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J. Biol. Chem., Vol. 280, Issue 40, 34316-34323, October 7, 2005

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Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase^*

Karen E. Christensen1, I. Ahmad Mirza2, Albert M. Berghuis3, and Robert E. MacKenzie4

From the Department of Biochemistry and the Department of Microbiology and Immunology, McGill University, Montréal, Québec H3G 1Y6, Canada

The mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) is believed to have evolved from a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. To enable us to investigate the roles of these ions, a homology model of human NMDMC was constructed based on the structures of three homologous proteins. The model supports the hypothesis that the absolutely required P_i can bind in close proximity to the 2'-hydroxyl of NAD through interactions with Arg¹⁶⁶ and Arg¹⁹⁸. The characterization of mutants of Arg¹⁶⁶, Asp¹⁹⁰, and Arg¹⁹⁸ show that Arg¹⁶⁶ is primarily responsible for P_i binding, while Arg¹⁹⁸ plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp¹⁹⁰ helps to properly position Arg¹⁶⁶. Mutants of Asp¹³³ suggest that the magnesium ion interacts with both P_i and the aspartate side chain and plays a role in positioning P_i and NAD. NMDMC uses P_i and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold.

Received for publication, May 11, 2005 , and in revised form, August 3, 2005.

The atomic coordinates and structure factors (code 1ZN4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by Canadian Institutes of Health Research (CIHR) Grant 29814. 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 Supported by the CIHR, Fonds de Recherche en Santé du Quebéc, and McGill University.

² Supported by the International Center for Diffraction Data.

³ Recipient of a Canada Research Chair in Structural Biology.

⁴ To whom correspondence should be addressed: Dept. of Biochemistry, McGill University, McIntyre Medical Sciences Bldg., 3655 Promenade Sir William Osler, Montréal, Québec H3G 1Y6, Canada. Tel.: 514-398-7270; Fax: 514-398-7384; E-mail: robert.mackenzie{at}mcgill.ca.

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