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J. Biol. Chem., Vol. 279, Issue 53, 55241-55246, December 31, 2004

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Synthesis of Adenylated Molybdopterin

AN ESSENTIAL STEP FOR MOLYBDENUM INSERTION^*

Angel Llamas, Ralf R. Mendel, and Günter Schwarz

From the Department of Plant Biology, Technical University of Braunschweig, Spielmannstrasse 7, D-3806 Braunschweig, Germany

The molybdenum cofactor (Moco) is part of the active site of all molybdenum (Mo)-dependent enzymes, except nitrogenase. Moco consists of molybdopterin (MPT), a phosphorylated pyranopterin with an enedithiolate coordinating Mo and it is synthesized by an evolutionary old multistep pathway. The plant protein Cnx1 from Arabidopsis thaliana catalyzes with its two domains (E and G) the terminal step of Moco biosynthesis, the insertion of Mo into MPT. Recently, the high-resolution MPT-bound structure of the Cnx1 G domain (Cnx1G) has been determined (Kuper, J., Llamas, A., Hecht, H. J., Mendel, R. R., and Schwarz, G. (2004) Nature 430, 803–806). Besides defining the MPT-binding site a novel and unexpected modification of MPT has been identified, adenylated MPT. Here we demonstrate that it is Cnx1G that catalyzes the adenylation of MPT. In vitro synthesized MPT was quantitatively transferred from Escherichia coli MPT synthase to Cnx1G. The subsequent adenylation reaction by Cnx1G was Mg²⁺- and ATP-dependent. Whereas Mn²⁺ could partially replace Mg²⁺, ATP was the only nucleotide accepted by Cnx1G. Consequently the formation of pyrophosphate was demonstrated, which was dependent on the ability of Cnx1G to bind MPT. Pyrophosphate, either formed in the reaction or added externally, inhibited the Cnx1G-catalyzed MPT adenylation reaction. Catalytically inactive Cnx1G mutant variants showed impaired MPT adenylation confirming that MPT-AMP is the reaction product of Cnx1G. Therefore Cnx1G is a MPT adenylyltransferase catalyzing the activation of MPT, a universal reaction in the Moco synthetic pathway because Cnx1G is able to reconstitute also bacterial and mammalian Moco biosynthesis.

Received for publication, August 26, 2004 , and in revised form, October 4, 2004.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Schw759/2-1 (to G. S.) and Me1266/12-3 (to R. R. M.) and European Union Grant HPRM-CT-2002-00244 (to R. R. M.). 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.

To whom correspondence should be addressed. Tel.: 49-531-391-5891; Fax: 49-531-391-8208; E-mail: g.schwarz{at}tu-bs.de.

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