NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene

Abstract

The recombinant human bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is unique in its absolute requirement for Mg2+ and inorganic phosphate. Both ions affect the affinity of the enzyme for NAD and have no effect on the binding of methylenetetrahydrofolate. The NAD cofactor can be replaced by NADP with a much higher KM and lower VMAX. Kinetic investigation using NADP supports the role of Mg2+ in dinucleotide binding and illustrates that the 2'-phosphate can substitute for phosphate in this process. The human NAD-dependent bifunctional enzyme has a 44% amino acid sequence identity with the dehydrogenase-cyclohydrolase domain of the yeast mitochondrial NADP-dependent trifunctional enzyme encoded by the MIS1 gene, compared to 37% identity with the corresponding domain of the cytosolic trifunctional enzyme. The sequence comparison and the kinetic properties suggest that the NAD bifunctional enzyme is the mammalian homolog of the yeast mitochondrial trifunctional enzyme, which has evolved a unique use of inorganic phosphate to change its dinucleotide specificity from NADP to NAD. Its role is proposed to be in providing formyltetrahydrofolate for the synthesis of formylmethionyl transfer RNA required for the initiation of protein synthesis in mitochondria.

Human Mitochondrial C1-Tetrahydrofolate Synthase

“…This bifunctional enzyme lacks the large C-terminal domain catalyzing the 10-formyl-THF synthetase activity and thus is unable to produce formate. When this enzyme was shown to be localized in mitochondria (14,15), MacKenzie and co-workers (18,19) proposed that mammalian mitochondria lack a trifunctional C 1 -THF synthase and that this bifunctional NAD-dependent dehydrogenase/cyclohydrolase is the mammalian homolog of the trifunctional mitochondrial enzyme. There are, however, several problems with this proposal.…”

“…Among adult differentiated tissues, NAD-dependent 5,10-methylene-THF dehydrogenase activity is detectable only in rat adrenal tissue (16), although the mRNA encoding this enzyme is present at low levels in all tissues examined (17). MacKenzie and co-workers (18,19) have argued that mammalian mitochondria lack a C 1 -THF synthase and that the bifunctional NAD-dependent dehydrogenase/cyclohydrolase is the mammalian homolog of the trifunctional mitochondrial enzyme.…”

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues

“…The NAD ϩ -dependent activity of MTHFD2 requires Mg 2ϩ and inorganic phosphate (P i ) (6). To explore redox cofactor specificity in MTHFD2L, we measured specific activity using saturating levels of NAD ϩ or NADP ϩ in the presence of Mg 2ϩ and/or P i (Fig.…”

“…The MTFHD2 isozyme has been named NAD ϩ -dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (6,10) but in fact exhibits dehydrogenase activity with NADP ϩ , albeit with a much higher K m and lower V max (6). In fact, the redox cofactor requirements of the two isozymes are quite similar: both exhibit lower K m values for NAD ϩ than for NADP (10).…”

“…1, reaction 2m). This enzyme, now referred to as MTHFD2, has been extensively characterized with respect to kinetics, substrate specificity, and expression profile (3,(5)(6)(7)(8)(9)(10).…”