The role of system-specific molecular chaperones in the maturation of molybdoenzymes in bacteria.

Biogenesis of prokaryotic molybdoenzymes is a complex process with the final step representing the insertion of a matured molybdenum cofactor (Moco) into a folded apoenzyme. Usually, specific chaperones of the XdhC family are required for the maturation of molybdoenzymes of the xanthine oxidase family in bacteria. Enzymes of the xanthine oxidase family are characterized to contain an equatorial sulfur ligand at the molybdenum center of Moco. This sulfur ligand is inserted into Moco while bound to the XdhC-like protein and before its insertion into the target enzyme. In addition, enzymes of the xanthine oxidase family bind either the molybdopterin (Mo-MPT) form of Moco or the modified molybdopterin cytosine dinucleotide cofactor (MCD). In both cases, only the matured cofactor is inserted by a proofreading process of XdhC. The roles of these specific XdhC-like chaperones during the biogenesis of enzymes of the xanthine oxidase family in bacteria are described.

Molybdopterin dinucleotide biosynthesis in Escherichia coli: identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides.

The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP:molybdopterin guanylyltransferase MobA and the CTP:molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.

Specific interactions between four molybdenum-binding proteins contribute to Mo-dependent gene regulation in Rhodobacter capsulatus.

The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA-binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.

MocA is a specific cytidylyltransferase involved in molybdopterin cytosine dinucleotide biosynthesis in Escherichia coli.

We have purified and characterized a specific CTP:molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP:molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase YagTSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37+/-0.01 min(-1) was obtained. A 1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23+/-0.02 microm for CTP and 1.17+/-0.18 microm for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.

A periplasmic aldehyde oxidoreductase represents the first molybdopterin cytosine dinucleotide cofactor containing molybdo-flavoenzyme from Escherichia coli.

Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions.

Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli.

Molybdenum insertion into the dithiolene group on the 6-alkyl side-chain of molybdopterin is a highly specific process that is catalysed by the MoeA and MogA proteins in Escherichia coli. Ligation of molybdate to molybdopterin generates the molybdenum cofactor, which can be inserted directly into molybdoenzymes binding the molybdopterin form of the molybdenum cofactor, or is further modified in bacteria to form the dinucleotide form of the molybdenum cofactor. The ability of various metals to bind tightly to sulfur-rich sites raised the question of whether other metal ions could be inserted in place of molybdenum at the dithiolene moiety of molybdopterin in molybdoenzymes. We used the heterologous expression systems of human sulfite oxidase and Rhodobacter sphaeroides dimethylsulfoxide reductase in E. coli to study the incorporation of different metal ions into the molybdopterin site of these enzymes. From the added metal-containing compounds Na(2)MoO(4), Na(2)WO(4), NaVO(3), Cu(NO(3))(2), CdSO(4) and NaAsO(2) during the growth of E. coli, only molybdate and tungstate were specifically inserted into sulfite oxidase and dimethylsulfoxide reductase. Other metals, such as copper, cadmium and arsenite, were nonspecifically inserted into sulfite oxidase, but not into dimethylsulfoxide reductase. We showed that metal insertion into molybdopterin occurs beyond the step of molybdopterin synthase and is independent of MoeA and MogA proteins. Our study shows that the activity of molybdoenzymes, such as sulfite oxidase, is inhibited by high concentrations of heavy metals in the cell, which will help to further the understanding of metal toxicity in E. coli.

Dedicated metallochaperone connects apoenzyme and molybdenum cofactor biosynthesis components.

The biogenesis of molybdenum-containing enzymes is a sophisticated process involving the insertion of a complex molybdenum cofactor into competent apoproteins. As for many molybdoenzymes, the maturation of trimethylamine-oxide reductase TorA requires a private chaperone. This chaperone (TorD) interacts with the signal peptide and the core of apo-TorA. Using random mutagenesis, we established that alpha-helix 5 of TorD plays a key role in the core binding and that this binding drives the maturation of TorA. In addition, we showed for the first time that TorD interacts with molybdenum cofactor biosynthesis components, including MobA, the last enzyme of cofactor synthesis, and Mo-molybdopterin, the precursor form of the cofactor. Finally we demonstrated that TorD also binds the mature molybdopterin-guanine dinucleotide form of the cofactor. We thus propose that TorD acts as a platform connecting the last step of the synthesis of the molybdenum cofactor just before its insertion into the catalytic site of TorA.

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 Me(2)SO 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.

Identification of a Rhodobacter capsulatus L-cysteine desulfurase that sulfurates the molybdenum cofactor when bound to XdhC and before its insertion into xanthine dehydrogenase.

The molybdenum cofactor (Moco) containing enzymes aldehyde oxidase and xanthine dehydrogenase (XDH) require for activity a sulfuration step that inserts a terminal sulfur ligand into Moco. XdhC was shown to be essential for the production of active XDH in Rhodobacter capsulatus but is itself not a subunit of the purified enzyme. XdhC binds stoichiometric amounts of Moco and is further able to transfer its bound Moco to XDH. Previous work suggested that XdhC particularly stabilizes the sulfurated form of Moco before the insertion into XDH. In this work, we identify an R. capsulatus l-cysteine desulfurase, NifS4, which is involved in the formation of the Mo=S ligand of Moco. We show that NifS4 interacts with XdhC and not with XDH. NifS4 mobilizes sulfur from l-cysteine by formation of a protein-bound persulfide intermediate and transfers this sulfur further to Moco. This reaction was shown to be more effective than the chemical sulfuration of Moco using sulfide as sulfur source. Further studies clearly showed that Moco is sulfurated before the insertion into XDH, while it is bound to XdhC. Conclusively, XdhC has a versatile role in R. capsulatus: binding of Moco, interaction with NifS4 for the sulfuration of Moco, protection of sulfurated Moco from oxidation, and further transfer to XDH.

Rhodobacter capsulatus XdhC is involved in molybdenum cofactor binding and insertion into xanthine dehydrogenase.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alphabeta)2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes.