Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, March 7, 2001, and in revised form, March 29, 2001
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ABSTRACT |
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It has been shown that conversion of precursor Z
to molybdopterin (MPT) by Escherichia coli MPT synthase
entails the transfer of the sulfur atom of the C-terminal
thiocarboxylate from the small subunit of the synthase to generate the
dithiolene group of MPT and that the moeB mutant of
E. coli contains inactive MPT synthase devoid of the
thiocarboxylate. The data presented here demonstrate that
L-cysteine can serve as the source of the sulfur for the
biosynthesis of MPT in vitro but only in the presence of a
persulfide-containing sulfurtransferase such as IscS, cysteine sulfinate desulfinase (CSD), or CsdB. A fully defined in
vitro system has been developed in which an inactive form of MPT
synthase can be activated by incubation with MoeB, Mg-ATP,
L-cysteine, and one of the NifS-like sulfurtransferases,
and the addition of precursor Z to the in vitro system
gives rise to MPT formation. The use of radiolabeled
L-[35S]cysteine has demonstrated that both
sulfurs of the dithiolene group of MPT originate from
L-cysteine. It was found that MPT can be produced from
precursor Z in an E. coli iscS mutant strain, indicating
that IscS is not required for the in vivo sulfuration of
MPT synthase. A comparison of the ability of the three
sulfurtransferases to provide the sulfur for MPT formation showed the
highest activity for CSD in the in vitro system.
In all molybdoenzymes with the exception of nitrogenase,
molybdenum is coordinated by the sulfur atoms of the dithiolene group present in the molybdenum cofactor
(Moco)1 (1). The biosynthetic
pathway for Moco is evolutionary conserved, since genes encoding highly
homologous proteins involved in the pathway have been found in archaea,
bacteria, higher plants, Drosophila, and higher animals
including humans. The reactions of the Moco biosynthetic pathway
comprise three stages, which are similar in all organisms utilizing
molybdoenzymes. In the first step, a guanine nucleotide is converted
into the metastable precursor Z. In the second step, the dithiolene
moiety is inserted into precursor Z, converting it to molybdopterin
(MPT) (Fig. 1). In the last step of Moco biosynthesis, molybdenum is
incorporated into MPT to form Moco. Additional modification of Moco
occurs in bacteria with the attachment of GMP, AMP, IMP, or CMP to the phosphate group of MPT.
Conversion of precursor Z to MPT requires the opening of a cyclic
phosphate to produce a terminal phosphate monoester as well as the
transfer of sulfur to generate the dithiolene group essential for
molybdenum ligation (2). This reaction is catalyzed by MPT synthase, a
tetrameric protein composed of two small MoaD subunits (8.8 kDa) and
two large MoaE subunits (16.8 kDa). The recently solved high resolution
crystal structure of E. coli MPT synthase has shown that the
C terminus of each small subunit is inserted into one of the large
subunits to form the active site (3). The small subunit of MPT synthase
shows high structural similarity to the eukaryotic protein ubiquitin.
In the activated form of MPT synthase, the C terminus of the small
subunit is converted to a glycine thiocarboxylate that acts as the
sulfur donor for the conversion of precursor Z to MPT (2, 3). Mass
spectroscopy has identified that MPT synthase in its inactive form is
lacking the thiocarboxylate at the C-terminal glycine of MoaD (2). Since the inactive form of MPT synthase could be purified from moeB mutant strains (2), it has been proposed that the MoeB protein is in fact MPT synthase sulfurase responsible for regenerating the active sulfur at the C-terminal glycine-carboxylate of the small
subunit of the synthase. This reaction has been shown to be
ATP-dependent (4); however, details of the mechanism of action of the sulfurase, including the identity of the sulfur donor for
the protein, remains as yet unknown.
This paper describes an in vitro system for the activation
of inactive MPT synthase isolated from a moeB mutant strain.
This activation was monitored by conversion of precursor Z to MPT
in vitro. The data strongly suggest that
L-cysteine is the likely physiological sulfur donor for the
dithiolene group of MPT and demonstrate that an additional protein
component is required for the transfer of sulfur from
L-cysteine to MPT synthase.
It is known that NifS, a well characterized pyridoxal
phosphate-dependent enzyme from Azotobacter
vinelandii, is involved in iron-sulfur cluster formation for
nitrogenase, converting L-cysteine to L-alanine
and elemental sulfur (5). In E. coli, three NifS-like proteins resembling A. vinelandii NifS in amino acid
sequence and catalytic properties have been identified (6-8). These
proteins, designated IscS, CSD, and CsdB, are described as pyridoxal
5'-phosphate-dependent enzymes that catalyze the
elimination of selenium and sulfur from L-selenocysteine
and L-cysteine, respectively, to form L-alanine (9). IscS is a cysteine desulfurase and is proposed to play a general
role in the formation of iron-sulfur clusters and additionally is
required for the biosynthesis of 4-thiouridine, thiamin and NAD (6, 10,
11). In contrast, CSD, encoded by csdA, has been shown to
act on L-selenocysteine, L-cysteine, and
L-cysteine sulfinate and is named cysteine sulfinate
desulfinase (7). CsdB shows much higher activity toward
L-selenocysteine than L-cysteine and is thus
similar to selenocysteine lyase in this respect (8). The exact
physiological functions of these enzymes remain to be elucidated. We
have cloned and purified all three NifS-like proteins from E. coli and demonstrated that in an in vitro system, all three proteins can transfer sulfur from L-cysteine for the
activation of inactive MPT synthase in an ATP-dependent
reaction, with CSD being the most effective of the three.
