The role of methionine synthases in fungal metabolism and virulence.

Methionine synthases (MetH) catalyse the methylation of homocysteine (Hcy) with 5-methyl-tetrahydrofolate (5, methyl-THF) acting as methyl donor, to form methionine (Met) and tetrahydrofolate (THF). This function is performed by two unrelated classes of enzymes that differ significantly in both their structures and mechanisms of action. The genomes of plants and many fungi exclusively encode cobalamin-independent enzymes (EC.2.1.1.14), while some fungi also possess proteins from the cobalamin-dependent (EC.2.1.1.13) family utilised by humans. Methionine synthase's function connects the methionine and folate cycles, making it a crucial node in primary metabolism, with impacts on important cellular processes such as anabolism, growth and synthesis of proteins, polyamines, nucleotides and lipids. As a result, MetHs are vital for the viability or virulence of numerous prominent human and plant pathogenic fungi and have been proposed as promising broad-spectrum antifungal drug targets. This review provides a summary of the relevance of methionine synthases to fungal metabolism, their potential as antifungal drug targets and insights into the structures of both classes of MetH.

Chemoproteomic Profiling of Cobalamin-Independent Methionine Synthases in Plants with a Covalent Probe.

Cobalamin-independent methionine synthases (MS) are zinc-binding methyltransferases that catalyze de novo methionine biosynthesis in higher plants, which are enzymes critically involved in seed germination and plant growth. Here, we report a highly selective sulfonyl fluoride-based probe for chemoproteomic profiling of MS enzymes in living systems of the model plant Arabidopsis thaliana, as implemented in in-gel-, mass spectrometry-, and imaging-based platforms. This probe holds promise for facilitating and accelerating fundamental research and industrial application of MS enzymes, particularly in the contexts of MS1/2-targeting herbicide screening and design.

The N-terminus of MTRR plays a role in MTR reactivation cycle beyond electron transfer.

In eucaryotic cells, methionine synthase reductase (MSR/MTRR) is capable of dominating the folate-homocysteine metabolism as an irreplaceable partner in electron transfer for regeneration of methionine synthase. The N-terminus of MTRR containing a conserved domain of FMN_Red is closely concerned with the oxidation-reduction process. Maternal substitution of I22M in this domain can bring about pregnancy with high risk of spina bifida. A new variation of Arg2del was identified from a female conceiving a fetus with spina bifida cystica. Although the deletion is far from the N-terminal FMN_Red domain, the biochemical features of the variant had been seriously investigated. Curiously, the deletion of arginine(s) of MTRR could not affect the electron relay, if only the FMN_Red domain was intact, but by degrees reduced the ability to promote MTR catalysis in methionine formation. Confirmation of the interaction between the isolated MTRR N-terminal polypeptide and MTR suggested that the native MTRR N-terminus might play an extra role in MTR function. The tandem arginines at the end of MTRR N-terminus conferring high affinity to MTR were indispensable for stimulating methyltransferase activity perhaps via triggering allosteric effect that could be attenuated by removal of the arginine(s). It was concluded that MTRR could also propel MTR enzymatic reaction relying on the tandem arginines at N-terminus more than just only implicated in electron transfer in MTR reactivation cycle. Perturbance of the enzymatic cooperation due to the novel deletion could possibly invite spina bifida in clinics.

Design, synthesis and biological activity of N5-substituted tetrahydropteroate analogs as non-classical antifolates against cobalamin-dependent methionine synthase and potential anticancer agents.

Cobalamin-dependent methionine synthase (MetH) is involved in the process of tumor cell growth and survival. In this study, a novel series of N5-electrophilic substituted tetrahydropteroate analogs without glutamate residue were designed as non-classical antifolates and evaluated for their inhibitory activities against MetH. In addition, the cytotoxicity of target compounds was evaluated in human tumor cell lines. With N5-chloracetyl as the optimum group, further structure research on the benzene substituent and on the 2,4-diamino group was also performed. Compound 6c, with IC50 value of 12.1 µM against MetH and 0.16-6.12 µM against five cancer cells, acted as competitive inhibitor of MetH. Flow cytometry studies indicated that compound 6c arrested HL-60 cells in the G1-phase and then inducted late apoptosis. The molecular docking further explained the structure-activity relationship.

