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INTRODUCTION |
Staphylococcus aureus is a Gram-positive opportunistic
pathogen that is a major cause of nosocomial infections. S. aureus was the first organism identified as having resistance to
penicillin and has since acquired resistance to most clinically
approved antibiotics (1). Methicillin-resistant strains of S. aureus (MRSA)1 are
multiply drug-resistant and pose serious challenges to modern medicine
and surgery. Current therapy for MRSA infections relies upon
intravenous treatment with vancomycin. There is concern that resistance
to vancomycin, which has emerged in Enterococcus faecalis, may transfer to MRSA, resulting in infections for which there may be no
effective therapy (2). Efforts are now being focused to identify new
cellular targets and to develop new chemotherapeutics effective against
MRSA. We are investigating the unique thiol metabolism of S. aureus and other Gram-positive bacteria in hopes of facilitating
this process.
Aerobic organisms maintain high levels of low molecular weight thiols
that in combination with specific disulfide reductases support the
reducing intracellular environment and the thiol/disulfide distribution
of other thiol-containing molecules. Glutathione (GSH,
-glutamyl-cysteinyl-glycine) is perhaps the best studied low
molecular weight thiol. It protects these organisms from oxygen toxicity by functioning as a slowly autooxidizing reserve of cysteine and as a cofactor in the detoxification of peroxides, epoxides, and
other products resulting from reaction with oxygen. It is also a
cofactor in the reduction of disulfides and ribonucleotides and in the
isomerization of protein disulfides (3-6). Glutathione reductase (GSR,
EC 1.6.4.2) catalyzes the NADPH-dependent reduction of
intracellular oxidized glutathione (GSSG) and thereby maintains the
high intracellular GSH/GSSG ratio (GSH/GSSG > 100). Together, GSH, GSSG, and GSR make up the primary thiol/disulfide redox system of
aerobic eukaryotes and Gram-negative bacteria (4, 5, 7, 8)
Many organisms do not produce GSH but instead employ alternative low
molecular weight thiols that participate in unique thiol/disulfide redox systems (5, 7-12). It was recently shown that
S.aureus, like many of the Gram-positive bacteria, produces
no GSH but rather produces millimolar levels of reduced CoA as its
predominant thiol (Fig. 1) (13). CoA,
which is required as a cofactor in many acyl transfer reactions, likely
has additional functions in S. aureus analogous to GSH. As a
first step toward understanding this novel thiol metabolism, we set out
to isolate and characterize the activity responsible for maintaining
CoA in its reduced form. Other organisms that utilize alternative
thiols produce an enzyme analogous to GSR whose preferred substrate is
the disulfide of the predominant thiol in the cell (14-16). All such
enzymes investigated belong to a widespread family of pyridine
nucleotide-disulfide oxidoreductases that includes GSR, lipoamide
dehydrogenase, and mercuric reductase. Most of these enzymes are
homodimeric flavoproteins with Mr of ~100,000
that utilize a conserved active site disulfide bond to effect
catalysis. We describe here the identification, purification, and
characterization of a CoA disulfide reductase (CoADR) from S. aureus that catalyzes the NADPH-dependent reduction of
CoA disulfide (Equation 1).
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(Eq. 1)
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In this and the following paper we highlight the similarities and
the striking differences between CoADR and other members of the
disulfide reductase superfamily.
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EXPERIMENTAL PROCEDURES |
Materials--
Reduced NADPH, riboflavin, flavin adenine
mononucleotide, flavin adenine dinucleotide (FAD), coenzyme A
disulfide, glutathionyl-coenzyme A mixed disulfide,
3'-dephospho-coenzyme A, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB),
lysostaphin, bicinchoninic acid solution, 4% copper sulfate
pentahydrate solution, 2',5'-ADP-Sepharose, and phenylmethylsulfonyl fluoride (PMSF) were from Sigma (Mississauga, ON, Canada).
3'-dephospho-CoA was oxidized to the disulfide by incubation at room
temperature overnight in Tris-HCl (20 mM), pH 9.0, containing copper (5 µM). EDTA was added to the solution
(to 10 µM) chelate the copper. 4-Phosphopantetheine was
formed by incubation of CoA with nucleotide pyrophosphatase. The thiol
was oxidized as described above, and the disulfide was purified by
HPLC. The purity of the disulfide was confirmed by HPLC and by thiol
analysis upon reaction with monobromobimane (mBBr) (described below).
All other chemicals were of reagent grade or better and were used
without further purification.