Bacterial Strains, Media, and Growth Conditions--
-E.
coli strains and plasmids used in this study are listed in Table
I. E. coli cell strains were
grown aerobically at 30 °C in LB medium. Cell strains containing
expression plasmids were grown in the presence of 150 µg/ml
ampicillin. For expression of pET15b-based plasmids, the Purification of the Reaction Components--
Precursor Z was
isolated from E. coli moaD cells using high
performance liquid chromatography (HPLC) with reverse phase and anion
exchange columns (12). Cloned MPT synthase was expressed in a pET15b
vector (Novagen) in E. coli moeB Cloning of the iscS, csdA, and csdB Genes from the E. coli
Genome--
The DNA fragments containing iscS,
csdA, and csdB were cloned from chromosomal
E. coli DNA by polymerase chain reaction. Oligonucleotide primers used were as follows. 1)
5'-CCATGGAATTACCGATTTATCTCGACTAC-3' and
5'-GGATCCTTAATGATGAGCCCATTCGATGCTGTTC-3' were used for
cloning iscS into the NcoI and BamHI
sites of pET15b. During cloning, the second amino acid of IscS was
changed from a lysine to a glutamate. The resulting plasmid was
designated pSL209. 2) Primers
5'-CGGTGCATCAAGCCGAGGAGTCATATGAACG-3' and
5'-GGATCCTTAATCCACCAATAATTCCAGCGCG-3' were used for cloning csdA into the NdeI and BamHI sites of
pET15b. The resulting plasmid was designated pSL215. 3) Primers
5'-CATATGATTTTTTCCGTCGACAAAGTGCGG-3' and
5'-GGATCCTTATCCCAGCAAACGGTGAATACGTTGC-3' were used for
cloning csdB into the NdeI and BamHI
sites of pET15b. The resulting plasmid was designated pSL213. The
corresponding restriction sites used for cloning are underlined.
Expression and Purification of IscS, CsdB, and CSD--
E.
coli BL21(DE3) cells containing the corresponding expression
plasmids were grown in 2 liters of LB medium to an
A600 of 0.6. At this point, protein
expression was induced by the addition of 100 µM
isopropyl-
For the purification of IscS, the cell pellet was resuspended in 50 mM Tris, 1 mM EDTA, pH 7.5, and lysed by
several passages through a French pressure cell. After centrifugation
at 17,000 × g for 25 min, nucleic acids were removed
by streptomycin sulfate addition, and IscS was precipitated with 45%
ammonium sulfate. After centrifugation, the protein was resolubilized
and dialyzed against 50 mM Tris, 1 mM EDTA, pH
7.5. The dialyzed sample was applied to a 25-ml Q-Sepharose FPLC column
equilibrated with 50 mM Tris, 1 mM EDTA, pH
7.5, and IscS was eluted with a linear gradient of 0-500
mM NaCl. The pool of fractions containing IscS was
concentrated to 1 ml and chromatographed on a Superose 12 FPLC column
equilibrated and eluted with 50 mM Tris, 1 mM
EDTA, 100 mM NaCl, pH 7.5. The yield of all three proteins
was ~20 mg/liter of E. coli culture.
Enzyme Assays--
Sulfite oxidase activity was assayed at room
temperature by monitoring the reduction of cytochrome c at
550 nm (14) using a Shimadzu 1601 spectrophotometer. One unit of
sulfite oxidase activity is defined as an absorbance change
( In Vitro Activation of Inactive MPT Synthase by MoeB--
For
the in vitro formation of MPT, 150-200 µM
precursor Z (in 10 mM sodium citrate buffer adjusted to pH
7.2); 175 nM to 78 µM inactive MPT synthase;
3.5-31 µM MoeB; 2.5 mM MgCl2;
2.5 mM ATP; 4 nM to 5 µM IscS,
CSD, or CsdB; and 2.5 mM L-cysteine were incubated in a total volume of 400 µl of 100 mM Tris, pH
7.2. For a standard incubation assay, all reactants were allowed to react at room temperature for 30 min under aerobic conditions. The reaction was stopped by the addition of acidic iodine and analyzed
for the production of form A afterward (16).
