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Originally published In Press as doi:10.1074/jbc.M704020200 on August 7, 2007

J. Biol. Chem., Vol. 282, Issue 39, 28493-28500, September 28, 2007
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Transfer of the Molybdenum Cofactor Synthesized by Rhodobacter capsulatus MoeA to XdhC and MobA*Formula

Meina Neumann{ddagger}, Walter Stöcklein§, and Silke Leimkühler{ddagger}1

From the Departments of {ddagger}Protein Analytics and §Analytical Biochemistry, Institute of Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany

Received for publication, May 16, 2007 , and in revised form, July 31, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The molybdenum cofactor (Moco) exists in different variants in the cell and can be directly inserted into molybdoenzymes utilizing the molybdopterin (MPT) form of Moco. In bacteria such as Rhodobacter capsulatus and Escherichia coli, MPT is further modified by attachment of a GMP nucleotide, forming MPT guanine dinucleotide (MGD). In this work, we analyzed the distribution and targeting of different forms of Moco to their respective user enzymes by proteins that bind Moco and are involved in its further modification. The R. capsulatus proteins MogA, MoeA, MobA, and XdhC were purified, and their specific interactions were analyzed. Interactions between the protein pairs MogA-MoeA, MoeA-XdhC, MoeA-MobA, and XdhC-MobA were identified by surface plasmon resonance measurements. In addition, the transfer of Moco produced by the MogA-MoeA complex to XdhC was investigated. A direct competition of MobA and XdhC for Moco binding was determined. In vitro analyses showed that XdhC bound to MobA, prevented the binding of Moco to MobA, and thereby inhibited MGD biosynthesis. The data were confirmed by in vivo studies in R. capsulatus cells showing that overproduction of XdhC resulted in a 50% decrease in the activity of bis-MGD-containing Me2SO reductase. We propose that, in bacteria, the distribution of Moco in the cell and targeting to the respective user enzymes are accomplished by specific proteins involved in Moco binding and modification.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The molybdenum cofactor (Moco)2 is an essential component of a diverse group of enzymes involved in important redox reactions in the global carbon, nitrogen, and sulfur cycles. Moco consists of a molybdenum atom coordinated to the dithiolene group of a tricyclic pyranopterin referred to as molybdopterin (MPT) (1). The biosynthesis of Moco is highly conserved in eukaryotes and prokaryotes (1) and can be divided into three general steps. In the first step, GTP is converted to the meta-stable intermediate Precursor Z (2, 3). In the second step, Precursor Z is further transformed by MPT synthase into MPT by generation of its characteristic dithiolene group (4, 5). In the third step, molybdate is inserted to the MPT dithiolene sulfurs, a reaction catalyzed by MogA and MoeA in Escherichia coli (6–8). MoeA mediates molybdenum ligation, whereas MogA helps to facilitate this step in an ATP-dependent manner (8). Recently, studies with the homologous Arabidopsis thaliana CNX1 protein G and E domains identified the formation of an MPT-AMP intermediate before the ligation of molybdate to the MPT moiety (9, 10). An unexpected observation in the crystal structure of the A. thaliana CNX1 protein G domain was the identification of copper bound to the MPT-AMP dithiolene sulfurs (11). Up to now, the function of this novel MPT ligand has been unknown, but it was speculated that copper might play a role in sulfur transfer to Precursor Z, in protection of the MPT dithiolene from oxidation, and/or in presentation of a suitable leaving group for molybdenum insertion (12). To date, copper-MPT-AMP has not been identified as an intermediate in the biosynthesis of Moco in E. coli (8).

After the insertion of molybdenum into MPT in E. coli, Moco either can be directly inserted into molybdoenzymes (such as YedY) binding the MPT form of Moco (13) or is further modified by attachment of GMP (14, 15), forming the bis-MPT guanine dinucleotide (bis-MGD) form of Moco found in enzymes of the Me2SO reductase family (16). In E. coli, the GMP attachment to Moco is catalyzed by the MobA and MobB proteins (17). Whereas MobA was shown to be essential for this reaction (18), the role of MobB still remains uncertain. From the crystal structure, it was postulated that MobB is an adapter protein that acts in concert with MobA to achieve the efficient biosynthesis and utilization of MGD (19).

