Characterization of Escherichia coli MoeB and Its Involvement in the Activation of Molybdopterin Synthase for the Biosynthesis of the Molybdenum Cofactor*

Amino acid sequence comparisons of Escherichia coli MoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of molybdopterin synthase might resemble the ubiquitin-activating step in the ubiquitin-targeted degradation of proteins in eukaryotes. To determine the exact role of MoeB in molybdopterin biosynthesis, the protein was purified after homologous overexpression. Using purified proteins, we have demonstrated the ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to gel filtration. Mass spectrometry of the complex revealed a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adenylate. However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed. There was also no evidence for a MoeB-bound sulfur during the sulfuration of MoaD. Amino acid substitutions were generated in every cysteine residue in MoeB. All of these exhibited activity comparable to the wild type, with the exception of mutations in cysteine residues located in putative Zn-binding motifs. For these cysteines, loss of activity correlated with loss of metal binding. In Escherichia coli , several loci ( moa, mob, mod, moe, and mog ) have been implicated in the pleiotrophy of the molybdenum enzymes. With the exception of mod , all of these are involved resulting designated tures

In Escherichia coli, several loci (moa, mob, mod, moe, and mog) have been implicated in the pleiotrophy of the molybdenum enzymes. With the exception of mod, all of these are involved in the biosynthesis of the molybdenum cofactor (1). Molybdenum cofactor contains a tricyclic pterin derivative termed molybdopterin (MPT) 1 that bears the cis-dithiolene group essential for molybdenum ligation. The dithiolene group is generated by MPT synthase, a heterotetrameric protein consisting of two large MoaE subunits (16.9 kDa) and two small MoaD subunits (8.8 kDa) (2). Mass spectrometry has identified a thiocarboxylate in the activated form of MoaD that serves as the sulfur donor for the synthesis of MPT from precursor Z. This reaction can be carried out in vitro with the purified components (3). The high-resolution crystal structure of MPT synthase revealed that the C terminus of each MoaD subunit is inserted into a MoaE subunit to form the active site (2). In addition, the small subunit of MPT synthase shows high structural similarity to the eukaryotic protein ubiquitin (2). The observation that an E. coli moeB mutant accumulates precursor Z indicated the absence of an active form of MPT synthase in the mutant (4). This led to the conclusion that MoeB is the MPT synthase sulfurase, the protein responsible for regenerating the thiocarboxylate group at the C terminus of MoaD in an ATP-dependent reaction (5).
Significant sequence similarities between MoeB and a number of other proteins have been identified (6). Particularly noteworthy is the amino acid sequence identity of 23% between MoeB and the eukaryotic ubiquitin-activating enzyme E1, encoded by Uba1 (5). As part of the process for ubiquitin-targeted degradation of proteins, Uba1 was shown to activate ubiquitin in an ATP-dependent reaction with the initial formation of an Uba1-ubiquitin adenylate complex. This reaction is followed by generation of a thioester linkage between the C-terminal glycine of ubiquitin and a cysteine residue of Uba1 (7). Ubiquitin is subsequently transferred to one of the ubiquitin carrier proteins (ubiquitin carrier protein (E2)/ubiquitin-protein isopeptide ligase (E3)) before its attachment to the target protein by the formation of an isopeptide bond with a lysine residue.
The functional similarity of MoeB to Uba1 (5,6,8) and of MPT synthase to ubiquitin (2) has led to the proposal that the mechanism of activation of MPT synthase by MoeB might resemble the process of ubiquitin activation by Uba1. It has been proposed that the initial formation of a MoaD-adenylate leads to a thioester linkage with MoeB before sulfuration of the C-terminal carboxyl group of MoaD to create the reactive thiocarboxylate (5,6). Significantly, MoeB contains several conserved cysteine residues, one of which has been postulated to be involved in the formation of a thioester linkage between MoaD and MoeB (5,6,8). It has been shown recently that the physiological sulfur donor for the formation of the dithiolene group of MPT is likely to be L-cysteine and that a NifS-like protein is involved in the mobilization and transfer of sulfur from cysteine to MPT synthase (9).