Generation of an IscS, CSD, or CsdB-bound Persulfide--
For
the generation of a IscS, CSD, or CsdB-bound persulfide, 3 mg of IscS,
CSD, or CsdB were incubated with 2 mM
L-cysteine for 5 min at 4 °C, gel-filtered using a PD10
column equilibrated with 100 mM Tris, pH 7.2, and
immediately added to the in vitro activation mixtures.
MPT Analysis and Quantification of MPT by Generation of Form A
(Dephospho)--
In vitro production of MPT was quantitated
by its conversion to the stable, fluorescent degradation product form
A. For this conversion, the incubation mixtures were adjusted to pH
2.5, and excess iodine was added as described in Refs. 16 and 17. The amount of form A was then quantitated by HPLC analysis by comparison with a standard sample obtained from purified human sulfite oxidase.
In Vitro Insertion of Radiolabeled 35S into
MPT--
For the in vitro insertion of 35S from
labeled L-cysteine into precursor Z, incubation mixtures
contained 73.8 µM MPT synthase, 31.3 µM
MoeB, 5.2 µM IscS, 400 µM precursor Z, 2 mM MgCl2, 2 mM ATP, 1 mM L-cysteine, 51 µM sulfite
oxidase, and 1 µCi of L-[35S]cysteine
(PerkinElmer Life Sciences) in 10 ml of 100 mM Tris, pH
7.2, with or without the addition of 20 mM
Na2MoO4. All components were preincubated for
30 min at room temperature before the addition of aposulfite oxidase.
Following a second 30-min incubation, the mixture was dialyzed against
50 mM sodium phosphate, 300 mM NaCl, pH 8.0. The tagged sulfite oxidase was purified from the mixture after the
method described in Temple et al. (13). Fractions containing
sulfite oxidase were pooled and concentrated to 1 ml using a Centriprep
10 filtration device (Amicon) and gel-filtered using a PD10 column
(Amersham Pharmacia Biotech) equilibrated with 100 mM Tris,
pH 7.2.
To produce form B from the cofactor bound to sulfite oxidase, 75 µl
of 1 M HCl was added to 600 µl of sulfite oxidase, prior to incubation for 30 min at 95 °C (16). The reaction mixture was
then treated with alkaline phosphatase (Roche Molecular Biochemicals), and the resulting dephosphorylated form B was purified by HPLC analysis at room temperature at a flow rate of 1 ml/min with an Alltech
C18 HPLC column equilibrated in 50 mM ammonium acetate, 10% methanol. The concentration of purified form B was determined using an extinction coefficient at 395 nm of 12,900 M
For the production of carboxamidomethyl-MPT (camMPT), 1400 µl of
sulfite oxidase was incubated anaerobically for 16 h in 17 ml of
10 mM potassium phosphate, pH 7.0, containing 80 mg of
iodoacetamide (Sigma), 100 µl of 100 mM sodium
dithionite, and 200 mg of SDS (18). After ultrafiltration using a PM-10
membrane, the effluent was applied to a 1-ml QAE-Sephadex column
(Sigma) equilibrated with water. The column was washed with 20 ml of
water, followed by 20 ml of 10 mM acetic acid, and camMPT
was eluted with 20 ml of 10 mM HCl. Fractions containing
camMPT were identified using a Beckman LS 1801 scintillation counter,
pooled, neutralized with NH4OH, and concentrated using a
SpeedVac system (Savant). Final purification of camMPT was achieved by
chromatography on a C18 HPLC column in 50 mM ammonium
acetate, 3% methanol. The concentration of camMPT was determined using
its extinction coefficient at 367 nm of 7340 M In Vitro Activation of Inactive MPT Synthase Purified from a moeB
Mutant Strain--
It has been shown earlier that MPT can be
synthesized in vitro by incubation of purified precursor Z
with the active form of MPT synthase (17, 19). After the transfer of
sulfur from MPT synthase to precursor Z, MPT synthase is present in an
inactive, desulfurated form lacking the C-terminal thiocarboxylate
group at the MoaD subunit of the protein. In order to define the sulfur transfer pathway involved in resulfuration of MPT synthase, the in vitro system was modified to include inactive MPT
synthase and purified MoeB protein. MoeB has been proposed to
regenerate the active sulfur at the glycine-carboxylate group of MPT
synthase in an ATP-dependent reaction and has been
designated as MPT synthase sulfurase (4). Precursor Z was purified from
a moaD mutant strain unable to convert the precursor
to MPT (12). Inactive, recombinant MPT synthase was purified from a
moeB mutant strain as described earlier (17). MoeB was
expressed in cells that contain a mutation in moaD
and purified afterward (experimental procedures). The activation of MPT
synthase was assayed by the ability of the synthase to convert
precursor Z to MPT in vitro.
For the production of MPT in vitro, precursor Z, inactive
MPT synthase, MoeB, and Mg-ATP were incubated at room temperature as
described under "Experimental Procedures." Acidic iodine treatment converts MPT to its oxidized fluorescent degradation product form A
(Fig. 1) (16). HPLC analysis revealed
that no form A was formed under these conditions (Fig.
