The mechanism of nucleotide-assisted molybdenum insertion into molybdopterin: A novel route towards metal cofactor assembly

doi:10.1074/jbc.M601415200

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Papers In Press, published online ahead of print April 24, 2006
J. Biol. Chem, 10.1074/jbc.M601415200
Submitted on February 14, 2006
Revised on April 7, 2006
Accepted on April 24, 2006

The mechanism of nucleotide-assisted molybdenum insertion into molybdopterin: A novel route towards metal cofactor assembly

Angel Llamas, Tanja Otte, Gerd Multhaup, Ralf Mendel, and Guenter Schwarz

Institute of Biochemistry, University of Cologne, Koeln D-50674

Corresponding Author: gschwarz{at}uni-koeln.de

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 (Mo) 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 for molybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn2+-/Mg2+-dependent hydrolysis of MPT-AMP, but only when molyddate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release of Moco that was quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is release as second product of the reaction. When comparing MPT-AMP 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 adenylated molybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to molybdopterin is proposed.

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