The active site of the molybdenum cofactor biosynthetic protein domain Cnx1G

Abstract 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 β5, α5, β6, and α6, whereas the negatively charged depression formed by the loop between β3 and α4, the N-terminal end of α2, the 3 10 helix, and the region between β6 and α6 is involved in catalysis.