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Methylation
Methylation
Methylation
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Methylation, in the chemical sciences, is the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These terms are commonly used in chemistry, biochemistry, soil science, and biology.

In biological systems, methylation is catalyzed by enzymes; such methylation can be involved in modification of heavy metals, regulation of gene expression, regulation of protein function, and RNA processing. In vitro methylation of tissue samples is also a way to reduce some histological staining artifacts. The reverse of methylation is demethylation.

In biology

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In biological systems, methylation is accomplished by enzymes. Methylation can modify heavy metals and can regulate gene expression, RNA processing, and protein function. It is a key process underlying epigenetics. Sources of methyl groups include S-methylmethionine, methyl folate, methyl B12, trimethylglycine.[1]

Methanogenesis

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Methanogenesis, the process that generates methane from CO2, involves a series of methylation reactions. These reactions are caused by a set of enzymes harbored by a family of anaerobic microbes.[2]

Cycle for methanogenesis, showing intermediates

In reverse methanogenesis, methane is the methylating agent.[3]

O-methyltransferases

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Main article: O-methyltransferase

A wide variety of phenols undergo O-methylation to give anisole derivatives. This process, catalyzed by such enzymes as caffeoyl-CoA O-methyltransferase, is a key reaction in the biosynthesis of lignols, percursors to lignin, a major structural component of plants.

Plants produce flavonoids and isoflavones with methylations on hydroxyl groups, i.e. methoxy bonds. This 5-O-methylation affects the flavonoid's water solubility. Examples are 5-O-methylgenistein, 5-O-methylmyricetin, and 5-O-methylquercetin (azaleatin).

Proteins

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Along with ubiquitination and phosphorylation, methylation is a major biochemical process for modifying protein function. The most prevalent protein methylations affect arginine and lysine residue of specific histones. Otherwise histidine, glutamate, asparagine, cysteine are susceptible to methylation. Some of these products include S-methylcysteine, two isomers of N-methylhistidine, and two isomers of N-methylarginine.[4]

Methionine synthase

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The methylation reaction catalyzed by methionine synthase

Methionine synthase regenerates methionine (Met) from homocysteine (Hcy). The overall reaction transforms 5-methyltetrahydrofolate (N5-MeTHF) into tetrahydrofolate (THF) while transferring a methyl group to Hcy to form Met. Methionine Syntheses can be cobalamin-dependent and cobalamin-independent: Plants have both, animals depend on the methylcobalamin-dependent form.

In methylcobalamin-dependent forms of the enzyme, the reaction proceeds by two steps in a ping-pong reaction. The enzyme is initially primed into a reactive state by the transfer of a methyl group from N5-MeTHF to Co(I) in enzyme-bound cobalamin ((Cob), also known as vitamine B12)), forming methyl-cobalamin (Me-Cob) that now contains Me-Co(III) and activating the enzyme. Then, a Hcy that has coordinated to an enzyme-bound zinc to form a reactive thiolate reacts with the Me-Cob. The activated methyl group is transferred from Me-Cob to the Hcy thiolate, which regenerates Co(I) in Cob, and Met is released from the enzyme.[5]

Heavy metals: arsenic, mercury, cadmium

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Biomethylation is the pathway for converting some heavy elements into more mobile or more lethal derivatives that can enter the food chain. The biomethylation of arsenic compounds starts with the formation of methanearsonates. Thus, trivalent inorganic arsenic compounds are methylated to give methanearsonate. S-adenosyl methionine is the methyl donor. The methanearsonates are the precursors to dimethylarsonates, again by the cycle of reduction (to methylarsonous acid) followed by a second methylation.[6] Related pathways are found in the microbial methylation of mercury to methylmercury.

Epigenetic methylation

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DNA methylation

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DNA methylation is the conversion of the cytosine to 5-methylcytosine. The formation of Me-CpG is catalyzed by the enzyme DNA methyltransferase. In vertebrates, DNA methylation typically occurs at CpG sites (cytosine-phosphate-guanine sites—that is, sites where a cytosine is directly followed by a guanine in the DNA sequence). In mammals, DNA methylation is common in body cells,[7] and methylation of CpG sites seems to be the default.[8][9] Human DNA has about 80–90% of CpG sites methylated, but there are certain areas, known as CpG islands, that are CG-rich (high cytosine and guanine content, made up of about 65% CG residues), wherein none is methylated. These are associated with the promoters of 56% of mammalian genes, including all ubiquitously expressed genes. One to two percent of the human genome are CpG clusters, and there is an inverse relationship between CpG methylation and transcriptional activity. Methylation contributing to epigenetic inheritance can occur through either DNA methylation or protein methylation. Improper methylations of human genes can lead to disease development,[10][11] including cancer.[12][13]

In honey bees, DNA methylation is associated with alternative splicing and gene regulation based on functional genomic research published in 2013.[14] In addition, DNA methylation is associated with expression changes in immune genes when honey bees were under lethal viral infection.[15] Several review papers have been published on the topics of DNA methylation in social insects.[16][17]

RNA methylation

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RNA methylation occurs in different RNA species viz. tRNA, rRNA, mRNA, tmRNA, snRNA, snoRNA, miRNA, and viral RNA. Different catalytic strategies are employed for RNA methylation by a variety of RNA-methyltransferases. RNA methylation is thought to have existed before DNA methylation in the early forms of life evolving on earth.[18]

