One Carbon Metabolism: Key Pathways and Disease Associations

One-Carbon Metabolism (OCM) is a series of core biochemical networks in organisms that revolve around the transfer and utilization of single carbon units (e.g., methyl, formyl, methylene). These seemingly tiny carbon units are the cornerstone of DNA synthesis, amino acid metabolism, epigenetic regulation and other life activities. One-carbon metabolism is involved in every stage of life, from embryonic development to cancer development, and from neurological function to immune response. In this article, we systematically analyze its metabolic pathways, regulatory mechanisms, and association with diseases to reveal the macroscopic significance of this microscopic world.

One-carbon metabolism (Petrova B et al., 2023).

The core of one-carbon metabolism: carriers and units

Chemical form of the one-carbon unit

• Methyl (-CH₃): often transferred via tetrahydrofolate (THF) as a carrier, involved in methylation reactions such as DNA methylation.
• Formyl (-CHO): involved in purine and thymidine synthesis, often transferred via formyltetrahydrofolate as a carrier.
• Methylene (-CH₂-): plays an important role in amino acid and nucleotide synthesis.
• Hypomethyl (-CH=): serves as a unit in the form of a double bond and is involved in amino acid synthesis and modification reactions.

Key cofactors

• Vitamin B9 (folate): precursor of THF, needs to be consumed through diet. Vitamin B9 (folate): precursor of THF, requires dietary intake.
• Vitamin B12 (cobalamin): involved in the methyl transfer reaction in the methionine cycle.The active form of B12 is cobalamin, which transfers methyl from 5-methyltetrahydrofolate to produce methionine.

A key component of carbon metabolism

Folate Cycle

Metabolism and activation of folic acid

• Folic acid source: Dietary folic acid (vitamin B9) is reduced to dihydrofolate (DHF) and tetrahydrofolate (THF) in turn by reductase (DHFR), which is the core carrier of one carbon unit.
• One carbon unit binding site: THF carries one carbon unit with different oxidation states (such as N5- methyl -THF and N10- formyl -THF) through N5 or N10.

Key enzymes and reactions

• Methyltetrahydrofolate reductase (MTHFR): Function: Catalyze the irreversible reduction of 5,10- methylene -THF to 5- methyl -THF, and provide methyl donors for methionine circulation. Regulation: With SAM's allosteric inhibition (negative feedback regulation), MTHFR C677T mutation (rs1801133) reduced the enzyme activity by 60% and increased the homocysteine level.

• Thymidine synthase (TYMS): The synthesis of thymidylate (dTMP) from 5,10- methylene -THF is a key step in DNA replication. Anti-folic acid drugs (such as 5- fluorouracil) block the proliferation of cancer cells by inhibiting TYMS.

Physiological significance

• Purine synthesis: 10- formyl -THF provides C2 and C8 carbon atoms.
• Thymine synthesis: 5,10- methylene -THF participates in the methylation of dUMP→dTMP.
• Methylation donor reserve: 5- methyl -THF provides methyl for the demethylation of homocysteine and maintains the stability of SAM library.

Serine-Glycine Pathway

Biosynthesis and metabolism of serine

• Synthetic route: Glycolysis branch: 3- phosphoglyceric acid is oxidized by phosphoglycerate dehydrogenase (PHGDH) to form 3- phosphohydroxypyruvate, and then transferred to amino group to form serine. Exogenous uptake: Cells can take serine directly from blood through transporters (such as ASCT2).
• One carbon unit's contribution: Serine hydroxymethyltransferase (SHMT): It catalyzes the reaction of serine with THF to produce glycine and 5,10- methylene -THF, providing > 50% one carbon unit for carbon metabolism. Subcellular localization: SHMT has two subtypes: mitochondria (SHMT2) and cytoplasm (SHMT1), which support different metabolic requirements.

Metabolic fate of glycine

• Oxidative decarboxylation: Glycine cleavage system (GCS) converts glycine into CO, NH and 5,10- methylene -THF, which further supports carbon metabolism.
• Glutathione synthesis: Glycine combines with glutamic acid and cysteine to produce glutathione (GSH), which maintains the antioxidant capacity of cells.

Metabolic reprogramming in cancer

• PHGDH amplification: overexpression of PHGDH in breast cancer and melanoma enhances serine synthesis to support carbon metabolism.
• SHMT2 is up-regulated: Mitochondrial SHMT2 is activated under hypoxia, and maintains nucleotide synthesis and NADPH regeneration through carbon metabolism.

Methionine Cycle

Homocysteine demethylation

• Methionine synthase (MS): Cofactor: Rely on vitamin B12 (methylcobalamin) to transfer the methyl group of 5- methyl -THF to homocysteine to generate methionine. Regulation: Vitamin B12 deficiency leads to homocysteine accumulation, megaloblastic anemia and nerve injury.
• Betaine-homocysteine methyltransferase (BHMT): Liver specificity: betaine (choline metabolite) is used to provide methyl, and homocysteine is converted into methionine.