All protein chromatography was performed on a fast phase liquid
chromatography system equipped with UV and conductivity flow cells, a
MonoQ 5/5 (anion exchange) column, a C 10/10 column containing 2',5'-ADP-Sepharose (5 ml), and a Superose 6HR 10/30 (gel filtration) column (Pharmacia Biotech Inc.). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a Mini Protean (Bio-Rad, Burlington, ON,
Canada) using a Tris-glycine buffer as described (17). Prestained Protein Markers (New England Biolabs (Mississauga, ON, Canada) were
used for SDS-PAGE molecular weight standards and contained maltose-binding protein-
-galactosidase (175 kDa), maltose-binding protein-paramycin (83 kDa), glutamic dehydrogenase (62 kDa), aldolase (47.5 kDa), and triose phosphate isomerase (32.5 kDa). Molecular weight
standards for gel filtration chromatography were from Bio-Rad and were
thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken
ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B-12 (1,350 kDa). Spectrophotometric measurements were performed on a thermostatted
Cary I spectrophotometer (Varian, Inc.) using quartz cuvettes (500 µl) (Hellma, Concord, ON, Canada). Concentration of protein samples
was carried out using Centricon filters (Amicon).
Media, Assays, and Buffers--
S. aureus strain
R8325-4, used in all studies, was from John Iandolo (Kansas State
University, Manhattan, KS) and was grown in trypticase soy broth
medium. Concentrations of FAD, NADPH, and CoA disulfide (and
dephospho-CoA disulfide) were measured spectrophotometrically at 450 nm
(
450 = 11,000 M
1
cm
1), 340 nm (
340 = 6220 M
1 cm
1), and 260 nm
(
260 = 33,600 M
1
cm
1), respectively. DTNB assays were performed in
Tris-HCl buffer (20 mM), pH 8.0, containing EDTA (1 mM) and were monitored at 412 nm for the thionitrobenzoate
anion (
412 = 13,600 M
1
cm
1 (18)). Protein concentrations were determined by
reaction with bicinchoninic acid and copper sulfate as described (19).
During purification CoADR activity in crude extracts was monitored by the NADPH (1 mM) and CoA disulfide (1 mM)-dependent reduction of DTNB (1 mM). For kinetic analysis of purified CoADR, the oxidation of NADPH was measured as the decrease in absorbance at 340 nm (
340 = 6,220 M
1
cm
1) and was carried out in Tris-HCl buffer (50 mM), pH 7.8, containing NaCl (50 mM).
Analysis of Thiols from S. aureus--
An analysis of the thiols
produced by S. aureus was carried out as described (20).
Briefly, cell pellets (250 mg) were resuspended in 50% acetonitrile in
Tris-HCl buffer (20 mM), pH 8.0, containing mBBr (2 mM) and incubated at 60 °C for 5 min in the dark.
Control samples were pretreated with N-ethylmaleamide (NEM)
(2 mM) under the same conditions before the addition of mBB
(to 2 mM). The cellular debris was removed by
centrifugation, and the samples were diluted in 10 mM
aqueous methane sulfonic acid for reverse phase HPLC analysis. Thiol
standards were prepared as described (20). To determine disulfide
content, cell extracts were first treated with NEM (2 mM)
to block the free thiols, then with dithiothreitol (3 mM)
to reduce the disulfides and titrate the remaining NEM, and finally
with mBBr to derivitize the resulting thiols. The thiol/disulfide ratio
within the cell was then calculated.
Cu2+ Catalyzed Oxidation of Cysteine, GSH, and
CoA--
To determine the relative stability of CoA to heavy metal
catalyzed oxidation, the rates of Cu2+ catalyzed oxidation
of cysteine, GSH, and CoA were compared. Each sample (2 ml) of thiol (1 mM) in Tris-HCl buffer (20 mM), pH 7.5, containing CuCl2 (1 µM) was incubated at
ambient temperature. Thiol determination was carried out at various
times by adding aliquots (100 µl) from each sample to a solution (900 µl) of DTNB (1 mM) in Tris-HCl buffer (20 mM), pH 8.0, containing EDTA (1 mM). The
absorbance of these samples at 412 nm were then measured, and the
concentration of remaining thiol was determined.
Identification of a Coenzyme A Disulfide Reductase from S. aureus--
An overnight culture (10 ml) of S. aureus was
centrifuged (5,000 × g for 10 min), resuspended in 3 ml of Tris-HCl buffer (20 mM), pH 8.0, containing
lysostaphin (5 µg/ml), and incubated at 37 °C for 30 min until the
suspension became viscous. Glass beads (1 g; 0.2 mm) and PMSF (to 1 mM) were added, and the mixture was vortexed for 2 min and
then centrifuged (15,000 × g for 10 min) to remove the
insoluble cellular debris. The resulting viscous lysate was dialyzed
exhaustively (3,600 Mr cutoff) against Tris-HCl buffer (20 mM), pH 8.0, containing EDTA (1 mM).
The dialysate (40 µl) was then assayed for the pyridine nucleotide (1 mM) and disulfide (1 mM)-dependent
reduction of DTNB (1 mM). A typical assay was 800 µl.