N6-methyladenosine (m6A) is the most common and abundant methylation modification in RNA molecules (mRNA) present in eukaryotes. 5-methylcytosine (5-mC) also commonly occurs in various RNA molecules. Recent data strongly suggest that m6A and 5-mC RNA methylation affects the regulation of various biological processes such as RNA stability and mRNA translation,[19] and that abnormal RNA methylation contributes to etiology of human diseases.[20]

In social insects such as honey bees, RNA methylation is studied as a possible epigenetic mechanism underlying aggression via reciprocal crosses.[21]

Protein methylation

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Protein methylation typically takes place on arginine or lysine amino acid residues in the protein sequence.[22] Arginine can be methylated once (monomethylated arginine) or twice, with either both methyl groups on one terminal nitrogen (asymmetric dimethylarginine) or one on both nitrogens (symmetric dimethylarginine), by protein arginine methyltransferases (PRMTs). Lysine can be methylated once, twice, or three times by lysine methyltransferases. Protein methylation has been most studied in the histones. The transfer of methyl groups from S-adenosyl methionine to histones is catalyzed by enzymes known as histone methyltransferases. Histones that are methylated on certain residues can act epigenetically to repress or activate gene expression.[23][24] Protein methylation is one type of post-translational modification.

Evolution

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Methyl metabolism is very ancient and can be found in all organisms on earth, from bacteria to humans, indicating the importance of methyl metabolism for physiology.[25] Indeed, pharmacological inhibition of global methylation in species ranging from human, mouse, fish, fly, roundworm, plant, algae, and cyanobacteria causes the same effects on their biological rhythms, demonstrating conserved physiological roles of methylation during evolution.[26]

In chemistry

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The term methylation in organic chemistry refers to the alkylation process used to describe the delivery of a CH3 group.[27]

Electrophilic methylation

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Methylations are commonly performed using electrophilic methyl sources such as iodomethane,[28] dimethyl sulfate,[29][30] dimethyl carbonate,[31] or tetramethylammonium chloride.[32] Less common but more powerful (and more dangerous) methylating reagents include methyl triflate,[33] diazomethane,[34] and methyl fluorosulfonate (magic methyl). These reagents all react via SN2 nucleophilic substitutions. For example, a carboxylate may be methylated on oxygen to give a methyl ester; an alkoxide salt RO− may be likewise methylated to give an ether, ROCH3; or a ketone enolate may be methylated on carbon to produce a new ketone.

Methylation of a carboxylic acid salt and a phenol using iodomethane

The Purdie methylation is a specific for the methylation at oxygen of carbohydrates using iodomethane and silver oxide.[35]

Purdie methylation

Eschweiler–Clarke methylation

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The Eschweiler–Clarke reaction is a method for methylation of amines.[36] This method avoids the risk of quaternization, which occurs when amines are methylated with methyl halides.

The Eschweiler–Clarke reaction is used to methylate amines.
The Eschweiler–Clarke reaction is used to methylate amines.

Diazomethane and trimethylsilyldiazomethane

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Diazomethane and the safer analogue trimethylsilyldiazomethane methylate carboxylic acids, phenols, and even alcohols:

The method offers the advantage that the side products are easily removed from the product mixture.[37]

Nucleophilic methylation

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Methylation sometimes involve use of nucleophilic methyl reagents. Strongly nucleophilic methylating agents include methyllithium (CH3Li)[38] or Grignard reagents such as methylmagnesium bromide (CH3MgX).[39] For example, CH3Li will add methyl groups to the carbonyl (C=O) of ketones and aldehyde.:

Methylation of acetone by methyl lithium

Milder methylating agents include tetramethyltin, dimethylzinc, and trimethylaluminium.[40]

See also

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Biology topics

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Organic chemistry topics

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Methylation

View on Grokipedia
from Grokipedia
Methylation is a fundamental biochemical process involving the covalent addition of a (–CH₃) to diverse molecular substrates, primarily catalyzed by methyltransferase enzymes that utilize S-adenosylmethionine (SAM) as the universal methyl donor. This reversible post-translational or alters the physicochemical properties of targets, such as charge, stability, and binding affinity, without changing the underlying genetic sequence. In , methylation manifests across the central dogma, profoundly influencing , cellular identity, and . , predominantly at the C5 position of cytosine residues in CpG dinucleotides (accounting for 60–80% of the approximately 32.28 million such sites in the ), serves as a heritable epigenetic mark that typically represses transcription by recruiting repressive protein complexes and compacting . , including various types such as the abundant N⁶-methyladenosine (m⁶A) on , regulates processes like splicing, nuclear , stability, and translation efficiency. , occurring mainly on and residues in histones and non-histone proteins, fine-tunes architecture, , and enzymatic activities. These modifications are dynamically balanced by "writers" (methyltransferases), "erasers" (demethylases), and "readers" (effector proteins that interpret the marks), enabling cells to respond to developmental cues, environmental stressors, and metabolic states. Aberrant methylation patterns, often driven by genetic , aging, or external factors like diet and toxins, contribute to diseases such as cancer (e.g., global hypomethylation promoting activation) and neurological disorders (e.g., altered in neurodegeneration). Ongoing research highlights methylation's therapeutic potential, including inhibitors of methyltransferases like for cancer treatment.