Enzymes containing the MPT form of Moco belong to either the sulfite oxidase or xanthine oxidase family, whereas enzymes binding the bis-MGD form of Moco belong to the Me2SO reductase family of molybdoenzymes. In Rhodobacter capsulatus, xanthine dehydrogenase (XDH; EC 1.17.1.4) is the only identified enzyme harboring the MPT form of the cofactor, whereas all other known molybdoenzymes bind the bis-MGD form of the cofactor (20). An essential role for the XdhC protein in the maturation of R. capsulatus XDH has been described, which entails binding of Moco and its insertion into the XdhB subunit of XDH (21). For all members of the xanthine oxidase family, the sulfurated form of Moco is essential for catalysis, where the equatorial of the two oxygen ligands of nascent Moco is replaced by sulfur (22). Previous work showed that XdhC specifically promotes the exchange of this ligand by interaction with the L-cysteine desulfurase NifS4, which transfers the sulfur to Moco bound to XdhC (23). It has remained so far unclear which protein of the Moco biosynthesis pathway acts as the direct Moco donor for XdhC. So far, R. capsulatus XdhC is the only protein identified in bacteria shown to be involved in the modification of Moco by exchange of an oxo ligand of Moco with sulfur. To investigate the question of targeting, distribution, and insertion of different forms of Moco into the specific molybdoenzymes in R. capsulatus, we cloned and purified the MogA, MoeA, and MobA proteins from R. capsulatus for the investigation of protein-protein interactions in the homologous system. A MobB homolog seems not to be present in R. capsulatus (24).

In this study, we show for the first time that the amounts of sulfurated Moco and bis-MGD produced in the cell are regulated at the protein level by protein-protein interactions. We show that both MobA and XdhC receive Moco from MoeA; however, by binding to MobA, XdhC prevents the Moco transfer to MobA, thus inhibiting MGD formation. This regulation ensures that enough Moco is abstracted from the major route of bis-MGD biosynthesis for further modification to the sulfurated form of Moco and thus ensures that enough of the MPT form of Moco is provided to produce an active XDH, an enzyme involved in the purine degradation pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Plasmids, Media, and Growth Conditions—E. coli BL21(DE3) cells were used for heterologous expression of the R. capsulatus proteins MobA, MoeA, and MogA. R. capsulatus XdhC was expressed in E. coli ER2566(DE3) cells and purified as described previously (21). The human sulfite oxidase Moco domain (hSO-MD) was expressed from plasmid pTG818 (25) in E. coli TP1000({Delta}mobAB) cells (18) to obtain Moco-containing hSO-MD or in E. coli RK5202(modC-) cells (26) to obtain MPT-containing hSO-MD. The hSO-MD variants were purified as described previously by Temple et al. (25). E. coli cultures were grown in LB medium under aerobic conditions at 30 °C. The E. coli S17-1 strain was used for conjugation of R. capsulatus KS36 (B10S({Delta}nifHDK::Spc)) (27) with plasmid pSL160, expressing xdhC under the control of the nifH promotor from plasmid pPHU231 (28–30). For control experiments, pSL143 harboring only the nifH promotor cloned into plasmid pPHU231 and pMN80 expressing mobA under the control of the nifH promotor were introduced into KS36 cells. R. capsulatus cells were grown in RCV minimal medium supplemented with (NH4)2SO4 as described previously (31). For expression of Me2SO reductase, Me2SO was added to a final concentration of 30 mM, and for induction of XdhC or MobA expression from the plasmids, (NH4)2SO4 was replaced with 1 g/liter serine as the nitrogen source. R. capsulatus cells were grown under anaerobic, phototrophic conditions and harvested at late log phase after 48 h of growth at 30 °C. When required, 1 mM Na2MoO4, 150 µg/ml ampicillin, 25 µg/ml kanamycin, and 5 µg/ml tetracycline (for E. coli) or 1 µg/ml tetracycline (for R. capsulatus) were added to the medium.

Cloning, Expression, and Purification of R. capsulatus MobA, MoeA, and MogA—DNA fragments containing the coding regions for R. capsulatus mobA, moeA, and mogA were amplified by PCR, and flanking restriction sites were introduced. The moeA and mogA genes were cloned into the NdeI-XhoI sites and mobA into the NdeI-SalI sites of pET28a (Novagen), resulting in plasmids pMN32, pMN53, and pMN56, respectively.

For expression of MoeA, MogA, and MobA, E. coli BL21(DE3) cells were transformed with plasmids pMN32, pMN53, and pMN56, respectively, and cell growth was started with 10 ml of overnight culture/liter of LB medium. The cells were grown at 30 °C, and expression of MogA and MobA was induced at A600 = 0.3–0.5 with 100 µM isopropyl beta-D-thiogalactopyranoside. Cell growth was continued for 5 h, and cells were harvested and resuspended in 50 mM NaH2PO4 and 300 mM NaCl (pH 8.0). After cell lysis, the soluble fraction was transferred onto a column with nickel-nitrilotriacetic acid (Ni-NTA; Qiagen Inc.). The resin was washed with 20 column volumes of phosphate buffer first containing 10 mM and then 20 mM imidazole. The proteins were eluted with phosphate buffer containing 250 mM imidazole and dialyzed against 100 mM Tris (pH 7.2). For MoeA, expression was induced with 300 µM isopropyl beta-D-thiogalactopyranoside; the shaking rate was increased to 210 rpm; and cells were harvested 3 h after induction of gene expression. After cell lysis, the soluble fraction was transferred onto nickel-triscarboxymethylethylenediamine (Macherey-Nagel). The column was washed with 40 column volumes of phosphate buffer containing 10 mM imidazole, and MoeA was eluted with the same buffer containing 250 mM imidazole and dialyzed against 100 mM Tris (pH 7.2).