Predicting the functional and structural consequences of nsSNPs in human methionine synthase gene using computational tools.

Methionine synthase encoded by the MTR gene is one of the key enzymes involved in the SAM (S- Adenosyl Methionine) cycle catalyzing the conversion of homocysteine to methionine. Methionine plays an important role in the DNA, RNA, protein, phospholipids, and neurotransmitters methylation. It also maintains serum homocysteine level and indirectly regulates de novo nucleotide synthesis and repair. The current study predicted the functional consequences of nsSNPs in human MTR gene using SIFT, PolyPhen2, PROVEAN, SNAP2, PMut, nsSNPAnalyzer, PhD-SNP, SNPs&GO, I-Mutant, MuPro, and iPTREE-STAB. The PTM sites within the protein were predicted using ModPred and the phylogenetic conservations of amino acids & conserved domains of protein were predicted using ConSurf and NCBI conserved domain search tool respectively. The protein 3D structure was generated using SPARKS-X and analyzed using RAMPAGE. Structural deviation was analyzed using TM-Score. STRING analysis was preformed to predict protein-protein interactions. D621G, G682D, V744L, V766E, and R1027W were predicted to be the most deleterious nsSNPs in MTR. R1027 was predicted to having the three PTM sites and G682 & V744 were predicted as highly conserved residues. D621G, G682D, V744L, V776E, and R1027W were predicted to be within conserved domains of methionine synthase. The G682D, V744L, V776E,  and R1027W were predicted to alter protein 3D structure. STRING predicted that methionine synthase interacting with 10 different proteins. The present study predicted D621G, G682D, V744L, V766E, and R1027W as functionally and structurally significant nsSNPs in human MTR gene. The present study can provide the significant information for further experimental analysis. Abbreviations: cblG: methylcobalamin deficiency G; MTR: 5-methyl tetrahydrofolate-homocysteine methyl transferase; MS: methionine synthase; SAM: S-adenosyl methionine; nsSNPs: non-synonymous single nucleotide polymorphisms; OMIM: online mendelian inheritance in man; NCBI: national center for biological information; SIFT: sorting intolerant from tolerant; PolyPhen2: polymorphism phenotyping 2; PROVEAN: protein variation effect analyzer; SNPs&GO: single nucleotide polymorphisms and gene ontology; PhD-SNP: predictor of human deleterious single nucleotide polymorphisms; RI: reliability index; PTM: post translational modification; SPDBV: Swiss PDB viewer; PDB: protein data bank; RMSD: root mean square deviation; STRING: search tool for the retrieval of interacting proteins.

Stabilizing intramolecular cobalt-imidazole coordination with a remote methyl group in the backbone of a cofactor B12-protein model.

This communication describes the stabilizing effect (ΔΔG° = -4 kJ mol-1) of a remote methyl group in the backbone of a cobalamin-enzyme mimic on intramolecular imidazole-cobalt coordination. For this purpose, two B12 derivatives with an appended imidazole base were synthesized and analysed with spectrophotometric pH titrations. Qualitative conformation analysis of the backbone structure suggests that a thermodynamically unfavoured gauche interaction in the base-off form of a model containing an (R)-configured CH3 group at position C176 of the linker between the corrin ring and the terminal imidazole ligand steers the base toward cobalt coordination.

Water-Mediated Carbon-Oxygen Hydrogen Bonding Facilitates S-Adenosylmethionine Recognition in the Reactivation Domain of Cobalamin-Dependent Methionine Synthase.

