Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo.

One-carbon flux into methionine and S-adenosylmethionine (AdoMet) is thought to be controlled at the methylenetetrahydrofolate reductase (MTHFR) step. Mammalian MTHFRs are inhibited by AdoMet in vitro, and it has been proposed that methyl group biogenesis is regulated in vivo by this feedback loop. In this work, we used metabolic engineering in the yeast Saccharomyces cerevisiae to test this hypothesis. Like mammalian MTHFRs, the yeast MTHFR encoded by the MET13 gene is NADPH-dependent and is inhibited by AdoMet in vitro. This contrasts with plant MTHFRs, which are NADH-dependent and AdoMet-insensitive. To manipulate flux through the MTHFR reaction in yeast, the chromosomal copy of MET13 was replaced by an Arabidopsis MTHFR cDNA (AtMTHFR-1) or by a chimeric sequence (Chimera-1) comprising the yeast N-terminal domain and the AtMTHFR-1 C-terminal domain. Chimera-1 used both NADH and NADPH and was insensitive to AdoMet, supporting the view that the C-terminal domain is responsible for AdoMet inhibition. Engineered yeast expressing Chimera-1 accumulated 140-fold more AdoMet and 7-fold more methionine than did the wild-type and grew normally. Yeast expressing AtMTHFR-1 accumulated 8-fold more AdoMet. This is the first in vivo evidence that the AdoMet sensitivity and pyridine nucleotide preference of MTHFR control methylneogenesis. (13)C labeling data indicated that glycine cleavage becomes a more prominent source of one-carbon units when Chimera-1 is expressed. Possibly related to this shift in one-carbon fluxes, total folate levels are doubled in yeast cells expressing Chimera-1.     

Quantitative analysis of pathways of methionine metabolism and their regulation in lemna

Individual rates of metabolism of the sulfur, methyl, and 4-carbon moieties of methionine were estimated in Lemna paucicostata Hegelm. 6746 growing under standard conditions, and used to quantitate pathways of methionine metabolism. Synthesis of S-adenosylmethionine (AdoMet) is the major pathway for methionine metabolism, with over 4 times as much methionine metabolized by this route as accumulates in protein. More than 90% of AdoMet is used for transmethylation. Methyl groups of choline, phosphatidylcholine, and phosphorylcholine are major end products of this pathway. Flux through methylthio recycling is about one-third the amount of methionine accumulating in protein. Spermidine synthesis accounts for at least 60% of the flux through methylthio recycling. The results obtained here, together with those reported for methionine-supplemented plants (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839), indicate that methionine supplementation reduced methylneogenesis by no more than the small amount expected from the reduced entry of sulfate sulfur into methionine (Giovanelli, Mudd, Datko, 1985 Plant Physiol 77: 450-455). Methionine supplementation had no significant effect on transmethylation or methylthio recycling. The combined data provide the first comprehensive estimates of the quantitative relationships of major pathways for methionine metabolism and their control in plants.     

The metabolic burden of creatine synthesis

Creatine synthesis is required in adult animals to replace creatine that is spontaneously converted to creatinine and excreted in the urine. Additionally, in growing animals it is necessary to provide creatine to the expanding tissue mass. Creatine synthesis requires three amino acids: glycine, methionine and arginine, and three enzymes: l-arginine:glycine amidinotransferase (AGAT), methionine adenosyltransferase (MAT) and guanidinoacetate methyltransferase (GAMT). The entire glycine molecule is consumed in creatine synthesis but only the methyl and amidino groups, respectively, from methionine and arginine. Creatinine loss averages approximately 2 g (14.6 mmol) for 70 kg males in the 20- to 39-year age group. Creatinine loss is lower in females and in older age groups because of lower muscle mass. Approximately half of this creatine lost to creatinine can be replaced, in omnivorous individuals, by dietary creatine. However, since dietary creatine is only provided in animal products, principally in meat and fish, virtually all of the creatine loss in vegetarians must be replaced via endogenous synthesis. Creatine synthesis does not appear to place a major burden on glycine metabolism in adults since this amino acid is readily synthesized. However, creatine synthesis does account for approximately 40% of all of the labile methyl groups provided by S-adenosylmethionine (SAM) and, as such, places an appreciable burden on the provision of such methyl groups, either from the diet or via de novo methylneogenesis. Creatine synthesis consumes some 20–30% of arginine’s amidino groups, whether provided in the diet or synthesized within the body. Creatine synthesis is, therefore, a quantitatively major pathway in amino acid metabolism and imposes an appreciable burden on the metabolism of methionine and of arginine.     

