A Sulfurtransferase Is Required in the Transfer of Cysteine Sulfur in the in Vitro Synthesis of Molybdopterin from Precursor Z in Escherichia coli*

Silke Leimkühler and K. V. RajagopalanDagger

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, March 7, 2001, and in revised form, March 29, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 lambda  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.

                              
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Table I
Bacterial strains and plasmids used in this work

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-(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).

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-beta -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.

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 (Delta 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.

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-1 cm-1.

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-1 cm-1. Radioactivity present in form B and camMPT was measured and compared with a standard curve recorded with L-[35S]cysteine.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 1.   Conversion of MPT to form A, form B, and camMPT.


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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).

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- 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.


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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.

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).

                              
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Table II
Abilities of E. coli NifS-like proteins to provide the sulfur for the formation of MPT

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.

                              
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Table III
In vitro insertion of radiolabeled 35S into MPT
The in vitro activation mixture for the incorporation of 35S into MPT contained 73.8 µM MPT synthase, 31.3 µM MoeB, 5.2 µM IscS, 400 µM precursor Z, 20 mM Na2MoO4, 2 mM Mg-ATP, 1 mM L-cysteine, 1 µCi of L-[35S]cysteine, and 51 µM sulfite oxidase.

Analysis of the Activities of Molybdoenzymes in the E. coli Strain CL100(iscS-)-- 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.

                              
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Table IV
Comparison of nitrate reductase and sulfite oxidase activities in E. coli strains MC1061 and CL100(iscS-)

                              
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Table V
Analysis of the MPT content of sulfite oxidase in comparison to the total MPT content of strains MC1061 and CL100(iscS-)

Analysis of the Ability of Extracts from Strains 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.

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-) 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.


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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).

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.


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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

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.

    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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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24. Deleted in proof
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