Fundamentals

Definition and Basic Mechanisms

Methylation is a fundamental in involving the transfer of a (CH₃) from a donor to a substrate, thereby modifying its structure or properties. This process is a specific type of where the replaces a or attaches to a nucleophilic site on the substrate. The basic mechanism of methylation typically proceeds via an , in which the substrate acts as a attacking the electrophilic carbon of the methyl donor, such as a methyl halide (CH₃X), leading to inversion of configuration if applicable and displacement of the (X). Alternatively, in cases where the methyl group is activated, an electrophilic attack by the methyl donor on a nucleophilic substrate can occur. The general reaction can be represented as: \ceR−Nu+CH3−X−>R−Nu−CH3+X−\ce{R-Nu + CH3-X -> R-Nu-CH3 + X-} where R-Nu represents the nucleophilic substrate and X is a leaving group like iodide./Chapters/Chapter_07:_Alkyl_Halides_and_Nucleophilic_Substitution/7.10:_The_SN2_Mechanism) Methylation reactions form various types of bonds depending on the substrate's nucleophilic site, including C-methylation (attachment to carbon, e.g., alkylation of enolates to form new C-C bonds), N-methylation (attachment to nitrogen, e.g., formation of N-methylamines from amines), O-methylation (attachment to oxygen, e.g., synthesis of anisole from phenol), and S-methylation (attachment to sulfur, e.g., formation of methyl sulfides from thiols)./09%3A_Substitution_and_Elimination_Reactions_of_Alkyl_Halides/9.14%3A_Biological_Methylating_Reagents) Thermodynamically, methylation reactions are influenced by bond dissociation energies (BDEs), which indicate bond stability under homolytic cleavage. For instance, the C-H BDE in is approximately 105 kcal/mol, while the C-C BDE in (representing a C-CH₃ bond) is about 90 kcal/mol, suggesting that C-CH₃ bonds are relatively weaker and less stable compared to C-H bonds. This difference affects the energetics of radical-mediated processes but underscores the overall feasibility of ionic methylation pathways in synthetic applications.

Methyl Group Donors and Acceptors

In biological systems, S-adenosylmethionine (SAM) serves as the primary and universal donor for a wide array of methyltransfer reactions, facilitated by its center which provides the electrophilic . This cofactor is essential for enzymatic processes involving the transfer of the methyl unit to diverse substrates, ensuring controlled and specific reactivity under physiological conditions. In , common synthetic methyl donors include (DMS) and methyl iodide (CH3I), which act as electrophilic agents in reactions. DMS, a diester of , is widely used in industrial-scale methylations due to its availability and reactivity, though it requires careful handling owing to its high and classification as a probable based on animal studies showing nasal and respiratory tumors. CH3I, a simple alkyl , offers superior reactivity as a methylating agent, often preferred in laboratory settings for its efficiency in SN2-type displacements, but it poses significant risks as a capable of causing delayed neuropsychiatric symptoms even at low exposure levels. For milder applications, such as selective O-methylation of under microwave-assisted conditions, (TMAH) functions as a quaternary ammonium-based donor, providing a safer alternative with reduced compared to traditional agents. The reactivity of these donors follows a general scale based on the electrophilicity of the methyl carbon and the quality of the : CH3I > DMS > SAM, reflecting the ease of nucleophilic attack where is an excellent , methyl intermediate, and the dimethylsulfonium moiety in SAM tuned for enzymatic specificity rather than high intrinsic reactivity. Stability varies accordingly; SAM is labile in aqueous solutions and prone to , necessitating biological regeneration systems, while DMS and CH3I are more stable but volatile and corrosive. Methyl group acceptors are typically nucleophilic sites that attack the electrophilic carbon of the donor. Common examples include carboxylates, which form methyl esters upon O-methylation; amines, leading to N-methylated products like tertiary amines or quaternary salts; thiols, yielding sulfides through S-methylation; and carbon nucleophiles such as enolates, enabling C-methylation at alpha positions of carbonyl compounds. Specificity in donor-acceptor interactions is governed by steric hindrance and electronic effects. Bulky donors like SAM favor less hindered nucleophiles due to its large moiety, while small, linear CH3I accommodates sterically demanding sites; electronically, hard nucleophiles (e.g., carboxylates) pair better with harder electrophiles like DMS, whereas softer thiols align with CH3I's polarizable leaving group, influencing selectivity in mixed-substrate reactions.
DonorRelative ReactivityPrimary ApplicationsToxicity/Stability Notes
S-Adenosylmethionine (SAM)Low (enzymatic control)Biological methylations (e.g., DNA, proteins)Labile in water; non-toxic in vivo
Dimethyl sulfate (DMS)MediumIndustrial O- and N-methylationsProbable carcinogen; corrosive, stable
Methyl iodide (CH3I)HighLaboratory alkylations (broad scope)Neurotoxin; volatile, stable
Tetramethylammonium (TMA)Low-mildSelective O-methylation of phenolsLower toxicity; stable solid

