Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase

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 Pi can bind in close proximity to the 2'-hydroxyl of NAD through interactions with Arg(166) and Arg(198). The characterization of mutants of Arg(166), Asp(190), and Arg(198) show that Arg(166) is primarily responsible for Pi binding, while Arg(198) plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp(190) helps to properly position Arg(166). Mutants of Asp(133) suggest that the magnesium ion interacts with both P-i and the aspartate side chain and plays a role in positioning Pi 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.