Cell biology of molybdenum in plants and humans

The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. In eukaryotes, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires iron, ATP and copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Cell biology of molybdenum in plants

The transition element molybdenum (Mo) is of essential importance for (nearly) all biological systems as it is required by enzymes catalyzing important reactions within the cell. The metal itself is biologically inactive unless it is complexed by a special cofactor. With the exception of bacterial nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of all other Mo-enzymes. In plants, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indispensable way. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins that also participate in Moco-insertion into the cognate apoproteins. Xanthine dehydrogenase and aldehyde oxidase, but not the other Mo-enzymes, require a final step of posttranslational activation of their catalytic Mo-center for becoming active.     

Cell biology of molybdenum

The transition element molybdenum (Mo) is of essential importance for (nearly) all biological systems as it is required by enzymes catalyzing diverse key reactions in the global carbon, sulfur and nitrogen metabolism. The metal itself is biologically inactive unless it is complexed by a special cofactor. With the exception of bacterial nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of all other Mo-enzymes. In eukaryotes, the most prominent Mo-enzymes are (1) sulfite oxidase, which catalyzes the final step in the degradation of sulfur-containing amino acids and is involved in detoxifying excess sulfite, (2) xanthine dehydrogenase, which is involved in purine catabolism and reactive oxygen production, (3) aldehyde oxidase, which oxidizes a variety of aldehydes and is essential for the biosynthesis of the phytohormone abscisic acid, and in autotrophic organisms also (4) nitrate reductase, which catalyzes the key step in inorganic nitrogen assimilation. All Mo-enzymes, except plant sulfite oxidase, need at least one more redox active center, many of them involving iron in electron transfer. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indispensable way. Moco as released after synthesis is likely to be distributed to the apoproteins of Mo-enzymes by putative Moco-carrier proteins. Xanthine dehydrogenase and aldehyde oxidase, but not sulfite oxidase and nitrate reductase, require the post-translational sulfuration of their Mo-site for becoming active. This final maturation step is catalyzed by a Moco-sulfurase enzyme, which mobilizes sulfur from l-cysteine in a pyridoxal phosphate-dependent manner as typical for cysteine desulfurases.     

Molybdenum metabolism in the alga Chlamydomonas stands at the crossroad of those in Arabidopsis and humans

Molybdenum (Mo) is a very scarce element whose function is fundamental in living beings within the active site of Mo-oxidoreductases, playing key roles in the metabolism of N, S, purines, hormone biosynthesis, transformation of drugs and xenobiotics, etc. In eukaryotes, each step from Mo acquisition until its incorporation into a biologically active molybdenum cofactor (Moco) together with the assembly of this Moco in Mo-enzymes is almost understood. The deficiency in function of a particular molybdoenzyme can be critical for the survival of the organism dependent on the pathway involved. However, incapacity in forming a functional Moco has a pleiotropic effect in the different processes involving this cofactor. A detailed overview of Mo metabolism: (a) specific transporters for molybdate, (b) the universal biosynthesis pathway for Moco from GTP, (c) Moco-carrier and Moco-binding proteins for Moco transfer and (d) Mo-enzymes, is analyzed in light of recent findings and three systems are compared, the unicellular microalga Chlamydomonas, the plant Arabidopsis and humans.     