Methylation in Biology

Methanogenesis

Methanogenesis is an anaerobic process carried out exclusively by , wherein (CO₂) or is reduced to (CH₄) through a series of methylation and reduction steps, culminating in the action of methyl-coenzyme M reductase (Mcr). This pathway relies heavily on methylation reactions to form key intermediates, such as methyl-coenzyme M (CH₃-S-CoM), which serves as the direct precursor to . In hydrogenotrophic methanogenesis, CO₂ is fixed and sequentially reduced via the Wood-Ljungdahl pathway to formyl- and then methyl-tetrahydromethanopterin (CH₃-H₄MPT), followed by transfer to coenzyme M. Acetoclastic methanogenesis involves the cleavage of to generate a methyl group that is transferred to coenzyme M, while methylotrophic pathways directly utilize methylated substrates like or methylamines for methyl group incorporation. Overall, the process can be represented by the net for CO₂ reduction: CO2+8H++8e−→CH4+2H2O\text{CO}_2 + 8\text{H}^+ + 8\text{e}^- \rightarrow \text{CH}_4 + 2\text{H}_2\text{O} with methylation intermediates playing a central role in conserving energy and channeling electrons. Central to these pathways are methyltransferases, which catalyze the transfer of methyl groups between cofactors and substrates in methanogenic archaea. The membrane-bound Mtr complex (MtrA-H), a multi-subunit enzyme, is particularly crucial in hydrogenotrophic and methyl-reducing methanogenesis, where it transfers the methyl group from CH₃-H₄MPT to coenzyme M (CoM-SH), forming CH₃-S-CoM while generating a sodium ion gradient for ATP synthesis. In methylotrophic methanogenesis, substrate-specific methyltransferases, such as MtaA (for methanol) or MttA (for trimethylamine), initially transfer methyl groups to corrinoid proteins (e.g., corrinoid/iron-sulfur protein, CFeSP), which then donate the methyl to CoM via activating enzymes. The final step in all pathways involves Mcr, a nickel-containing enzyme that reduces CH₃-S-CoM with coenzyme B (CoB-SH) to release CH₄ and the heterodisulfide CoM-S-S-CoB, which is subsequently reduced to regenerate the thiols. These enzymes ensure efficient methyl handling under strictly anaerobic conditions. The detailed pathway begins with methyl group formation: in CO₂-reducing methanogenesis, CO₂ is activated by formylmethanofuran dehydrogenase and reduced stepwise to CH₃-H₄MPT via a series of dehydrogenases and reductases, incorporating eight electrons from H₂ or formate. This methyl intermediate is then transferred by the Mtr complex to CoM, forming CH₃-S-CoM. In acetoclastic routes, acetate is activated to acetyl-CoA by the acetyl-CoA synthetase complex, cleaved to CH₃-H₄MPT and CO, with the methyl group routed to CoM. For methylotrophic processes, exogenous methyl donors like methanol (CH₃OH) are oxidized to formaldehyde and then methylated onto H₄MPT or directly transferred via corrinoid-mediated steps. Methane release occurs when Mcr reduces CH₃-S-CoM, with electrons from electron-bifurcating hydrogenases (e.g., Eha or Ehb) driving the overall reduction. These methylation steps not only facilitate carbon flow but also couple catabolism to energy conservation through ion gradients. Ecologically, methanogenesis plays a pivotal role in the global by producing , a potent to which methanogenesis contributes the majority (approximately 60%) of total global emissions (as of the ), from both sources like wetlands (about 40% of total) and anthropogenic sources such as guts and landfills. thrive in extremophilic anaerobic habitats, including guts where they convert H₂ and CO₂ from into CH₄ (accounting for about 30% of anthropogenic emissions), freshwater and marine sediments, and hypersaline environments. In wetlands, methylotrophic methanogens dominate where methylated compounds from decaying fuel up to 90% of local production, influencing and feedback loops. This process links anaerobic decomposition to biogeochemical cycling, with methanogens mitigating H₂ accumulation while exacerbating effects.