Purification of CoADR from S. aureus--
An overnight culture
of S. aureus was grown in trypticase soy broth (10 ml) at
37 °C and was used as an inoculum (0.4 ml) for each of ten 2-liter
flasks containing trypticase soy broth (1 liter). These cells were
shaken (180 rpm) for 12 h at 37 °C before being harvested by
centrifugation (7000 × g for 15 min). All subsequent
handling of the sample prior to chromatography was carried out at
4 °C. The cell pellet was resuspended in a minimum volume of
Tris-HCl buffer (20 mM), pH 8.0, containing PMSF (1 mM) and lysostaphin (5.0 mg), incubated at 37 °C with agitation for 1 h (or until viscous), passed twice through a
French pressure cell operating at 1,500 lb/in2, and then
centrifuged (15,000 × g for 20 min) to remove
insoluble cellular debris. The supernatant was brought to 50%
saturation with (NH4)2SO4, stirred
for 15 min, and centrifuged (15,000 × g, 10 min). The
resulting supernatant was brought to 80% saturation with
(NH4)2SO4 and centrifuged, and the
pellet containing the CoADR activity was dissolved in a minimum volume
of TE buffer containing PMSF (1 mM). The resulting solution
was dialyzed exhaustively (3,500 Mr cutoff)
against TE buffer containing PMSF (1 mM).
All chromatography was carried out at room temperature on a Pharmacia
fast phase liquid chromatography. The dialyzed
(NH4)2SO4 fraction was applied (1.0 ml/min) to a 2',5'-ADP-Sepharose affinity column equilibrated with
Tris-HCl buffer (20 mM), pH 8.0. The column was washed (25 ml) and then eluted with a linear gradient (35 ml) of NaCl (0-4
M) in the same buffer. The fractions (1 ml) exhibiting
CoADR activity were pooled, concentrated and diluted in Tris-HCl buffer
(20 mM), pH 9.0, twice to reduce conductivity, and applied
(1.0 ml/min) to a MonoQ HR 5/5 anion exchange column equilibrated with
the same buffer. The column was washed (5 ml) and then eluted with a
linear gradient (25 ml) of NaCl (0.3-0.6 mM) in Tris-HCl
buffer (20 mM), pH 9.0. The purity of fractions showing
CoADR activity was determined by SDS-PAGE (5% stacking gel; 12%
running gel) and silver staining.
Determination of Native Molecular Weight--
Purified CoADR (25 µg) was applied to a Superose 6 HR 10/30 gel exclusion column (0.5 ml/min) equilibrated with Tris-HCl buffer (20 mM), pH 8.0, containing NaCl (1 M) and then eluted isocratically in the
same buffer. Fractions containing CoADR were identified by UV
absorbance and by measuring enzyme activity. The native molecular
weight of CoADR was calculated by comparison of its elution
volume to that of protein standards as described (21).
Identification of Flavin Cofactor--
The visible absorption
spectrum of purified CoADR is indicative of a flavoprotein. To identify
the bound flavin cofactor, CoADR (5 µg or 100 pmol in 0.1 ml) was
heated to 98 °C for 10 min and then centrifuged (15,000 × g, 10 min) to remove the denatured protein. The supernatant
was then separated by reverse phase HPLC as described previously (15).
The elution of the released cofactor was then compared with that of
riboflavin, flavin adenine mononucleotide, and FAD (100 pmol each).
Quantitation of Active Site Thiols--
A solution of oxidized
CoADR (9.5 µM) in Tris-HCl buffer (20 mM), pH
8.0, containing EDTA (1 mM) was incubated with NADPH (0.2 mM) for 10 min at ambient temperature before being diluted (1:1) with the same buffer containing urea (8 M) and DTNB
(0.2 mM). The absorbance at 412 nm was then measured and
compared with that of a similar reaction in which CoADR was not
incubated with NADPH. The number of thiols liberated per FAD
(
450 = 11,300 M
1
cm
1) was then calculated. To determine the identity of
the two thiols detected, oxidized CoADR (9.5 µM) in 100 µl of Tris-HCl buffer (20 mM), pH 8.0, containing NADPH
(20 µM) and CoA disulfide (200 µM),
3'-dephospho-CoA disulfide (200 µM), or CoASSG (200 µM) was incubated (incubation 1) at 37 °C for 10 min,
and then the reaction was filtered through a Centricon 30 (30,000 Mr cutoff) to separate CoADR from the low
molecular weight reaction products. The extensively washed CoADR
fraction was resuspended in 100 µl of Tris-HCl buffer (20 mM), pH 8.0, containing EDTA (1 mM) and NADPH
(0.2 mM) and incubated for 10 min at 37 °C (incubation
2). The reaction product was either 1) filtered as above with the thiol
content of the high and low molecular weight fraction determined by
DTNB assay or 2) reacted directly with NEM and/or mBBr and then
separated on HPLC as described above to identify any low molecular
weight thiols present. The results were then compared with similar
reactions in which oxidized CoADR was incubated alone or in the
presence of either CoA disulfide or NADPH but not both.