Generation and function of S-adenosylmethionine (SAM)

• SAM synthesis: Methionine and ATP are catalyzed by SAM Synthase (MAT) to form SAM, which consumes three high-energy phosphate bonds.
• Methylation reaction: DNA methylation: DNA methyltransferase (DNMT) uses SAM as donor to catalyze CpG island methylation (epigenetic silencing). Histone modification: Histone methyltransferase (such as EZH2) catalyzes H3K27me3 by SAM, which inhibits gene transcription. Neurotransmitter synthesis: SAM participates in the methylation modification of dopamine and norepinephrine.

Transsulfur pathway of homocysteine

• Cysteine β-Synthase (CBS): With the help of vitamin B6, it catalyzes homocysteine and serine to form cystathionine, which is finally converted into cysteine and α -ketobutyric acid.
• Pathological significance: CBS deficiency leads to homocysteine, which is characterized by thrombosis and mental retardation.

Choline Metabolism

• Choline uptake and oxidation: Dietary source: Egg yolk and liver are rich in choline, which is absorbed by the intestine and enters the central nervous system through the blood-brain barrier. Oxidation pathway: choline is gradually oxidized to betaine under the catalysis of choline dehydrogenase (CHDH) and betaine aldehyde dehydrogenase (BADH).
• Methyl donor function of betaine: BHMT reaction: betaine transfers methyl to homocysteine through BHMT to produce methionine and dimethylglycine. Physiological significance: Lipid metabolism: betaine promotes liver lipid oxidation and prevents nonalcoholic fatty liver (NAFLD). Osmotic protection: betaine in kidney is used as osmotic agent to maintain cell volume stability.
• Choline metabolism and epigenetics: Synthesis of phosphatidylcholine: choline participates in the synthesis of phosphatidylcholine (the main component of cell membrane), and its metabolic intermediates (such as glycerophosphate choline) can regulate histone acetylation. Maintenance of methyl pool: When folic acid is deficient, choline metabolism becomes an "emergency pathway" for methyl donors.

Regulation Hub: Triangular Relationship among Nutrition, Genes and Epigenetics

The steady state of one carbon metabolism is determined by the dynamic balance of nutrient input, genetic variation and epigenetic regulation. The imbalance of this triangle relationship can lead to many pathological conditions such as cancer and neurodegenerative diseases.

Nutrition input

Cascade effect of folic acid deficiency

• Obstacle in nucleotide synthesis: insufficient folic acid leads to the decrease of 5,10- methylene -THF, which inhibits the synthesis of dTMP, causes uracil to be mistakenly incorporated into DNA (about 2-3 times increased), and induces DNA double-strand breaks.
• Epigenetic disorder: the decrease of SAM production (by 30-50%) leads to the overall hypomethylation of the genome (for example, the hypomethylation rate of LINE-1 repeats increases by 20%) and activates proto-oncogenes (such as MYC).
• Immune surveillance escape: CD8⁺ T cell proliferation was inhibited (IL-2 secretion decreased by 40%), NK cell killing activity decreased, and tumor immune editing ability was enhanced (Thabet RH et al., 2024).

Double pathology of vitamin B12 deficiency

• B12 deficiency leads to the inactivation of methionine synthase (MS), and 5- methyl -THF cannot be utilized, which leads to the stagnation of folic acid cycle (the accumulation of intracellular 5-MTHF increases by 300%) and inhibits purine synthesis.
• Megaloblastic anemia: the mean volume of red blood cells (MCV) is more than 100 fl, and the myelogram shows giant promyelocytic cells.
• Subacute combined degeneration: Demyelination of posterior and lateral cords of spinal cord, characterized by disappearance of vibration perception and gait ataxia (Mucha P et al., 2024).

Heritable variation

• MTHFR C677T mutation: C677T is a single nucleotide polymorphism (SNP) in MTHFR gene, which means that cytosine (C) at gene position 677 is replaced by thymine (T). The MTHFR activity of TT genotype individuals is only 30% of CC genotype, which leads to a decrease of 5-MTHF by 50% and an increase of homocysteine by > 15 μM m.. The risk of ER-negative breast cancer in TT genotype carriers increased by 1.5 times (OR=1.52, 95%CI 1.1-2.1), and the sensitivity to methotrexate increased (Petrone I et al., 2021).
• SHMT2 overexpression: SHMT2 is highly expressed in tumor tissues of patients with BLCA, and its high expression is related to poor survival and prognosis. SHMT2 can promote the growth of BLCA cells and regulate the cell cycle, especially by promoting the transformation from G1 phase to S phase. In addition, overexpression of SHMT2 inhibited apoptosis, while knocking down SHMT2 significantly increased apoptosis. SHMT2 may affect the growth and apoptosis of BLCA cells through STAT3 signaling pathway, especially by enhancing the phosphorylation of STAT3 to promote cell proliferation. The 5-year survival rate of patients with high expression of SHMT2 decreased by 40% (from 65% to 25%) (Zhang P et al., 2021).