MGD Formation by MobA—Moco was obtained after heat treatment of purified hSO-MD expressed in TP1000 cells, and MPT was obtained after heat treatment of purified hSO-MD expressed in RK5202 cells as described previously by Temple et al. (25) and Neumann et al. (21). 100 µM MobA was incubated with 160 µM Moco or MPT before excess Moco/MPT was removed by gel filtration. 1 mM Na2MoO4 was included in incubation mixtures containing Moco. 1 mM MgCl2 and 1 mM GTP were added, and the mixtures were incubated for 60 min at room temperature in a total volume of 400 µl of 100 mM Tris (pH 7.2) before the protein was denatured and analyzed for the presence of Moco or MGD by conversion to Form A (as described below).

MPT and Moco Binding by MogA, MoeA, and MobA—KD values were determined by ultrafiltration as described previously (21). Samples contained 6 µM MogA, MoeA, or MobA and 0–24 µM Moco or MPT.

Moco, MPT, and MGD Analysis—The Moco/MPT content of the purified proteins was quantified after conversion to Form A as described previously (21). To separate Form A obtained from Moco/MPT and Form A-GMP obtained from MGD, the protocol originally described by Joshi and Rajagopalan (32) was used with some modifications. MGD was converted to Form A-GMP and Moco/MPT to Form A by overnight treatment with acidic iodine at room temperature (33). Form A was separated from Form A-GMP by chromatography on Q-Sepharose (GE Healthcare). 400 µl of Q-Sepharose was equilibrated with H2O; the oxidized samples were loaded; and Form A was eluted with 10 mM acetic acid. Form A-GMP was eluted with 50 mM HCl and converted to Form A by the addition of MgCl2, nucleotide pyrophosphatase, and alkaline phosphatase at pH 8.0. The pH of the samples was adjusted to pH 5.3 by the addition of 10 µl of 50% acetic acid before application to a C18 reversed-phase high pressure liquid chromatography column (4.6 x 250-mm Hypersil ODS, 5-µm particle size) equilibrated in 5 mM ammonium acetate and 15% methanol. In-line fluorescence was monitored by an Agilent 1100 series detector with excitation at 383 nm and emission at 450 nm.

Surface Plasmon Resonance (SPR) Measurements—All binding experiments were performed with the SPR-based instrument BiacoreTM 2000 on CM5 sensor chips at 25 °C and a flow rate of 10 µl/min using BiaControl 2.1 and BiaEvaluation 3.0 software (Biacore AB) as described previously (23). The proteins were immobilized after dilution in 10 mM acetate buffer at pH 4 (bovine serum albumin, MoeA, and MogA) or pH 5 (XdhC, XDH, and MobA). For control experiments, the N-terminal His6 tags of MoeA, MogA, and MobA were cleaved using the thrombin CleanCleave kit (Sigma). Cleavage was controlled by SDS-PAGE.

In Vitro Transfer of Moco from MoeA to XdhC—For production of Moco, 15 µM MPT obtained from hSO-MD was incubated with 30 µM MogA, 30 µM MoeA, 37.5 µM Na2MoO4, 1 mM MgCl2, and 1 mM ATP in a volume of 200 µl of 100 mM Tris (pH 7.2). After 15 min of incubation at room temperature, 8 µM XdhC was added, and the mixture was further incubated for 20 min before MoeA and MogA were removed by Ni-NTA chromatography. Single components were left out for control experiments. Free Moco in the XdhC fraction was removed by an additional gel filtration step using NICK columns (GE Healthcare). 400 µl of the XdhC fraction was treated with acidic iodine, and bound Moco was quantified as Form A fluorescence as described above.

The same setup as described above was used to analyze the competition of Moco transfer from MoeA to XdhC in the presence of MobA. To avoid free Moco in the assay, these mixtures contained 15 µM XdhC and either 15 µM or 150 µM MobA in addition to 1 mM GTP.

In Vitro Transfer of Moco from MobA to XdhC—25 µM MobA was incubated with 100 µM Moco and 1 mM Na2MoO4 for 10 min at room temperature, and excess Moco was removed by gel filtration. Moco bound to MobA was quantified after conversion to Form A. 10 µM XdhC was added to the Moco-loaded MobA fraction before MobA and XdhC were separated by Ni-NTA chromatography. Free Moco in the XdhC fraction was further removed by gel filtration. 400 µl of the XdhC fraction was treated with acidic iodine, and released Moco was converted to Form A and quantified as described above.