Kinetic Characterization of the Substrate Specificity and Kinetic
Mechanism of CoADR--
Kinetic measurements were performed in a 1-cm
pathlength quartz cuvette maintained at 37 °C. Each assay (0.3 ml)
was carried out in Tris-HCl buffer (50 mM), pH 7.8, containing NaCl (50 mM), CoADR (2-10 nM;
subunit concentration determined using the calculated extinction
coefficient for CoADR
452 = 12,800 M
1 cm
1), NADPH (2-200
µM), and CoA disulfide (2-200 µM),
3'-dephospho-CoA A disulfide (10-500 µM),
4,4'-diphosphopantethine (2 µM to 2 mM), pantethine (10 µM to 100 mM), glutathione
disulfide (10 µM to 100 mM), cystine (10 µM to 100 mM), CoA-glutathione mixed
disulfide (10 µM to 1 mM), or
H2O2 (10 µM to 10 mM)
(EDTA (10 µM) was present in the stock solution of
3'dephospho-CoA disulfide and was thus present in the enzyme assays for
this substrate up to 0.5 µM. We have found that EDTA up
to 10 mM has no effect on the observed kinetics of the
reaction catalyzed by CoADR.). Enzyme and NADPH were combined in buffer
and equilibrated to 37 °C, and the reaction was initiated by the
addition of the disulfide substrate or H2O2 (reduction of each substrate was monitored at
7 different substrate concentrations). The activity of CoADR was monitored at 340 nm as the
decrease in absorbance resulting from the oxidation of NADPH. To study
the kinetic mechanism of CoADR a two-substrate kinetic analysis was
performed under identical conditions except for the concentrations of
enzyme and substrate used: [CoADR] = 1.5 nM, [NADPH] = 4-30 µM (in reduction of CoA disulfide) and 6-10
µM (for reduction of 3'-dephospho-CoA; [CoA disulfide] = 4-80 µM; and [3'-dephospho-CoA] = 60-400
µM. All kinetic measurements were recorded in the linear
range, and kinetic constants were calculated from a nonlinear least
squares fit of the initial velocity data to the Michaelis-Menton
equation using the program HYPERO (22).
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RESULTS |
Analysis of Thiols from S. aureus--
Bimane derivatives of low
molecular weight thiols separated by reverse phase HPLC are shown in
Fig. 2. As described previously (13),
S. aureus produces CoA, H2S, and a small amount
of cysteine. CoA is measured as the combination of 3'-dephospho-CoA and
CoA, because the former is a hydrolysis product of CoA that arises during the acidic storage conditions prior to separation by the HPLC.
Total CoA was estimated at 1.1 ± 0.1 µmol/g (dry residual weight). H2S is commonly detected in bacterial extracts
(13) and may derive from iron-sulfur proteins. The large peak running at 18 min was isolated and characterized by 1H NMR as
bismethylbimane (data not shown). This compound apparently arises from
reaction of mBBr with an unknown cellular factor that is inactivated by
NEM. If GSH was present, it was
0.02 µmol/g (dry residual weight).
The predominant disulfide observed was CoA disulfide, and the ratio of
reduced CoA/oxidized CoA was 450 ± 50.

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Fig. 2.
Reverse phase HPLC of S. aureus
thiols. A, thiol sample, cell extracts were
reacted with mBBr. B, control sample, cell extracts were reacted first with NEM and then with mBBr. Peaks that
appear in chromatogram A but not chromatogram B
are bimane derivatives of cellular thiols. C, bimane
derivatives of standard thiols (Stds). BMB, bis
methylbimane; MSH, mycothiol.
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Cu2+ Catalyzed Oxidation of Cysteine, GSH, and
CoA--
Biological thiols found as predominant cellular thiols, such
as GSH,
-glutamylcysteine, and mycothiol have proved to be
kinetically resistant to metal catalyzed auto oxidation (12, 15). The relative resistance to Cu2+ catalyzed air oxidation for the
intracellular thiols cysteine and CoA were compared with that for GSH
(Fig. 3). CoA oxidized 4-fold less
rapidly than GSH, whereas GSH oxidized 180-fold less rapidly than
cysteine. Cysteine proved to be the least stable.

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Fig. 3.
Cu2+ catalyzed autooxidation of
cysteine, glutathione, and coenzyme A. Each thiol (1 mM) was incubated with CuCl2 (1 µM). At various times samples were removed, and total
free thiol was measured by reaction with DTNB.
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Identification and Purification of CoADR from S. aureus--
S. aureus grows aerobically, yet maintains
millimolar levels of reduced CoA and an intracellular CoASH/CoA
disulfide ratio of >100. A comparison of enzyme activity from an
S. aureus extract incubated with various disulfide
substrates and pyridine nucleotide cofactors was performed to determine
whether any specific disulfide reductases were present. The extract
catalyzed the reduction of DTNB slowly in the presence of either NADH
or NADPH. However, in the presence of both NADPH and CoA disulfide this
reduction was 3-fold greater than that observed for NADPH alone and
8-fold greater than that observed for NADH alone. No disulfide enhanced the rate of NADH-dependent reduction of DTNB, nor did GSSG,
cystine, or pantethine enhance the NADPH-dependent
reduction of DTNB. These results suggested that S. aureus
produces a CoADR that catalyzes specifically the reduction of CoA
disulfide by NADPH and does not produce detectable glutathione
reductase.