2A). This finding indicated
that MoeB by itself was not able to sulfurate inactive MPT synthase in vitro. In contrast, when a crude cell extract prepared
from a moeB mutant strain was included in the in
vitro incubation mixture, form A was obtained (Fig.
2B). To identify the component in the crude extract
necessary for activation of MPT synthase, the extract was separated
into a protein fraction and a low molecular weight fraction by gel
filtration and ultrafiltration, respectively. As shown in Fig. 2,
C and D, neither the protein fraction nor the low
molecular weight fraction alone was able to provide the missing
component in the in vitro system. This result led to the conclusion that an as yet unidentified protein component as well as a
low molecular weight substance are necessary for the activation of
inactive MPT synthase by MoeB, with the low molecular weight substance
presumably providing the sulfur source for the sulfuration of the
synthase.
In order to identify possible physiological sulfur donors, different
sulfur sources were added to the in vitro system containing precursor Z, MPT synthase, MoeB, and Mg-ATP and tested for their ability to activate MPT synthase in the presence of the protein fraction of the moeB Three NifS-like Sulfurtransferases Can Catalyze the Activation of
MPT Synthase--
With the identification of L-cysteine as
the likely physiological sulfur donor for the sulfuration of MPT
synthase, it was of further interest to identify the sulfurtransferase
required for the mobilization of this sulfur. Since this protein has to act as an L-cysteine desulfurase, it appeared that a
NifS-like protein might be involved in this reaction. Three NifS-like
proteins have been identified in the E. coli genome
sequence, designated IscS, CSD, and CsdB (6-8). While all three
enzymes can desulfurate L-cysteine, they displayed
different substrate specificities. IscS has the highest activity with
L-cysteine, whereas CSD, described as a sulfinate
desulfinase, prefers L-cysteine sulfinate as substrate (7).
CsdB has a much higher activity with L-selenocysteine than
L-cysteine and is regarded as the E. coli
counterpart of mammalian selenocysteine lyase (8). To test the relative
abilities of the three proteins to utilize sulfur from
L-cysteine for the sulfuration of MPT synthase in
vitro, IscS, CSD, and CsdB were cloned from the E. coli
genome and purified after expression in BL21(DE3) cells as described
under "Experimental Procedures." The effectiveness of the three
enzymes for in vitro MPT production was examined using
reaction mixtures containing L-cysteine as a sulfur source,
one of the three NifS-like sulfurtransferases, MoeB, inactives MPT
synthase, precursor Z, and Mg-ATP. The sulfurtransferase activity was
assessed by the amount of MPT produced in vitro by equivalent amounts of the sulfurtransferases. As shown in Table II, the catalytic activities of IscS,
CSD, and CsdB varied markedly. The mixture containing CSD produced the
highest amount of MPT (34.04 nmol of MPT/nM CSD). IscS
produced much less MPT (29.5%) compared with CSD (10.05 nmol of
MPT/nM IscS), whereas CsdB showed the lowest activity, with
about 1.2% MPT formed compared with CSD (0.40 nmol MPT/nM
CsdB). The differences in the activities of the three enzymes in the
transfer of sulfur from L-cysteine to activate MPT synthase
are in conformity with their specific activities estimated by the
production of elemental sulfur from L-cysteine as reported
by Mihara et al. (8). In sum, these experiments delineate a
sulfur transfer pathway from L-cysteine to MPT synthase,
which in turn converts precursor Z to MPT in the presence of Mg-ATP.
All components described above are essential for the in
vitro assembly of MPT, since in the absence of either MoeB, MPT
synthase, Mg-ATP, L-cysteine, or a sulfurtransferase no MPT
was formed (data not shown).
Direct Evidence for the Transfer of Sulfur from
L-Cysteine to the Dithiolene Group of MPT--
In order to
determine whether both sulfur atoms of the dithiolene group of MPT
originate from L-cysteine, radiolabeled
L-[35S]cysteine was added to the in
vitro activation mixture consisting of IscS, MoeB, MPT synthase,
precursor Z, Mg-ATP, and L-[35S]cysteine. The
mixture was incubated under aerobic conditions for 30 min at room
temperature (see "Experimental Procedures"). In order to stabilize
the MPT produced, aposulfite oxidase was added after the 30-min
incubation. We have previously reported that in vitro
synthesized MPT can reconstitute a cofactor-free form of recombinant
human sulfite oxidase, and in the presence of molybdate, active sulfite
oxidase is obtained (17). Since the cloned sulfite oxidase contains a
His6 tag, it can be easily purified from the in
vitro incubation mixture (13). In order to determine whether both
sulfurs of the dithiolene group of MPT originate from
L-cysteine, the cofactor bound to sulfite oxidase was
converted to either form B or camMPT. CamMPT is an alkylated product of MPT that retains both sulfur atoms of the dithiolene group
(Fig. 1) (20). In contrast, form B is a fluorescent derivative formed
by air oxidation (16), which retains only the sulfur atom on C-2' of
the dithiolene group of MPT (Fig. 1). Sulfite oxidase isolated after
incubation with L-[35S]cysteine was divided
into two fractions, one of which was incubated for 30 min at 95 °C
for the production of form B, and the other fraction was denatured with
SDS in the presence of iodoacetamide for the formation of camMPT. As
shown in Table III, analysis of the
radioactivity present in purified form B and camMPT revealed a ratio of
1:1.73, correlating with the number of sulfur atoms present in the two
MPT derivatives. This ratio remained the same in the presence or
absence of sodium molybdate in the in vitro incubation
mixture (data not shown). These results showed conclusively that both
sulfur atoms present in the dithiolene group of MPT are derived from
MPT synthase.