This article describes the characterization of the mechanism of action of E. coli MoeB. Studies on cysteine to alanine mutants of MoeB revealed that all of them retained activity, with the exception of mutations in the cysteine residues of the two conserved Zn-binding sequences. No evidence was found for the formation of a thioester intermediate between MPT synthase and MoeB or for the covalent binding of the cysteine-derived sulfur to MoeB. We propose a model for the role of MoeB in the activation of MPT synthase in which MoeB is only involved in the formation of an acyl-adenylate of MoaD, a mechanism that only partially resembles the first step of the ubiquitin-targeted degradation of proteins in eukaryotes.

EXPERIMENTAL PROCEDURES
Bacterial Strains, Media, and Growth Conditions-E. coli moeB Ϫ (DE3) and moaD Ϫ mutant strains used in this study are isogenic mutants described previously (9,10). E. coli BL21(DE3) cells and pET15b were obtained from Novagen. Cell strains containing expression plasmids were grown aerobically at 30°C in LB medium in the presence of 150 g/ml ampicillin or 50 g/ml carbenicillin. Superdex 75, Superose 12, and Phenyl-Sepharose resins were purchased from Amersham Pharmacia Biotech, and Q-Sepharose resin was purchased from Sigma. Inductively coupled plasma emission spectroscopy was performed by Garrat Callahan Company (Millbrae, CA).
Purification of the Reaction Components-Precursor Z was isolated from E. coli moaD Ϫ cells by high performance liquid chromatography (HPLC) using reverse phase and anion exchange columns (11). Cloned MPT synthase was expressed from a pET15b vector (Novagen) in E. coli moeB Ϫ (DE3) cells, and the protein was purified by ammonium sulfate precipitation and gel filtration as described by Rudolph et al. (2). E. coli cysteine sulfinate desulfinase (CSD) was expressed in a pET15b vector in BL21(DE3) cells and purified by nickel-nitrilotriacetic acid chromatography (9).
Cloning, Expression, and Purification of Wild Type MoeB-The gene encoding E. coli moeB was cloned from genomic DH5␣ DNA by polymerase chain reaction. The published gene sequence (12) was used to design primers that permitted cloning into the NcoI and BamHI sites of the multiple cloning region of the pET15b expression vector. The resulting plasmid was designated pMW15eB. For expression, 6-liter cultures of BL21(DE3) cells transformed with pMW15eB were grown in LB medium containing carbenicillin to an A 600 of 0.6. The cultures were then induced by the addition of 0.1 mM isopropyl-␤-D-thiogalactopyranoside. After 4 h of aerobic growth at 30°C, the cells were harvested, resuspended with a total of 60 ml of 50 mM Tris, 2 mM EDTA, pH 8.0, and frozen at Ϫ20°C.
The cell suspension was lysed by two passes through a French pressure cell, and the resulting extract was centrifuged at 17,000 ϫ g. All subsequent steps were carried out in buffers containing 5 mM DTT. The volume of the extract was increased to 800 ml with suspension buffer, and 88 ml of 2% w/v streptomycin sulfate was added. After centrifugation, solid ammonium sulfate (243 g/liter) was slowly added to the supernatant. The precipitate was removed by centrifugation, and a second aliquot of ammonium sulfate (48.5 g/liter) was added to the supernatant. After centrifugation, the pellet was suspended in 50 mM Tris, pH 8.0, and dialyzed overnight against the same buffer. The dialyzed sample was applied to a Q-Sepharose column (50-ml bed volume) equilibrated with 50 mM Tris, pH 8.0, and MoeB was eluted with a linear gradient of 0.25-0.7 M NaCl using an Amersham Pharmacia Biotech FPLC system. Fractions containing MoeB were pooled and dialyzed overnight against 50 mM Tris, pH 7.5. The sample was then divided in half, and each half was brought to 15% saturation with ammonium sulfate immediately before injection onto a Phenyl-Sepharose column (25-ml bed volume) equilibrated in the same buffer. MoeB was subsequently eluted from the column with a 15% to 0% saturated ammonium sulfate gradient. After concentration, the protein was chromatographed on a 100-ml Superose 12 column. Fractions containing pure MoeB were pooled and dialyzed against 25 mM Tris, 10 mM NaCl, and 5 mM DTT, pH 7.5.