The purification of CoADR from S. aureus is described in
Table I. The crude extract was first
subjected to a 50-80% ammonium sulfate precipitation. This fraction
was dialyzed and applied to a 2',5'-ADP-Sepharose affinity column,
which provided a ~250-fold purification. CoADR from the ADP column
was contaminated by three other proteins that were efficiently removed
by MonoQ anion exchange chromatography. CoADR eluted as two separate
peaks of activity. The second peak (shown by the arrow in
Fig. 4B) had a higher specific activity than the first and was the only fraction retained for further
study. The difference between the first and second peak is unknown but
could be due to modification(s) (such as loss of an FAD or deamidation)
incurred during the purification process. However, no efforts have been
made to investigate these speculations.
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Table I
Purification of coenzyme A disulfide reductase from S. aureus
CoADR activity was monitored through various fractionations of a cell
extract from 10 liters of S. aureus cells. A unit was the
amount of enzyme required to catalyze the reduction of 2 µmol of DTNB
(or 1 µmol of CoA disulfide) in 1 min in the presence of DTNB, CoA
disulfide, and NADPH (0.1 mM each).
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Fig. 4.
Purification of CoADR from S. aureus. A, chromatogram for the elution of CoADR from
a 2',5'-ADP-Sepharose column. B, chromatogram for the
elution of CoADR from the MonoQ column. The arrows mark
where the CoADR activity eluted. C, silver-stained SDS-PAGE
analysis of different fractions containing CoADR activity: I, 50-80% (NH4)2SO4
fraction; II, 2',5' ADP active eluate; and III,
MonoQ active eluate. D, the native molecular weight of CoADR was determined by gel filtration chromatography and a plot of the
parameter K versus the log of the molecular weight of
various protein standards. The parameter, K, is defined as
(Ve Vo)/Vs, where
Ve is the elution volume,
Vo is the column void volume, and
Vs is the volume of the stationary phase
(21).
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Physical Characterization--
The known pyridine
nucleotide-disulfide oxidoreductases are multimeric, most being
homodimers, and have a FAD noncovalently bound to each subunit (14, 16,
24, 25). Purified CoADR migrates as a single polypeptide of ~50,000
apparent molecular weight according to SDS-PAGE (Fig. 4C).
The native molecular weight of CoADR was estimated by its relative
elution through a Superose 6 HR 10/30 gel filtration column. A plot of
the parameter, K, verses the log of the molecular weight of
various protein standards is shown in Fig. 4D. The
parameter, K, is defined as (Ve
Vo)/Vs, where
Ve is the elution volume,
Vo is the column void volume, and
Vs is the volume of the stationary phase (21).
Native CoADR elutes between chicken ovalbumin (44 kDa) and bovine gamma
globulin (158 kDa) from the Superose gel filtration column, and the
K value calculated for CoADR (0.49) can be extrapolated from
the standard plot to a Mr of approximately
90,000. This suggests that native CoADR is a homodimer.
The visible absorbance spectrum of purified CoADR is typical of that of
a flavoenzyme (Fig. 5A) and
shows a
max at 375 and 452 nm, having a shoulder at 478 nm. The flavin bound to CoADR could be released from the enzyme by
thermal denaturation and was identified as FAD by reverse phase HPLC
chromatography (Fig. 5B). In addition to the peak
corresponding to FAD, an additional peak running at 24 min was
observed. This peak is likely a degradation product of FAD because it
was obserevd for FAD run alone (data not shown) and is present in the
control chromatogram (Fig. 5A). Quantitation of the flavin
released from CoADR revealed that 90 ± 10 pmol or 0.9 ± 0.1 FAD molecules/subunit CoADR were released. An extinction coefficient
for a native CoADR subunit in Tris-HCl buffer (20 mM), pH
9.0, at 452 nm was estimated as 12,800 M
1
cm
1 from the extinction coefficient for FAD
(
450 = 11,300 M
1
cm
1) using the following equation.
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(Eq. 2)
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Fig. 5.
A, the absorption spectrum of purified
CoADR. CoADR shows absorbance peaks typical of flavoproteins
( max at 276, 375, and 452 nm). B,
identification of the flavin cofactor of CoADR by reverse phase HPLC.
a, flavin standards: riboflavin, flavin adenine mononucleotide (FMN), and FAD. b, the flavin
released from thermally denatured CoADR.