The C-terminal domain of cobalamin-dependent methionine synthase (MetH) has an essential role in catalyzing the reactivation of the enzyme following the oxidation of its cobalamin cofactor. This reactivation occurs through reductive methylation of the cobalamin using S-adenosylmethionine (AdoMet) as the methyl donor. Herein, we examine the molecular recognition of AdoMet by the MetH reactivation domain utilizing structural, biochemical, and computational approaches. Crystal structures of the Escherichia coli MetH reactivation domain in complex with AdoMet, the methyl transfer product S-adenosylhomocysteine (AdoHcy), and the AdoMet analogue inhibitor sinefungin illustrate that the ligands exhibit an analogous conformation within the solvent-exposed substrate binding cleft of the enzyme. AdoMet binding is stabilized by an intramolecular sulfur-oxygen chalcogen bond between the sulfonium and carboxylate groups of the substrate and by water-mediated carbon-oxygen hydrogen bonding between the sulfonium cation and the side chains of Glu1097 and Glu1128 that bracket the substrate binding cleft. AdoMet and sinefungin exhibited similar binding affinities for the MetH reactivation domain, whereas AdoHcy displayed an affinity for the enzyme that was an order of magnitude lower. Mutations of Glu1097 and Glu1128 diminished the AdoMet/AdoHcy binding selectivity ratio to approximately 2-fold, underscoring the role of these residues in enabling the enzyme to discriminate between the substrate and product. Together, these findings indicate that Glu1097 and Glu1128 in MetH promote high-affinity recognition of AdoMet and that sinefungin and potentially other AdoMet-based methyltransferase inhibitors can abrogate MetH reactivation, which would result in off-target effects associated with alterations in methionine homeostasis and one-carbon metabolism.

The folate-binding module of Thermus thermophilus cobalamin-dependent methionine synthase displays a distinct variation of the classical TIM barrel: a TIM barrel with a `twist'.

Methyl transfer between methyltetrahydrofolate and corrinoid molecules is a key reaction in biology that is catalyzed by a number of enzymes in many prokaryotic and eukaryotic organisms. One classic example of such an enzyme is cobalamin-dependent methionine synthase (MS). MS is a large modular protein that utilizes an SN2-type mechanism to catalyze the chemically challenging methyl transfer from the tertiary amine (N5) of methyltetrahydrofolate to homocysteine in order to form methionine. Despite over half a century of study, many questions remain about how folate-dependent methyltransferases, and MS in particular, function. Here, the structure of the folate-binding (Fol) domain of MS from Thermus thermophilus is reported in the presence and absence of methyltetrahydrofolate. It is found that the methyltetrahydrofolate-binding environment is similar to those of previously described methyltransferases, highlighting the conserved role of this domain in binding, and perhaps activating, the methyltetrahydrofolate substrate. These structural studies further reveal a new distinct and uncharacterized topology in the C-terminal region of MS Fol domains. Furthermore, it is found that in contrast to the canonical TIM-barrel ß8α8 fold found in all other folate-binding domains, MS Fol domains exhibit a unique ß8α7 fold. It is posited that these structural differences are important for MS function.

A proteomic approach to the mechanisms underlying activation of aluminium resistance in roots of Urochloa decumbens.

