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

Karen E Christensen,I Ahmad Mirza,Albert M Berghuis,Robert E Mackenzie

doi:10.1074/jbc.m505210200

Abstract

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 Arg166 and Arg198. The characterization of mutants of Arg166, Asp190, and Arg198 show that Arg166 is primarily responsible for Pi 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 Pi and the aspartate side chain and plays a role in positioning Pi and NAD. NMDMC uses Pi and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold.

Highlights

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is thought to have evolved from a trifunctional NADP-dependent methylene-THF dehydrogenase-methenyl-THF cyclohydrolase-formyl-THF synthetase (DCS) through the loss of the synthetase domain and the change in cofactor specificity from NADP to NAD [4]
NMDMC is unique in its absolute requirement for magnesium and inorganic phosphate ions for NAD-dependent dehydrogenase activity and magnesium ions for NADP-dependent dehydrogenase activity [3, 7]
The homology model of NMDMC is in good agreement with the template structures

Summary

EXPERIMENTAL PROCEDURES

Materials—(R,S)-tetrahydrofolate was synthesized according to the method of Drury et al [14] and stored in sealed glass vials at 4 °C. Oligonucleotide primers were obtained from Sigma Genosys, Qiagen Operon, and Integrated DNA Technologies, Inc. NAD, imidazole, and rifampicin were purchased from Sigma. The DC domain of the human NADP-dependent DCS structure has been solved with NADP [10] and with NADP and three different folate analogues [11]. The E. coli NADP-dependent DC structure has been solved in the absence of ligands [12]. The S. cerevisiae NAD-dependent dehydrogenase has been solved both without ligands and in complex with NAD [13]. The three template structures were obtained from the Protein Data Bank (PDB): the DC domain of the human NADP-dependent DCS in complex with NADP and a folate analogue (PDB ID: 1DIB), the E. coli NADP-dependent DC (PDB ID: 1B0A), and the S. cerevisiae NAD-dependent dehydrogenase in complex with NAD (PDB ID: 1EE9). The sequence similarity among these proteins is not high as the pair-wise

TABLE TWO

TABLE THREE

NMDMC Homology Model

The Phosphate Binding Site of NMDMC

TABLE SIX

The Magnesium Binding Site

DISCUSSION