaks containing
rhodanese activity were eluted. The relative amount of rhodanese
in each peak varied, but two peaks eluting near 160 and 185 mM NaCl
typically contained most of the total rhodanese activity. The third
peak eluted in 1 M NaCl. The major protein found in each peak exhibited
similar electrophoretic mobility (approximately 12 kDa) and was
therefore tentatively identified as an isoform of GlpE. Rhodaneses can
be isolated in a sulfur-free form, a persulfide-containing form
(63, 67), and oxidized, less-active forms (3, 16,
24). The first two peaks were pooled and concentrated by
rechromatography and step elution. The resulting protein was more than
95% homogeneous, as visualized by protein staining of an SDS-15%
polyacrylamide gel (Fig. 2). From a 500-ml culture, 1.3 mg of GlpE was
purified 25-fold to a specific activity of 270 U/mg (Table
4).
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-mercaptoethanol was found to reactivate oxidized enzyme. Loss of activity occurred less rapidly when 750 mM sodium chloride or ammonium sulfate was present.
Molecular mass of GlpE. The molecular mass of the GlpE monomer calculated from the deduced amino acid sequence (12.1 kDa) was confirmed previously by SDS-PAGE (12.1 kDa) (72). Gel filtration chromatography was used to determine the apparent molecular mass of GlpE under nondenaturing conditions. GlpE chromatographed as a dimer (22.5 kDa) under a variety of buffer conditions (data not shown). Addition of cysteine or dithiothreitol, although able to activate oxidized enzyme, did not appreciably change the apparent size of the enzyme (data not shown).
Catalytic properties of GlpE.
The well-characterized bovine
liver rhodanese utilizes a double-displacement (ping-pong) mechanism
(63, 67). Data from activity measurements of purified GlpE
with various concentrations of thiosulfate at fixed concentrations of
cyanide were fit to the equation describing this type of mechanism.
Figure 3A shows the data and the fit
obtained when points exhibiting substrate inhibition by thiosulfate
were excluded. Figure 3B is a secondary double-reciprocal plot of the
apparent maximum velocities in Fig. 3A versus cyanide concentration.
The regression of the data to the equation describing a
double-displacement mechanism yielded Km values
for thiosulfate and cyanide of 78 and 17 mM, respectively. The
kcat based on GlpE functioning as a dimer was
230 s1.
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),
5 mM (
), 10 mM (
), and 50 mM (
). (B) Secondary
double-reciprocal plot of apparent Vmax from the
data in panel A versus cyanide concentration. Data, excluding the
points exhibiting substrate inhibition, were fit to the equation
describing a double-displacement mechanism.
-mercaptoethanol were unable to replace thiosulfate as the sulfur
donor for GlpE (data not shown).
Several previously characterized rhodaneses, including the bovine
liver enzyme (64) and the accessible rhodanese of
E. coli, are specifically inhibited by certain
anions (3). In contrast, little or no inhibition by anions
was observed for GlpE. Addition of sodium phosphate, sodium acetate,
sodium chloride, sodium sulfate, or potassium phosphate at an ionic
strength of 0.3 resulted in 20% or less inhibition of rhodanese
activity (data not shown). However, as shown in Fig. 3A, substrate
inhibition by thiosulfate was observed, particularly at low cyanide
concentrations. This type of inhibition was previously described for
the bovine liver rhodanese (64) and the accessible rhodanese
of E. coli (3).
Chemical inactivation of GlpE by DTNB.
Catalysis by the bovine
liver rhodanese requires a cysteine residue. The cysteine forms a
persulfide linkage to the sulfane sulfur from thiosulfate in an
enzyme-sulfur intermediate (63, 67). GlpE contains two
cysteine residues, one of which has been identified as the active site
of GlpE, based on sequence alignment with other sulfurtransferases
(Cys-65) (Fig. 1). GlpE was incubated with the cysteine-specific
modifying reagent DTNB to verify that a cysteine residue is required
for rhodanese activity. Incubation of GlpE with DTNB in a 1:2 molar
ratio resulted in a greater-than-90% loss of activity (Fig.
4).
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Thioredoxin acts as a sulfur acceptor substrate for GlpE.
It
has been shown that reduced dithiols such as dithiothreitol (1,
53), dihydrolipoate (50), and E. coli
thioredoxin 1 (48) serve as sulfur-acceptor substrates for
bovine liver rhodanese. Thioredoxin 1 acts as a substrate with an
affinity near that determined for cyanide (apparent
Kms of 18.5 µM [48]) and 63 µM [64], respectively). To test thioredoxin 1 as a
sulfur acceptor substrate for GlpE, the assay method described for
bovine rhodanese was used (48). (See Materials and Methods
for the basis for this assay.) A mixture containing thioredoxin 1, thioredoxin reductase, and NADPH was allowed to equilibrate. Then GlpE,
ammonium thiosulfate, or both were added. Addition of either GlpE or
thiosulfate alone resulted in no significant oxidation of NADPH.
However, addition of both GlpE and thiosulfate resulted in a marked
increase in the rate of NADPH oxidation (Fig.
