The Molybdenum Cofactor Biosynthetic Protein Cnx1 Complements Molybdate-Repairable Mutants, Transfers Molybdenum to the Metal Binding Pterin, and Is Associated with the Cytoskeleton

Abstract

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 Mechanism of Nucleotide-assisted Molybdenum Insertion into Molybdopterin

“…Later in E. coli the mod locus encoding a high affinity molybdate uptake system (33) and the mogA gene essential for Moco biosynthesis (34) have been identified. In contrast to the single-locus defect in bacterial MogA plant, fungal and mammalian molybdate-repairable mutants showed a mutation in one of the two conserved domains of proteins like Cnx1 (14), CnxE (35), or gephyrin (15), respectively. The two-domain nature of Cnx1 and its homologues pointed to a functional "cooperation" between both domains such as product-substrate-channeling.…”

“…In eukaryotes a defect in any of the two conserved domains of Cnx1 or homologous proteins causes a molybdate-repairable phenotype (14,35,48). This finding suggests that in the presence of excess molybdate the nucleotide-asssisted metal insertion step might be bypassed by direct chelation of molybdenum to the MPT dithiolate.…”

“…In E. coli, mogA mutants are molybdate-repairable but moeA mutants are not. Together with the inability of Cnx1E to reconstitute E. coli MoeA function (14) one can argue that the metal insertion step is different between eukaryotes and bacteria, which might be due to the synthesis of a bis-MPT type cofactor in prokaryotes. Recently, it was shown that recombinant MoeA is also able to transfer molybdenum to MPT (49) but using a much higher concentration of both MoeA and molybdate (49) that does not reflect the physiological situation.…”

“…Eukaryotic molybdenum insertion is catalyzed by Cnx1 in plants (14) and gephyrin in mammals (15). Both proteins exhibit two conserved functional domains (E and G) homologous to the Escherichia coli proteins MoeA (16) and MogA (17), respectively.…”

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

“…The results obtained demonstrate that Arabidopsis MoBP proteins interact with both Cnx1 and apoNR, which are known to be cytosolic proteins (23,24). Therefore we assumed that likewise MoBPs are localized in the cytosol of the cell.…”