Formation of MGD by MobA in the Presence of XdhC—To analyze the influence of XdhC on Moco binding to MobA, 10 µM MobA was incubated with 0, 1, 10, and 100 µM XdhC for 10 min at room temperature before the addition of 1 mM GTP, 1 mM MgCl2, 1 mM Na2MoO4, and 80 µM Moco. In another set of experiments, XdhC was added after incubation of 115 µM MobA with 190 µM Moco and 1 mM Na2MoO4 for 10 min at room temperature. Unbound Moco was removed by gel filtration, and 50 µl of the Moco-loaded MobA fraction was incubated with 1 mM Na2MoO4, 1 mM GTP, and increasing amounts of XdhC (0, 1.16, 11.6, and 116 µM) in a total volume of 400 µl in 100 mM Tris (pH 7.2). The incubation mixtures were incubated for 90 min under anaerobic conditions, and the MGD content of all samples was quantified as described above.


Figure 1
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  		FIGURE 1.
Purification and analysis of the activities of MobA, MoeA, and MogA after heterologous expression in E. coli. A, 12% SDS-PAGE of the purification stages of MobA, MoeA, and MogA. Lane I, 1 µl of E. coli BL21(DE3)xpMN56 (MobA) extract after cell lysis; lane II, 5 µg of purified MobA; lane III, 1 µl of E. coli BL21(DE3)xpMN32 (MoeA) extract after cell lysis; lane IV, 3 µg of purified MoeA; lane V, 1 µl of E. coli BL21(DE3)xpMN53 (MogA) extract after cell lysis; lane VI, 3 µg of purified MogA. B, MobA-catalyzed conversion of MPT or Moco to MGD. 100 µM MobA was incubated with 160 µM MPT (bars I and II) or 160 µM Moco (bars III and IV) and 1 mM MgGTP, and samples were analyzed for the formation of MGD (see "Experimental Procedures"). MPT and Moco bound to MobA were separated from produced MGD by conversion to Form A and Form A-GMP, respectively. Subsequently, Form A-GMP was converted to Form A, and Form A fluorescence was quantified in MobA samples incubated with MPT (bar I), MPT that was converted to MGD (bar II), Moco (bar III), and Moco that was converted to MGD (bar IV). ND, no Form A detected.

 
Analysis of XDH and Me2SO Reductase Activities in R. capsulatus Crude Extracts—Plasmids were mobilized from E. coli S17-1 into R. capsulatus KS36 by filter mating as described previously (31). Crude extracts were obtained after cell lysis by sonification and subsequent removal of cell debris by centrifugation. Protein concentrations were determined following the method of Bradford (34). XDH activity was measured as described previously (24). The specific XDH activity (units/mg) is defined as the reduction of 1 µmol of NAD+/min/mg of enzyme. Me2SO reductase activity was measured as described by McEwan et al. (35) with dithionite-reduced benzyl viologen as the electron donor. Me2SO reductase activity (units/mg) is defined as the reduction of 1 µmol of Me2SO/min/mg of protein.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification and Analysis of the Functional Activities of R. capsulatus MobA, MoeA, and MogA—For purification of R. capsulatus MobA, MoeA, and MogA, fusion proteins were generated each containing an N-terminal His6 tag (see "Experimental Procedures"). After heterologous expression in E. coli BL21(DE3) cells, the soluble fractions of MobA, MoeA, and MogA were purified by affinity chromatography (see "Experimental Procedures"). After elution, one major band was displayed for MobA, MoeA, and MogA on Coomassie Brilliant Blue R-stained SDS-polyacrylamide gels, corresponding to molecular masses of 22.1, 44.6, and 20.3 kDa, respectively (Fig. 1A). This procedure yielded ~6.5 mg of MobA/liter of E. coli culture, 1.5 mg of MoeA/liter of E. coli culture, and 13.3 mg of MogA/liter of E. coli culture.

To show the functionality of R. capsulatus MoeA and MogA, Moco was produced from MPT in vitro and inserted into human aposulfite oxidase following the procedure described by Nichols and Rajagopalan (8) for E. coli MoeA and MogA. In contrast to the E. coli proteins, for which maximum human sulfite oxidase reconstitution was observed using a MogA/MoeA ratio of 1:10, the best human sulfite oxidase reconstitution was obtained when R. capsulatus MogA and MoeA where used in a ratio of at least 1:1.4 (data not shown). Thus, for all further assays, R. capsulatus MogA and MoeA were mixed in a 1:1 ratio.

To test the functionality of R. capsulatus MobA, its ability to produce MGD from either MPT or Moco in the presence of MgGTP was analyzed. Moco was extracted from hSO-MD expressed in E. coli TP1000 cells (25), whereas MPT was obtained from hSO-MD expressed in RK5202(modC-) cells (26). After the addition of MgGTP to the incubation mixture, MGD was formed only from Moco (Fig. 1B, bars II and IV). Bound MPT and the remaining Moco eluted in the Moco/MPT fraction (bars I and III). These results show that molybdenum insertion into MPT has to precede MGD formation. Thus, for all further experiments, Moco was used as a precursor for the formation of MGD.