E. coli moeB Ϫ (DE3) cells containing the mutant forms of pMW15eB were grown in 3 liters of LB medium containing ampicillin and induced with isopropyl-␤-D-thiogalactopyranoside as described for the wild type. For the purification of the MoeB variants, the cell pellet was resuspended in 50 mM Tris and 1 mM EDTA, pH 7.5, and lysed as described above. After streptomycin sulfate treatment, each MoeB variant was precipitated by the addition of 277 g/liter ammonium sulfate. After centrifugation, the protein was resolubilized and dialyzed against 50 mM Tris, 1 mM EDTA, and 5 mM DTT, pH 7.5. The dialyzed sample was applied to a 25-ml Q-Sepharose FPLC column equilibrated with 50 mM Tris, 1 mM EDTA, and 5 mM DTT, pH 7.5, and the mutant protein was eluted with a linear gradient of 0 -1 M NaCl. The pool of fractions containing MoeB was concentrated to 1 ml and chromatographed on a Superose 12 column equilibrated and eluted with 50 mM Tris, 1 mM EDTA, 5 mM DTT, and 100 mM NaCl, pH 7.5.
In Vitro Activation of Inactive MPT Synthase with MoeB-For the in vitro formation of MPT, inactive MPT synthase, MoeB, MgCl 2 , ATP, precursor Z, and CSD-bound persulfide were incubated in a total volume of 400 l of 100 mM Tris, pH 7.2, as described previously (9). For the generation of CSD-bound persulfide, CSD was incubated for 5 min with L-cysteine at 4°C, followed by passage through a PD10 gel filtration column (Amersham Pharmacia Biotech) to remove excess L-cysteine as described by Leimkü hler and Rajagopalan (9). In vitro production of MPT was quantitated by conversion to form A, its stable, fluorescent degradation product. For this conversion, the incubation mixtures were adjusted to pH 2.5 and excess iodine was added (13,14). Form A was quantitated by subsequent HPLC analysis with an Alltech C18 HPLC column equilibrated with 50 mM ammonium acetate and 10% methanol, pH 6.8 (9). Polyacrylamide Gel Electrophoresis-For polyacrylamide gel electrophoresis, eluates of a Superdex 75 gel filtration column were heated at 95°C in buffer containing 2% SDS and 5% ␤-mercaptoethanol. Perfect Protein Markers from Novagen were used as molecular mass standards. Electrophoresis was carried out on 15% polyacrylamide Ready Gels (Bio-Rad), and the gels were stained with Coomassie Brilliant Blue R (Sigma).
Mass Spectrometry-Mass spectrometric data were acquired on a Micromass Quattro LC triple quadrupole mass spectrometer (Altrincham) equipped with a pneumatically assisted electrostatic ion source operating at atmospheric pressure and in a positive ion mode. The protein samples in 25 mM Tris, pH 7.2, were analyzed in 50% aqueous acetonitrile containing 1% formic acid by loop injection into a stream of 50% aqueous acetonitrile flowing at 10 ml/min. The native protein samples were electrosprayed in 0.01 M ammonium acetate. Spectra were acquired in the multichannel analyzer mode from m/z 1000 -1800 (scan time, 5 s). The mass scale was calibrated using the multiply charged envelope of myoglobin (16,951.48 Da). The raw mass spectra were transformed to a molecular mass scale using a maximum entropybased method (MaxEnt) that uses the MemSys5 program (MaxEnt Solutions Ltd., Cambridge, United Kingdom) and is part of the Mass-Lynx software suite.

