Evidence for MoeA-dependent formation of the molybdenum cofactor from molybdate and molybdopterin in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The function of the MoeA protein in the biosynthesis of the molybdenum cofactor (MoCo) was analyzed in vitro, using purified His(6)-MoeA from Escherichia coli, molybdopterin (MPT) isolated from buttermilk xanthine oxidase and molybdate. The formation of MoCo was monitored by the reconstitution of nitrate reductase activity in extracts of the Neurospora crassa nit-1 mutant. Formation of MoCo from MPT and molybdate required MoeA and L-cysteine or glutathione. The reaction proceeded at micromolar molybdate levels and was time- and MoeA concentration-dependent. A physical interaction between MoeA and MPT was demonstrated by HPLC analysis of MoeA-bound MPT.     

Mutational analysis of the gephyrin-related molybdenum cofactor biosynthetic gene cnxE from the lower eukaryote Aspergillus nidulans

We report the identification of a number of mutations that result in amino acid replacements (and their phenotypic characterization) in either the MogA-like domain or domains 2 and 3 of the MoeA-like region of the Aspergillus nidulans cnxE gene. These domains are functionally required since mutations that result in amino acid substitutions in any one domain lead to the loss or to a substantial reduction in all three identified molybdoenzyme activities (i.e., nitrate reductase, xanthine dehydrogenase, and nicotinate hydroxylase). Certain cnxE mutants that show partial growth with nitrate as the nitrogen source in contrast do not grow on hypoxanthine or nicotinate. Complementation between mutants carrying lesions in the MogA-like domain or the MoeA-like region, respectively, most likely occurs at the protein level. A homology model of CnxE based on the dimeric structure of E. coli MoeA is presented and the position of inactivating mutations (due to amino acid replacements) in the MoeA-like functional region of the CnxE protein is mapped to this model. Finally, the activity of nicotinate hydroxylase, unlike that of nitrate reductase and xanthine dehydrogenase, is not restored in cnxE mutants grown in the presence of excess molybdate.     

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.     

In vivo interactions between gene products involved in the final stages of molybdenum cofactor biosynthesis in Escherichia coli

The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin. Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli. Numerous interactions among these proteins have been identified. Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA. Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins.     

In vitro molybdenum ligation to molybdopterin using purified components

We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA(-) cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.     

Molybdenum cofactor biosynthesis in Escherichia coli. Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide.

The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor. The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin. The molybdenum cofactor in a number of E. coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain. In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E. coli. Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E. coli cells. Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide.     

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.     

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.     

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.     

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.     

The crystal structure of Escherichia coli MoaB suggests a probable role in molybdenum cofactor synthesis

The crystal structure of Escherichia coli MoaB was determined by multiwavelength anomalous diffraction phasing and refined at 1.6-A resolution. The molecule displayed a modified Rossman fold. MoaB is assembled into a hexamer composed of two trimers. The monomers have high structural similarity with two proteins, MogA and MoeA, from the molybdenum cofactor synthesis pathway in E. coli, as well as with domains of mammalian gephyrin and plant Cnx1, which are also involved in molybdopterin synthesis. Structural comparison between these proteins and the amino acid conservation patterns revealed a putative active site in MoaB. The structural analysis of this site allowed to advance several hypothesis that can be tested in further studies.     

Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis

Biosynthesis of the molybdenum cofactor, a chelate of molybdenum or tungsten with a novel pterin, occurs in virtually all organisms including humans. In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin. Escherichia coli molybdopterin synthase, the protein responsible for adding the dithiolene to a desulfo precursor termed precursor Z, is a dimer of dimers containing the MoaD and MoaE proteins. The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus. Using an intein expression system for preparation of thiocarboxylated MoaD, the mechanism of the molybdopterin synthase reaction was examined. A stoichiometry of 2 molecules of thiocarboxylated MoaD per conversion of a single precursor Z molecule to molybdopterin was observed. Examination of several synthase variants bearing mutations in the MoaE subunit identified Lys-119 as a residue essential for activity and Arg-39 and Lys-126 as other residues critical for the reaction. An intermediate of the synthase reaction was identified and characterized. This intermediate remains tightly associated with the protein and is the predominant product formed by synthase containing the K126A variant of MoaE. Mass spectral data obtained from protein-bound intermediate are consistent with a monosulfurated structure that contains a terminal phosphate group similar to that present in molybdopterin.     

Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis

The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.     

The crystal structure of Escherichia coli MoeA, a protein from the molybdopterin synthesis pathway.

MoeA is involved in synthesis of the molybdopterin cofactor, although its function is not yet clearly defined. The three-dimensional structure of the Escherichia coli protein was solved at 2.2 A resolution. The locations of highly conserved residues among the prokaryotic and eukaryotic MoeA homologs identifies a cleft in the dimer interface as the likely functional site. Of the four domains of MoeA, domain 2 displays a novel fold and domains 1 and 4 each have only one known structural homolog. Domain 3, in contrast, is structurally similar to many other proteins. The protein that resembles domain 3 most closely is MogA, another protein required for molybdopterin cofactor synthesis. The overall similarity between MoeA and MogA, and the similarities in a constellation of residues that are strongly conserved in MoeA, suggests that these proteins bind similar ligands or substrates and may have similar functions.     

nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii

Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active molybdopterin cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits.     

MoeA,an enzyme in the molybdopterin synthesis pathway,is required for rifamycin SV production in Amycolatopsis mediterranei U32

Rifamycin SV contains one amide nitrogen atom at its C(7)N moiety. Earlier labeling studies suggested that nitrogen might be incorporated from a pathway involved in a molybdenum-dependent nitrate reductase. However, no genetic evidence is available thus far. The structural gene moeA, which is involved in molybdopterin synthesis in various organisms, has been cloned from rifamycin SV-producing Amycolatopsis mediterranei strain U32. The amino acid sequence deduced from the moeA gene showed significant similarity to members of the MoeA protein family and contains all the structural features that are highly conserved in the putative functional domains of MoeA proteins. Southern hybridization showed that there is only one moeA gene in the A. mediterranei genome. To further investigate the possible physiological function of the moeA gene, a double crossover gene replacement was achieved by inserting an aparmycin resistance gene into moeA in the A. mediterranei U32 chromosome. Phenotype analysis showed that the moeA gene is required for A. mediterranei growth in a minimal medium with nitrate as sole nitrogen source, possibly because nitrate reductase activity is diminished due to disruption of the moeA gene. Compared to the wild type strain, moeA-disrupted mutants lost 95% of their rifamycin SV production capacity in complex fermentation media. The results demonstrate that the moeA gene is necessary for rifamycin SV production in A. mediterranei, and that the nitrogen assimilation pathway involved in nitrate reductase is the major pathway for the genesis of the amide nitrogen atom in the rifamycin SV molecule.     

Role of the C-terminal Gly-Gly motif of Escherichia coli MoaD, a molybdenum cofactor biosynthesis protein with a ubiquitin fold

In Escherichia coli, the MoaD protein plays a central role in the conversion of precursor Z to molybdopterin (MPT) during molybdenum cofactor biosynthesis. MoaD has a fold similar to that of ubiquitin and contains a highly conserved C-terminal Gly-Gly motif, which in its active form contains a transferrable sulfur in the form of a thiocarboxylate group. During MPT biosynthesis, MoaD cycles between two different heterotetrameric complexes, one with MoaE to form MPT synthase and the other with MoeB, a protein similar to E1 in the ubiquitin pathway, to regenerate its transferrable sulfur. To determine the specific roles of each of the two terminal Gly residues with regard to the MoaD cycle, variants at the penultimate (Gly80) or terminal (Gly81) residues of both MoaD and thiocarboxylated MoaD were created. These variants were analyzed to determine their effects on complex formation with MoaE and MoeB, formation of the MoaD-acyl-adenylate complex, transfer of sulfur to precursor Z to form MPT, and total cofactor biosynthesis. The combined results show that while conservative substitutions at Gly80 had little effect on any of the processes that were examined, the terminal MoaD residue (Gly81) is important for transfer of sulfur to precursor Z and essential for formation of the MoaD-AMP complex. These results further our understanding of the mechanistic similarities of the MoaD-MoeB reaction to that of the ubiquitin-E1 system.