Analysis of Protein-Protein Interactions by SPR Measurements—To identify possible protein-protein interactions between XdhC, MogA, MoeA, and MobA, SPR measurements were employed for real-time detection of specific interactions using the purified proteins. XdhC, MoeA, MogA, and MobA were immobilized via amine coupling to a CM5 chip, and interactions were analyzed with each protein partner. The results obtained by SPR measurements for the protein pairs listed in Table 1 showed the tightest interaction between MoeA and XdhC, with KD values of 1.45 and 1.15 µM depending on the immobilized protein. In contrast, no significant interaction between XdhC and MogA was obtained with either immobilized protein partner, whereas in another set of experiments, MoeA bound to MogA with KD values of 6.2 and 6.0 µM (Table 1). However, this also shows that XdhC preferentially interacts with MoeA and not with MogA. The interaction between MoeA and MobA was also investigated. When MoeA was immobilized, MobA interacted with a KD of 3.5 µM, whereas when MobA was immobilized, the KD was significantly higher (27 µM) (Table 1). The second value for immobilized MobA shows that the dissociation constant between both proteins was negatively influenced by the immobilization of MobA. Because both XdhC and MobA interacted with MoeA and might compete for the same binding site, a possible interaction between XdhC and MobA was also investigated. As shown in Table 1, an interaction between both proteins was identified with KD values of 1.75 and 1.04 µM for immobilized XdhC and MobA, respectively. In contrast to the MoeA-MobA interaction, the dissociation constant for this protein pair was not influenced by the immobilization of MobA, showing that MobA was immobilized in a functional form, partly impairing the binding site for MoeA.


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  		TABLE 1
Analysis of protein-protein interactions between MobA, MoeA, MogA, and XdhC by SPR measurements

 
Control experiments showed that MoeA did not interact with immobilized XDH, supporting the idea that Moco transfer to XDH is mediated by XdhC, rather than a direct transfer of Moco from MoeA to XDH. Additional control experiments were performed to exclude a possible influence of the N-terminal His6 tags fused to MoeA, MogA, and MobA on the dissociation constants. For this purpose, the His6 tags of MogA, MoeA, and MobA were cleaved by thrombin treatment, and the interaction of these proteins with immobilized XdhC was compared with the sensograms obtained for the His6-tagged proteins. Although untagged MogA also showed no interaction with XdhC (data not shown), comparison of the binding curves of His6-tagged and untagged MoeA and MobA with immobilized XdhC showed no significant difference in the Biacore sensograms (supplemental Fig. S1), clearly showing that the His6 tag had no influence on the protein-protein interactions or the dissociation constants.

In Vitro Transfer of Moco from MoeA to XdhC—To investigate whether Moco bound to MoeA can directly be transferred to XdhC, Moco was produced in vitro by incubation of MogA and MoeA with MPT, Formula, and MgATP before XdhC was added. To ensure that the incubation mixture did not contain free MPT, which can be bound directly by XdhC, a MogA/MPT ratio of 2:1 was chosen. After incubation for 20 min, MogA and MoeA were removed by Ni-NTA chromatography, and the amount of Moco bound to XdhC was quantified after the conversion of Moco to Form A (see "Experimental Procedures"). The results in Fig. 2 show that Moco was specifically transferred to XdhC in incubation mixtures containing MoeA, MgATP, and Formula (bar III). The inclusion of MogA resulted in higher Moco saturation of XdhC (bar I) as a result of increased Moco production in the incubation mixture. The results in Fig. 2 also show that Moco was specifically transferred to XdhC and not MPT because the omission of Formula from the incubation mixtures did not give rise to Form A fluorescence (bar V).

Because an interaction between MoeA and both MobA and XdhC was seen by SPR measurements, we were interested in whether XdhC and MobA directly compete for Moco produced by MoeA. To analyze this, GTP and increasing amounts of MobA were added to an incubation mixture containing MPT, MoeA, MogA, MgATP, Formula, and XdhC. The results in Fig. 3A show that a 10-fold excess of MobA in the presence of GTP resulted in a drastically decreased Moco saturation of XdhC (bar II versus bar I). In contrast, omission of GTP from the incubation mixtures with equimolar concentrations of XdhC and MobA resulted in an increase in the Moco content of XdhC (bar III versus bar I). This shows a rapid conversion of Moco to MGD when GTP is present, and thus, the Moco concentration is reduced in the incubation mixture. In the absence of GTP, Moco is not converted to MGD; thus, a 10-fold excess of MobA results in only a slightly reduced Moco content of XdhC. These results show that MobA and XdhC compete for Moco produced by MoeA and, in addition, that binding of Moco to XdhC is only slightly reduced by the concentration of MobA in the presence of GTP.