Monoamine oxidase A from human placenta and monoamine oxidase B from bovine liver both have one FAD per subunit.

Choline and C1 metabolism pathways intersect at the formation of methionine from homocysteine. Hepatic S-adenosylmethionine (AdoMet) concentrations are decreased in animals ingesting diets deficient in choline, and it has been suggested that this occurs because the availability of methionine limits AdoMet synthesis. If the above hypothesis is correct, changes in hepatic AdoMet concentrations should relate in some consistent manner to changes in hepatic methionine concentrations. Rats were fed on a choline-deficient or control diet for 1-42 days. Hepatic choline concentrations in control animals were 105 nmol/g, and decreased to 50% of control after the first 7 days on the choline-deficient diet. Hepatic methionine concentrations decreased by less than 20%, with most of this decrease occurring between days 3 and 7 of choline deficiency. Hepatic AdoMet concentrations decreased by 25% during the first week, and continued to decrease (in total, by over 60%) during each subsequent week during which animals consumed a choline-deficient diet. Hepatic S-adenosylhomocysteine (AdoHcy) concentrations increased by 50% when animals consumed a choline-deficient diet. AdoHcy is formed when AdoMet is utilized as a methyl donor. In summary, choline deficiency can deplete hepatic stores of AdoMet under dietary conditions that only minimally decrease the availability of methionine within liver. Thus decreased availability of methionine may not have been the only mechanism whereby choline deficiency lowers hepatic AdoMet concentrations. We suggest that increased utilization of AdoMet might also have occurred.     

Effect of nitrous oxide and methionine on hepatic S-adenosylmethionine levels and in vivo histidine oxidation in rats.

Nitrous oxide (N2O) decreased in vivo oxidation of histidine in rats fed a basal diet marginally deficient in methionine, although hepatic levels of S-adenosylmethionine (AdoMet) were not significantly altered. Excess dietary methionine increased hepatic levels of AdoMet and increased histidine oxidation. However, it did not protect histidine oxidation when the rats were treated with N2O. Parenteral administration of methionine greatly increased hepatic levels of AdoMet and increased histidine oxidation in normal and N2O treated rats. This indicates that when hepatic levels of AdoMet are greatly elevated by administration of methionine, N2O does not affect in vivo histidine oxidation.     

Transmethylation, transsulfuration, and aminopropylation reactions of S-adenosyl-L-methionine in vivo

S-Adenosylmethionine (AdoMet) is metabolized through three main pathways, i.e. (a) transfer of its methyl group to a variety of methyl acceptors, (b) decarboxylation followed by aminopropylation leading to polyamine synthesis, and (c) cleavage of the bond between the sulfur atom and carbon 4 of the amino acid chain, resulting in formation of methylthioadenosine and homoserine thiolactone. In this study the metabolism of AdoMet through these pathways was studied after intravenous administration to rats of [1-14C]-, [3,4-14C]-, [methyl-14C]-, and [35S]AdoMet at various doses. The relative utilization of AdoMet and methionine was also investigated. The results show that intravenously administered AdoMet is efficiently metabolized in vivo up to the highest tested dose (250 mumol X kg-1 body weight), about two-thirds of the metabolized compound being utilized via transmethylation and cleavage to methylthioadenosine and one-third via decarboxylation. The efficient incorporation of the methyl group of AdoMet into muscle creatine indicates unambiguously that the compound is taken up and metabolized by the liver. Moreover, intravenously administered AdoMet is shown to be a better precursor than methionine both in creatine formation and in the utilization of the sulfur atom in transsulfuration reactions.     

