Identification and biochemical characterization of molybdenum cofactor-binding proteins from Arabidopsis thaliana.
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.
Kruse
Tobias
T
Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
Gehl
Christian
C
Geisler
Mirco
M
Lehrke
Markus
M
Ringel
Phillip
P
Hallier
Stephan
S
Hänsch
Robert
R
Mendel
Ralf R
RR
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
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