Fig. 1

MTHFD2 exhibits NADP⁺-dependent dehydrogenase activity with 5,10-CH₂-THF pentaglutamate. a Compartmentation of mammalian one-carbon metabolism. MTHFD1 is the cytoplasmic trifunctional C₁-THF synthase that catalyzes 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase activities. In mammalian mitochondria, bifunctional MTHFD2 or MTHFD2L enzymes catalyze 5,10-methenyl-THF cyclohydrolase and 5,10-methylene-THF dehydrogenase activities, and monofunctional MTHFD1L catalyzes the 10-formyl-THF synthetase reaction. SHMT1 and SHMT2 represent cytoplasmic and mitochondrial serine hydroxymethyltransferase isozymes, respectively. Gray ovals represent putative metabolite transporters. b, c Purified MTHFD2 was assayed for NAD⁺- and NADP⁺-dependent 5,10-CH₂-THF dehydrogenase activity with saturating concentrations of CH₂-H₄PteGlu₁ or CH₂-H₄PteGlu₅ (insets, 5,10-CH₂-THF dehydrogenase activity of MTHFD2L; data from ref. 11). CH₂-H₄PteGlu₁ and CH₂-H₄PteGlu₅ concentrations were 354 and 429 μM, respectively. NAD⁺ and NADP⁺ concentrations were 1.0 and 6.0 mM, respectively. NAD⁺-dependent reactions also included 5 mM MgCl₂ and 25 mM P _i . NADP⁺-dependent reactions included only 5 mM MgCl₂. Each column represents the mean ± S.E. of triplicate determinations