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J. Biol. Chem., Vol. 281, Issue 27, 18343-18350, July 7, 2006
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1


¶2
From the
Institute of Plant Biology, Technical University Braunschweig, 38106 Braunschweig, Germany,
Institute of Chemistry and Biochemistry, Freie Universitaet Berlin, 14195 Berlin, and ¶Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany
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 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 molybdate 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.
Received for publication, February 14, 2006 , and in revised form, April 7, 2006.
* This work was supported by the Deutsche Forschungsgemeinschaft (to G. S. and R. R. M.), the European Union (to R. R. M.), and the Fonds der Chemischen Industrie (to G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Current address: Dept. de Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, Córdoba 14071, Spain.
2 To whom correspondence should be addressed: Inst. für Biochemie, Universität zu Köln, Otto-Fischer-Str. 12-14, 50674 Köln, Germany. Tel.: 49-221-470-6432; Fax: 49-221-470-6731; E-mail: gschwarz{at}uni-koeln.de.
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