One-Carbon Metabolism (Folate Cycle)

1. 1

   Dihydrofolate (DHF) + NADPH + H⁺ → tetrahydrofolate (THF) + NADP⁺

   Dihydrofolate reductase (DHFR) 1.5.1.3 ST-0160 Irreversible NADP+ NADPH THF (folate)
   In Dihydrofolate (DHF) NADPH H+

   Out Tetrahydrofolate (THF) NADP+
   › Notes

   Dihydrofolate reductase (DHFR) regenerates reduced THF after DHF is produced by thymidylate synthase. NADPH is required; this is an essential, effectively irreversible reductive regeneration step and the pharmacologic target of methotrexate, trimethoprim, and pyrimethamine.

2. 2

   L-serine + THF ⇌ glycine + 5,10-methylene-THF + H₂O

   Serine hydroxymethyltransferase (SHMT1/SHMT2) 2.1.2.1 ST-0161 Reversible PLP (B6) THF (folate) H2O
   In Serine Tetrahydrofolate (THF)

   Out Glycine 5,10-Methylene-THF H2O
   › Notes

   Serine hydroxymethyltransferase (SHMT1 cytosolic; SHMT2 mitochondrial) uses PLP (vitamin B6) to transfer the serine β-carbon to THF. This reversible reaction is a major source of 5,10-methylene-THF.

3. 3

   5,10-Methylene-THF + NADP⁺ ⇌ 5,10-methenyl-THF + NADPH + H⁺

   Methylene-THF dehydrogenase (MTHFD1) 1.5.1.5 ST-0162 Reversible NADP+ NADPH THF (folate)
   In 5,10-Methylene-THF NADP+

   Out 5,10-Methenyl-THF NADPH H+
   › Notes

   The dehydrogenase activity of cytosolic trifunctional MTHFD1 (or mitochondrial MTHFD2/MTHFD2L) interconverts these folate species. Cofactor preference is enzyme- and compartment-specific; MTHFD1 uses NADP⁺, whereas mitochondrial MTHFD2 commonly uses NAD⁺.

4. 4

   5,10-Methenyl-THF + H₂O ⇌ 10-formyl-THF

   Methenyl-THF cyclohydrolase (MTHFD1) ST-0163 Reversible THF (folate) H2O
   In 5,10-Methenyl-THF H2O

   Out 10-Formyl-THF
   › Notes

   The cyclohydrolase activity of MTHFD1 (or mitochondrial MTHFD enzymes) converts the methenyl to the formyl oxidation state. This reversible hydration supplies 10-formyl-THF for purine biosynthesis.

5. 5

   THF + formate + ATP ⇌ 10-formyl-THF + ADP + Pᵢ

   10-Formyl-THF synthetase 6.3.4.3 ST-0164 Irreversible ATP THF (folate) Pi
   In Tetrahydrofolate (THF) Formate ATP

   Out 10-Formyl-THF ADP Pi
   › Notes

   10-Formyl-THF synthetase activity of MTHFD1 activates formate onto THF. ATP is consumed; in the direction of formate incorporation this is an essentially irreversible, energy-coupled reaction.

6. 6

   5,10-Methylene-THF + dUMP → dTMP + DHF

   Thymidylate synthase (TYMS) 2.1.1.45 ST-0165 Irreversible THF (folate)
   In 5,10-Methylene-THF dUMP

   Out dTMP Dihydrofolate (DHF)
   › Notes

   Thymidylate synthase transfers and reduces a one-carbon unit to form dTMP, generating DHF. This is the committed one-carbon donation step for de novo thymidylate synthesis and is effectively irreversible; THF must be regenerated by DHFR for sustained DNA synthesis.

7. 7

   10-Formyl-THF + glycinamide ribonucleotide (GAR) → formyl-GAR + THF

   GAR transformylase (GART) 2.1.2.2 ST-0166 Irreversible/directional THF (folate)
   In 10-Formyl-THF Glycinamide ribonucleotide (GAR)

   Out Formylglycinamide ribonucleotide (FGAR) Tetrahydrofolate (THF)
   › Notes

   GAR transformylase (the phosphoribosylglycinamide formyltransferase domain of GART) donates one formyl group during de novo purine synthesis. This reaction is a committed folate-dependent input into the purine pathway after early purine assembly has begun.

