Cystathionine as a precursor of methionine in Escherichia coli and Aerobacter aerogenes

Balish, Edward (Argonne National Laboratory, Argonne, Ill.), and Stanley K. Shapiro. Cystathionine as a precursor of methionine in Escherichai coli and Aerobacter aerogenes. J. Bacteriol. 92:1331-1336. 1966.-Cystathionine has been shown to be a precursor of methionine biosynthesis in Escherichia coli and Aerobacter aerogenes. A double enzyme assay was developed to show the formation of homocysteine from cystathionine. The results obtained support the concept that cystathionine serves as a precursor of methionine via the intermediate formation of homocysteine. The latter compound is methylated by the homocysteine methyltransferase of these microorganisms. Sulfhydryl and keto acid assays were used to demonstrate cystathionase activity. Methionine represses both homocysteine methyltransferase formation and cystathionase formation. However, the presence of methionine in reaction mixtures resulted in product inhibition of homocysteine methyltransferase activity, but not of cystathionase activity.     

In vitro inactivation of methionine synthase by nitrous oxide

Nitrous oxide (N2O) is commonly used as an anesthetic agent. Prolonged exposure to N2O leads to megaloblastic anemia in humans and to loss of methionine synthase activity in vertebrates. We now report that purified preparations of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) from both Escherichia coli and pig liver are irreversibly inactivated during turnover in buffers saturated with N2O. Inactivation by N2O occurs only in the presence of all components required for turnover: homocysteine, methyltetrahydrofolate, adenosylmethionine, and a reducing system. Reisolation of the inactivated E. coli enzyme after turnover in the presence of N2O resulted in significant losses of bound cobalamin and of protein as compared to controls where the enzyme was subjected to turnover in N2-equilibrated buffers before reisolation. However, N2O inactivation was not associated with major changes in the visible absorbance spectrum of the remaining enzyme-bound cobalamin. We postulate that N2O acts by one-electron oxidation of the cob(I)alamin form of the enzyme which is generated transiently during turnover with the formation of cob(II)alamin, N2, and hydroxyl radical. Generation of hydroxyl radical at the active site of the enzyme could explain the observed irreversible loss of enzyme activity.     

Formation and utilization of methionine by rat liver cells in culture

The enzyme N5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells.     

Methylation demand: a key determinant of homocysteine metabolism

Elevated plasma homocysteine is a risk factor for cardiovascular disease and Alzheimer's disease. To understand the factors that determine the plasma homocysteine level it is necessary to appreciate the processes that produce homocysteine and those that remove it. Homocysteine is produced as a result of methylation reactions. Of the many methyltransferases, two are, normally, of the greatest quantitative importance. These are guanidinoacetate methyltransferase (that produces creatine) and phosphatidylethanolamine N-methyltransferase (that produces phosphatidylcholine). In addition, methylation of DOPA in patients with Parkinson's disease leads to increased homocysteine production. Homocysteine is removed either by its irreversible conversion to cysteine (transsulfuration) or by remethylation to methionine. There are two separate remethylation reactions, catalyzed by betaine:homocysteine methyltransferase and methionine synthase, respectively. The reactions that remove homocysteine are very sensitive to B vitamin status as both the transsulfuration enzymes contain pyridoxal phosphate, while methionine synthase contains cobalamin and receives its methyl group from the folic acid one-carbon pool. There are also important genetic influences on homocysteine metabolism.     

Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.     

Cloning and expression of the metE gene in Escherichia coli

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.     

The interaction of folic acid derivatives in the methylation of homocysteine

1. The cobalamin-independent synthesis of methionine from serine and homocysteine by ultrasonic extracts of E. coli with tetrahydropteroyltriglutamate as cofactor was inhibited competitively by tetrahydropteroylmonoglutamate and derivatives which were readily converted into this compound. 2. The potency of these inhibitors was directly related to their ability to function as cofactors or substrates in the alternative, cobalamin- dependent mechanism for homocysteine methylation. 3. The cobalamin-dependent and -independent mechanisms of homocysteine methylation were both inhibited by reduced derivatives of aminopterin in a similar manner. 4. It was tentatively concluded that the inhibition was due to a competitive interaction between the folates for N(5)N(10)-methylenetetrahydrofolate reductase.     