RESULTS
Expression and Purification of MoeB-Homologous expression of MoeB cloned from genomic E. coli DNA yielded a protein with an approximate monomeric mass of 25 kDa as the major soluble protein after cell lysis (Fig. 1). This value corresponds closely to the calculated molecular mass of 26,719 Da for MoeB. The protein was purified by fractionated ammonium sulfate precipitation followed by chromatography on Q-Sepharose, phenyl-Sepharose, and Superose 12 columns as shown in Fig. 1. By this procedure, ϳ50 mg of MoeB can be obtained from Involvement of MoeB in the Biosynthesis of MPT a 6-liter E. coli culture. After Superose 12 gel filtration chromatography, the purified protein displayed a single band on Coomassie Brilliant Blue R-stained SDS gels, and gel filtration experiments showed that in its native state, MoeB is a dimer with a molecular mass of about 52 kDa (data not shown).
Site-directed Mutagenesis of the E. coli MoeB Protein-From the amino acid sequence alignment shown in Fig. 2, it can be seen that E. coli MoeB shares significant sequence identities with E. coli ThiF, a protein involved in thiamine biosynthesis (15), Anabaena HesA, a protein involved in Fe/S protein synthesis during heterocyst formation (16), and Saccharomyces cerevisiae Uba4, a protein sharing similarities to the ubiquitinactivating enzyme Uba1 (6). These proteins and S. cerevisiae Uba1 (data not shown) (8) all contain a highly conserved nucleotide binding motif at the N terminus, represented by the sequence GXGXXG. In addition, two CXXC motifs were identified near the C terminus of MoeB, which are also highly conserved in ThiF, HesA, and Uba4 ( Fig. 2) but are not present in Uba1 (8).
The sequence similarity of E. coli MoeB and Uba1 from a variety of sources has led to the proposal that the mechanisms of ATP-dependent MPT synthesis and ATP-dependent ubiquitination are similar (2,5,6). The process of ubiquitin-dependent protein degradation is initiated by the adenylation of the carboxylate of the C-terminal glycine of ubiquitin, followed by attachment of ubiquitin to a cysteine of the activating enzyme Uba1 to form a thioester. It has been shown that the active site cysteines of E1-like enzymes in eukaryotes are located ϳ10 -20 amino acid residues from a metal-binding motif (6).
Several conserved cysteine residues have been identified in the E. coli MoeB protein (Fig. 2). To ascertain which E. coli MoeB cysteine residue was involved in the formation of the putative thioester linkage with MoaD, site-directed mutagenesis was performed to replace each MoeB cysteine residue with an alanine (Fig. 1). Appleyard et al. (8) have reported that mutation of residue Cys-263 in Aspergillus nidulans CnxF (the MoeB equivalent in this organism) to a tyrosine results in a loss of function of the protein for MPT biosynthesis. Accordingly, the C128Y mutant was generated in E. coli MoeB. In addition, the C142A/C187A double mutant was created.
To characterize the MoeB mutants and determine their activities in comparison to the wild type protein, all MoeB variants were expressed and purified from an E. coli moeB Ϫ strain. The activities of the purified proteins were analyzed by their ability to activate inactive MPT synthase in vitro in mixtures containing precursor Z, Mg-ATP, and CSD-bound persulfide (9). CSD, described as sulfinate desulfinase (17), has been shown to form an internal persulfide by transfer of sulfur from free L-cysteine to a thiol group within itself. This CSD-persulfide can act as the in vitro sulfur donor for the sulfuration of MPT synthase in an ATP-dependent reaction requiring the MoeB protein (9). The formation of activated MPT synthase is demonstrated by its ability to convert precursor Z to MPT in vitro. Subsequent acidic iodine treatment converts the generated MPT to its oxidized fluorescent degradation product, form A, which is quantitated by HPLC analysis (13).