Analysis of the Activities of Molybdoenzymes in the E. coli Strain
CL100(iscS Analysis of the Ability of Extracts from Strains
CL100(iscS
It was of further interest to determine whether the two remaining
sulfurtransferases, CSD and CsdB, are sufficient to provide the sulfur
for the conversion of larger amounts of precursor Z to MPT. For this
purpose, the extracts of CL100(iscS The Sulfur Is Transferred as a Protein-bound Persulfide--
In
A. vinelandii, it has been shown that the two proteins NifS
and IscS form protein-bound persulfides by transfer of sulfur from free
L-cysteine to an cysteine thiol group of the protein (6,
21). This protein-bound persulfide acts as the sulfur donor for the
sulfuration of the corresponding substrates of these proteins. To
determine whether a persulfide bound to E. coli IscS, CSD,
or CsdB can act as the sulfur donor for the sulfuration of inactive MPT
synthase in vitro, IscS, CSD, and CsdB were incubated with
L-cysteine as described under "Experimental
Procedures," and excess L-cysteine was removed by gel
filtration. Reaction mixtures containing the putative
persulfide-containing proteins IscS, CSD or CsdB, MPT synthase, MoeB,
precursor Z, and Mg-ATP were tested for their abilities to produce MPT
without the addition of L-cysteine as a sulfur source. The
amount of MPT produced in the in vitro incubation mixtures
was again determined by conversion of MPT to form A. As shown in Fig.
5, form A production was observed in all
three incubation mixtures, indicating that the sulfur for the
sulfuration of MPT synthase is indeed being transferred from a
sulfurtransferase in the form of a protein-bound persulfide. However,
Fig. 5 shows that the abilities of the three sulfurtransferases to
transfer the sulfur are significantly different. The in
vitro assay containing the CSD-bound persulfide produced the
highest amount of form A (Fig. 5A). IscS-persulfide produced
only 37% of the amount of form A in comparison with CSD (Fig.
5C), and CsdB-bound persulfide produced only 2% form A
compared with CSD (Fig. 5B). These results are in agreement
with the results shown in Table II, where CSD showed the highest
catalytic activity in the desulfuration of L-cysteine for
the production of MPT, followed by IscS and CsdB. These findings
indicate that CSD has a high ability to interact with MoeB/MPT synthase
for the regeneration of the glycine-thiocarboxylate group of the
synthase.
The studies presented here delineate an in vitro system
using purified components for studying the mechanism of assembly of the
dithiolene group of MPT. Using this system, L-cysteine was identified as the likely physiological sulfur donor. Additionally, it
has been demonstrated that any of three NifS-like sulfurtransferases, namely IscS, CSD, and CsdB, is capable of mobilizing and transferring sulfur from L-cysteine to precursor Z. The minimal
requirement for the activation of inactive MPT synthase was shown
to be MoeB, Mg-ATP, L-cysteine, and a sulfurtransferase.
After the addition of excess precursor Z, the reaction was shown to be
catalytic rather than stoichiometric, since with the amounts of
precursor used up to 30 times more MPT was produced than MPT synthase
present in the system (data not shown), showing clearly that the
components of the in vitro system are getting turned over
during the reaction.
Since the biosynthesis of Moco has been most extensively studied in
E. coli, the identification of a novel protein component separate from the products of the previously identified mo
loci involved in MPT formation was a somewhat surprising observation. The well characterized genetic loci moa, mob,
mod, moe, and mog were identified in
E. coli by selection for chlorate resistance. During this
selection, mutant strains were obtained that are deficient in nitrate
reductase activity (22). Since a sulfurtransferase involved in the
mobilization of L-cysteine-bound sulfur for the biosynthesis of Moco was not identified by selecting for
chlorate-resistant mutants, it must be concluded that either a mutation
in the sulfurtransferase impairs the viability of the cell or that a
number of sulfurtransferases within the cell are capable of this activity.