The mechanisms of extreme Al-resistance in Urochloa decumbens are not established. Full resistance expression requires a lag time of 72-96h and is preceded by a sensitive phase (24-48h) with Al-induced root growth inhibition. The aim here was to identify key processes of the activation phase of Al-resistance analysing both root exudates and comparative root proteome. Samples were taken after 0, 24 and 96h exposure to 0 or 200µM Al. Al-induced stimulation of citrate and oxalate efflux was limited to the sensitive phase. Only 11 proteins revealed Al-induced abundance differences; six were identified. After 24h, phenylalanine ammonium lyase (PAL), methionine synthase (MS), and deoxymugineic acid synthase (DMAS) decreased, while acid phosphatase (APase) abundance increased. Coincident with growth recovering, PAL and MS, but not DMAS, returned to initial levels. After 96h, γ­carbonic anhydrase (γ­CA) and adenylate kinase (AK) along with two unidentified proteins were more abundant. In conclusion, few protein changes characterize the initial response to Al in signalgrass. During the alarm phase, changes are related to P-mobilization, downregulation of Fe-acquisition, reduction of phenolic biosynthesis, and small stimulation of organic acid exudation. After recovering (resistant phase), biosynthesis of phenolics and methionine, but not Fe-mobilization are re-established. Full expression of Al-resistance is characterized by enhanced γ­CA mediating mitochondrial complex I assembly and increased AK abundance indicating higher root respiration and better provision of ADP and Mg2+ to ATP synthase, respectively. The unidentified proteins and the specific role of γ­CA in Al resistance of U. decumbens will centre future research.

Intraprotein transmethylation via a CH3-Co(iii) species in myoglobin reconstituted with a cobalt corrinoid complex.

Myoglobin reconstituted with a cobalt tetradehydrocorrin derivative, rMb(Co(TDHC)), was investigated as a hybrid model to replicate the reaction catalyzed by methionine synthase. In the heme pocket, Co(I)(TDHC) is found to react with methyl iodide to form the methylated cobalt complex, CH3-Co(III)(TDHC), although it is known that a similar nucleophilic reaction of a cobalt(i) tetradehydrocorrin complex does not proceed effectively in organic solvents. Furthermore, we observed a residue- and regio-selective transmethylation from the CH3-Co(III)(TDHC) species to the Nε2 atom of the His64 imidazole ring in myoglobin at 25 °C over a period of 48 h. These findings indicate that the protein matrix promotes the model reaction of methionine synthase via the methylated cobalt complex. A theoretical calculation provides support for a plausible reaction mechanism wherein the axial histidine ligation stabilizes the methylated cobalt complex and subsequent histidine-flipping induces the transmethylation via heterolytic cleavage of the Co-CH3 bond in the hybrid model.

In Silico Analysis of Sequence-Structure-Function Relationship of the Escherichia coli Methionine Synthase.

The molecular evolution of various metabolic pathways in the organisms can be employed for scrutinizing the molecular aspects behind origin of life. In the present study, we chiefly concerned about the sequence-structure-function relationship between the Escherichia coli methionine synthase and their respective animal homologs by in silico approach. Using homology prediction technique, it was observed that only 79 animal species showed similarity with the E. coli methionine synthase. Also, multiple sequence alignment depicted only 25 conserved patterns between the E. coli methionine synthase and their respective animal homologs. Based on that, Pfam analysis identified the protein families of 22 conserved patterns among the attained 25 conserved patterns. Furthermore, the 3D structure was generated by HHpred and evaluated by corresponding Ramachandran plot specifying 93% of the ϕ and ψ residues angles in the most ideal regions. Hence, the designed structure was established as a good quality model for the full length of E. coli methionine synthase.

Co(II)/Co(I) reduction-induced axial histidine-flipping in myoglobin reconstituted with a cobalt tetradehydrocorrin as a methionine synthase model.

A conjugate between apomyoglobin and cobalt tetradehydrocorrin was prepared to replicate the coordination behavior of cob(I)alamin in methionine synthase. X-ray crystallography reveals that the tetra-coordinated Co(I) species is formed through the cleavage of the axial Co-His93 ligation after the reduction of the penta-coordinated Co(II) cofactor in the heme pocket.

Unraveling vitamin B12-responsive gene regulation in algae.