For the in vitro production of MPT, the assays contained precursor Z, inactive MPT synthase, CSD-persulfide, Mg-ATP, and wild type or mutated MoeB. As shown in Fig. 3, the majority of the MoeB mutants exhibit activities comparable to that of wild type MoeB. The exceptions are mutations C172A, C175A, C244A, and C247A in the highly conserved Zn-binding motifs. Because all other cysteine to alanine mutations showed MPT production equivalent to that of the wild type, it can be concluded that none of the cysteine residues within MoeB are required for the sulfur transfer reaction for the activation of MPT synthase. The C128Y variant exhibited no activity as seen a Inductively coupled plasma emission spectroscopy was performed by Garrat Callahan Company. b -, below the limit of detection. for the equivalent mutant in A. nidulans (8). However, because the C128A mutant was completely active, it can be postulated that insertion of a tyrosine at this position disrupts the structure of the protein.
Inductively coupled plasma emission spectroscopy was performed on purified MoeB and the mutated variants C172A, C175A, C244A, and C247A to determine the Zn content of the proteins. Table I shows that whereas the wild type protein contains a near stoichiometric amount of Zn, no Zn was identified in the mutated variants analyzed. These results agree with the previous suggestion that the two conserved CXXC motifs are responsible for the binding of Zn (5). The residual activities exhibited by the C244A and C247A variants (Fig. 3) are probably a reflection of the traces of other metals (e.g. Cu) found in the purified proteins. This suggests that other metals may be able to at least partially fulfill the role of Zn in the activity of MoeB.
Incubation of MoeB and MPT Synthase with Sulfide and Mg-ATP-It was shown earlier that in an in vitro system, inorganic sulfide can circumvent the requirement for L-cysteine and a sulfurtransferase in the activation of MPT synthase (9). To investigate the possibility that a sulfur atom is attached to MoeB during the activation of MPT synthase, purified MoeB was incubated with inorganic sulfide for 30 min at room temperature. Excess sulfide was then removed by gel filtration. The filtered protein was incubated with inactive MPT synthase, Mg-ATP, and precursor Z for 30 min at room temperature under aerobic conditions to determine the ability of sulfide-treated MoeB to produce MPT in vitro. As shown in Table  II, no form A was detected after acidic iodine treatment of the incubation mixture, implying that MoeB is not directly sulfurated by inorganic sulfide. Inclusion of Mg-ATP in the incubation mixture before gel filtration did not alter the results (Table  II). Similarly, direct treatment of MPT synthase with sulfide in the presence or absence of Mg-ATP did not produce a sulfurated form of MPT synthase because no MPT was formed by the gel-filtered sample after the addition of precursor Z (Table II)  filtration, MPT was formed after the addition of precursor Z (Table II). In addition, the sulfide-dependent activation of MPT synthase by MoeB is ATP-dependent since MPT formation was only observed when Mg-ATP was included in the mixture before gel filtration (Table II).
Purification of the MoeB-MoaD Adenylate Complex by Gel Filtration-The data presented above suggested that MoeB itself is not sulfurated during the sulfur transfer reaction for the activation of MPT synthase and that its role is limited to As described previously (11,14), the fluorescence peak eluting at about 4 min was identified as compound Z, the oxidized product of precursor Z. Form A elutes at about 8.5 min, as verified by its absorption spectrum (data not shown). generation of the acyl adenylate of MoaD. To purify the proposed adenylate complex between MoeB and MPT synthase, MoeB was incubated with inactive MPT synthase and Mg-ATP for 30 min at room temperature before the mixture was applied to a gel filtration column, and the eluting fractions were analyzed by SDS-PAGE. Fig. 4A shows that MoeB and MoaD coelute and are well separated from the MoaE subunit during gel filtration, indicating that the MoaD subunit is released from the MPT synthase tetramer during the formation of the MoeB-MoaD adenylate complex. The clear separation of the MoeB-MoaD adenylate complex from the 33.9-kDa MoaE dimer indicates that the complex is a tetramer with a mass of 71.8 kDa. We propose that the two MoaD bands visualized in SDS-PAGE ( Fig. 2A) represent the difference in molecular mass between the adenylated (9.1 kDa) and the nonadenylated (8.8 kDa) forms of MoaD. In the absence of Mg-ATP, no separation of the MoaD subunit from the MoaE subunit was observed, revealing that the formation of the MoeB-MoaD complex is ATP-dependent (Fig. 4B). Because of the similar molecular masses of the MPT synthase tetramer (51.6 kDa) and the MoeB dimer (53.4 kDa), these proteins have similar elution profiles during gel filtration (Fig. 4B).