Molybdenum Metabolism in Plants

Abstract: Among the micronutrients essential for plant growth and for microsymbionts, Mo is required in minute amounts. However, since Mo is often sequestered by Fe- or Al-oxihydrox-ides, especially in acidic soils, the concentration of the water-soluble molybdate anion available for uptake by plants may be limiting for the plant, even when the total Mo content of the soil is sufficient. In contrast to bacteria, no specific molybdenum uptake system is known for plants, but since molybdate and sulfate behave similarly and have similar structure, uptake of molybdate could be mediated unspecifically by one of the sulfate transporters. Transport into the different plant organs proceeds via xylem and phloem. A pterin-bound molybdenum is the cofactor of important plant enzymes involved in redox processes: nitrate reductase, xanthine dehydrogenase, aIdehyde oxidase, and probably sulfite oxidase. Biosynthesis of the molybdenum cofactor (Moco) starts with a guanosine-X-phos-phate. Subsequently, a sulfur-free pterin is synthesized, sulfur is added, and finally molybdenum is incorporated. In addition to the molybdopterin enzymes, small molybdopterin binding proteins without catalytic function are known and are probably involved in the storage of Moco. In symbiotic systems the nitrogen supply of the host plant is strongly influenced by the availability of Mo in soil, since both bacterial nitrogenase and NADPH-dependent nitrate reductase of mycorrhizal fungi are Mo enzymes.     

The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton

Molybdenum (Mo) plays an essential role in the active site of all eukaryotic Mo-containing enzymes. In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism. Mo is bound to a unique metal binding pterin (molybdopterin [MPT]), thereby forming the active Mo cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria. Here, we describe the function of the two-domain protein Cnx1 from Arabidopsis in the final step of Moco biosynthesis. Cnx1 is constitutively expressed in all organs and in plants grown on different nitrogen sources. Mo-repairable cnxA mutants from Nicotiana plumbaginifolia accumulate MPT and show altered Cnx1 expression. Transformation of cnxA mutants and the corresponding Arabidopsis chl-6 mutant with cnx1 cDNA resulted in functional reconstitution of their Moco deficiency. We also identified a point mutation in the Cnx1 E domain of Arabidopsis chl-6 that causes the molybdate-repairable phenotype. Recombinant Cnx1 protein is capable of synthesizing Moco. The G domain binds and activates MPT, whereas the E domain is essential for activating Mo. In addition, Cnx1 binds to the cytoskeleton in the same way that its mammalian homolog gephyrin does in neuronal cells, which suggests a hypothetical model for anchoring the Moco-synthetic machinery by Cnx1 in plant cells.     

Molybdoenzymes and molybdenum cofactor in plants

The transition element molybdenum (Mo) is essential for (nearly) all organisms and occurs in more than 40 enzymes catalysing diverse redox reactions, however, only four of them have been found in plants. (1) Nitrate reductase catalyses the key step in inorganic nitrogen assimilation, (2) aldehyde oxidase(s) have been shown to catalyse the last step in the biosynthesis of the phytohormone abscisic acid, (3) xanthine dehydrogenase is involved in purine catabolism and stress reactions, and (4) sulphite oxidase is probably involved in detoxifying excess sulphite. Among Mo-enzymes, the alignment of amino acid sequences permits domains that are well conserved to be defined. With the exception of bacterial nitrogenase, Mo-enzymes share a similar pterin compound at their catalytic sites, the molybdenum cofactor. Mo itself seems to be biologically inactive unless it is complexed by the cofactor. This molybdenum cofactor combines with diverse apoproteins where it is responsible for the correct anchoring and positioning of the Mo-centre within the holo-enzyme so that the Mo-centre can interact with other components of the enzyme's electron transport chain. A model for the three-step biosynthesis of Moco involving the complex interaction of six proteins will be described. A putative Moco-storage protein distributing Moco to the apoproteins of Mo-enzymes will be discussed. After insertion, xanthine dehydrogenase and aldehyde oxidase, but not nitrate reductase and sulphite oxidase, require the addition of a terminal sulphur ligand to their Mo-site, which is catalysed by the sulphur transferase ABA3.     

Plant molybdoenzymes and their response to stress

Molybdenum-containing enzymes catalyse basic reactions in the nitrogen, sulphur and carbon metabolism. Mo-enzymes contain at their catalytic sites an organometallic structure termed the molybdenum cofactor or Moco. In higher plants, Moco is incorporated into the apoproteins of four enzymes: nitrate reductase (EC 1.6.6.1-3; NR), xanthine dehydrogenase (EC 1.1.1.204; XDH), aldehyde oxidase (EC 1.2.3.1; AO) and sulphite oxidase (EC1.8.3.1; SO). Molybdoenzymes in plants are key enzymes in nitrate assimilation, purine metabolism, hormone biosynthesis, and most probably in sulphite detoxification. They are considered to be involved in stress acclimation processes and, therefore, elucidation of the mechanisms of their response to environmental stress conditions is of agricultural importance for the improvement of plant stress tolerance. Here we would like to give a brief functional and biochemical characteristic of the four plant molybdoenzymes and to focus mainly on their sensitivity to environmental stress factors.     

