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.     

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.     

Molybdenum enzymes and molybdenum cofactor in mycobacteria

When intracelluar pathogens enter the host macrophages where in addition to oxidative and antibiotic mechanisms of antimicrobial activity, nutrients are deprived. Human pathogen Mycobacterium tuberculosis is one of macrophage parasitisms, which can replicate and persist for decades in dormancy state in virulent environments. It is very successful in escaping the killing mechanisms of macrophage. Molybdenum (Mo) enzymes involve in the global carbon, sulfur, and nitrogen cycles by catalyzing important redox reactions. There are several Mo enzymes in mycobacteria and they exert several important physiological functions, such as dormancy regulation, the metabolism of energy sources, and nitrogen source. Pterin-based Mo cofactor (Moco) is the common cofactor of the Mo enzymes in mycobacteria but the cofactor biosynthesis is nearly an untapped area. The present article discusses the physiological function of Mo enzymes and the structural feature of the genes coding for Moco biosynthesis enzymes in mycobacteria.     

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.     

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.     

Molybdoenzymes and Molybdenum Cofactor in Plants

The transition element molybdenum is essential for (nearly) all organisms and occurs in more than 30 enzymes catalyzing diverse redox reactions; however, only three Mo-enzymes have been found in plants so far. (1) Nitrate reductase catalyzes the key step in inorganic nitrogen assimilation, (2) aldehyde oxidase(s) recently have been shown to catalyze the last step in the biosynthesis of the phytohormones indole acetic acid and abscisic acid, respectively, and (3) xanthine dehydrogenase is involved in purine catabolism. These enzymes are homodimeric proteins harboring an electron transport chain that involves different prosthetic groups (FAD, heme, or Fe-S, Mo). Among different Mo-enzymes, the alignment of amino acid sequences helps to define regions that are well conserved (domains) and other regions that are highly variable in sequence (interdomain hinge regions). The existence of additional plant Mo-enzymes (like sulfite oxidase) also has to be considered. In this review we focus on structure-function relationships and stress the functional importance of the enzymes for the plant. With the exception of bacterial nitrogenase, Mo-enzymes share a similar pterin compound at their catalytic sites, the molybdenum cofactor. Molybdenum 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-center within the holo-enzyme so that the Mo-center can interact with other components of the enzyme's electron transport chain. The organic moiety of the molybdenum cofactor is a unique pterin named molybdopterin. The core structure of molybdopterin is conserved in all organisms. Accordingly, its biosynthetic pathway seems to be conserved because a similar set of cofactor genes has been found in bacteria and higher plants. We describe a model for the biosynthesis of the plant molybdenum cofactor involving the complex interaction of seven proteins.     

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.     

Crystal structures of human gephyrin and plant Cnx1 G domains: comparative analysis and functional implications

The molybdenum cofactor (Moco) consists of a unique and conserved pterin derivative, usually referred to as molybdopterin (MPT), which coordinates the essential transition metal molybdenum (Mo). Moco is required for the enzymatic activities of all Mo-enzymes, with the exception of nitrogenase and is synthesized by an evolutionary old multi-step pathway that is dependent on the activities of at least six gene products. In eukaryotes, the final step of Moco biosynthesis, i.e. transfer and insertion of Mo into MPT, is catalyzed by the two-domain proteins Cnx1 in plants and gephyrin in mammals. Gephyrin is ubiquitously expressed, and was initially found in the central nervous system, where it is essential for clustering of inhibitory neuroreceptors in the postsynaptic membrane. Gephyrin and Cnx1 contain at least two functional domains (E and G) that are homologous to the Escherichia coli proteins MoeA and MogA, the atomic structures of which have been solved recently. Here, we present the crystal structures of the N-terminal human gephyrin G domain (Geph-G) and the C-terminal Arabidopsis thaliana Cnx1 G domain (Cnx1-G) at 1.7 and 2.6 A resolution, respectively. These structures are highly similar and compared to MogA reveal four major differences in their three-dimensional structures: (1) In Geph-G and Cnx1-G an additional alpha-helix is present between the first beta-strand and alpha-helix of MogA. (2) The loop between alpha 2 and beta 2 undergoes conformational changes in all three structures. (3) A beta-hairpin loop found in MogA is absent from Geph-G and Cnx1-G. (4) The C terminus of Geph-G follows a different path from that in MogA. Based on the structures of the eukaryotic proteins and their comparisons with E. coli MogA, the predicted binding site for MPT has been further refined. In addition, the characterized alternative splice variants of gephyrin are analyzed in the context of the three-dimensional structure of Geph-G.     