In order to identify the sulfurtransferase required for MPT formation
in vivo, a mutant strain with an in frame deletion in the
iscS gene was tested for its ability to form MPT. This
iscS mutant strain was reported to have decreased levels of
the activity of Fe-S-containing enzymes (10). Additionally, it lacks
4-thiouridine in its tRNA and requires thiamin and nicotinic acid for
growth in minimal media (11). These observations implied that
IscS has a general role in sulfur mobilization for the biosynthesis of
Fe-S clusters, 4-thiouridine tRNA, and thiamine (11). However, it was
reported that several Fe-S cluster-containing enzymes tested in the
iscS mutant exhibited some residual activity, indicating that other proteins are at least partially able to replace IscS in its
role for Fe-S cluster formation in vivo (10). Our data have
shown that nitrate reductase, a molybdoenzyme requiring Fe-S clusters
for its activity, is completely inactive in the iscS mutant
strain. However, expression of recombinant sulfite oxidase in an
iscS mutant strain yielded 10% of the activity in
comparison with the corresponding parental strain. Correspondingly, the
cofactor content of the iscS mutant strain was determined to
be very low, and no precursor Z accumulation could be demonstrated in
this strain. It is concluded that the low level of MPT in this strain is due to the impaired activity of MoaA, an Fe-S cluster-containing enzyme involved in the synthesis of precursor Z (23), and not due to an
inability to convert precursor Z to MPT. Since the crude extract of the
iscS mutant exhibited the ability to convert externally added precursor Z to MPT to the same level as the parental strain, it
was apparent that the other sulfurtransferases in the cell are fully
capable of providing the sulfurs for the sulfuration of MPT synthase.
These findings also indicate that IscS is not involved in the sulfur
transfer process for MPT formation in vivo.
The exact physiological functions of the two E. coli
NifS-like proteins CSD and CsdB are not known to date. It has been
assumed that like IscS, they are involved in iron-cluster formation or the biosynthesis of selenophosphate in the cell (9). To determine the
role of CSD and CsdB in the biosynthesis of MPT in vivo, the ability of mutant strains in csdB and csdA to
produce MPT must be investigated. Since CSD showed the highest
catalytic activity for MPT formation in vitro and CsdB is
considered more as a selenocysteine lyase than as a cysteine
desulfurase, CSD appears more likely to be involved in the mobilization
of sulfur from L-cysteine for the synthesis of MPT. In sum,
the data presented here show that the utilization of the sulfur atom of
cysteine for MPT synthesis requires a persulfide-containing protein.
While all NifS-like proteins tested in this study can serve the purpose
in the established in vitro system, it is possible that also
other persulfide-containing proteins are able to serve the same
function. Nevertheless, the data presented in this study establish the
requirement for a persulfide-containing protein, which acts as the
sulfur donor for MPT biosynthesis. In future studies, it has to be
investigated which persulfide-containing protein is the physiological
sulfur donor for MPT formation in the cell.
Recently, we could show that MoeB does not serve the function as the
immediate sulfur donor for the formation of the thiocarboxylate group
in MoaD.2 The proposed mechanism of MPT synthase
activation by sulfur transfer suggests that MoeB primes the small
subunit of MPT synthase by the formation of a MoeB-MoaD adenylate
complex for subsequent sulfuration but is itself not a carrier of the
sulfur atom derived from L-cysteine.2
The results shown above using 35S-labeled
L-cysteine demonstrated that the same sulfur transfer
pathway is involved in the incorporation of both sulfurs of the
dithiolene group of MPT. It was shown previously that purified active
MPT synthase and precursor Z are sufficient for the formation of MPT
in vitro (17, 19). In the activated form of MPT synthase,
the C terminus of the small MoaD subunit is present as a
thiocarboxylate, which serves as the sulfur donor for MPT formation.
The high resolution crystal structure of MPT synthase has shown that in
the heterotetrameric protein, the C terminus of each MoaD subunit is
inserted into one of the MoaE subunits to form the active site (3). The
newly formed MPT remains tightly bound to the synthase in the absence
of proteins that are able to bind MPT with higher affinity (17).
However, details of the mechanism catalyzed by MPT synthase, including the insertion of two sulfur atoms into precursor Z for the formation of
MPT without the need for resulfuration of the MoaD subunit, are unknown
at present. One possibility is that the two active sites of an MPT
synthase tetramer are able to act cooperatively and that each MoaD
subunit of the tetramer provides one sulfur atom for the formation of
the MPT dithiolene group. This reaction would require that the
precursor is getting transferred between the two active sites of each
MoaE subunit. In future studies, the reconstitution system presented
here should help in understanding the mechanism of sulfur incorporation
carried out by MPT synthase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DE3
lysogenization kit from Novagen was used to integrate the gene for T7
RNA polymerase into the chromosome of the E. coli strains.
To determine nitrate reductase activity, cells were grown aerobically
in LB medium supplemented with 15 mM NaNO3.