Photosynthetic microalgae play a vital role in primary productivity and biogeochemical cycling in both marine and freshwater systems across the globe. However, the growth of these cosmopolitan organisms depends on the bioavailability of nutrients such as vitamins. Approximately one-half of all microalgal species requires vitamin B12 as a growth supplement. The major determinant of algal B12 requirements is defined by the isoform of methionine synthase possessed by an alga, such that the presence of the B12-independent methionine synthase (METE) enables growth without this vitamin. Moreover, the widespread but phylogenetically unrelated distribution of B12 auxotrophy across the algal lineages suggests that the METE gene has been lost multiple times in evolution. Given that METE expression is repressed by the presence of B12, prolonged repression by a reliable source of the vitamin could lead to the accumulation of mutations and eventually gene loss. Here, we probe METE gene regulation by B12 and methionine/folate cycle metabolites in both marine and freshwater microalgal species. In addition, we identify a B12-responsive element of Chlamydomonas reinhardtii METE using a reporter gene approach. We show that complete repression of the reporter occurs via a region spanning -574 to -90 bp upstream of the METE start codon. A proteomics study reveals that two other genes (S-Adenosylhomocysteine hydrolase and Serine hydroxymethyltransferase2) involved in the methionine-folate cycle are also repressed by B12 in C. reinhardtii. The strong repressible nature and high sensitivity of the B12-responsive element has promising biotechnological applications as a cost-effective regulatory gene expression tool.

Structural analysis of a fungal methionine synthase with substrates and inhibitors.

The cobalamin-independent methionine synthase from Candida albicans, known as Met6p, is a 90-kDa enzyme that consists of two (ßα)8 barrels. The active site is located between the two domains and has binding sites for a zinc ion and substrates L-homocysteine and 5-methyl-tetrahydrofolate-glutamate3. Met6p catalyzes transfer of the methyl group of 5-methyl-tetrahydrofolate-glutamate3 to the L-homocysteine thiolate to generate methionine. Met6p is essential for fungal growth, and we currently pursue it as an antifungal drug design target. Here we report the binding of L-homocysteine, methionine, and several folate analogs. We show that binding of L-homocysteine or methionine results in conformational rearrangements at the amino acid binding pocket, moving the catalytic zinc into position to activate the thiol group. We also map the folate binding pocket and identify specific binding residues, like Asn126, whose mutation eliminates catalytic activity. We also report the development of a robust fluorescence-based activity assay suitable for high-throughput screening. We use this assay and an X-ray structure to characterize methotrexate as a weak inhibitor of fungal Met6p.

Design, synthesis, and enzyme kinetics of novel benzimidazole and quinoxaline derivatives as methionine synthase inhibitors.

Methionine synthase catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, producing methionine and tetrahydrofolate. Benzimidazole and deazatetrahydrofolates derivatives have been shown to inhibit methionine synthase by competing with the substrate 5-methyltetrahydrofolate. In this study, a novel series of substituted benzimidazoles and quinoxalines were designed and assessed for inhibitory activity against purified rat liver methionine synthase using a radiometric enzyme assay. Compounds 3g, 3j, and 5c showed the highest activity against methionine synthase (IC50: 20 µM, 18 µM, 9 µM, respectively). Kinetic analysis of these compounds using Lineweaver-Burk plots revealed characteristics of mixed inhibition for 3g and 5c; and uncompetitive inhibition for 3j. Docking study into a homology model of the rat methionine synthase gave insights into the molecular determinants of the activity of this class of compounds. The identification of these drug-like inhibitors could lead the design of the next generation modulators of methionine synthase.

Mechanistic insights for formation of an organometallic Co-C bond in the methyl transfer reaction catalyzed by methionine synthase.