Characterization of a mutant of Chlamydomonas reinhardtii deficient in the molybdenum cofactor

Molybdenum (Mo) is an essential micronutrient for almost all organisms. In eukaryotes, it forms a part of the molybdenum cofactor (Moco), which is required for numerous enzymes involved in carbon, nitrogen and sulfur metabolism. Mo is taken up by cells in the form of molybdate and recently molybdate transporters have been identified in Arabidopsis thaliana and Chlamydomonas reinhardtii. Here, we report the characterization of a novel mutant (DB6) of C. reinhardtii generated by random insertional mutagenesis that is unable to assimilate nitrate as a nitrogen source because it lacks functional nitrate reductase (NR). Besides lacking NR, DB6 also lacks xanthine dehydrogenase activity; a common requirement of both enzymes is Moco. DB6 displays a ‘molybdate‐repairable’ phenotype—growth on nitrate is partially restored by supplementing media with high levels of molybdate. This phenotype is typically associated with mutants defective in either molybdate transport or insertion of Mo into the pterin precursor of Moco. Mo content was found to be significantly lower in DB6 than in the wild‐type strain, AOXR1, which suggests that DB6 is defective in Mo uptake. Genetic complementation with a variety of candidate genes that include the known molybdate transporter MOT1 and DNA that spans the site of mutation was unable to recover the wild‐type phenotype. Taken together, our results indicate that DB6 is a novel molybdate transport‐deficient mutant.     

Insights into molybdenum cofactor deficiency provided by the crystal structure of the molybdenum cofactor biosynthesis protein MoaC

BACKGROUND: The molybdenum cofactor (Moco) is an essential component of a large family of enzymes involved in important transformations in carbon, nitrogen and sulfur metabolism. The Moco biosynthetic pathway is evolutionarily conserved and found in archaea, eubacteria and eukaryotes. In humans, genetic deficiencies of enzymes involved in this pathway trigger an autosomal recessive and usually deadly disease with severe neurological symptoms. The MoaC protein, together with the MoaA protein, is involved in the first step of Moco biosynthesis. RESULTS: MoaC from Escherichia coli has been expressed and purified to homogeneity and its crystal structure determined at 2 A resolution. The enzyme is organized into a tightly packed hexamer with 32 symmetry. The monomer consists of an antiparallel, four-stranded beta sheet packed against two long alpha helices, and its fold belongs to the ferredoxin-like family. Analysis of structural and biochemical data strongly suggests that the active site is located at the interface of two monomers in a pocket that contains several strictly conserved residues. CONCLUSIONS: Asp128 in the putative active site appears to be important for catalysis as its replacement with alanine almost completely abolishes protein activity. The structure of the Asp128-->Ala variant reveals substantial conformational changes in an adjacent loop. In the human MoaC ortholog, substitution of Thr182 with proline causes Moco deficiency, and the corresponding substitution in MoaC severely compromises activity. This residue is located near the N-terminal end of helix alpha4 at an interface between two monomers. The MoaC structure provides a framework for the analysis of additional dysfunctional mutations in the corresponding human gene.     

