Dual role of the molybdenum cofactor biosynthesis protein MOCS3 in tRNA thiolation and molybdenum cofactor biosynthesis in humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.     

Splice-specific functions of gephyrin in molybdenum cofactor biosynthesis

Gephyrin is a multifunctional protein involved in the clustering of inhibitory neuroreceptors. In addition, gephyrin catalyzes the last step in molybdenum cofactor (Moco) biosynthesis essential for the activities of Mo-dependent enzymes such as sulfite oxidase and xanthine oxidoreductase. Functional complexity and diversity of gephyrin is believed to be regulated by alternative splicing in a tissue-specific manner. Here, we investigated eight gephyrin variants with combinations of seven alternatively spliced exons located in the N-terminal G domain, the central domain, and the C-terminal E domain. Their activity in Moco synthesis was analyzed in vivo by reconstitution of gephyrin-deficient L929 cells, which were found to be defective in the G domain of gephyrin. Individual domain functions were assayed in addition and confirmed that variants containing either an additional C5 cassette or missing the C6 cassette are inactive in Moco synthesis. In contrast, different alterations within the central domain retained the Moco synthetic activity of gephyrin. The recombinant gephyrin G domain containing the C5 cassette forms dimers in solution, binds molybdopterin, but is unable to catalyze molybdopterin (MPT) adenylylation. Determination of Moco and MPT content in different tissues showed that besides liver and kidney, brain was capable of synthesizing Moco most efficiently. Subsequent analysis of cultured neurons and glia cells demonstrated glial Moco synthesis due to the expression of gephyrins containing the cassettes C2 and C6 with and without C3.1.     

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.     

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.     

Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA

Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.     

Insight into the role of Escherichia coli MobB in molybdenum cofactor biosynthesis based on the high resolution crystal structure

Two proteins, which are co-transcribed in Escherichia coli (MobA and MobB), are involved in the attachment of a nucleotide moiety to the molybdenum cofactor to form active molybdopterin guanine dinucleotide. Although not essential for this process, the dimeric MobB increases the activation of molybdoenzymes, incorporating this cofactor by a mechanism that is not understood. The structure of MobB has been elucidated in two crystal forms, one of which has provided a model at 1.9-A resolution with Rwork and Rfree values of 21.5 and 28.7%, respectively. The MobB subunit displays an alpha/beta-fold arranged into a major and minor domain, the latter of which is inserted between the major and minor domains of the partner subunit, creating an elongated dimer constructed around a 16-stranded beta-sheet. Structural homologues have been identified, and they include a number of nucleotide-binding proteins. Comparisons indicate that although the phosphate-binding regions are highly conserved, MobB lacks the elements of structure required to interact with and efficiently bind a nucleotide base. In the present structure, a sulfate is bound to the Walker A phosphate-binding motif of MobB. The possibility that MobB forms a complex with the nucleotide-binding MobA, the protein with which it is co-transcribed, is explored, and modeling suggests that such a MobA:MobB complex is feasible. This hypothesis is supported by recent biochemical evidence indicating that MobB interacts with several proteins involved in various stages of molybdenum cofactor biosynthesis including MobA. We propose therefore that MobB is an adapter protein that acts in concert with MobA to achieve the efficient biosynthesis and utilization of molybdopterin guanine dinucleotide.     

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.     

Rescue of molybdenum cofactor biosynthesis in gephyrin-deficient mice by a Cnx1 transgene

Gephyrin is a bifunctional protein which is essential for both synaptic clustering of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (MoCo) in peripheral tissues. Mice deficient in gephyrin die early postnatally and display a loss of glycine receptors (GlyRs) and many GABA(A) receptor (GABA(A)R) subtypes from postsynaptic sites. In addition, the activities of the MoCo-dependent enzymes xanthine dehydrogenase and sulfite oxidase are reduced to background levels in the liver and intestine of these animals. To genetically separate the different consequences of gephyrin deficiency, we expressed a transgene of the plant homolog Cnx1, known to rescue mammalian MoCo deficiency, on the background of gephyrin knockout mice. Cnx1 partially restored sulfite oxidase activity in the liver of the transgenic animals, whereas early lethality and the loss of GlyR clustering were unaltered. Our data suggest that the loss of neurotransmitter receptor clustering at inhibitory synapses causes the early lethality of gephyrin deficient mice.     

Biology of the molybdenum cofactor

The transition element molybdenum (Mo) is an essential micronutrient for plants where it is needed as a catalytically active metal during enzyme catalysis. Four plant enzymes depend on molybdenum: nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. However, in order to gain biological activity and fulfil its function in enzymes, molybdenum has to be complexed by a pterin compound thus forming the molybdenum cofactor. In this article, the path of molybdenum from its uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes will be reviewed.     

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.     

Chlamydomonas reinhardtii CNX1E reconstitutes molybdenum cofactor biosynthesis in Escherichia coli mutants

We have isolated and characterized the Chlamydomonas reinhardtii genes for molybdenum cofactor biosynthesis, namely, CNX1G and CNX1E, and expressed them and their chimeric fusions in Chlamydomonas and Escherichia coli. In all cases, the wild-type phenotype was restored in individual mutants as well as in a CNX1G CNX1E double mutant. Therefore, CrCNX1E is the first eukaryotic protein able to complement an E. coli moeA mutant.     

Escherichia coli MoeA and MogA. Function in metal incorporation step of molybdenum cofactor biosynthesis

Escherichia coli MoeA and MogA are required for molybdenum cofactor biosynthesis and are believed to function in the addition of molybdenum to the dithiolene of molybdopterin to form molybdenum cofactor. Here we show that moeA(-) and mogA(-) cells are able to synthesize molybdopterin, but both are deficient in molybdenum incorporation and, as a consequence, are deficient in the formation of molybdopterin-guanine dinucleotide. Human sulfite oxidase expressed in E. coli moeA(-) could be activated in vitro in the presence of MoeA and low concentrations of molybdate. Sulfite oxidase purified from the moeA(-) lysate was also activated, although to a lesser extent than observed in the presence of lysate. MogA was incapable of activating sulfite oxidase expressed in E. coli mogA(-). These results demonstrate that molybdenum insertion into molybdopterin is required for molybdopterin-guanine dinucleotide formation, and that MoeA facilitates molybdenum incorporation at low levels of molybdate, but MogA has an alternative function, possibly as a carrier for molybdopterin during molybdenum incorporation.     

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.