The neurotransmitter receptor-anchoring protein gephyrin reconstitutes molybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells

Abstract

The molybdenum cofactor (Moco), a highly conserved pterin compound complexing molybdenum, is required for the enzymatic activities of all molybdenum enzymes except nitrogenase. Moco is synthesized by a unique and evolutionarily old pathway that requires the activities of at least six gene products. Some of the proteins involved in bacterial, plant, and invertebrate Moco biosynthesis show striking homologies to the primary structure of gephyrin, a polypeptide required for the clustering of inhibitory glycine receptors in postsynaptic membranes in the rat central nervous system. Here, we show that gephyrin binds with high affinity to molybdopterin, the metabolic precursor of Moco. Furthermore, gephyrin expression can reconstitute Moco biosynthesis in Moco-deficient bacteria, a molybdenum-dependent mouse cell line, and a Moco-deficient plant mutant. Conversely, inhibition of gephyrin expression by antisense RNA expression in cultured murine cells reduces their Moco content significantly. These data indicate that in addition to clustering glycine receptors, gephyrin also is involved in Moco biosynthesis and illustrate the remarkable conservation of its function in Moco biosynthesis throughout phylogeny.

Isoform Heterogeneity of the Human Gephyrin Gene (GPHN), Binding Domains to the Glycine Receptor, and Mutation Analysis in Hyperekplexia

“…Indeed, GPHN expression is not restricted to rat brain and spinal cord but is also found in liver, kidney, lung, and retina (28 -30). Gephyrin knock-out mice and reconstitutive cell culture assays have demonstrated that gephyrin expression in nonneuronal tissue is a requirement for the biosynthesis of molybdenum cofactor (20,31). Striking homologies are observed between the primary structure of GPHN and proteins involved in bacterial, plant, and invertebrate molybdenum cofactor biosynthesis.…”

Mechanistic Studies of Human Molybdopterin Synthase Reaction and Characterization of Mutants Identified in Group B Patients of Molybdenum Cofactor Deficiency

“…Similarly, the MOCS2 locus encodes the two subunits of MPT synthase and has been shown to be bicistronic with overlapping reading frames encoding MOCS2A and MOCS2B, the congeners of E. coli MoaD and MoaE (7). In the last step of Moco biosynthesis in humans, molybdenum is incorporated into MPT by the two-domain protein gephyrin (8,9). Based on the presence or absence of precursor Z in clinical samples, Moco deficiency was originally divided into two complementation groups.…”

Molecular architecture of glycinergic synapses

“…Surprisingly, the analysis of gephyrin's primary structure revealed signiWcant sequence homologies of the amino-and carboxy-terminal thirds to proteins involved in the biosynthesis of the molybdenum cofactor (moco) (Prior et al 1992;Stallmeyer et al 1999), a pterin cofactor required for the activity of molybdenum-dependent oxidoreductases, namely sulWte oxidase, xanthine dehydrogenase and aldehyde oxidase all of which are predominantly expressed in liver but also in brain (Mendel and Schwarz 2002;Schwarz and Mendel 2006). The amino terminal part of gephyrin (amino acids 1-181), now called the G-domain, shows homologies to the MogA protein, whereas the carboxy terminal part (amino acids 318-736), now called E-domain, is homologous to the MoeA protein of E. coli.…”

“…The amino terminal part of gephyrin (amino acids 1-181), now called the G-domain, shows homologies to the MogA protein, whereas the carboxy terminal part (amino acids 318-736), now called E-domain, is homologous to the MoeA protein of E. coli. In plants and invertebrates these two polypeptides were fused to a single protein named cnx1 (Arabidopsis thaliana) and cinnamon (Drosophila melanogaster), respectively (Stallmeyer et al 1999). In vertebrates the two domains with homology to the aforementioned proteins are connected by an extensive central region (C-domain), which does not display homologies to known proteins in the databases (Fig.…”

“…Indeed, molybdopterin (a precursor of moco) binding of gephyrin and its orthologs was demonstrated (Schwarz and Mendel 2006;Smolinsky et al 2008) and its role in the moco biosynthetic pathway is well established (Reiss et al 2001). Curiously, the G-domain of rat gephyrin could functionally replace the corresponding protein of Arabidopsis mutants lacking the cnx1 protein (Stallmeyer et al 1999) and conversely, transgenic expression of cnx1 in gephyrin-deWcient mice could partially restore the activity of moco-dependent enzymes (Grosskreutz et al 2003). …”