To obtain further evidence for the formation of the MoeB-MoaD adenylate complex, the effect of excess pyrophosphate on the formation of the complex was examined. For this purpose, MoeB, MPT synthase, and Mg-ATP were incubated for 15 min at room temperature before the addition of pyrophosphate. The mixture was incubated for another 15 min before it was applied to a gel filtration column. As shown in Fig. 4C, the inclusion of pyrophosphate in the ATP-containing incubation mixture gave an elution profile virtually identical to that observed from an incubation mixture containing no ATP. This indicates that the MPT synthase tetramer was reformed under these conditions. Thus, pyrophosphate is able to reverse the formation of the MoeB-MoaD adenylate complex by reversing the adenylation step. In accordance with the ability of sulfide to serve as the in vitro sulfur donor for the production of the activated MPT synthase (shown above), the addition of sulfide to the incubation mixture consisting of MoeB, MPT synthase, and Mg-ATP also resulted in the breakage of the adenylate bond between MoaD and MoeB (Fig. 4D). After the formation of the C-terminal MoaD thiocarboxylate group, the MPT synthase tetramer is formed and thus is not well separated from the MoeB dimer by gel filtration. (Fig. 4D).
The MoeB-MoaD Adenylate Complex Forms MPT in the Absence of Added Mg-ATP-In extension of these findings, it was of further interest to examine the ability of the gel-filtered MoeB-MoaD adenylate complex to form MPT in vitro in the absence of Mg-ATP. For this purpose, a sample of gel-filtered MoeB-MoaD adenylate complex including the MoaE dimer was incubated with CSD, L-cysteine, and precursor Z. After acidic iodine treatment of the mixture, form A was readily detected (Fig. 5A), indicating that the MoeB-MoaD adenylate complex does not require the presence of additional Mg-ATP for the conversion of precursor Z to MPT. As expected, when MoeB and MPT synthase were incubated without the addition of Mg-ATP before gel filtration, no MPT was formed (Fig. 5B). In addition, the pyrophosphate-treated incubation mixture also produced no MPT (Fig. 5C), providing further evidence that the formation of the MoeB-MoaD adenylate complex is reversed by pyrophosphate. As already shown in Table II, the inclusion of sulfide in the incubation mixture results in active MPT synthase capable of converting precursor Z to MPT (Fig. 5D).
Mass Spectrometry of the Adenylated MoaD Subunit-To obtain direct evidence for the MoeB-catalyzed formation of MoaD adenylate and the sulfide-dependent generation of MoaD thiocarboxylate from the adenylate, the reaction mixtures were analyzed by mass spectrometry. The mass spectrum of an incubation mixture of MoeB with inactive MPT synthase in the presence of Mg-ATP yielded masses for MoaE and MoeB of 16,852 and 26,563 Da, respectively (data not shown). Two other peaks with molecular masses of 8,744 and 9,073 Da were also observed (Fig. 6A). Whereas the 8,744-Da peak corresponds to the inactive form of MoaD with the C-terminal carboxyl group, the 9,073-Da peak corresponds to the adenylated form of MoaD. The difference in molecular mass of 329 Da corresponds exactly to the expected molecular mass of the adenylate group. However, the majority of MoaD was determined to be present in its inactive nonadenylated form, indicating that the MoaDadenylate complex is not stable and that the majority of the complex is hydrolyzed under the acid conditions used during mass spectrometry. These data validate the conclusion that the two MoaD bands seen in Fig. 2A represent free MoaD and its acyl adenylate. The acyl adenylate is stable when bound to MoeB but is susceptible to hydrolysis upon release.