Betaine:homocysteine methyltransferase from rat liver: purification and inhibition by a boronic acid substrate analog.

Betaine:homocysteine methyltransferase (BHMT) from rat liver has been highly purified by an efficient procedure requiring only two chromatographic steps: Sephadex G-100 chromatography and fast protein liquid chromatography chromatofocusing. A 170-fold purification and 7.5% overall yield were achieved. Chromatofocusing yielded three active forms of BHMT with pI values near 8.0, 7.6, and 7.0. The subunit molecular weight of each active form is 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native enzyme has a molecular weight of 270,000 as determined by exclusion chromatography. The stability of the purified enzyme was found to be potentiated by the presence of 1 mM dimethylglycine and 1 mM homocysteine. Boronate analogs of betaine (pinanyl N,N,N-trimethylaminomethaneboronate) (4) and dimethylglycine (pinanyl N,N-dimethylaminomethaneboronate) were synthesized from pinanyl iodomethaneboronate (3) and trimethylamine or dimethylamine, respectively. The free acid of the betaine analog (5) was reversibly generated from (4). The inhibition of BHMT by (5) appears competitive with a Ki = 45 microM. Since the Km for betaine measured with the purified enzyme is near 0.1 mM, the boronic acid analog of betaine appears to function effectively as a substrate analog inhibitor of BHMT. The analog does not appear to act as a methyl donor to homocysteine when (5) is substituted for betaine in the enzyme reaction. In addition, an enzyme assay based upon C3-cyano reverse phase HPLC detection of the o-phthalaldehyde derivative of methionine was developed as an alternative to the standard radiochemical assay. Betaine:homocysteine methyltransferase in the picomole range can be quantitated using this assay as indicated by a linear response of enzyme activity to protein concentration.     

Reduced activity of Arabidopsis thaliana HMT2, a methionine biosynthetic enzyme, increases seed methionine content

In the S- methylmethionine cycle of plants, homocysteine methyltransferase (HMT) catalyzes the formation of two molecules of methionine from homocysteine and S- methylmethionine, and methionine methyltransferase (MMT) catalyzes the formation of methionine from S- methylmethionine using S- adenosylmethionine as a methyl group donor. Somewhat surprisingly, two independently isolated knockdown mutations of HMT2 (At3g63250), one of three Arabidopsis thaliana genes encoding homocysteine methyltransferase, increased free methionine abundance in seeds. Crosses and flower stalk grafting experiments demonstrate that the maternal genotype at the top of the flower stalk determines the seed S- methylmethionine and methionine phenotype of hmt2 mutants. Uptake, transport and inter-conversion of [¹³C] S- methylmethionine and [¹³C]methionine in hmt2 , mmt and wild-type plants show that S- methylmethionine is a non-essential intermediate in the movement of methionine from vegetative tissue to the seeds. Together, these results support a model whereby elevated S- methylmethionine in hmt2 vegetative tissue is transported to seeds and either directly or indirectly results in the biosynthesis of additional methionine. Manipulation of the S- methylmethionine cycle may provide a new approach for improving the nutritional value of major grain crops such as rice, as methionine is a limiting essential amino acid for mammalian diets.     