Quantitative evaluation and regulation of S-adenosylmethionine-dependent transmethylation in sheep tissues

The overall rates of S-adenosylmethionine (AdoMet)-dependent transmethylation were estimated in various tissues from the initial rate of S-adenosylhomocysteine (AdoHcy) plus AdoMet accumulation after blocking hydrolysis of AdoHcy. The rates were found to differ widely among the tissues of sheep and the highest rate was in the pancreas, being 600 times higher than that in the muscle. Sheep liver possessed approximately 75% of total-body capacity for transmethylation although the transmethylation rate was approximately half that in rat liver. The minimum estimate of daily requirement of AdoMet for transmethylation for adult sheep was approximately 18 mmol, far in excess of methionine intake. Methionine loading elevated AdoMet levels only in the tissues with a high or moderate rate of transmethylation. The kinetic properties of major methyltransferases in sheep liver along with tissue distribution of AdoMet and AdoHcy suggest that transmethylation rate is subject to physiological regulation by tissue levels of AdoMet and AdoHcy.     

Regulation of S-adenosylmethionine levels in Saccharomyces cerevisiae

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine biosynthesis. Methionine can be activated by ATP to give rise to the universal methyl donor, S-adenosylmethionine (AdoMet). Previously, a chimeric MTHFR (Chimera-1) comprised of the yeast Met13p N-terminal catalytic domain and the Arabidopsis thaliana MTHFR (AtMTHFR-1) C-terminal regulatory domain was constructed (Roje, S., Chan, S. Y., Kaplan, F., Raymond, R. K., Horne, D. W., Appling, D. R., and Hanson, A. D. (2002) J. Biol. Chem. 277, 4056-4061). Engineered yeast (SCY4) expressing Chimera-1 accumulated more than 100-fold more AdoMet and 7-fold more methionine than the wild type. Surprisingly, SCY4 showed no appreciable growth defect. The ability of yeast to hyperaccumulate AdoMet was investigated by studying the intracellular compartmentation of AdoMet as well as the mode of hyperaccumulation. Previous studies have established that AdoMet is distributed between the cytosol and the vacuole. A strain expressing Chimera-1 and lacking either vacuoles (vps33 mutant) or vacuolar polyphosphate (vtc1 mutant) was not viable when grown under conditions that favored AdoMet hyperaccumulation. The hyperaccumulation of AdoMet was a robust phenomenon when these cells were grown in medium containing glycine and formate but did not occur when these supplements were replaced by serine. The basis of the nutrient-dependent AdoMet hyperaccumulation effect is discussed in relation to homocysteine biosynthesis and sulfur metabolism.     

Perturbation of methionine metabolism in sheep with nitrous-oxide-induced inactivation of cobalamin

Exposure of sheep to 36% nitrous oxide for 8 days (2-hr per day) led to 90%, 82% and 74% inhibition of 5-methyltetrahydrofolate-homocysteine methyltransferase in the liver, heart and brain, respectively, while there was no significant decrease in the activity of methylmalonyl-CoA mutase. There was also no change of betaine-homocysteine methyltransferase activity. The level of plasma methionine in nitrous-oxide-exposed sheep fell to 30% of its initial value. S-Adenosylmethionine level was reduced to 50% of the control value in the liver, and was also significantly decreased in the heart, but not in the brain. Excretion of formiminoglutamic acid and homocystine was also observed in the urine of sheep exposed to nitrous oxide. These results demonstrate that inhibition of 5-methyltetrahydrofolate-homocysteine methyltransferase causes a pronounced perturbation of methionine metabolism in sheep, suggesting that dietary methionine plus methionine synthesized from the methyl groups of betaine are not sufficient to meet the methyl needs for biological methylation reactions in this species and, in turn, emphasizing the role of 5-methyltetrahydrofolate-homocysteine methyltransferase in methionine synthesis in the sheep.     

Adaptation of adenosylmethionine metabolism and methionine recycling to variations in dietary methionine in the rat

