Molybdoenzymes are involved in a large number of enzymatic reactions in the nitrogen, carbon and sulfur cycles. They occur in all the kingdoms of life. With the exception of nitrogenase, all molybdoenzymes carry the molybdenum cofactor (Moco), where the molybdenum atom is coordinated to the unique dithiolene moiety of a conserved tricyclic pyranopterin cofactor called molybdopterin (MPT). Depending on the remaining ligands of the molybdenum center, molybdoenzymes are classified into three families: (a) the xanthine oxidase (XO) family, characterized by a cyanolyzable equatorial sulfur ligand coordinated to the molybdenum atom; (b) the sulfite oxidase family, with two oxo ligands at the molybdenum center; and 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 ironsulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (abc) 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 · [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. Abbreviations ICP-OES, inductively coupled plasma optical emission spectroscopy; MCD, molybdopterin cytosine dinucleotide; MGD, molybdopterin guanine dinucleotide; Moco, molybdenum cofactor; MPT, molybdopterin; Tat, twin arginine protein transport; XDH, xanthine dehydrogenase; XO, xanthine oxidase.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (␣␤) 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 enzymespecific 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.Xanthine oxidoreductase is a complex metalloflavoprotein that catalyzes the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, using a water molecule as the ultimate source of oxygen incorporated into the product (1). The enzyme exists in two forms; the xanthine dehydrogenase form (XDH; EC1.17.1.4) 2 uses NAD ϩ as electron acceptor, whereas the xanthine oxidase (EC1.17.3.2) form uses O 2 as electron acceptor (2). Xanthine oxidoreductases are found both in eukaryotes and prokaryotes, with the enzymes isolated from bovine milk and Rhodobacter capsulatus being functionally and structurally the best characterized (3, 4). R. capsulatus XDH is a cytoplasmic enzyme with an (␣␤) 2 heterodimeric structure, with the two subunits encoded by the xdhA and xdhB genes, respectively (5 This report describes the detailed analysis for the requirement of XdhC to produce active XDH during heterologous expression in E. coli TP1000 cells. Analysis of XDH expressed under different aeration levels in the presence or absence of XdhC showed that, especially under aerobic conditions, XdhC is required to produce active XDH containing the terminal sulfur ligand of Moco. In addition, we purified and characterized XdhC after heterologous expression in E. coli. We could show that XdhC binds Moco/MPT in stoichiometric amounts and is able to insert bound Moco into Moco-free apo-XDH. In addition, a specific interaction between XdhC and XdhB was identified. We showed that XdhC acts as a Moco-binding protein, which protects the sulfurated form of Moco from oxidation. We propose that sulfurated Moco is inserted into apo-XDH by the aid of XdhC to produce active XDH. This is the first example of a system-specific protein involved in maturation of a molybdoenzyme for which Moco binding could be shown. Bacterial Strains, Pla...

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 apoTorA. Using random mutagenesis, we established that ␣-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.

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 Yag-TSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl 2 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 M for CTP and 1.17 ؎ 0.18 M 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.

Molybdenum is the only second-row transition metal that is required by most living organisms, and the few species that do not require molybdenum use tungsten, which lies immediately below molybdenum in the Periodic Table [1,2]. Molybdenum-and tungsten-containing enzymes often catalyse the same types of reaction; while molybdenum-containing enzymes are found in all aerobic organisms, including humans, tungstencontaining enzymes are found only in obligate, typically thermophilic, anaerobic bacteria and archaea [3,4]. The molybdenum cofactor (Moco) is the essential component of a group of redox enzymes which catalyse a variety of transformations at carbon, sulfur and nitrogen atoms. More than 40 molybdenum and tungsten enzymes have been identified in bacteria, archaea, plants and animals to date [5,6] 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.Abbreviations FeVco, iron-vanadium cofactor; hSO, human sulfite oxidase; hSO-MD, human sulfite oxidase Moco domain; ICP-OES, inductively coupled plasma-optical emission spectrometry; MGD, molybdopterin guanine dinucleotide cofactor; Moco, molybdenum cofactor; MPT, molybdopterin; Wco, tungsten cofactor.