title: Identification and biochemical characterization of molybdenum cofactor-binding proteins from Arabidopsis thaliana.
abstract: The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K(D) values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.
authors.affiliation: Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Kruse
authors.forename: Christian
authors.initials: C
authors.lastname: Gehl
authors.forename: Mirco
authors.initials: M
authors.lastname: Geisler
authors.forename: Markus
authors.initials: M
authors.lastname: Lehrke
authors.forename: Phillip
authors.initials: P
authors.lastname: Ringel
authors.forename: Stephan
authors.initials: S
authors.lastname: Hallier
authors.forename: Robert
authors.initials: R
authors.lastname: Hänsch
authors.forename: Ralf R
authors.initials: RR
authors.lastname: Mendel
mesh_terms: D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000378:metabolism; D001665:Binding Sites; D037281:Calnexin / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase / Q000378:metabolism; D011485:Protein Binding; D011621:Pteridines / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D037281:Calnexin; D002352:Carrier Proteins; D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D011621:Pteridines; C080351:CNX1 protein, Arabidopsis; C022261:molybdopterin