5A). Oxidation of NADPH was dependent on
the presence of thioredoxin 1 and thioredoxin reductase as well as GlpE
and thiosulfate. To quantify the affinity of thioredoxin 1 for the
sulfur transferred from GlpE, the thioredoxin 1 concentration was
varied while the thiosulfate concentration was held at 80 mM (Fig. 5B).
Regression of the resulting data yielded an estimate of the apparent
Km for thioredoxin 1 of 34 µM and an apparent Vmax of 1 nmol of NADPH oxidized per min, almost
15 times faster than the maximum rate of NADPH oxidation observed with
the bovine liver rhodanese (48). This apparent maximum rate
corresponds to a turnover number of 0.5 mol of NADPH oxidized per mol
of GlpE dimer per min.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Comparison of GlpE with other sulfurtransferases. The well-characterized rhodanese from bovine liver mitochondria is approximately twice the size of GlpE and functions as a monomer. The bovine liver enzyme, however, contains two approximately 15-kDa domains. The two domains are structurally similar, although they exhibit only limited sequence similarity to each other (54). Hence, it is thought that duplication of an ancestral rhodanese gene has given rise to the genes that encode the two-domain rhodaneses. This hypothesis is supported by the identification of GlpE and Sud, a polysulfide sulfurtransferase from Wolinella succinogenes (29, 30), as single-domain sulfurtransferases having sequence similarity to the catalytic domain of the bovine liver enzyme (Fig. 1). Like the bovine enzyme, GlpE and Sud require a cysteine residue for activity and utilize a double-displacement reaction mechanism.
Although GlpE possesses some of the characteristics of the previously identified accessible rhodanese from E. coli (molecular weights of 12,000 and 14,000, accessibility of enzyme to substrates added to intact cells, and release from cells by freeze-thaw) (3), it is not likely that these two enzymes are identical. First, the catalytic properties of the two enzymes are distinct. The Kms for SSO32 are 78 and 5 mM for GlpE and the accessible rhodanese, respectively. The
Kms for CN are about the same (17 and 24 mM, respectively), but the kcats differ
(115 s1 per GlpE monomer versus 260 s1 per
monomer for the accessible rhodanese) (3). Also, GlpE does
not exhibit anion-specific inhibition as described for accessible rhodanese and bovine liver rhodanese. Ki values
for sulfate and acetate versus thiosulfate were 36 and 45 mM,
respectively, for accessible rhodanese (3) and 40 and 167 mM, respectively, for bovine rhodanese (64). Specific
inhibition of bovine rhodanese by anions has been linked to two
positively charged residues (R248 and K249) located adjacent to the
active-site cysteine and an arginine residue (R186) within the CH2A
motif (36). None of these residues are conserved in GlpE
(see Fig. 1). These basic residues have also been implicated in the
recognition of thiosulfate as a substrate. Substitution of R186 in the
bovine liver rhodanese with isoleucine increases the
Km for thiosulfate from 4 to 70 mM. Substitution
of K249 with alanine results in a protein that has almost no activity
with thiosulfate as the substrate, but the variant can utilize a number
of organic thiosulfonates almost as well as the wild-type protein
(41). Lack of conservation of these residues in GlpE may be
the basis for the lower affinity of GlpE for thiosulfate compared with
other rhodaneses. The relatively low affinity of GlpE for both
thiosulfate and cyanide suggests that these compounds are not the
physiological substrates of the enzyme.
Finally, GlpE and accessible rhodanese differ in their response to
reducing agents. Addition of cysteine not only activated oxidizedaccessible rhodanese but also resulted in a shift from dimeric
to monomeric form (3). Although GlpE is activated by cysteine, it does not exhibit this shift to monomeric form in the
presence of cysteine or dithiothreitol. The gene encoding GlpE is only
one of a number of genes that have been identified in E. coli that encode rhodanese like proteins (23, 58). One of the other paralogs is likely the previously characterized accessible rhodanese. Deletion of the glpE gene did not significantly
decrease total rhodanese activity measured in cell extracts, verifying that GlpE is not the only protein possessing rhodanese activity in
E. coli (data not shown). Other organisms also contain
multiple sulfurtransferase genes. For example, disruption of the
rhdA (rhodanese) gene of Azotobacter vinelandii
did not result in a discernible phenotype or abolish all rhodanese
activity (13). Finally, among the 21 completely sequenced
genomes in the COG database (57), 8 genomes contain multiple
sulfurtransferase genes.
It is of interest that expression of both the accessible rhodanese and
GlpE is subject to catabolite repression (3, 12, 71) and
repressed during anaerobic growth (3) (data not shown).