Metabolic Pathways

One-carbon metabolism encompasses interconnected pathways that facilitate the transfer of single-carbon units for biosynthetic processes, primarily through the folate and methionine cycles, which shuttle methyl groups essential for cellular function. The folate cycle involves the interconversion of tetrahydrofolate (THF) derivatives, starting with the reduction of methylene-THF to 5-methyl-THF by methylenetetrahydrofolate reductase (MTHFR), providing the methyl donor for methionine regeneration. The methionine cycle, in turn, utilizes S-adenosylmethionine (SAM) as the primary methyl donor, which is formed from methionine and ATP by methionine adenosyltransferase (MAT); after donating its methyl group, SAM becomes S-adenosylhomocysteine (SAH), which is hydrolyzed to homocysteine, closing the cycle. These pathways are conserved across eukaryotes and prokaryotes, ensuring efficient methyl group economy for amino acid, nucleotide, and polyamine synthesis. Central to the methionine cycle is the remethylation of homocysteine to methionine, primarily catalyzed by B12-dependent methionine synthase (MTR). This enzyme transfers a methyl group from 5-methyl-THF to homocysteine, regenerating THF for the folate cycle: Homocysteine+5-CH3-THF→Methionine+THF\text{Homocysteine} + 5\text{-CH}_3\text{-THF} \rightarrow \text{Methionine} + \text{THF} MTR is a modular protein with distinct domains: an N-terminal cobalamin-binding region that activates vitamin B12 as methylcobalamin, a central catalytic domain facilitating methyl transfer via redox reactions involving cob(II)alamin intermediates, and a C-terminal domain for homocysteine binding. The mechanism proceeds through oxidative activation of methylcobalamin, followed by nucleophilic attack by homocysteine on the cobalt-methyl bond, with reactivation by methionine synthase reductase to prevent inactivation. An alternative pathway, particularly in liver and kidney, employs betaine-homocysteine methyltransferase (BHMT), a zinc-dependent enzyme that uses betaine (trimethylglycine) as the methyl donor, converting it to dimethylglycine without requiring folate or B12. BHMT's catalytic mechanism involves zinc coordination to homocysteine's sulfur, polarizing the betaine methylene group for transfer. Regulation of these pathways maintains metabolic balance, with SAM acting as a key allosteric inhibitor. High SAM levels feedback-inhibit MTHFR, preventing futile cycling and conserving , while also inhibiting BHMT to prioritize folate-dependent remethylation under nutrient sufficiency. Nutritional deficiencies disrupt this ; or B12 shortages impair activity, leading to accumulation (), which is linked to cardiovascular and neurological disorders. For instance, B12 deficiency traps as 5-methyl-THF, halting the cycle and exacerbating elevation. Recent advances highlight dysregulation in contexts, particularly cancer metabolism. Overexpression of MAT2A, the isoform producing SAM in non-hepatic tissues, supports rapid proliferation by enhancing methyl availability for synthesis and epigenetic modifications in tumors like and gastric cancers. Studies from the 2020s also underscore dietary influences; for example, diets rich in one-carbon nutrients (e.g., , ) from traditional sources like fermented improve global methylation status and mitigate metabolic imbalances in at-risk populations.

Heavy Metal Detoxification

Methylation serves as a key biological mechanism for detoxifying by enzymatically adding methyl groups to reduce , enhance volatility, or facilitate . This process primarily involves S-adenosylmethionine (SAM) as the methyl donor, transferring methyl groups to metal ions or compounds via specialized methyltransferases. In organisms ranging from microbes to humans, methylation transforms bioavailable inorganic forms into less soluble or more excretable organic species, playing a critical role in mitigating environmental and risks associated with metals like and mercury. For , detoxification occurs through sequential methylation of inorganic [As(III)] to methylated forms, primarily catalyzed by the arsenite S-adenosylmethionine methyltransferase (ArsM in microbes or AS3MT in humans). This SAM-dependent pathway converts As(III) to monomethylarsonite (MMA(III)), then dimethylarsenite (DMA(III)), and ultimately volatile trimethylarsine (TMA), which dissipates from cells, reducing intracellular accumulation. Pentavalent products like dimethylarsenate (DMA(V)) and trimethylarsine oxide (TMAO) are significantly less toxic—up to 1,000-fold compared to As(III)—as they do not readily bind thiols or disrupt cellular processes. In microbial communities, ArsM homologs are widespread, enabling arsenic volatilization as a survival strategy under oxidative conditions. Microbial arsenic methylation drives biogeochemical cycling, influencing environmental speciation and human exposure. In anaerobic soils like rice paddies, methylating outcompete demethylators in younger fields, producing toxic DMA and dimethymonothioarsenate (DMMTA) that accumulate in grains, exacerbating in regions like the and . This leads to dietary risks, with methylated detected in human urine from consumption. Recent studies highlight how and microbial balance govern these dynamics, with high methylator-to-demethylator ratios predicting elevated in crops and straighthead in . Genetic variations in the AS3MT modulate detoxification efficiency, affecting the proportion of methylated metabolites excreted in . Rare protein-altering variants, identified in multi-population studies, reduce dimethylarsenic percentage (DMA%) by 6–10%, leading to higher retention of toxic inorganic forms and increased risks. A across Bangladeshi, Mexican, and Native American cohorts confirmed these variants' consistent impact, independent of ancestry or exposure levels, underscoring AS3MT's role in personalized susceptibility. Mercury methylation, predominantly microbial, contrasts by increasing toxicity through biomagnification but serves as a detox strategy in some contexts by converting inorganic Hg(II) to methylmercury (MeHg). The hgcAB gene cluster encodes HgcA (a corrinoid methyltransferase) and HgcB (a ferredoxin), facilitating SAM-dependent transfer of a methyl group to Hg(II), forming the lipophilic MeHg that can volatilize or enter food webs. HgcA's cobalt center and HgcB's iron-sulfur clusters enable electron transfer for methylation, with the process enhanced under anaerobic conditions in sediments and soils. While this reduces Hg(II) toxicity in methylating microbes, MeHg bioaccumulates in aquatic ecosystems, concentrating up to a million-fold in predatory fish. Health implications of mercury methylation are severe due to MeHg's , readily crossing the blood-brain barrier and to cause developmental delays, cognitive deficits, and cardiovascular effects in humans. Environmental factors like dissolved and sulfate-reducing amplify methylation rates, linking industrial Hg emissions to global contamination. Recent advances identify over 60 methylating strains, with hgcAB abundance correlating directly to MeHg production in diverse habitats, including rice paddies. Cadmium detoxification via methylation is less common than for or mercury, primarily relying on conjugation with (GSH) to form excretable complexes rather than direct methylation. GSH chelates Cd(II), enabling vacuolar sequestration or efflux in cells, with SAM indirectly supporting GSH synthesis through metabolic pathways. This mechanism predominates in and mammals exposed to Cd, reducing but without the volatility seen in arsenic methylation.