Synthesis of adenylated molybdopterin: an essential step for molybdenum insertion

The molybdenum cofactor (Moco) is part of the active site of all molybdenum (Mo)-dependent enzymes, except nitrogenase. Moco consists of molybdopterin (MPT), a phosphorylated pyranopterin with an enedithiolate coordinating Mo and it is synthesized by an evolutionary old multistep pathway. The plant protein Cnx1 from Arabidopsis thaliana catalyzes with its two domains (E and G) the terminal step of Moco biosynthesis, the insertion of Mo into MPT. Recently, the high-resolution MPT-bound structure of the Cnx1 G domain (Cnx1G) has been determined (Kuper, J., Llamas, A., Hecht, H. J., Mendel, R. R., and Schwarz, G. (2004) Nature 430, 803-806). Besides defining the MPT-binding site a novel and unexpected modification of MPT has been identified, adenylated MPT. Here we demonstrate that it is Cnx1G that catalyzes the adenylation of MPT. In vitro synthesized MPT was quantitatively transferred from Escherichia coli MPT synthase to Cnx1G. The subsequent adenylation reaction by Cnx1G was Mg(2+)- and ATP-dependent. Whereas Mn(2+) could partially replace Mg(2+), ATP was the only nucleotide accepted by Cnx1G. Consequently the formation of pyrophosphate was demonstrated, which was dependent on the ability of Cnx1G to bind MPT. Pyrophosphate, either formed in the reaction or added externally, inhibited the Cnx1G-catalyzed MPT adenylation reaction. Catalytically inactive Cnx1G mutant variants showed impaired MPT adenylation confirming that MPT-AMP is the reaction product of Cnx1G. Therefore Cnx1G is a MPT adenylyltransferase catalyzing the activation of MPT, a universal reaction in the Moco synthetic pathway because Cnx1G is able to reconstitute also bacterial and mammalian Moco biosynthesis.     

Probing the role of copper in the biosynthesis of the molybdenum cofactor in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The crystal structure of Cnx1G, an enzyme involved in the biosynthesis of the molybdenum cofactor (Moco) in Arabidopsis thaliana, revealed the remarkable feature of a copper ion bound to the dithiolene unit of a molybdopterin intermediate (Kuper et al. Nature 430:803-806, 2004). To characterize further the role of copper in Moco biosynthesis, we examined the in vivo and/or in vitro activity of two Moco-dependent enzymes, dimethyl sulfoxide reductase (DMSOR) and nitrate reductase (NR), from cells grown under a variety of copper conditions. We found the activities of DMSOR and NR were not affected when copper was depleted from the media of either Escherichia coli or Rhodobacter sphaeroides. These data suggest that while copper may be utilized during Moco biosynthesis when it is available, copper does not appear to be strictly required for Moco biosynthesis in these two organisms.     

Molybdoproteomes and evolution of molybdenum utilization

The trace element molybdenum (Mo) is utilized in many life forms, and it is a key component of several enzymes involved in nitrogen, sulfur, and carbon metabolism. With the exception of nitrogenase, Mo is bound in proteins to a pterin, thus forming the molybdenum cofactor (Moco) at the catalytic sites of molybdoenzymes. Although a number of molybdoenzymes are well characterized structurally and functionally, evolutionary analyses of Mo utilization are limited. Here, we carried out comparative genomic and phylogenetic analyses to examine the occurrence and evolution of Mo utilization in bacteria, archaea and eukaryotes at the level of (i) Mo transport and Moco utilization trait, and (ii) Mo-dependent enzymes. Our results revealed that most prokaryotes and all higher eukaryotes utilize Mo whereas many unicellular eukaryotes including parasites and most yeasts lost the ability to use this metal. In addition, eukaryotes have fewer molybdoenzyme families than prokaryotes. Dimethylsulfoxide reductase (DMSOR) and sulfite oxidase (SO) families were the most widespread molybdoenzymes in prokaryotes and eukaryotes, respectively. A distant group of the ModABC transport system, was predicted in the hyperthermophilic archaeon Pyrobaculum. ModE-type regulation of Mo uptake occurred in less than 30% of Moco-utilizing organisms. A link between Mo and selenocysteine utilization in prokaryotes was also identified wherein the selenocysteine trait was largely a subset of the Mo trait, presumably due to formate dehydrogenase, a Mo- and selenium-containing protein. Finally, analysis of environmental conditions and organisms that do or do not depend on Mo revealed that host-associated organisms and organisms with low G + C content tend to reduce their Mo utilization. Overall, our data provide new insights into Mo utilization and show its wide occurrence, yet limited use of this metal in individual organisms in all three domains of life.     