Figure 2
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  		FIGURE 2.
Analysis of the in vitro transfer of Moco produced by the MogA-MoeA complex to XdhC and MobA. 15 µM MPT, 30 µM MoeA, and 30 µM MogA were incubated with 37.5 µM Na2MoO4 and 1 mM MgATP before 8 µM XdhC was added. Subsequently, MoeA and MogA were removed by Ni-NTA chromatography from the incubation mixture, and free Moco was removed from the XdhC fraction by gel filtration. The Moco content of XdhC was quantified after its conversion to Form A. Single components were omitted from the incubation mixtures as controls. ND, no Form A detected.

 


Figure 3
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  		FIGURE 3.
Analysis of the influence of increasing MobA concentrations on the in vitro transfer of Moco from MoeA to XdhC. A, incubation mixtures contained 15 µM MPT, 30 µM MogA, 30 µM MoeA, 1 mM MgATP, and 37.5 µM Na2MoO4 before 15 µM XdhC and increasing amounts of MobA and GTP were added. MobA, MoeA, and MogA were removed by Ni-NTA chromatography, and free Moco was removed by gel filtration. Moco bound to XdhC was quantified after its conversion to Form A. Bar I, equimolar amounts of MobA and XdhC; bar II, 1:10 XdhC/MobA ratio; bar III, equimolar amounts of XdhC and MobA in the absence of GTP; bar IV, a 1:10 XdhC/MobA ratio in the absence of GTP; bars V–VII, GTP, MobA, or XdhC was omitted from the incubation mixture, respectively. B, 25 µM MobA was incubated with 100 µM Moco, and excess Moco was removed by gel filtration. The Moco content of MobA was quantified after its conversion to Form A (bar VIII). 10 µM XdhC was added, and MobA was removed by Ni-NTA chromatography. The Moco content of XdhC was quantified after its conversion to Form A (bar IX). ND, no Form A detected.

 
Because SPR measurements also showed an interaction between XdhC and MobA, it was of further interest to determine whether Moco can be directly transferred from MobA to XdhC. For these investigations, MobA was incubated with Moco; excess Moco was removed by gel filtration; and XdhC was added. Subsequently, MobA and XdhC were separated by Ni-NTA chromatography, and the Moco content of XdhC was quantified. The results in Fig. 3B show that Moco remained bound to MobA in this incubation mixture and was not further transferred to XdhC.

Dissociation Constants for MPT and Moco Binding to MogA, MoeA, and MobA—To determine the dissociation constants for binding of Moco and MPT to MogA, MoeA, and MobA, the purified proteins were incubated with varying concentrations of Moco or MPT for 15 min at 4 °C before unbound Moco/MPT was separated by ultrafiltration using a membrane with a molecular mass cutoff of 10 kDa. The Moco/MPT concentration in the flow-through fraction was quantified after conversion to the stable, fluorescent oxidation product Form A (see "Experimental Procedures").

Quantification of Moco and MPT after ultrafiltration in the presence and absence of MogA, MoeA, or MobA allowed the determination of KD values for Moco and MPT binding to these proteins (Table 2). The amount of Moco or MPT bound to MogA, MoeA, or MobA and the free MogA, MoeA, or MobA concentrations were calculated according to the determined free and total Moco/MPT concentrations in relation to the total protein concentration. Fitting revealed a function according to the law of mass action for a ratio of Moco/MPT to MogA, MoeA, or MobA of 1:1 (Table 2). The KD values in Table 2 show that MogA bound MPT 30 times more tightly than Moco, whereas MobA bound Moco ~4 times more tightly than MPT, and MoeA bound both cofactor forms to the same extent. The binding of Moco to MoeA appears to be weaker in comparison with the binding of Moco to MobA or XdhC (3.6 µM) (21). In total, the KD values determined for MobA and XdhC were in the same range, whereas the ones determined for MogA showed the weakest binding.


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  		TABLE 2
KD values for MPT and Moco binding to R. capsulatus MogA, MoeA, and MobA

 
XdhC Inhibits the Binding of Moco to MobA—To analyze whether increasing concentrations of XdhC also influence Moco binding to MobA, purified MobA was incubated with XdhC prior to the addition of Moco and MgGTP. To ensure saturation of the proteins with Moco, the cofactor was added in excess to the incubation mixture. The formation of MGD was analyzed by its conversion to Form A from Form A-GMP as described under "Experimental Procedures." The results in Fig. 4A show that MGD formation by MobA was inhibited by increasing amounts of XdhC. At equimolar concentrations of XdhC and MobA, MGD production was reduced by 70%, whereas a 10-fold excess of XdhC resulted in an 80% reduction. To elucidate the nature of the negative effect of XdhC on MGD production, we analyzed whether XdhC prevents the binding of Moco to MobA. For this purpose, MobA was preincubated with Moco; unbound Moco was removed from the mixture; and XdhC and MgGTP were subsequently added. As shown in Fig. 4B, increasing amounts of XdhC had no influence on the formation of MGD by Moco-saturated MobA. These results show that, in the course of binding to MobA, XdhC prevents the binding of Moco, so no MGD can be formed.