Bacterial strains and plasmids used in this work
(DE3) cells,
and the protein was purified by ammonium sulfate precipitation and gel
filtration after the procedure described in Ref. 3. Cloned MoeB was
expressed in a pET15b vector (Novagen) in E. coli
moaD
(DE3) cells, and the protein was purified
by ammonium sulfate precipitation, ion exchange chromatography,
hydrophobic interaction chromatography, and gel
filtration.2 Human sulfite
oxidase was cloned into a pTrc-His vector (Amersham Pharmacia Biotech),
generating an N-terminal fusion to a His6 tag, expressed in
E. coli moaA
, MC1061, and
CL100(iscS
) cells, and purified by
Ni2+-nitrilotriacetic acid chromatography (13).
-D-thiogalactoside. After 4 h, cells were harvested by centrifugation at 5000 × g. The
His6 tag-containing CsdB and CSD were purified by
Ni2+-nitrilotriacetic acid chromatography. The cell pellets
were resuspended in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, and lysed by several passages through a
French pressure cell. After centrifugation at 17,000 × g for 25 min, imidazole was added to the supernatant to a
final concentration of 10 mM. The supernatant was then
combined with 1.5 ml of Ni2+-nitrilotriacetic acid resin
(Qiagen) per liter of cell growth, and the slurry was equilibrated with
gentle stirring at 4 °C for 30 min. The slurry was poured into a
column and washed with 2 column volumes of 10 mM imidazole,
50 mM sodium phosphate, 300 mM NaCl, pH 8.0, followed by a wash with 10 column volumes of the same buffer with 20 mM imidazole. The His-tagged proteins were eluted with 100 mM imidazole in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. Fractions containing CsdB or CSD were
combined and dialyzed against 50 mM Tris, 1 mM
EDTA, pH 7.5.
A) of 1 per min. Nitrate reductase activity was assayed
in extracts at room temperature with benzyl viologen as electron donor
after the method described in Ref. 15. One unit of nitrate reductase
activity is described as the production of 1 µmol of nitrate/min/mg
of protein.
1
cm
1.
1 cm
1.
Radioactivity present in form B and camMPT was measured and compared
with a standard curve recorded with
L-[35S]cysteine.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Conversion of MPT to form A, form B, and
camMPT.
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[in a new window]
Fig. 2.
Analysis of form A obtained after the
in vitro activation of inactive MPT synthase with MoeB
and moeB extract. HPLC elution
profiles of a reversed phase C18 column after the in vitro
treatment of 3.4 µM inactive MPT synthase with 2.5 mM Mg-ATP, 200 µM precursor Z with the
inclusion of 15 µM MoeB (A), 15 µM MoeB and 10 µl (4 mg/ml)
moeB
extract (B), 15 µM MoeB and 20 µl (2 mg/ml) of the high molecular
weight fraction of moeB
extract
(C), and 15 µM MoeB and 20 µl of the low
molecular weight fraction of moeB
extract (D).
As described previously (12, 17), the fluorescence peak eluting at
about 4 min was identified as compound Z, the oxidized product of
precursor Z. Form A elutes at about 10 min as verified by its
absorption spectrum (data not shown).
crude extract. As shown
in Fig. 3, among sulfide, thiosulfate, thiocyanate, L-cystine, and L-cysteine,
significant form A formation could only be observed in the presence of
sulfide or L-cysteine (Fig. 3, A and
E). However, as shown in Fig. 3F, the formation of form A was also observed when inorganic sulfide was present in the
incubation mixture in the absence of the protein fraction of
moeB
extract. These results indicated that
while L-cysteine is the likely physiological sulfur donor
for the activation of MPT synthase, inorganic sulfide is able to serve
the same function in vitro. Since in the presence of
L-cysteine an additional protein component is required for
the sulfur transfer process, it was apparent that an as yet
unidentified sulfurtransferase is required for the transfer of sulfur
from L-cysteine to MPT synthase in a reaction requiring MoeB as well.
View larger version (18K):
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Fig. 3.
Identification of the physiological sulfur
donor required for the in vitro activation of inactive
MPT synthase with MoeB and the high molecular weight fraction of
moeB extract. HPLC elution profiles
of a reversed phase C18 column after the in vitro incubation
of 3.4 µM inactive MPT synthase with 2.5 mM
Mg-ATP, 200 µM precursor Z, 15 µM MoeB, 20 µl (2 mg/ml) of the high molecular weight fraction of
moeB
extract after the addition of 250 µM sodium sulfide (A), 250 µM sodium
thiosulfate (B), 250 µM sodium thiocyanate
(C), 250 µM L-cystine
(D), 250 µM L-cysteine
(E), or 250 µM sodium sulfide (F)
without the addition of the high molecular weight fraction of
moeB
extract.