Weanling rats were fed a casein-based diet supplemented to give dietary methionine (Met) concentrations of 0.41, 0.61, and 1.50%. After 2 weeks of feeding, the rats received intraperitoneally 800 nCi of 2-14C-labeled and/or methyl-3H-labeled L-Met. The animals were killed 20 min, 1 hr, or 2 hr after the isotope injection and the specific radioactivity of adenosylmethionine (AdoMet) as well as the total acid-soluble radioactivity was analyzed in the liver and skeletal muscle. Met concentrations of the liver and skeletal muscle were increased 20-fold by the diet containing 1.50% of Met. In the liver, but not in skeletal muscle, accumulation of AdoMet closely followed changes in Met concentration. Within 2 hr after intraperitoneal injection, the rate of disappearance of 3H label from the acid-soluble fraction was slow in both tissues; increasing in the liver and decreasing in skeletal muscle with increasing dietary Met concentration. At the same time, disappearance of 14C label was slow in both tissues in the rats fed the toxic Met diet, and also in the liver of the rats fed the Met-deficient diet. Decline of the specific radioactivity of the AdoMet pool with respect to 3H label was similar to that of 14C label in the skeletal muscle at all dietary Met concentrations. In the liver, the rate of disappearance of 14C label from the AdoMet pool was markedly increased and that of the 3H label slightly decreased with increasing dietary Met supply. Met deprivation resulted in rapid disappearance of 3H label from the hepatic AdoMet pool, whereas the disappearance of the 14C label was very slow. The results indicate that hepatic Met recycling is very effective with deficient or adequate dietary Met concentrations. In skeletal muscle, the capacity to catabolize extra Met is very limited and continuous flow of Met to liver takes place. Unlike in the liver, in skeletal muscle the transsulfuration route is not adaptable to changes in Met supply and plays a minor role in Met catabolism. The approach used to determine the efficacy and adaptation of methionine salvage pathways by following simultaneously the decline of the specific radioactivities of the methyl group and the methionyl carbon chain of AdoMet following intraperitoneal injection of double-labeled Met has several advantages over that used in literature reports. It offers a reliable means of observing these metabolic pathways in whole animals without disruption of metabolite fluxes.     

A substrate switch: a new mode of regulation in the methionine metabolic pathway

We propose a simple mathematical model of liver S -adenosylmethionine (AdoMet) metabolism. Analysis of the model has shown that AdoMet metabolism can operate under two different modes. The first, with low metabolic rate and low AdoMet concentration, serves predominantly to supply the cell with AdoMet, the substrate for various cellular methylation reactions. The second, with high metabolic rate and high AdoMet concentration, provides an avenue for cleavage of excess methionine and can serve as a source of cysteine when its increased synthesis is necessary. The switch that triggers interconversion between the "low" and "high" modes is methionine concentration. Under a certain set of parameters both modes may coexist. This behavior results from the kinetic properties of (i) the two isoenzymes of AdoMet synthetase, MATI and MATIII, that catalyse AdoMet production; one is inhibited by AdoMet, whereas the other is activated by it, and (ii) glycine- N -methyltransferase that displays highly cooperative kinetics that is different from that of other AdoMet-dependent methyltransferases. Thus, the model provides an explanation for how different cellular needs are met by regulation of this pathway. The model also correctly identifies a critical role for glycine N -methyltransferase in depleting excess methionine in the high mode, thus avoiding the toxicity associated with elevated levels of this essential amino acid.     

Reverse methionine biosynthesis from S-adenosylmethionine in eukaryotic cells

The intracellular ratio between methionine and its activated form S-adenosylmethionine (AdoMet) is of crucial importance for the one-carbon metabolism. AdoMet recycling into methionine was believed to be largely achieved through the methyl and the thiomethyladenosine cycles. We show here that in yeast, AdoMet recycling actually occurs mainly through the direct AdoMet-dependent remethylation of homocysteine. Compelling evidences supporting this result were obtained owing to the identification and functional characterization of two new genes, SAM4 and MHT1, that encode the yeast AdoMet-homocysteine methyltransferase and S-methylmethionine-homocysteine methyltransferase, respectively. Homologs of the Sam4 and Mht1 proteins exist in other eucaryotes, indicating that such enzymes would be universal and not restricted to the bacterial or fungal kingdoms. New pathways for AdoMet or S-methylmethionine-dependent methionine synthesis are presented.     