Evolutionary Perspectives

Methylation processes trace their origins to the (LUCA), where one-carbon played a central role in carbon assimilation and production, with methyl groups essential for pathways like the route that fixed CO₂ using H₂ in anaerobic hydrothermal environments approximately 4 billion years ago. In , emerged as an early innovation around 3.5 billion years ago, relying on methylation steps via enzymes such as methyl-coenzyme M reductase to convert methylated substrates into , a process that dominated the . SAM synthetases, which generate the universal methyl donor S-adenosylmethionine (SAM), are highly conserved across all domains of life—bacteria, archaea, and eukaryotes—reflecting their presence in LUCA and involvement in at least 10–20 ancient SAM-binding proteins for methylation of nucleic acids and other biomolecules. While core metabolic methylation machinery remains broadly conserved, epigenetic components like DNA methyltransferases (DNMTs) diverged significantly, with eukaryotes developing specialized DNMT1 for maintenance methylation and DNMT3 for de novo activity, whereas prokaryotes primarily employ them in defense-related restriction-modification systems. These methylation mechanisms conferred adaptive advantages by enhancing metabolic flexibility in nutrient-limited settings, such as the early anoxic , where one-carbon pathways allowed efficient recycling of scarce carbon resources and co-evolved with cycles to supply methyl groups for and stress responses. Comparative genomic analyses reveal of methanogenic methyltransferases, such as and Hmd genes, among archaeal lineages like Euryarchaeota and TACK superphylum, facilitating the spread of beyond its ancestral host. biomarkers, including biologically derived inclusions from 3.4–3.5 billion-year-old rocks, corroborate these ancient origins, while 2020s phylogenomic studies document dynamic of methylation enzymes, including independent losses of DNMTs in lineages like , highlighting ongoing adaptations across life's domains.

Epigenetic Methylation

DNA Methylation

refers to the covalent addition of a to the fifth carbon of residues in DNA, predominantly at CpG dinucleotides, resulting in (5mC). This modification serves as a key epigenetic mechanism that influences , structure, and genomic stability without changing the underlying sequence. In vertebrates, patterns are dynamically established and maintained during development, contributing to and tissue-specific gene regulation. Aberrant methylation is implicated in various diseases, particularly cancer, where it can lead to inappropriate or activation. The primary mechanism of DNA methylation involves DNA methyltransferases (DNMTs), which transfer a methyl group from S-adenosylmethionine (SAM), the universal methyl donor, to . Maintenance methylation is primarily catalyzed by , which recognizes hemimethylated DNA after replication and methylates the newly synthesized strand to preserve existing patterns. De novo methylation, establishing new patterns, is mediated by DNMT3A and DNMT3B, often in complex with their regulatory subunit DNMT3L. The enzymatic reaction can be represented as: Cytosine+SAM→5-methylcytosine+SAH\text{Cytosine} + \text{SAM} \rightarrow 5\text{-methylcytosine} + \text{SAH} where SAH is S-adenosylhomocysteine. This process typically occurs at CpG sites, though non-CpG methylation is prevalent in specific contexts, such as CHG and CHH motifs in plants for transposon silencing and gene body methylation in mammalian neurons. Biologically, DNA methylation facilitates gene silencing by recruiting methyl-binding proteins like MeCP2, which in turn associate with histone deacetylases and other repressors to compact chromatin. It plays essential roles in genomic imprinting, where parent-of-origin-specific methylation marks at imprinting control regions (ICRs) silence one allele, ensuring monoallelic expression of genes like IGF2 and H19. In X-chromosome inactivation (XCI), methylation spreads across the inactive X chromosome in female mammals, maintaining dosage compensation by silencing most X-linked genes through promoter hypermethylation following Xist RNA coating. During embryonic development, global demethylation occurs in the zygote and inner cell mass, followed by waves of de novo methylation to establish tissue-specific patterns. Detection of DNA methylation relies on methods that distinguish 5mC from unmethylated cytosine. Bisulfite sequencing, the gold standard, treats DNA with , which deaminates unmethylated cytosines to uracils (read as thymines during sequencing) while leaving 5mC intact, allowing site-specific resolution at single-base level. Genome-wide approaches include methylation arrays, such as Illumina's Infinium platforms, which interrogate hundreds of thousands of CpG sites via hybridization probes. These techniques have revealed dynamic methylation landscapes, including active demethylation via ten-eleven translocation (TET) enzymes, discovered in 2009, which oxidize 5mC to (5hmC) and further to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), facilitating and erasure of methylation marks. In cancer, aberrant hypermethylation of CpG islands in tumor suppressor gene promoters, such as p16INK4a and MLH1, leads to their transcriptional silencing, promoting oncogenesis by disabling cell cycle control and DNA repair. This epigenetic alteration is observed in up to 80% of solid tumors and leukemias, often cooperating with genetic mutations. Therapeutic strategies target these aberrations with DNA methyltransferase inhibitors like azacitidine, a cytidine analog incorporated into RNA and DNA, where it covalently traps DNMTs during methylation attempts, depleting enzyme levels and inducing global hypomethylation to reactivate silenced genes. Azacitidine is FDA-approved for myelodysplastic syndromes and acute myeloid leukemia, with response rates around 50% in clinical trials. Recent advances highlight non-CpG methylation's role in the brain, where it accumulates in neuronal gene bodies during postnatal development, influencing synaptic plasticity and binding to MeCP2 for repression, and in plants, where it constitutes over 30% of total methylation for heterochromatin maintenance.

