The active site of the molybdenum cofactor biosynthetic protein domain Cnx1G.
The final step of molybdenum cofactor biosynthesis in plants is catalyzed by the two-domain protein Cnx1. The G domain of Cnx1 (Cnx1G) binds molybdopterin with high affinity and transfers molybdenum to molybdopterin. Here, we describe the functional and structural characterization of structure-based Cnx1G mutants. For molybdopterin binding residues Thr542 and Ser573 were found to be important because different mutations of those residues resulted in 7- to 26-fold higher k(D) values for molybdopterin binding. Furthermore, we showed that the terminal phosphate of molybdopterin is directly involved in protein-pterin interactions as dephosphorylated molybdopterin binds with one magnitude of order lower affinity to the wild-type protein. Molybdopterin binding was not affected in mutants defective in Ser476, Asp486, or Asp515. However, molybdenum insertion was completely abolished, indicating their important role for catalysis. Based on these results we propose the binding of molybdopterin to a large depression in the structure of Cnx1G formed by beta5, alpha5, beta6, and alpha6, whereas the negatively charged depression formed by the loop between beta3 and alpha4, the N-terminal end of alpha2, the 3(10) helix, and the region between beta6 and alpha6 is involved in catalysis.
Kuper
Jochen
J
Department of Plant Biology, Technical University of Braunschweig, 38028, Braunschweig, Germany.
Winking
Jan
J
Hecht
Hans-Jürgen
HJ
Mendel
Ralf R
RR
Schwarz
Günter
G
D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D037281:Calnexin / Q000737:chemistry* / Q000378:metabolism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D005998:Glycine; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; C080351:CNX1 protein, Arabidopsis; D011994:Recombinant Proteins; D037281:Calnexin; D005998:Glycine