Figure 4
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  		FIGURE 4.
Analysis of the influence of XdhC on MGD formation by MobA. A, 10 µM MobA was incubated with the indicated amounts XdhC before the addition of 1 mM GTP, 1 mM MgCl2, 1 mM Na2MoO4, and 80 µM Moco. B, 115 µM MobA was incubated with 190 µM Moco and 1 mM Na2MoO4, before free Moco was removed by gel filtration. The Moco-loaded MobA fraction (11.6 µM) was further incubated with 1 mM Na2MoO4, 1 mM MgGTP, and increasing amounts of XdhC. The MGD produced was quantified after its conversion to Form A.

 
Analysis of the Dependence of XDH and Me2SO Reductase Activities in R. capsulatus KS36 Cells on Overproduced XdhC—After the identification of the direct competition of XdhC and MobA for available Moco in vitro, we investigated whether the same effects occur in vivo. To examine the in vivo consequences of high XdhC concentrations on bis-MGD formation in the cell, a plasmid expressing the xdhC gene under the control of the strong nifH promotor was introduced into R. capsulatus KS36 cells by conjugation. This plasmid allows the induction of xdhC gene expression under nitrogenase-derepressing conditions in medium containing serine as the sole nitrogen source. To analyze the influence of bis-MGD formation when large amounts of XdhC are present, cells were grown in RCV minimal medium containing serine and Me2SO, and after cell lysis, the crude extract was assayed for Me2SO reductase and XDH activities. The results in Table 3 show that XDH activity remained unaffected by the presence of XdhC, whereas Me2SO reductase activity was reduced by >50% when XdhC was overexpressed. The effects of high MobA concentrations on Me2SO reductase and XDH activities were also analyzed as a control. For this purpose, a plasmid expressing the mobA gene under the control of the nifH promotor was constructed and transferred into R. capsulatus KS36 cells. The results in Table 3 show that Me2SO reductase and XDH activities remained unaffected by high levels of MobA in the cell, showing that mono-oxo-Moco biosynthesis is not inhibited by high concentrations of MobA at physiological GTP concentrations. The data confirm the results obtained in the in vitro studies and show that high XdhC concentrations in the cell result in decreased MGD production.


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  		TABLE 3
Effect of the presence of overproduced XdhC on the activities of XDH and Me2SO reductase in R. capsulatus KS36 cells

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
For R. capsulatus XDH, the XdhC protein has been identified as a chaperone that is involved in the maturation of XDH (21, 23, 36). In contrast to proteins that bind the bis-MGD form of Moco such as R. capsulatus Me2SO reductase, MobA is not essential for the generation of active XDH, which contains the MPT form of Moco (24). However, for XDH to be active, the sulfurated form of Moco has to be produced by exchange of the equatorial oxygen ligand with sulfur. This reaction is catalyzed by the L-cysteine desulfurase NifS4 while Moco is bound to XdhC (23). XdhC further protects the Mo = S group from oxidation and is involved in the insertion of sulfurated Moco into XDH. Interactions of both MobA and XdhC with MoeA were identified, and the MoeA-MobA and MoeA-XdhC pairs showed KD values in the same range. Most R. capsulatus molybdoenzymes such as Me2SO reductase bind bis-MGD, and under derepressing conditions, Me2SO reductase is present in large amounts in the cell (37, 38). In contrast, XDH is constitutively expressed and represents only a minor fraction of the total cell proteins (20). We present here a fully defined in vitro system for studying the mechanism of Moco biosynthesis and targeting. Our in vitro experiments have shown that MobA and XdhC obtain Moco produced by MoeA; however, most of the Moco supplied by MoeA is converted to MGD when MobA is present (Fig. 5). In addition, MobA is unable to transfer Moco to XdhC by binding Moco tightly and thereby enabling the biosynthesis of MGD to ensure the supply for all bis-MGD-containing enzymes. To prevent all available Moco in the cell from being converted to MGD, XdhC has to ensure that at least some Moco is converted to its sulfurated form for further transfer to XDH. We have shown that XdhC not only binds to MoeA but also interacts with MobA and thereby prevents the binding of Moco to MobA, simultaneously inhibiting MGD formation. Our results have shown that XdhC has a crucial role to guarantee the Moco supply for R. capsulatus XDH. The in vitro studies were confirmed by in vivo studies in R. capsulatus showing that overproduction of XdhC results in a decrease in Me2SO reductase activity. In contrast, overproduction of MobA does not influence the activities of Me2SO reductase and XDH. Because the xdhC gene in R. capsulatus is coexpressed with xdhAB, XdhC is in general present only in low amounts in the cell. Because no induction of xdh gene expression under certain growth conditions has been reported so far (20), the cellular role of XdhC is to abstract a minor part of the total amount of synthesized Moco in the cell for further modification to the sulfurated form. The other part of Moco is converted to MGD by MobA. Because XDH requires only a small part of the total Moco concentration in the cell, a mutation of R. capsulatus mobA does not result in an increase in XDH activity, as reported previously by Leimkühler and Klipp (24). In addition, previous results also showed that mobA gene expression is constitutive at low levels, and no increase in mobA expression was observed even under conditions of high MGD demand (24). This suggests a high turnover rate of the MobA-catalyzed MGD production, but also implies that MobA is not directly involved in the insertion of produced MGD into the respective MGD-binding molybdoenzymes and that other proteins might mediate the transfer of MGD from MobA to the target enzyme (39, 40). This observation was confirmed by overproduction of MobA in R. capsulatus, which does not give rise to a higher activity of bis-MGD-containing Me2SO reductase. In addition, the production of Moco is restricted in R. capsulatus by linking part of the genes for Moco biosynthesis to expression of the genes for Me2SO reductase (41), explaining why Me2SO reductase is not increased by the overexpression of MobA because the amount of produced Moco is the limiting factor.