RNA Methylation

RNA methylation refers to the addition of methyl groups to RNA molecules, a key epitranscriptomic modification that dynamically regulates RNA processing, stability, and function without altering the genetic code. The most prevalent form is N6-methyladenosine (m6A), primarily occurring on messenger RNA (mRNA) in eukaryotic cells, where it influences gene expression at multiple levels. Other notable modifications include 5-methylcytosine (m5C) on transfer RNA (tRNA), which supports tRNA stability and translation accuracy. These modifications are reversible and orchestrated by a machinery of enzymes that install, remove, and interpret them, paralleling epigenetic mechanisms in DNA but with greater dynamism suited to RNA's transient nature. The core mechanism of RNA methylation involves the transfer of a methyl group from S-adenosylmethionine (SAM), the universal methyl donor, to specific nucleotide residues via SAM-dependent methyltransferases. For m6A, this occurs at the nitrogen-6 position of adenosine, typically in the consensus sequence RRACH (R = purine, A = adenosine, C = cytosine, H = A/C/U), often near stop codons or 3' untranslated regions of mRNA. m6A was first identified in the 1970s as an abundant modification in eukaryotic mRNA, but the full enzymatic machinery was elucidated in the 2010s, marking the rise of epitranscriptomics. Writers, such as the METTL3-METTL14-WTAP core complex, catalyze m6A installation in the nucleus, while erasers like fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) remove it in a demethylation reaction dependent on α-ketoglutarate and iron. Readers, including YTH domain-containing proteins (e.g., YTHDF1/2/3 and YTHDC1/2), bind m6A-modified RNA to mediate downstream effects, such as nuclear export or decay. Similarly, m5C on tRNA is installed by NOP2/Sun domain family member 2 (NSUN2) and recognized by readers like ALYREF to facilitate mRNA export. Functionally, m6A promotes mRNA decay by recruiting YTHDF2 to target transcripts for deadenylation and degradation, thereby fine-tuning ; it also enhances efficiency via YTHDF1 recruitment to the mRNA cap and influences through interactions with splicing factors like SRSF3. In tRNA, m5C stabilizes the molecule against endonucleolytic cleavage and ensures precise codon recognition during protein synthesis. These roles extend to viral RNAs, where host m6A machinery can methylate transcripts to restrict replication, but viral proteins sometimes evade or hijack this for immune escape. Recent research has expanded to circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs), revealing m6A and m5C modifications that regulate circRNA stability in neuronal differentiation and lncRNA-mediated interactions in cancer, fueling the epitranscriptomics boom with advanced mapping techniques like m6A-seq. Dysregulation of methylation links to , with FTO variants associated with altered m6A levels contributing to through hypothalamic regulation of appetite genes like . In cancer, upregulated METTL3 drives oncogenic mRNA stabilization, while ALKBH5 loss promotes tumor growth; thus, m6A inhibitors targeting writers or erasers show therapeutic promise, such as METTL3 small-molecule blockers enhancing sensitivity in models. Ongoing studies highlight potential in targeting lncRNA m6A for precision , underscoring RNA methylation's role in etiology and intervention.

Protein Methylation

Protein methylation refers to the covalent addition of one or more methyl groups to specific residues, primarily and , within proteins, serving as a key that regulates diverse cellular processes. This modification occurs on both and non-histone proteins, with residues capable of mono-, di-, or tri-methylation and residues typically undergoing mono- or di-methylation in asymmetric or symmetric forms. The primary enzymes catalyzing methylation are the protein arginine methyltransferases (PRMTs), a family of nine enzymes divided into types I (producing asymmetric di-methylarginine, e.g., PRMT1), type II (symmetric di-methylarginine, e.g., PRMT5), and type III (monomethylarginine, e.g., PRMT7). methylation is predominantly mediated by SET-domain-containing methyltransferases, including families like SET1/ for H3K4, SUV39 for H3K9, and EZ for H3K27, which exhibit varying degrees of processivity depending on the substrate and cellular context. In , methylation patterns form part of the "histone code," where specific marks dictate structure and transcriptional outcomes by serving as docking sites for reader proteins. Trimethylation of at 4 (H3K4me3) is a hallmark of active promoters, facilitating recruitment of remodelers like the complex and promoting gene activation. In contrast, trimethylation of H3 at 27 (H3K27me3), deposited by the Polycomb repressive complex 2 (PRC2), correlates with transcriptional repression and heterochromatin , often at developmental genes. These opposing marks enable bivalent states, balancing activation and repression during cell differentiation. Non-histone targets extend this regulatory layer; for example, SET7/9 methylates at 372 to stabilize it and enhance DNA damage-induced transcription, while methylation of at 142 by SET7/9 promotes its degradation, linking protein methylation to epigenetic . The biochemical mechanism of protein methylation is strictly dependent on S-adenosylmethionine (SAM), where the enzyme's positions the substrate residue to accept the from SAM, yielding S-adenosylhomocysteine as a byproduct and enabling precise control over modification extent. Reversibility is achieved through demethylation: -specific demethylase 1 (LSD1), a flavin adenine dinucleotide-dependent amine oxidase, removes mono- and di-methyl groups from via oxidative deamination, generating and . The jumonji C (JmjC)-domain-containing (JMJD) family, comprising over 30 members, employs a 2-oxoglutarate and Fe(II)-dependent mechanism to demethylate mono-, di-, or tri-methyl across a broader substrate range, including via JMJD3. These pathways ensure dynamic turnover of methyl marks in response to cellular signals. Functionally, protein methylation orchestrates by modulating positioning and accessibility—, for instance, recruits ATP-dependent remodelers to open for transcription—while in non-histone contexts, it fine-tunes pathways, such as PRMT1-mediated arginine methylation of transcription factors altering their localization and activity. Dysregulation of these processes contributes to neurodegeneration; hypomethylation of at residues, driven by reduced PRMT activity, promotes tau hyperphosphorylation, aggregation, and formation in models. Recent insights from the 2020s reveal non-histone methylation's role in immunity, where PRMT1 methylates heterogeneous nuclear ribonucleoprotein A1 to regulate splicing and responses in macrophages. Additionally, methylation influences liquid-liquid phase-separated condensates, as seen in SET7/9-modified proteins that modulate condensate assembly for efficient in immune and stress responses.

