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J. Biol. Chem., Vol. 282, Issue 47, 33925-33934, November 23, 2007

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NAD Kinases Use Substrate-assisted Catalysis for Specific Recognition of NAD^*

Guillaume Poncet-Montange¹, Liliane Assairi^¶, Stefan Arold, Sylvie Pochet^¶||, and Gilles Labesse²

From the Atelier de Bioet Chimie Informatique Structurale, Centre de Biochimie Structurale, UMR5048, CNRS, Universités Montpellier 1 et 2, 29 rue de Navacelles, F34090 Montpellier, France, INSERM, U554, 29 rue de Navacelles, F34090 Montpellier, France, ^¶INSERM, U759, Institut Curie, 91405 Orsay, France, and ^||Unité de Chimie Organique, Institut Pasteur, CNRS, URA 2128, 75724 Paris, France

Here we describe the crystal structures of the NAD kinase (LmNADK1) from Listeria monocytogenes in complex with its substrate NAD, its product NADP, or two synthesized NAD mimics. We identified one of the NAD mimics, di-adenosine diphosphate, as a new substrate for LmNADK1, whereas we showed that the closely related compound di-5'-thioadenosine is a novel non-natural inhibitor for this enzyme. These structures suggest a mechanism involving substrate-assisted catalysis. Indeed, sequence/structure comparison and directed mutagenesis have previously shown that NAD kinases (NADKs) and the distantly related 6-phosphofructokinases share the same catalytically important GGDGT motif. However, in this study we have shown that these enzymes use the central aspartate of this motif differently. Although this acidic residue chelates the catalytic Mg²⁺ ion in 6-phosphofructokinases, it activates the phospho-acceptor (NAD) in NADKs. Sequence/structure comparisons suggest that the role of this aspartate would be conserved in NADKs and the related sphingosine and diacylglycerol kinases.

Received for publication, February 16, 2007 , and in revised form, July 17, 2007.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7.

The atomic coordinates and structure factors (code 2I1W, 2I29, 2I2A, 2I2B, 2I2C, 2I2D, 2I2E, 2I2F, and 2Q5F) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ Present address: Université Montpellier 2 and INSERM, U710, Montpellier, F-34095 France and EPHE, Paris, F-75007 France.

² To whom correspondence should be addressed. Tel.: 33-4-67-41-77-12; Fax: 33-4-67-41-79-13; E-mail: labesse{at}cbs.cnrs.fr.

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