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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{sup -} 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 themore » 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.« less

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.

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 thatmoeA − 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 themoeA − 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.

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.