Methylation in Chemistry

Electrophilic Methylation

Electrophilic methylation refers to a class of reactions in where a methyl cation equivalent (CH₃⁺) is transferred from an electrophilic donor to a nucleophilic substrate, most commonly involving oxygen or atoms as the nucleophilic sites. This process is fundamental for introducing methyl groups in ethers, esters, and amines, enabling the modification of functional groups under controlled conditions. The electrophilic nature of the methyl donor ensures regioselective attack at electron-rich centers, distinguishing it from other strategies. Common reagents for electrophilic methylation include methyl halides such as (CH₃I) and (CH₃Br), which act as direct sources of the methyl . exhibits the highest reactivity among these due to the excellent leaving group ability of , following the order CH₃I > CH₃Br > CH₃Cl, while and are far less effective. (CH₂N₂) serves as another key reagent, generating the methyl through loss of , particularly suited for sensitive substrates. ((CH₃)₂SO₄) is also widely employed, offering high reactivity for O- and N-methylations via sequential transfer of its two methyl groups. Side reactions, such as over-methylation in polyfunctional molecules or competing pathways with longer-chain halides, can occur, necessitating careful control of and reaction conditions. In applications, electrophilic methylation is prominently used for the esterification of carboxylic acids, exemplified by the reaction with , which proceeds under mild, neutral conditions without requiring additional catalysts beyond trace . R-COOH+CH2N2→R-COOCH3+N2\text{R-COOH} + \text{CH}_2\text{N}_2 \rightarrow \text{R-COOCH}_3 + \text{N}_2 This method is highly efficient for labile compounds, yielding methyl esters in high purity. For O-methylation, are converted to derivatives using in the presence of a base like , while N-methylation of amines employs similar conditions to form tertiary amines selectively. finds utility in industrial-scale etherifications, such as in the synthesis of derivatives. These methods offer advantages in versatility and compatibility with acid-sensitive substrates, enabling clean transformations at . However, limitations include poor selectivity in multifunctional systems and the inherent hazards of the reagents: is explosive and highly toxic, requiring specialized handling, while is carcinogenic and corrosive. Methyl halides, though less hazardous, pose risks of volatility and lachrymatory effects, prompting the development of safer alternatives in modern synthesis.

Nucleophilic Methylation

Nucleophilic methylation refers to reactions in which a or its synthetic equivalent serves as a , attacking electrophilic centers to form new carbon-carbon bonds, most commonly in C-methylation processes. This approach is fundamental in for introducing methyl groups at carbonyl carbons or other electrophiles like alkyl halides. Key reagents for generating the methyl nucleophile include organometallic species such as the methyl (CH₃MgBr), prepared by reacting methyl bromide with magnesium in or THF, and (CH₃Li), obtained from methyl iodide and metal in or . These compounds exhibit strong nucleophilicity due to the polarized carbon-metal bond, where the methyl group behaves as R⁻. strategies, such as those employing 1,3-dithiane anions, provide masked nucleophilic equivalents that can facilitate selective methylation by inverting the reactivity of typically electrophilic carbonyls, allowing addition to alkyl halides or other electrophiles in a controlled manner. A primary application is the to aldehydes for synthesizing secondary alcohols, which can serve as precursors to ketones upon oxidation. The reaction proceeds via nucleophilic attack on the carbonyl carbon, forming an intermediate that yields the alcohol upon aqueous : R-CHO+CH3MgBr→R-CH(OMgBr)CH3→H3O+R-CH(OH)CH3\text{R-CHO} + \text{CH}_3\text{MgBr} \rightarrow \text{R-CH(OMgBr)CH}_3 \xrightarrow{\text{H}_3\text{O}^+} \text{R-CH(OH)CH}_3
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