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Thiol Titration of Active Site--
The deduced amino acid
sequence of CoADR contains two cysteine residues, and only one of these
(Cys43) lies in the putative active site region (36). To
determine whether CoADR utilizes a thiol-disulfide exchange mechanism,
a thiol titration of the active site of the enzyme was performed. CoADR
in its oxidized state and reduced with NADPH was denatured in the
presence of DTNB. The denatured oxidized enzyme had 0.9 ± 0.1 reactive thiol/subunit, whereas denatured CoADR that had been reduced
with NADPH had 3.2 ± 0.2 reactive thiols/subunit. This suggests
that CoADR in its oxidized state has one reduced cysteine and that upon
reduction with NADPH a disulfide bond was reduced. However, the 3.2 thiols/subunit measured also suggested that one of the thiols produced
upon reduction with NADPH was not an enzymic cysteine (because CoADR
only has two cysteines/subunit) (36).
CoADR and NADH peroxidase from E. faecalis both have a
single cysteine in their active site, thus CoADR might form a mixed disulfide intermediate with the substrate thiol similar to the sulfenic
acid formed during reduction of hydrogen peroxide by NADH peroxidase.
To address this question, CoADR was incubated in the presence of
limiting NADPH and an excess of disulfide (incubation 1) and then
separated from the low molecular weight reaction products by
filtration. The resulting oxidized enzyme was then reduced with an
excess of NADPH (incubation 2), and the reaction products were
separated into low and high molecular weight fractions and characterized for their thiol content. Both the low and high molecular weight fractions contained thiol, 0.8 ± 0.1 mol of thiol/subunit of enzyme in the low molecular weight fraction (Table
II) and 2.2 ± 0.2 mol thiol/subunit
enzyme in the high molecular weight fraction. This suggested that the
disulfide bond reduced by NADPH had both a high and a low molecular
weight component. Enzyme treated only with NADPH during incubation 1, washed extensively, treated again with NADPH during incubation 2, and
then size fractionated produced only one thiol/subunit in the high
molecular weight fraction and no thiols in the low molecular weight
fraction, providing further evidence for a low molecular weight
component to the reduced disulfide. A thiol analysis of the low
molecular weight thiol carried out by mBBr modification and separation
on HPLC (as described earlier) identified the low molecular weight
thiol as reduced disulfide substrate (CoA, 3'-dephospho-CoA, and CoA in
the reduction of CoA disulfide, 3'-dephospho-CoA disulfide, and
CoASSG). No substrate-derived thiols were detected in the high
molecular weight fraction. Enzyme alone or treated with CoA disulfide
during incubation 1 had results similar to that found for the enzyme
treated with NADPH and CoA disulfide, suggesting that the enzyme is
isolated from cells as the CoA mixed disulfide. Together, these data
suggest that CoADR in the oxidized state forms a stable mixed disulfide with half of the disulfide substrate and that this mixed disulfide is
reduced upon treatment with NADPH. In addition, these data suggest the
mixed disulfide is preferentially formed with that half of the
substrate resembling CoA because no GSH was detected in the CoASSG
sample.
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Table II
Analysis of active site thiols of CoADR
CoADR was oxidized by pretreatment (incubation 1) with a 10:1 molar
ratio of disulfide:NADPH at 37 °C for 10 min. Oxidized CoADR was
then filtered from the low molecular weight reaction products, washed
extensively, and then reduced with NADPH (200 µM)
(incubation 2). The reaction products were either 1) filtered as above
with the thiol content (SH/subunit) of the high and low molecular
weight fraction determined by DTNB assay or 2) reacted directly with
NEM and/or mBBr and then separated on HPLC as described under
"Experimental Procedures" to identify any low molecular weight
thiols (RSH) present.
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Kinetic Characterization of CoADR--
CoADR is highly specific
for its physiological substrates, CoA disulfide and NADPH, and at the
optimum pH 7.8 is saturated by micromolar concentrations of each. The
Km for NADPH, at a saturating CoA disulfide
concentration, was 2 µM, and the Km
for CoA disulfide, at a saturating NADPH concentration, was 11 µM. The results of the kinetic analysis of the CoADR
catalyzed reduction of various disulfide substrates by NADPH are shown
in Table III. Although CoADR reduces CoA
disulfide preferentially, it also reduces 3'-dephospho-CoA disulfide
(Km = 40 µM), 4,4'-diphosphopantethine
(Km = 80 µM), and CoASSG
(Km = 1100 µM). Activity of CoADR upon
pantethine, cystine, GSSG, or peroxide was below the detectability of
the assay (
A340
0.0001 min
1)
and thus has an upper limit of 0.1%, the activity observed for CoA
disulfide. A two-substrate kinetic analysis of the
NADPH-dependent reduction of CoA disulfide and
3'-dephospho-CoA disulfide by CoADR suggests that the reaction proceeds
by a sequential rather than a simple ping-pong kinetic mechanism (Fig.
6).
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Table III
Steady-state kinetic analysis of the substrate specificity of CoADR
The rate of NADPH oxidation catalyzed by CoADR in the presence of
various disulfide substrates was spectrophotometrically followed. The
reactions were carried out in Tris-HCl buffer (50 mM), pH
7.8, containing NaCl (50 mM), and NADPH (200 µM). CoA disulfide was maintained at 120 µM
to determine the kinetic parameters for NADPH. Kinetic parameters for
reactions having rates below that detectable by the assay
(A260 0.0001/min) were not determined (ND).