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.     

Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation

Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea and eukaryotes, including humans. Genetic deficiencies of enzymes involved in Moco biosynthesis in humans lead to a severe and usually fatal disease. Moco contains a tricyclic pyranopterin, termed molybdopterin (MPT), that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of MPT is generated by MPT synthase, which consists of a large and small subunits. The 1.45 A resolution crystal structure of MPT synthase reveals a heterotetrameric protein in which the C-terminus of each small subunit is inserted into a large subunit to form the active site. In the activated form of the enzyme this C-terminus is present as a thiocarboxylate. In the structure of a covalent complex of MPT synthase, an isopeptide bond is present between the C-terminus of the small subunit and a Lys side chain in the large subunit. The strong structural similarity between the small subunit of MPT synthase and ubiquitin provides evidence for the evolutionary antecedence of the Moco biosynthetic pathway to the ubiquitin dependent protein degradation pathway.     

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.     

The tetrahydropyranopterin structure of the sulfur-free and metal-free molybdenum cofactor precursor

The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase. Moco is synthesized by a unique and evolutionary old multi-step pathway with two intermediates identified so far, the sulfur-free and metal-free pterin derivative precursor Z and molybdopterin, a pterin with an enedithiolate function essential for Mo ligation. The latter pterin component is believed to form a tetrahydropyranopterin similar to the one found for Moco in the crystal structure of Mo as well as tungsten (W) enzymes. Here we report the spectroscopic characterization and structure elucidation of precursor Z purified from Escherichia coli overproducing MoaA and MoaC, two proteins essential for bacterial precursor Z synthesis. We have shown that purified precursor Z is as active as precursor Z present in E. coli cell extracts, demonstrating that no modifications during the purification procedure have occurred. High resolution electrospray ionization mass spectrometry afforded a [M + H]+ ion compatible with a molecular formula of C10H15N5O8P. Consequently 1H NMR spectroscopy not allowed structural characterization of the molecule but confirmed that this intermediate undergoes direct oxidation to the previously well characterized non-productive follow-up product compound Z. The 1H chemical shift and coupling constant data are incompatible with previous structural proposals and indicate that precursor Z already is a tetrahydropyranopterin system and carries a geminal diol function in the C1' position.     

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.     

Elucidation of the dual role of Mycobacterial MoeZR in molybdenum cofactor biosynthesis and cysteine biosynthesis

The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.     

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.     

The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein gephyrin

BACKGROUND: Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in archaea, eubacteria, and eukaryotes. In humans, genetic abnormalities in the biosynthetic pathway result in Moco deficiency, which is accompanied by severe neurological symptoms and death shortly after birth. The Escherichia coli MoeA and MogA proteins are involved in the final step of Moco biosynthesis: the incorporation of molybdenum into molybdopterin (MPT), the organic pyranopterin moiety of Moco. RESULTS: The crystal structure of E. coli MoeA has been refined at 2 A resolution and reveals that the highly elongated MoeA monomer consists of four clearly separated domains, one of which is structurally related to MogA, indicating a divergent evolutionary relationship between both proteins. The active form of MoeA is a dimer, and a putative active site appears to be localized to a cleft formed between domain II of the first monomer and domains III and IV of the second monomer. CONCLUSIONS: In eukaryotes, MogA and MoeA are fused into a single polypeptide chain. The corresponding mammalian protein gephyrin has also been implicated in the anchoring of glycinergic receptors to the cytoskeleton at inhibitory synapses. Based on the structures of MoeA and MogA, gephyrin is surmised to be a highly organized molecule containing at least five domains. This multidomain arrangement could provide a structural basis for its functional diversity. The oligomeric states of MoeA and MogA suggest how gephyrin could assemble into a hexagonal scaffold at inhibitory synapses.     

The biosynthesis of the molybdenum cofactors in Escherichia coli

The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5′-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years.     

Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins

The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1 , a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA . The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum cofactor locus chl6 . As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E. coli Moco protein MoeA, the C-terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli , is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.