The mass spectrum of the reaction mixture incubated in the absence of ATP provided further support that the 9,073-Da species is the adenylated form of MoaD. Fig. 6B shows clearly that only the 8,744-Da peak of inactive MoaD was present in this incubation mixture. However, after the addition of sulfide to a mixture of inactive MPT synthase, MoeB, and Mg-ATP, the molecular mass of the major peak was determined to be 8,759.5 Da (Fig. 6C). The ϳ16-Da increase in the mass of MoaD corresponds to that expected for the substitution of a single sulfur for an oxygen, as reported previously for activated MPT synthase (4). The complete absence of the 8,744-Da peak in this incubation mixture shows that the addition of sulfide converts all of the MoaD to the thiocarboxylated species. In contrast, no thiocarboxylated MoaD is formed when inactive MPT synthase, MoeB, and sulfide are incubated in the absence of Mg-ATP (Fig.  6D). The data presented in Fig. 6C also show that in the presence of Mg-ATP, all of the MoaD in the mixture was present as the adenylate since, as shown in Fig. 6D, sulfide does not form thiocarboxylate from nonadenylated MoaD. In sum, the results clearly show that in the presence of Mg-ATP, MoeB catalyzes the formation of a MoaD adenylate complex with a molecular mass of 9073 Da that can be detected by mass spectrometry. DISCUSSION The fact that E. coli moeB mutant strains contain an inactive, desulfo form of MPT synthase led to the proposal that MoeB is involved in sulfuration of MPT synthase. Accordingly, MoeB has been designated as MPT synthase sulfurase (4,5). The sequence homology between MoeB and the ubiquitin-activating enzyme Uba1 (5) and the structural similarity between MoaD and ubiquitin (2) suggested that MoeB might catalyze the adenylation of MoaD, followed by the formation of a thioester linkage between the two proteins. Cleavage of this bond by sulfur would then lead to the formation of the reactive thiocarboxylate group of MoaD that is required for MPT biosynthesis (5,6,8). In general, E1-like enzymes of the ubiquitin pathway in eukaryotes contain an active site cysteine residue involved in the formation of the thioester linkage to ubiquitin (6).
The present studies on the mechanism of action of the E. coli MoeB protein show that MoeB binds the MoaD subunit of MPT synthase and catalyzes the formation of an adenylated MoaD species, activating the C-terminal glycine carboxylate group for thiolation. Based on the data obtained from cysteine to alanine substitutions within MoeB, we suggest that MoeB itself is not a carrier of sulfur in the sulfur transfer process for the formation of the thiocarboxylate group in MoaD. Residue Cys-187 in E. coli MoeB corresponded to the location of a possible active site cysteine residue (6). However, analysis of the activity of the generated MoeB variants C187A and C142A/C187A showed that these exhibited an in vitro enzyme activity comparable to that of the wild type protein, excluding the possibility of involvement of Cys-187 in the sulfur transfer reaction. To mimic the C263Y mutant in A. nidulans cnxF (8), the equivalent E. coli MoeB residue Cys-128 was mutated to tyrosine or alanine. Whereas the C128Y mutation inactivated MoeB completely, the C128A mutation retained full activity, indicating that Cys-128 is not essential for enzyme activity and that substitution with a tyrosine disrupts the structure of the protein in a manner resulting in loss of protein activity. Substitution of all other MoeB cysteine residues with alanine generated variants that retained full enzyme activity, with the exception of cysteine to alanine substitutions in the two CXXC Zn-binding motifs, which abolished the ability of MoeB to bind Zn. Although the exact role of Zn for MoeB function is unknown, it is likely that the metal is primarily involved in structural stabilization of the protein as opposed to playing a direct role in enzyme catalysis, a statement supported by the observation that other metals can partially replace Zn for enzyme activity. In addition, the structure of the heterotetrameric MoeB-MoaD adenylate complex has been recently solved. 2 The crystal structure revealed that the Zn-binding site is quite distant from the active site, supporting the suggestion of a structural role for the metal. In agreement with our data, the most striking feature of the structure is the presence of a MoaD acyl adenylate at the active site. Formation of a thioester intermediate was also not observed.