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Fig. 6.
Two-substrate analysis of the kinetic
mechanism of CoADR. Enzyme and NADPH were incubated at 37 °C,
and the reactions were initiated by the addition of the disulfide
substrate. The kinetic parameters for each set of data were determined
by fitting the initial velocity data to the Michaelis-Menton equation
using HYPERO (22). The equation for each line in these figures
is simply the reciprocal of the Michaelis-Menton equation carrying the
measured kinetic parameters. Each line results from varying the
concentration of CoA disulfide at constant NADPH concentrations: 1 ( ), 6 ( ), 8 ( ), 10 ( ), and 30 µM ( ).
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DISCUSSION |
Although cysteine is ubiquitous, it rarely is the predominant
cellular thiol of aerobic organisms. High intracellular levels of
cysteine apparently are toxic because rapid metal catalyzed autooxidation results in the production of cystine and hydrogen peroxide (27). Glutathione is resistant to metal catalyzed oxidation (Fig. 3) (10) and provides cells with a reserve of cysteine and a
stable intracellular redox environment. It was shown that S. aureus does not produce GSH but instead produces CoA as its predominant cellular thiol (Fig. 2) (13). Like GSH, CoA is stable to
metal catalyzed oxidation (Fig. 3), is found at millimolar levels in
the cell and is maintained in its reduced form (CoASH/CoASSCoA
450). This thiol/disulfide ratio is likely an underestimate because the
calculated disulfide content would be contaminated by cellular
thioesters, which liberate thiol upon treatment with dithiothreitol
(28). CoA thus represents a stable intracellular thiol/disulfide redox
buffer in S. aureus. The current understanding of cysteine
biosynthesis, however, does not provide an efficient route for
conversion of CoA to cysteine. If indeed aerobes store cysteine in a
form that is resistant to oxidation, the manner by which S. aureus stores cysteine is not obvious.
The thiol/disulfide ratio of thiols in equilibrium with air is
~10
16, yet aerobes maintain a cytoplasmic
thiol/disulfide ratio of 102-103 (29).
S. aureus maintains this ratio, at least in part, using the
enzyme CoADR, which catalyzes specifically the
NADPH-dependent reduction of CoA disulfide. CoADR is
purified over 3000-fold from S. aureus by 2',5'-ADP affinity
and anion exchange chromatography, resulting in a 25% recovery of the
active enzyme (Table I). Chromatography with 2',5'-ADP-Sepharose, which
mimics the adenyldiphosphate moiety of NADPH, is the most
effective step in the purification of CoADR, and the
concentration of NaCl (~2 M) necessary to elute CoADR from this column reflects the high affinity CoADR has for this cofactor
(Km (NADPH) = 2 µM). Native CoADR has
an apparent Mr of 90,000 ± 10,000 according to gel filtration chromatography (Fig. 4D) and
runs as a single 49-kDa band on an SDS-PAGE gel (Fig. 4C).
These data are consistent with a subunit Mr of
49,200 calculated from the deduced primary structure of CoADR (36) and
suggest that CoADR is a homodimer. The absorption spectrum (Fig.
5A) and HPLC analysis (Fig. 5B) show CoADR to be
a flavoprotein that noncovalently binds a single FAD to each 49-kDa
subunit.
The known pyridine nucleotide-disulfide oxidoreductases utilize a pair
of active site cysteine residues to catalyze a thiol disulfide exchange
reaction with the disulfide substrate (23, 24). In human erythrocyte
GSR, Cys58, the interchange cysteine, reduces the substrate
disulfide resulting in the formation of a transient mixed disulfide
intermediate. Cys63, the charge transfer cysteine, is
adjacent to the enzyme bound FAD and reduces the mixed disulfide
effecting the release of substrate and the formation of cystine 58-63.
The deduced primary structure of CoADR does not have the conserved
CXXXXC motif observed for other disulfide oxidoreductases
(36). CoADR instead has a single cysteine residue (Cys43)
that is homologous to the active site cysteine residue of the SFXXC motif of NADH oxidase and NADH peroxidase from
E. faecalis (30). These enzymes, also members of the
pyridine nucleotide-disulfide oxidoreductase superfamily, utilize this
single catalytic cysteine to form a stable sulfenic acid (Cys-SOH)
during the reduction of O2 and
H2O2. We have shown that the NADPH reduction of
oxidized CoADR results in the production of two thiols/enzyme subunit, of which half originates from an enzymic cysteine residue (presumably Cys43) and half originates from the disulfide substrate
(Table II). These results suggest that reduction of disulfides by CoADR
occurs by a thiol-disulfide exchange reaction but involves only a
single cysteine residue that forms a stable mixed disulfide (Enz-SSR) analogous to the sulfenic acid formed by NADH peroxidase. A proposed catalytic mechanism for CoADR is shown in Equation 2.