GlpE and thioredoxin 1. GlpE, like bovine liver rhodanese (48), is capable of transferring sulfur from thiosulfate to thioredoxin 1 with relatively high affinity. GlpE has an apparent Km for thioredoxin 1 of 34 µM when thiosulfate is present near its Km. Thus, the affinity for thioredoxin 1 is 500 times higher than that determined for cyanide (17 mM). Sulfurtransferases from other organisms may also have the ability to utilize dithiol proteins such as thioredoxin as sulfur acceptors. For example, a partially purified rhodanese isolated from Thiobacillus novellus was capable of producing sulfide from thiosulfate in the absence of cyanide. The physiological sulfur acceptor substrate present in the partially purified preparation had a molecular mass of between 10 and 20 kDa (22) and so may have been a protein similar to thioredoxin. Interestingly, the two structural motifs, designated CH2A and CH2B, that flank the active site in the superfamily of transfer proteins (Fig. 1) have been suggested to play a role in protein-protein interactions. These interactions may mediate recognition of protein substrates by the tyrosine and dual-specificity phosphatases (18). It is intriguing to speculate that the CH2A and CH2B motifs of the sulfurtransferases play a similar role in recognition of dithiol protein substrates. E. coli contains multiple dithiol proteins besides thioredoxin 1 that could fulfill this function. A second thioredoxin, the gene product of trxC, has recently been characterized (44). In addition to the two thioredoxins, E. coli possesses three isoforms of glutaredoxin, proteins structurally and functionally similar to the thioredoxins (6, 37). Given the overlap of function that has been demonstrated for the thioredoxin/glutaredoxin family of proteins, it is possible that these other proteins also serve as sulfur acceptor substrates for sulfurtransferases.
A number of cytoplasmic proteins in E. coli, including elongation factor Tu (25), DnaK (17), and the small dithiol proteins thioredoxin 1 and 2 (40, 44), and many others (39), are released from cells by most methods used for isolating periplasmic proteins. Retention of proteins such as DnaK and thioredoxins by lysozyme-generated spheroplasts, however, indicates a cytoplasmic location (17, 40, 44, 59). GlpE also is a cytoplasmic protein, since it is retained by spheroplasts. Recently, it was shown that release of thioredoxin, DnaK, and elongation factor Tu by mild osmotic shock required the presence of the gated mechanosensitive ion channel MscL (2, 8). Preliminary results from our laboratory suggest that MscL also facilitates release of GlpE by osmotic shock (data not shown).Physiological role of GlpE. The physiological role of rhodaneses is still in question. Cyanide detoxification has been proposed as a possible role (61), but given the low affinity for cyanide (Km = 17 mM), this seems unlikely for GlpE.
Rhodaneses may play various roles in sulfur metabolism (66). The expression of RhdA, a rhodanese-like protein from Synechococcus sp. strain PCC7942, is induced by sulfur starvation (35). However, nothing has thus far implicated GlpE in metabolism of sulfur in E. coli. Growth on various sulfur sources or on limiting sulfur had no apparent effect on the expression of a single-copy glpE-lacZ transcriptional fusion (data not shown). To our knowledge, the only phenotype identified to date for a rhodanese-like enzyme is that of CysA from Saccharopolyspora erythraea (15). Disruption of the gene encoding CysA results in cysteine auxotrophy but, as described above for glpE, does not have a significant effect on the total rhodanese activity. The pathway for cysteine biosynthesis in S. erythraea differs from that established for E. coli and involves thiosulfate as an intermediate. Thus, it is possible that CysA functions to synthesize thiosulfate (15). Although thiosulfate is not an obligate intermediate in aerobic cysteine biosynthesis in enteric bacteria, thiosulfate might be an intermediate in anaerobic biosynthesis of cysteine (19, 31). Rhodanese has also been proposed to mobilize sulfur for the formation or repair of iron-sulfur clusters. The iron-sulfur clusters of ferredoxins (10, 51), succinate dehydrogenase (9), and mitochondrial NADH dehydrogenase (52) could be reconstituted by incubation with various combinations of bovine liver rhodanese, thiosulfate, dihydrolipoate, and iron ions. Interestingly, thioredoxin 1 has also been proposed to play a role in the formation or repair of iron-sulfur clusters (14). The participation of sulfurtransferases in the formation of iron-sulfur clusters has been questioned, however, since there is strong evidence that NifS/IscS and associated proteins are involved in mobilization of sulfur from cysteine for synthesis of iron-sulfur clusters (20, 28, 47, 73). Although GlpE has now been identified as a sulfurtransferase, its physiological function and possible association with the metabolism of glycerol-P remain to be elucidated. The relationship between GlpE and thioredoxin that was discovered during this study might help clarify these issues.| |
ACKNOWLEDGMENTS |
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We thank Ali T. van Loo-Bhattacharya and Janet Donahue for skillful technical assistance. We also thank E. V. Koonin for pointing out the similarity between GlpE and phospho- and sulfurtransferases and C. H. Williams, Jr., for providing thioredoxin reductase.
This work was supported by grant MCB-9118757 from the National Science Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. Phone: (540) 231-7060. Fax: (540) 231-9070. E-mail: tilarson{at}vt.edu.
Present address: Department of Horticultural Science, North
Carolina State University, Raleigh, NC 27695-7609.
Present address: National Cancer Institute/Surgery Branch,
National Institutes of Health, Bethesda, MD 20892.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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