Epigenetic regulation

• Molecular switch of DNA methylation: WH domain specifically recognizes unmethylated CpG islands, SAM domain forms nuclear aggregates through phase separation, and DNMT3A is recruited to achieve targeted methylation. The deletion of SAMD1 in mouse embryonic stem cells resulted in abnormal demethylation of pluripotent genes (such as Nanog) (the methylation rate decreased by 40%), and the differentiation potential was impaired (Stielow B et al., 2021).
• Dynamic balance of histone modification: SAMS-4 can enhance the modification of H3K4me3 in the absence of SAMS-1, and SAMS-1 is very important for heat shock response. Nematodes without SAMS-1 can survive better during heat shock. In sams-1 deficient nematodes, SAMS-4 enhanced the expression of heat shock protein (HSP-16.2) by enhancing the modification of H3K4me3 (up to 2 times), thus enhancing the heat tolerance. The survival rate of heat stress increased by 60%, but the mortality rate of Pseudomonas aeruginosa infection also increased by 50% (due to the inhibition of antimicrobial peptide expression). The decrease of SAM level leads to the decrease of H3K27me3 modification and the decrease of chromatin openness of immune-related genes (such as abu-1) (Godbole AA et al., 2023).

Physiology and Pathology

Physiological function

• Embryonic development: ART-induced hypomethylation may affect embryonic development, especially in some gene regions, and female embryos have a greater impact. After folic acid supplementation, female embryos seem to benefit more than male embryos, especially in correcting the unique DNA methylation changes of ART. Appropriate folic acid supplementation is beneficial to the pregnancy outcome of ART, and can alleviate some adverse effects related to ART, such as reducing embryo development delay and improving DNA methylation pattern (Rahimi S et al., 2019).
• Immune response: MAT2A is an enzyme involved in carbon metabolism, which affects the latency of HIV-1 by regulating S- adenosylmethionine (SAM)-mediated metabolic pathway. MAT2A knockout enhanced the reactivation of latent HIV-1, while its overexpression inhibited the reactivation of latent HIV-1 infection. MAT2A controls the latent state of HIV-1 by regulating DNA and histone methylation in HIV-1 5'-LTR region. After MAT2A knockout, the down-regulation of these methylation markers contributes to the reactivation of HIV-1. It was also found that the level of SAM in plasma was positively correlated with the level of HIV-1 DNA in peripheral blood mononuclear cells (PBMC) of HIV-infected people receiving antiretroviral therapy, suggesting that SAM may become a biomarker of HIV latent pool (Yang X et al., 2021).

Disease association

• Metabolic reprogramming: SHIN1 is a compound that specifically inhibits SHMT1/2, and it has a highly specific inhibitory effect on LUAD cells, especially through overexpression of SHMT2. Ser90 phosphorylation of SHMT2 stabilizes SHMT2 by reducing the homology of STIP1 and ubiquitination and degradation mediated by STUB1. Ser90 dephosphorylation of SHMT2 can reduce the level of SAM in LUAD cells, which in turn leads to the decrease of m6A level in whole RNA, but has no effect on total protein or DNA methylation. SHMT2-Ser90 dephosphorylation accelerates the degradation of oncogene RNA by reducing m6A modification, thus inhibiting the occurrence of tumor (Han T et al., 2024).
• Therapeutic target: FPGS deficiency will lead to the accumulation of folic acid and methotrexate in cells, which is related to the drug resistance of methotrexate and 5- fluorouracil. C1 is an anti-folic acid drug independent of FPGS, which can selectively bind to and inhibit dihydrofolate reductase (DHFR), thus inhibiting the growth of tumor cells. Compared with methotrexate, C1 inhibits DHFR activity more effectively in FPGS-deficient cells. C1 has a stronger inhibitory effect on FPGS-deficient tumor cells, and can be used in combination with FPGS-dependent folic acid drugs to improve the therapeutic effect in clinical application. Studies have shown that the combination of C1 and traditional anti-folic acid drugs such as methotrexate or 5-FU can effectively overcome drug resistance, especially in tumor cells with FPGS deficiency (van der Krift F et al., 2023).
• Cardiovascular disease: the increase of serum homocysteine level may aggravate the damage of endothelial function through oxidative stress and the mechanism of affecting vascular dilation/contraction. The serum homocysteine level is skewed in patients, and the level of most patients is less than 30 μ mol/L. The patients with higher homocysteine level, especially higher than 9 μmol/L, have a significantly higher incidence of CMED. The serum homocysteine level of patients supplemented with B vitamins is low, and the incidence of CMED is high. For statins, the relationship between serum homocysteine level and CMED is more significant in patients who do not use statins (Ahmad A et al., 203).

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