Methionine synthase (MetH) catalyzes the transfer of a methyl group from methyltetrahydrofolate (CH3-H4Folate) to the cob(I)alamin intermediate to form an organometallic Co-C bond, a reaction similar to that of CH3-H4Folate:corrinoid/iron-sulfur protein (CFeSP) methyltransferase (MeTr). How precisely it is formed remains elusive because the displacement of a methyl group from the tertiary amine is not a facile reaction. To understand the electronic structure and mechanistic details of the MetH-cob(I)alamin:CH3-H4Folate reaction complex, we applied quantum mechanics/molecular mechanics (QM/MM) computations. The hybrid QM/MM calculations reveal the traditionally assumed SN2 mechanism for formation the CH3-cob(III)alamin resting state where the activation energy barrier for the SN2 reaction was found to be ~8-9 kcal/mol, which is comparable with respect to the determined experimental rate constant. However, the possibility of an electron transfer (ET) based radical mechanism consistent with the close-lying diradical states observed from triplet and open-shell singlet states has also been suggested as an alternative, where first an electron transfer from His-on cob(I)alamin to the pterin ring of the protonated CH3-H4Folate takes place, forming the Co(II)(d(7))-pterin radical (π*)(1) diradical state, followed by a methyl radical transfer. Although the predicted energy barrier for the ET-mediated radical reaction is comparable to that of the SN2 pathway, the major advantage of ET is that a methyl radical can be transferred at a longer distance, which does not require the close proximity of two binding modules of MetH as does the SN2 type. In addition, based on the energy barrier of the transition state (TS) in both the protonated (~8-9 kcal/mol) and the unprotonated N5 (39 kcal/mol) species of the CH3-H4Folate, it can be inferred that the protonation event must takes place either prior to or during the methyl transfer reaction in a ternary complex. The results of the present study including mechanistic insights can have implications to a broad class of corrinoid-methyltransferases, which utilize a CH3-H4Folate substrate or its related analogues as methyl donor.

Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia.

The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin  (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.

Can the local enzyme scaffold act as an H-donor for a Co(I)H bond formation? The curious case of methionine synthase-bound cob(I)alamin.

The density functional calculations and analysis of the existing X-ray crystallographic data have been carried out to gain mechanistic insight into the reactivation cycle of methionine synthase (MetH) enzyme. The calculations were carried out on the cobinamide-type model complexes of cob(I)alamin (Co(I)Cbx) testing H2O and PhOH as possible ß-axial ligands. The PhOH motif was used to mimic the tyrosine (Y1139) residue that has been found in the active site of the MetH-bound cob(II)alamin (Co(II)Cbx). The calculations indicate that the ß-axial PhOH ligand forms stronger Co(I)H bonds than H2O ligand due to its better H-donor capacity. The calculated redox tuning of Co(I)H interactions on the reduction potential of Co(II)/Co(I) couple (60-800 mV vs standard hydrogen electrode (SHE)), irrespective of the ß-axial ligand considered, is significantly higher than the biological redox gap between the reduction potential of Co(II)/Co(I) couple and that of the biological reducing agents (50 mV vs SHE). The analysis of existing crystallographic data for the reactivation conformation of MetH enzyme (1K7Y (@3.0 Å); 1K98 (@3.8 Å) and 3IVA (@2.7 Å)) indicates that the Y1139 residue and the ß-axial H2O ligand in the MetH-bound Co(II)Cbx complex are equidistant from the Co(II) ion (Y1139Co(II)=3.97 Å; H2OCo(II)=3.96 Å). Taking into account that the Y1139-induced Co(I)H linkages are thermodynamically more stable than the H2O-induced ones, the present calculations suggest that the Y1139 residue may serve as the ß-axial ligand in the reactivation conformation of MetH enzyme.

Navigating the B(12) road: assimilation, delivery, and disorders of cobalamin.

The reactivity of the cobalt-carbon bond in cobalamins is the key to their chemical versatility, supporting both methyl transfer and isomerization reactions. During evolution of higher eukaryotes that utilize vitamin B12, the high reactivity of the cofactor coupled with its low abundance pressured development of an efficient system for uptake, assimilation, and delivery of the cofactor to client B12-dependent enzymes. Although most proteins suspected to be involved in B12 trafficking were discovered by 2009, the recent identification of a new protein reveals that the quest for elucidating the intracellular B12 highway is still far from complete. Herein, we review the biochemistry of cobalamin trafficking.

Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

The folate and vitamin B12-dependent enzyme methionine synthase (MS) is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin) from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.