Figure 5
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  		FIGURE 5.
Model for the role of XdhC and MobA in the biosynthesis of Moco and MGD. Moco is produced from MPT by the MogA-MoeA complex, catalyzing the ATP-dependent ligation of the molybdenum atom to MPT. Synthesized Moco can subsequently be transferred either to MobA, converting it into bis-MGD (which is inserted into enzymes of the Me2SO (DMSO) reductase family), or to XdhC, forming the sulfurated form of Moco by exchange of an oxo ligand with sulfur (which is inserted into XDH). XdhC interacts with MobA and thereby inhibits the transfer of Moco to MobA. Direct Moco transfer from MobA to XdhC was not detected. Modification of Moco and trafficking to the specific target proteins are tightly regulated by the direct binding of Moco to both XdhC and MobA.

 
In addition to the newly identified MoeA-XdhC and XdhC-MobA interactions, the SPR measurements partly confirmed the interactions identified previously for E. coli MoeA, MogA, MobB, and MobA in a bacterial two-hybrid approach (42). Whereas the E. coli two-hybrid studies revealed a major role for MobB in the interactions between MogA, MoeA, and MobA, no MobB homolog was so far identified in R. capsulatus. In addition, in the two-hybrid system, interactions between E. coli MobA-MoeA and MogA-MoeA were identified only in strains producing active Moco; however, the interaction of these protein pairs was only slightly increased. In contrast, we identified interactions between the analyzed protein pairs in the absence of Moco. Thus, differences seem to exist in the interaction of proteins in Moco biosynthesis in E. coli and R. capsulatus.

Most molybdoenzymes in R. capsulatus and E. coli contain the bis-MGD form of the cofactor; however, the formation of bis-MGD is one of the most enigmatic steps in Moco biosynthesis. It is still not known whether the two MGD molecules assemble on MobA or after insertion into target proteins such as Me2SO reductase and nitrate reductase A. Here, we have shown that molybdenum ligation to MPT has to precede MGD formation.

So far, XdhC is the only protein for which a role in Moco binding and insertion into its specific target protein has been identified. Because organisms like E. coli contain several different bis-MGD-containing enzymes, it remains to be elucidated whether factors exist that ensure the trafficking of MGD or bis-MGD into specific enzymes like trimethylamine-N-oxide reductase and nitrate reductase under certain growth conditions. It remains speculative whether chaperones like TorD for trimethylamine-N-oxide reductase and NarJ for nitrate reductase A are involved in targeting the right amount of cofactor to the acceptor protein in a manner similar to XdhC. Because an interaction between MobA and NarJ was identified using an E. coli two-hybrid assay (40), this implies that NarJ might be involved in a similar reaction.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant LE1171/3-3 (to S. L.) and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed. Tel.: 49-331-977-5603; Fax: 49-331-977-5419; E-mail: sleim{at}uni-potsdam.de.

2 The abbreviations used are: Moco, molybdenum cofactor; MPT, molybdopterin; bis-MGD, bismolybdopterin guanine dinucleotide; XDH, xanthine dehydrogenase; hSO-MD, human sulfite oxidase Moco domain; Ni-NTA, nickel-nitrilotriacetic acid; SPR, surface plasmon resonance. Back


   ACKNOWLEDGMENTS
 
We thank K. V. Rajagopalan (Duke University) for critical reading of the manuscript, helpful discussions, and providing plasmid pTG818 and Bernd Masepohl (Ruhr-Universität Bochum, Bochum, Germany) for providing plasmids containing the coding regions of mobA, moeA, and mogA.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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