Partial purification and properties of the isozymes of S-adenosylmethionine synthetase from sheep liver

Three forms of AdoMet synthetase were separated from sheep liver. The apparent molecular weights of the native isozymes were 122,000, 62,400 and 70,800 for the alpha-, beta 1- and beta 2-form, respectively and beta 1 was the predominant form. The alpha-form exhibited negative cooperativity with [S] 0.5 values of 31 microM for methionine and 62 microM for ATP; while the two beta-forms exhibited positive cooperativity with [S]0.5 values for methionine of 82 microM and 70 microM and those for ATP of 572 microM and 505 microM for the beta 1- and beta 2-form, respectively. Dimethylsulfoxide markedly stimulated the activities of the two beta-forms at low methionine concentrations. However, at high methionine levels, it inhibited the activity of the beta 2-form but not that of the beta 1-form. The effect of dimethylsulfoxide on the alpha-form was not significant. AdoMet was inhibitory at high concentrations. However, it had a slight stimulatory effect on the two beta-forms at low concentrations when methionine level was also low. These results suggest that AdoMet synthetase is a regulatory enzyme and the reaction rate in vivo can be directly influenced by substrate and product concentrations.     

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA methyltransferase that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N(1) of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two enzymes in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD.     

An allosteric mechanism for switching between parallel tracks in mammalian sulfur metabolism

Methionine (Met) is an essential amino acid that is needed for the synthesis of S-adenosylmethionine (AdoMet), the major biological methylating agent. Methionine used for AdoMet synthesis can be replenished via remethylation of homocysteine. Alternatively, homocysteine can be converted to cysteine via the transsulfuration pathway. Aberrations in methionine metabolism are associated with a number of complex diseases, including cancer, anemia, and neurodegenerative diseases. The concentration of methionine in blood and in organs is tightly regulated. Liver plays a key role in buffering blood methionine levels, and an interesting feature of its metabolism is that parallel tracks exist for the synthesis and utilization of AdoMet. To elucidate the molecular mechanism that controls metabolic fluxes in liver methionine metabolism, we have studied the dependencies of AdoMet concentration and methionine consumption rate on methionine concentration in native murine hepatocytes at physiologically relevant concentrations (40–400 µM). We find that both [AdoMet] and methionine consumption rates do not change gradually with an increase in [Met] but rise sharply (~10-fold) in the narrow Met interval from 50 to 100 µM. Analysis of our experimental data using a mathematical model reveals that the sharp increase in [AdoMet] and the methionine consumption rate observed within the trigger zone are associated with metabolic switching from methionine conservation to disposal, regulated allosterically by switching between parallel pathways. This regulatory switch is triggered by [Met] and provides a mechanism for stabilization of methionine levels in blood over wide variations in dietary methionine intake.     

Enzymatic methyl esterification of the protein in bovine crystalline lens in culture

In the present paper a study on the enzymatic methyl esterification of proteins in the bovine lens has been presented. The data obtained show that the synthesis of the methyl donor (AdoMet) as well as the methyl esterification of proteins are operative in the lens incubated in the presence of L-(methyl-14C) methionine and the amount of labelled methyl esters decreases during cell ageing. Furthermore, the Authors suggest the usefulness of this model system in the evaluation of the overall AdoMet metabolism in aging.     

Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5'-deoxy-5'-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.     

Mutations in human glycine N-methyltransferase give insights into its role in methionine metabolism

Methylation is an essential process in the body. Methyl groups in the form of S-adenosylmethionine are used for the synthesis of many essential compounds (e.g., creatine, phosphatidylcholine, and the methylation of DNA in gene expression). Glycine N-methyltransferase (GNMT) is an abundant enzyme in liver. It catalyzes the methylation of glycine by using S-adenosylmethionine (AdoMet) to form N-methylglycine (sarcosine) with the concommitant production of S-adenosylhomocysteine (AdoHcy). It plays an important role in the economy of methyl groups in the body. The function of GNMT has been hypothesized to provide an alternative route for the conversion of excess AdoMet to AdoHcy in order to preserve the AdoMet/AdoHcy ratio. GNMT is also inhibited by a specific form of folate, 5-methyltetrahydrofolate pentaglutamate. As such, GNMT participates in a regulatory scheme that links the de novo synthesis of methyl groups to the availability of dietary methionine. This hypothesis can now be tested in man. We report here for the first time two Italian sibs who are compound heterzygotes in the gene that encodes GNMT. Both have evidence of mild liver disease. Each bears the same two missense mutations, one in exon 1 (Leu49Pro) and the second in exon 4 (His176Asn). Restriction enzyme analysis of panels of diverse DNA samples indicates that these mutations are not attributable to a common polymorphism.     

Betaine Homocysteine Methyltransferase Is Active in the Mouse Blastocyst and Promotes Inner Cell Mass Development

Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.