A widespread riboswitch candidate that controls bacterial genes involved in molybdenum cofactor and tungsten cofactor metabolism

We have identified a highly conserved RNA motif located upstream of genes encoding molybdate transporters, molybdenum cofactor (Moco) biosynthesis enzymes, and proteins that utilize Moco as a coenzyme. Bioinformatics searches have identified 176 representatives in gamma-Proteobacteria, delta-Proteobacteria, Clostridia, Actinobacteria, Deinococcus-Thermus species and DNAs from environmental samples. Using genetic assays, we demonstrate that a Moco RNA in Escherichia coli associated with the Moco biosynthetic operon controls gene expression in response to Moco production. In addition, we provide evidence indicating that this conserved RNA discriminates against closely related analogues of Moco. These results, together with extensive phylogenetic conservation and typical gene control structures near some examples, indicate that representatives of this structured RNA represent a novel class of riboswitches that sense Moco. Furthermore, we identify variants of this RNA that are likely to be triggered by the related tungsten cofactor (Tuco), which carries tungsten in place of molybdenum as the metal constituent.     

Mutational analysis of Escherichia coli MoeA: two functional activities map to the active site cleft

The molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, ligation of metal to molybdopterin (the organic component of the cofactor) to form the active cofactor. In Escherichia coli, the MoeA protein mediates ligation of Mo to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The X-ray crystal structures for both proteins have been previously described as well as two essential MogA residues, Asp49 and Asp82. Here we describe a detailed mutational analysis of the MoeA protein. Variants of conserved residues at the putative active site of MoeA were analyzed for a loss of function in two different, previously described assays, one employing moeA- crude extracts and the other utilizing a defined system. Oddly, no correlation was observed between the activity in the two assays. In fact, our results showed a general trend toward an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified component assay. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. On the basis of these results, two different functional areas were assigned to regions at or near the MoeA active site cleft.     

蝶呤型钼辅因子生物合成、运输及组装——潜在的药物靶标

钼辅因子作为氧化还原反应中的重要分子,参与硫、氮、碳的氧化还原代谢.钼辅因子主要分为两类:以铁硫簇为基础的铁钼辅因子和以亚钼蝶呤为基础的钼辅因子.钼-二-亚钼蝶呤-鸟苷二核苷钼辅因子(Mo-bis-MGD)是蝶呤型钼辅因子的重要成员之一,是硝酸盐还原酶的重要辅因子.膜结合硝酸盐还原酶介导的硝酸盐还原为细菌提供了氮源和能...     

Crystal structure of the gephyrin-related molybdenum cofactor biosynthesis protein MogA from Escherichia coli

Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in archaea, eubacteria, and eukaryotes, including humans. Genetic deficiencies of enzymes involved in this biosynthetic pathway trigger an autosomal recessive disease with severe neurological symptoms, which usually leads to death in early childhood. The MogA protein exhibits affinity for molybdopterin, the organic component of Moco, and has been proposed to act as a molybdochelatase incorporating molybdenum into Moco. MogA is related to the protein gephyrin, which, in addition to its role in Moco biosynthesis, is also responsible for anchoring glycinergic receptors to the cytoskeleton at inhibitory synapses. The high resolution crystal structure of the Escherichia coli MogA protein has been determined, and it reveals a trimeric arrangement in which each monomer contains a central, mostly parallel beta-sheet surrounded by alpha-helices on either side. Based on structural and biochemical data, a putative active site was identified, including two residues that are essential for the catalytic mechanism.     

A Novel Role for Arabidopsis Mitochondrial ABC Transporter ATM3 in Molybdenum Cofactor Biosynthesis

The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to ∼50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.     

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.     

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.