Abilities of E. coli NifS-like proteins to provide the sulfur for the
formation of MPT
In vitro insertion of radiolabeled 35S into MPT
)--
The data presented above have shown the
requirement for an NifS-like protein to mobilize the sulfur atom of
cysteine for the biosynthesis of MPT in vitro. Thus, it was
of further interest to determine the in vivo roles of the
three sulfurtransferases IscS, CSD, and CsdB for the biosynthesis of
MPT. Lauhon and Kambampati (11) reported the successful construction of
an E. coli strain with an in-frame deletion of the
iscS gene (Table I). To determine whether IscS is required
for the synthesis of MPT in vivo, we analyzed the activities
of different molybdoenzymes in the E. coli strain
CL100(iscS
) and the corresponding parental
strain MC1061. As shown in Table IV,
nitrate reductase activity was detected in the strain MC1061 but not in
strain CL100(iscS
). In contrast, analysis of
activity of human sulfite oxidase expressed in these strains revealed
that active sulfite oxidase is produced in strain
CL100(iscS
), but only to the extent of 10% in
comparison with the parental strain MC1061 (Table IV). Measurement of
the cofactor content of purified sulfite oxidase revealed that the
lower activity of sulfite oxidase in
CL100(iscS
) corresponded with its cofactor
content (Table V). In addition, the
amount of total MPT determined in whole cells of these two strains
showed a significantly lower amount of MPT in
CL100(iscS
) in comparison with MC1061 (Table
V). It therefore appeared that the reduced activity of sulfite oxidase
in strain CL100(iscS
) is based on an impaired
ability of this strain to produce MPT.
Comparison of nitrate reductase and sulfite oxidase activities in E. coli strains MC1061 and CL100(iscS)
Analysis of the MPT content of sulfite oxidase in comparison to the
total MPT content of strains MC1061 and CL100(iscS)
) and MC1061 to Convert Added Precursor Z to
MPT--
It remained possible that the reduced ability of
CL100(iscS
) to produce MPT is based on the
limited ability of the strain to produce the sulfurated form of MPT
synthase. It has been shown that mutant strains in
moaD or moeB, which lack MPT synthase or produce an unsulfurated form of the synthase, respectively, accumulate precursor Z (12). Analysis of the precursor Z content of
CL100(iscS
) revealed no such accumulation
(data not shown), indicating that all precursor Z produced in
CL100(iscS
) is completely converted to
MPT by a sulfurated form of MPT synthase. Thus, it could be concluded
that the inability of strain CL100(iscS
) to
produce larger amounts of MPT is due to a reduced ability of this
strain to synthesize precursor Z.
) and MC1061 were
tested for their ability to convert externally added precursor Z to MPT
in vitro. As shown in Fig. 4,
the same amounts of form A were formed after the addition of precursor Z to extracts of CL100(iscS
) and MC1061. This
finding showed that either CSD or CsdB in strain CL100(iscS
) is sufficient for producing a
sulfurated form of MPT synthase, which in turn can convert all
precursor Z present to MPT. Conclusively, the reduced ability of strain
CL100(iscS
) to produce precursor Z may be due
to the inability of this strain to provide the Fe-S clusters for MoaA,
a protein required for the synthesis of precursor Z from a guanosine
nucleotide.
View larger version (15K):
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Fig. 4.
Analysis of the ability of
CL100(iscS ) and MC1061 extracts to
convert externally added precursor Z to MPT. Shown are HPLC
elution profiles of form A obtained after the in vitro
incubation of 1.7 µM inactive MPT synthase, 3 µM MoeB, 200 µM precursor Z, and 2.5 mM Mg-ATP with 50 µl of MC1061 extract (2 mg/ml)
(A) and 50 µl of CL100(iscS
)
extract (2 mg/ml) (B).
View larger version (13K):
[in a new window]
Fig. 5.
Analysis of the ability of CSD, CsdB, and
IscS to transfer the sulfur for the activation of MPT synthase as a
protein-bound persulfide. HPLC elution profiles of form A obtained
after the in vitro incubation of 5.4 µM
inactive MPT synthase, 3.5 µM MoeB, 200 µM
precursor Z, 2.5 mM ATP, and 4.2 µM CSD-SSH
(A), 4.2 µM CsdB-SSH (B), or 4.2 µM IscS-SSH (C). For the formation of CSD,
CsdB, or IscS-bound persulfide, the sulfurtransferases were incubated
for 5 min with 2 mM L-cysteine at 4 °C.
After the cysteine treatment, the proteins were gel-filtered and
immediately added to the in vitro activation mixture.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank C. Lauhon (University of Wisconsin)
for providing strains MC1061 and CL100(iscS)
and M. Wuebbens for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was financially supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to S. L.) and additionally by National Institutes of Health Grant GM44283 (to K. V. R.).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. Tel.: 919-681-8845;
Fax: 919-684-5470; E-mail: raj@biochem.duke.edu.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M102072200
2 S. Leimkühler, M. M. Wuebbens, and K. V. Rajagopalan, submitted.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Moco, molybdenum cofactor; MPT, molybdopterin; HPLC, high performance liquid chromatography; camMPT, carboxamidomethyl-MPT; CSD, cysteine sulfinate desulfinase.
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REFERENCES |
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