8. 8

   10-Formyl-THF + AICAR → formyl-AICAR + THF

   AICAR transformylase (ATIC) 2.1.2.3 ST-0167 Irreversible/directional THF (folate)
   In 10-Formyl-THF AICAR

   Out FAICAR Tetrahydrofolate (THF)
   › Notes

   AICAR transformylase (ATIC) donates the second formyl group required for inosine monophosphate synthesis. This reaction is followed by cyclization to IMP by the IMP cyclohydrolase activity of ATIC.

9. 9

   5,10-Methylene-THF + NADPH + H⁺ → 5-methyl-THF + NADP⁺

   Methylenetetrahydrofolate reductase (MTHFR) 1.5.1.20 ST-0168 Irreversible NADP+ NADPH FAD THF (folate)
   In 5,10-Methylene-THF NADPH H+

   Out 5-Methyl-THF NADP+
   › Notes

   Methylenetetrahydrofolate reductase (MTHFR) uses FAD as a prosthetic group and NADPH-derived reducing power to generate 5-methyl-THF. This is an effectively irreversible, regulated branch-point reaction that commits one-carbon units to methionine regeneration rather than nucleotide synthesis.

10. 10

    5-Methyl-THF + homocysteine → THF + methionine

    Methionine synthase (MTR) 2.1.1.13 ST-0169 Irreversible/directional Cobalamin (B12) THF (folate)
    In 5-Methyl-THF Homocysteine

    Out Tetrahydrofolate (THF) Methionine
    › Notes

    Methionine synthase (5-methyltetrahydrofolate–homocysteine methyltransferase) requires methylcobalamin (vitamin B12) and transfers the methyl group through a cobalamin intermediate. The reaction regenerates THF and prevents trapping of cellular folate as 5-methyl-THF.

Showing all 10 steps.

Overview

The folate cycle uses tetrahydrofolate (THF) derivatives to carry and distribute one-carbon units at oxidation states ranging from methyl to formyl. These units support de novo purine synthesis, dTMP synthesis, methionine regeneration, serine/glycine interconversion, histidine catabolism, and formylation of initiator methionyl-tRNA in mitochondria. The cycle is particularly important in rapidly dividing tissues because nucleotide synthesis depends on 5,10-methylene-THF and 10-formyl-THF.

Cellular location

Folate-mediated one-carbon metabolism operates in both cytosol and mitochondria, with compartment-specific folate pools linked by export of one-carbon units such as formate. Cytosolic reactions are crucial for nucleotide synthesis and remethylation of homocysteine; mitochondrial serine catabolism supplies much of the one-carbon flux in proliferating cells. High flux occurs in liver, bone marrow, intestinal epithelium, placenta, and other tissues with active proliferation or methyl-group demand.

Net energetics

The folate cycle has no universal single net stoichiometry because THF carries one-carbon units among several linked pathways. Regeneration of THF from DHF costs 1 NADPH per DHF, formation of 10-formyl-THF from formate costs 1 ATP, and reduction of 5,10-methylene-THF to 5-methyl-THF consumes 1 NADPH equivalent. One 5,10-methylene-THF is oxidized to DHF for each dTMP formed, whereas two 10-formyl-THF molecules are used per IMP synthesized.

Clinical significance

Folate deficiency impairs dTMP and purine synthesis, producing defective DNA replication and megaloblastic anemia, particularly in bone marrow. Vitamin B12 deficiency impairs methionine synthase, causes methyl-folate trapping and functional folate deficiency, and also produces neurologic disease through mechanisms not corrected by folate alone. Antifolate drugs exploit the dependence of rapidly proliferating cells or microorganisms on DHFR and folate-dependent nucleotide synthesis; excessive folate supplementation can partially correct anemia while masking hematologic evidence of B12 deficiency.