Transfer of the Molybdenum Cofactor Synthesized by Rhodobacter capsulatus MoeA to XdhC and MobA*

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.

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 Me 2 SO 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 Me 2 SO 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
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(⌬mobAB) cells (18) to obtain Moco-containing hSO-MD or in E. coli RK5202(modC Ϫ ) 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(⌬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 (NH 4 ) 2 SO 4 as described previously (31). For expression of Me 2 SO reductase, Me 2 SO was added to a final concentration of 30 mM, and for induction of XdhC or MobA expression from the plasmids, (NH 4 ) 2 SO 4 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 Na 2 MoO 4 , 150 g/ml ampicillin, 25 g/ml kanamycin, and 5 g/ml tetracycline (for E. coli) or 1 g/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 ampli-fied 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 A 600 ϭ 0.3-0.5 with 100 M isopropyl ␤-D-thiogalactopyranoside. Cell growth was continued for 5 h, and cells were harvested and resuspended in 50 mM NaH 2 PO 4 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 M isopropyl ␤-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 M MobA was incubated with 160 M Moco or MPT before excess Moco/MPT was removed by gel filtration. 1 mM Na 2 MoO 4 was included in incubation mixtures containing Moco. 1 mM MgCl 2 and 1 mM GTP were added, and the mixtures were incubated for 60 min at room temperature in a total volume of 400 l 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). 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 l of Q-Sepharose was equilibrated with H 2 O; 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 MgCl 2 , 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 l of 50% acetic acid before application to a C 18 reversed-phase high pressure liquid chromatography column (4.6 ϫ 250-mm Hypersil ODS, 5-m 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 Biacore TM 2000 on CM5 sensor chips at 25°C and a flow rate of 10 l/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 His 6 tags of MoeA, MogA, and MobA were cleaved using the thrombin CleanCleave kit (Sigma). Cleavage was controlled by SDS-PAGE.
In In Vitro Transfer of Moco from MobA to XdhC-25 M MobA was incubated with 100 M Moco and 1 mM Na 2 MoO 4 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 M 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 l of the XdhC fraction was treated with acidic iodine, and released Moco was converted to Form A and quantified as described above. Analysis of XDH and Me 2 SO 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 mol of NAD ϩ /min/mg of enzyme. Me 2 SO reductase activity was measured as described by McEwan et al. (35) with dithionite-reduced benzyl viologen as the electron donor. Me 2 SO reductase activity (units/mg) is defined as the reduction of 1 mol of Me 2 SO/min/mg of protein.  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.

Purification and Analysis of the Functional
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 Ϫ ) 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 (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 K D of 3.5 M, whereas when MobA was immobilized, the K D was significantly higher (27 M) ( 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 K D values of 1.75 and 1.04 M 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.
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 His 6 tags fused to MoeA, MogA, and MobA on the dissociation constants. For this purpose, the His 6 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 His 6 -tagged proteins. Although untagged MogA also showed no interaction with XdhC (data not shown), comparison of the binding curves of His 6 -tagged and untagged MoeA and MobA with immobilized XdhC showed no significant difference in the Biacore sensograms (supplemental Fig. S1), clearly showing that the His 6 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, MoO 4 2Ϫ , 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 Table 2). The K D values in Table 2 show that MogA bound MPT 30 times more tightly than Moco, whereas MobA bound Moco ϳ4 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 M) (21). In total, the K D values determined for MobA and XdhC were in the same range, whereas the ones determined for MogA showed the weakest binding.
XdhC Inhibits the Binding of Moco to MobA-To analyze whether increasing concentrations of XdhC also influence   b B max describes the maximum saturation of the respective protein revealed by a 1:1 fitting procedure following the law of mass action. c K D values were determined as described previously (21) Table 3 show that XDH activity remained unaffected by the presence of XdhC, whereas Me 2 SO reductase activity was reduced by Ͼ50% when XdhC was overexpressed.
The effects of high MobA concentrations on Me 2 SO 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 Me 2 SO 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.

DISCUSSION
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 Me 2 SO 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 ϭ 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 K D values in the same range. Most R. capsulatus molybdoenzymes such as Me 2 SO reductase bind bis-MGD, and under derepressing conditions, Me 2 SO 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   (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 Me 2 SO 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 Me 2 SO reductase (41), explaining why Me 2 SO reductase is not increased by the overexpression of MobA because the amount of produced Moco is the limiting factor. 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 Me 2 SO 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.