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(Eq. 3)
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(Eq. 4)
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In the reduced enzyme Cys43 is a free thiol. Upon
encounter with CoA disulfide the Cys43 thiolate anion
attacks one sulfur atom of the substrate, displacing a molecule of CoA
and forming a Cys43-CoA mixed disulfide. This oxidized
state is stable and is the enzyme form isolated from cells (reduction
of this sample with NADPH releases CoA; Table II). NADPH binds the
oxidized enzyme and reduces the mixed disulfide via the FAD cofactor,
regenerating the reduced enzyme and another molecule of CoA. However,
the sequential kinetics observed from a two-substrate kinetic analysis
(Fig. 6) suggests that CoADR does not utilize a simple two-site
ping-pong mechanism. The specific sequence of binding events and the
flow of electrons cannot be addressed from the present data but will have to await future stopped flow kinetic studies.
CoADR is a very specific for NADPH (Km = 2 µM) and CoA disulfide (Km = 11 µM), suggesting that these compounds are natural
substrates for this enzyme. A kinetic survey of disulfide substrates
(Table III) demonstrates the specificity of CoADR and the
contribution of the 3'-phosphoryl, the adenyl, and the 4-phosphoryl groups to the binding and turnover of CoA disulfide. This analysis shows that CoADR recognizes only substrates containing the
4-phosphopantethiene moiety and is most proficient in the reduction of
CoA disulfide. A comparison of the kinetic constants for
3'-dephospho-CoA disulfide reveals that loss of the 3' phosphate
moieties from CoA disulfide results in a 10-fold increase in
Km with no detectable change in
kcat. A similar comparison for 4,4'
diphosphopantethine and 3' dephospho-CoA disulfide demonstrates that
loss of the adenyl moiety results in a 2-3-fold decrease in
kcat and little change in Km.
The 4 and/or 4' phosphate moieties are essential for substrate binding
and turnover because reduction of pantethine by CoADR is not
detectable. CoADR does reduce CoASSG, although poorly
(Km = 1.4 mM), but does not reduce GSSG,
cystine, O2, or H2O2 (despite the
homology to E. faecalis NADH oxidase and peroxidase; Ref.
36). It should be noted that upon reduction of CoASSG CoADR selectively
forms a mixed disulfide with CoA (Table II). Thus, the active site is
designed to form a mixed disulfide with the more tightly bound portion
of the substrate.
Although CoADR apparently belongs to a new separate subfamily of
disulfide reductases (36), CoADR is physically and functionally similar
to the pyridine nucleotide-disulfide oxidoreductase superfamily. Indeed, all of the low molecular weight disulfide reductases appear to
belong to this family. Enzymes studied include trypanothione reductase
(14, 31), bis-
-glutamyl-cysteine reductase (15), and
4,4'-diphosphopantethine reductase (16). Although each appears to
catalyze the same chemical reaction, the NAD(P)H reduction of a
disulfide bond, each enzyme is specific for the disulfide of the
predominant thiol in the cell. CoADR is related functionally to the
Bacillus megaterium 4',4'-diphosphopantethine reductase (4PPR). Although both CoADR and 4PPR each prefer substrates having the
4'-phosphopantethienyl moiety, these enzymes are quite different. 4PPR
has a Km approximately 10-fold higher for CoA
disulfide (7.6 mM) than for its preferred substrate
4,4'-diphosphopantethine (0.67 mM) (16), which itself is 2 orders of magnitude higher than the Km for turnover
of CoA disulfide by CoADR. 4PPR is also 20% larger. It is interesting
that B. megaterium produces an enzyme of such specificity,
because B. megaterium likely has a thiol content similar to
that of B. subtilis, which like S. aureus
produces CoA as its predominant thiol. The B. megaterium enzyme was identified and isolated from spores and may have evolved to
function in this dormant state. If so, there may be an additional enzyme such as CoADR that is expressed in vegetative cells.
GSH plays many different roles in cellular metabolism. How these
functions occur in organisms lacking GSH represents an enigma in the
field of thiol biology. In organisms like S. aureus, which produce CoA as a predominant thiol, many GSH-mediated functions are
presumably mediated by CoA and enzymes that utilize CoA as cofactor.
Enzymes present in pathogens that utilize cofactors structurally
distinct from their eukaryotic counterparts may be potential drug
targets because cofactor discrimination could be exploited in the
design of selective drugs. Indeed, successes in this area have been
reported in the design of inhibitors to trypanothione reductase
(32-35). The disparate disulfide specificities, structures (36), and
catalytic mechanisms of CoADR and GSR suggest that CoADR could be a
useful target for developing new drugs. The study of bacterial thiol
metabolism will likely unveil many such promising targets.
S. B. delCardayré thanks the
members of the Davies lab for useful discussions and insight regarding
this work.