Some mutants of Saccharomyces cerevisiae inhibited by adenoylmethionine and adenosylhomocysteine

These investigations have established the existence of a novel type of non-nutritional mutant (ai) which is inhibited in the presence of two naturally occurring cellular compounds. The inhibition is complete at an extracellular concentration at least as low as 0.05 μmole/ml of either adenosylhomocysteine or adenosylmethionine. It is suggested that adenosylhomocysteine is the true inhibitor. The ai mutants are phenotypically indistinguishable from the wild type in the absence of inhibitors. The results have shown that, if any direct effect on the methionine biosynthetic pathway exists, it is a secondary rather than the primary effect of the inhibitors. The ai mutation does not involve the loss of the adenosylmethionine (or methylmethionine): homocysteine methyltransferase. In addition, the ai mutants accumulate, maintain, and utilize adenosylmethionine and methionine in a manner similar to the parental strain. No genetic relationship could be detected between the ai-1 mutation and several different markers affecting methionine biosynthesis. The ai-1 mutation was also shown to be genetically recessive. Methionine partially reverses the inhibition caused by adenosylmethionine or adenosylhomocysteine. Neither methylmethionine nor homocysteine reversed the inhibition, which showed that the homocysteine methyltransferase cannot catalyze the synthesis of sufficient methionine under these conditions to simulate the effects of extracellularly supplied methionine. If adenine is present, methionine does not cause reversal of inhibition due to adenosylmethionine or adenosylhomocysteine. From the data presented, it is clear that the ai mutation involves some metabolic control mechanism, though the alteration does not appear to be associated primarily with the biosynthesis of methionine.     

The microbial biosynthesis of methionine

1. The enzymes leading to the methylation of homocysteine have been examined in three micro-organisms: a cobalamin-producing bacterium, Bacillus megaterium; a yeast, Candida utilis; and a basidiomycete fungus, Coprinus lagopus. The yeast and the fungus contain negligible endogenous cobalamin. 2. Extracts of each organism catalyse C(1)-transfer from serine to homocysteine with a polyglutamate folate coenzyme. 3. The enzymes generating the methyl group of methionine from C-3 of serine have similar properties in each case, but different mechanisms of homocysteine transmethylation from 5-methyltetrahydrofolates were found. 4. B. megaterium contains an enzyme with properties suggestive of a vitamin B(12)-dependent homocysteine transmethylase, whereas Cand. utilis and Cop. lagopus transfer the methyl group by a reaction characteristic of the cobalamin-independent mechanism established for Escherichia coli. 5. The specificity of each transmethylase for a 5-methyltetrahydropteroylpolyglutamate is consistent with the results of analyses of endogenous folates in these organisms, which showed only conjugated forms. 6. None of the extracts catalysed methionine production from S-adenosylmethionine and homocysteine. 7. These results are compared with results now available for methionine synthesis in other organisms, which show a considerable diversity of mechanisms.     

Formation and utilization of methionine by rat liver cells in culture

Summary The enzymeN ⁵-methyltetrahydrofolate: homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells. Presented in part at the Conference on Differentiation and Carcinogenesis in Liver Cell Cultures sponsored by the New York Academy of Sciences, October 11, 1979 (see reference 1).     

Regulation of hepatic betaine-homocysteine methyltransferase by dietary methionine

The hepatic activity of betaine-homocysteine methyltransferase is a complex function of the content of methionine in the diet. Enzyme levels are lower in the livers of rats fed a 0.3% methionine diet than in livers of animals maintained on either methionine-free or excessivemethionine (1.0%) rations. The finding that activities are increased at both extremes of the spectrum of dietary methionine intake suggests the possibility that the betaine-homocysteine methyltransferase reaction may function both to maintain tissue concentrations of methionine when intake of this amino acid is limited and to remove homocysteine when methionine intake is excessive.     

The reductive acetyl coenzyme A pathway: sequence and heterologous expression of active methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum.

The methyltransferase (MeTr) from Clostridium thermoaceticum transfers the N5-methyl group of (6S)-methyltetrahydrofolate to the cobalt center of a corrinoid/iron-sulfur protein in the acetyl coenzyme A pathway. MeTr was purified to homogeneity and shown to lack metals. The acsE gene encoding MeTr was sequenced and actively expressed in Escherichia coli at a level of 9% of cell protein. Regions in the sequence of MeTr and the E. coli cobalamin-dependent methionine synthase were found to share significant homology, suggesting that they may represent tetrahydrofolate-binding domains.     