MoeB shares significant sequence similarities to the ThiF protein in the E. coli thiamine biosynthetic pathway. In this pathway, ThiF, ThiS, ThiI, and IscS participate in the generation of the thiazole moiety of thiamine, and L-cysteine has been identified as the sulfur donor (18). ThiS bears structural homology to MoaD (19), and its C-terminal thiocarboxylate acts as the direct sulfur donor for the generation of the thiamine thiazole moiety. ThiS thiocarboxylate synthesis requires adenylation by ThiF (20), and ThiI appears to act as a sulfurtransferase, accepting sulfur from IscS and transferring it further on to ThiS (15). During the generation of ThiS thiocarboxylate, sulfur transfer to ThiF was not observed (18). Given the mechanistic similarities between the synthesis of the thiazole moiety of thiamin and the dithiolene group of MPT, the pathway of sulfur transfer in both systems is likely to be similar. Recently, our laboratory reported that L-cysteine is the likely physiological sulfur donor for the formation of the dithiolene group of MPT and that NifS-like sulfurtransferases are capable of mobilizing sulfur from cysteine for the sulfuration of MPT synthase in an in vitro system (9). However, in contrast to the role of IscS in thiamine biosynthesis (18), the protein is not essential for the biosynthesis of MPT since an iscS mutant strain produces active MPT synthase (9). Unlike the thiamine pathway, in which ThiI appears to act as an intermediate between IscS and ThiS, no such requirement has been identified for the biosynthesis of MPT.
In summary, the data presented above indicate that the transfer of sulfur for the activation of MPT synthase proceeds as shown in Fig. 7. MoeB activates MPT synthase by adenylating the C-terminal carboxylate group of MoaD. During this process, MoaD must dissociate from its complex with MoaE to form a stable adenylate complex with MoeB (Fig. 5A). In the adenylate complex, MoaD is susceptible to sulfuration, which most likely proceeds in vivo by the action of a NifS-like sulfurtransferase (9), transferring sulfur from L-cysteine to MoaD. However, it is not yet known how the sulfurtransferase interacts with the MoeB-MoaD complex to perform the sulfur transfer reaction. The nonspecificity of this reaction is somewhat surprising. In vitro, inorganic sulfide is able to serve the same function, suggesting the possibility that nascent sulfide is produced in the active site for addition to the activated MoaD intermediate. After the formation of the reactive MoaD thiocarboxylate group, MoaD thiocarboxylate dissociates from MoeB and reassociates with MoaE to form active MPT synthase, which is able to convert precursor Z to MPT. These observations suggest that the partitioning of MoaD between MoaE and MoeB is governed by the carboxylate versus thiocarboxylate status of the C-terminal Gly of MoaD. The basis for this selectivity is under investigation.
The data presented here show that the interaction of MoeB with MoaD resembles only the first step of the ubiquitin-targeted degradation of proteins in eukaryotes, i.e. the ATP-dependent activation of ubiquitin by Uba1. The similarity in the mechanism of ATP-dependent cofactor sulfuration and ATP-dependent protein conjugation mirrors the structural similarities seen between the components of the two systems (2,19). This implies that the eukaryotic system has evolved further to include the formation of thioester intermediates with proteins involved in the transfer of ubiquitin to target proteins.