The Mechanism of Nucleotide-assisted Molybdenum Insertion into Molybdopterin

The molybdenum cofactor (Moco) is synthesized by an ancient and conserved biosynthetic pathway. In plants, the two-domain protein Cnx1 catalyzes the insertion of molybdenum into molybdopterin (MPT), a metal-free phosphorylated pyranopterin carrying an ene-dithiolate. Recently, we identified a novel biosynthetic intermediate, adenylated molybdopterin (MPT-AMP), which is synthesized by the C-terminal G domain of Cnx1. Here, we show that MPT-AMP and molybdate bind in an equimolar and cooperative way to the other N-terminal E domain (Cnx1E). Tungstate and sulfate compete formolybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn -/ Mg -dependent hydrolysis of MPT-AMP but only when molybdate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release ofMoco thatwas quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is release as second product of the reaction. When comparing MPTAMP hydrolysis with the formation of Moco and AMP a 1.5-fold difference in reaction rates were observed. Together with the strict dependence of the reaction on molybdate the formation of adenylatedmolybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to molybdopterin is proposed.