S-adenosyl methionine alters the DNA contacts of the EcoKI methyltransferase.

The EcoKI methyltransferase methylates two adenines on opposite strands of its bipartite DNA recognition sequence AAC(N6)GTGC. The enzyme has a strong preference for hemimethylated DNA substrates, but the methylation state of the DNA does not influence its binding affinity. Methylation interference was used to compare the contacts made by the EcoKI methyltransferase with unmodified, hemimethylated or fully modified DNAs. Contacts were seen at or near the N7 position of guanine, in the major groove, for all of the guanines in the EcoKI recognition sequence, and at two guanines on the edge of the intervening spacer sequence. The presence of the cofactor and methyl donor S-adenosyl methionine had a striking effect on the interference pattern for unmodified DNA which could not be mimicked by the presence of the cofactor analogue S-adenosyl homocysteine. In contrast, S-adenosyl methionine had no effect on the interference patterns for either kind of hemimethylated DNA, or for fully modified DNA. Differences between the interference patterns for the unmodified DNA and any of the three forms of methylated DNA provide evidence that methylation of the target sequence influences the conformation of the protein-DNA interface, and illustrate the importance of S-adenosyl methionine in the distinction between unmodified and methylated DNA by the methyltransferase.     

Biosynthesis of methionine in mouse cells cultured in vitro

In the mouse cell-lines cultured in vitro, viz. L-cells and mouse embryo fibroblasts, the methylation of homocysteine to methionine is carried out by vitamin B12-dependent 5-methyltetrahydrofolate:L-homocysteine methyltransferase only. In these cells grown in the standard Eagle medium, the activity of another methyltransferase, which utilizes betaine as the methyl donor, was not detected. The high activity of the vitamin B12-dependent methionine synthetase is typical for mouse cells from the logarithmic phase of growth. In L-cells 60%, and in the mouse fibroblasts 30% of the enzyme exist in the holo-form; the ratio between the holo- and apoenzyme activity remains stable in cells from logarithmic and stationary cultures. The level of the activity of methionine synthetase strongly depends on the presence of vitamin B12, folate and methionine in the culture medium and is greater after prolonged contact of the cells with these agents.     

Methionine metabolism in mammals. Adaptation to methionine excess

We conducted a systematic evaluation of the effects of increasing levels of dietary methionine on the metabolites and enzymes of methionine metabolism in rat liver. Significant decreases in hepatic concentrations of betaine and serine occurred when the dietary methionine was raised from 0.3 to 1.0%. We observed increased concentrations of S-adenosylhomocysteine in livers of rats fed 1.5% methionine and of S-adenosylmethionine and methionine only when the diet contained 3.0% methionine. Methionine supplementation resulted in decreased hepatic levels of methyltetrahydrofolate-homocysteine methyltransferase and increased levels of methionine adenosyltransferase, betaine-homocysteine methyltransferase, and cystathionine synthase. We used these data to simulate the regulatory locus formed by the enzymes which metabolize homocysteine in livers of rats fed 0.3% methionine, 1.5% methionine, and 3.0% methionine. In comparison to the model for the 0.3% methionine diet group, the model for the 3.0% methionine animals demonstrates a 12-fold increase in the synthesis of cystathionine, a 150% increase in flow through the betaine reaction, and a 550% increase in total metabolism of homocysteine. The concentrations of substrates and other metabolites are significant determinants of this apparent adaptation.     

Betaine-homocysteine methyltransferase in the fungus Aspergillus nidulans.

A betaine:homocysteine methyltransferase activity was demonstrated in the cell-free extracts from the fungus $\text{Aspergillus}\text{nidulans}$. Among methionine-requiring mutants which do not grow on homocysteine one class responds to betaine indicating that this compound can serve as a methyl donor in methionine synthesis $\text{in vivo}$. Mutants of the second class which grow only on methionine were shown to have betaine: homocysteine — and methyltetrahydrofolate: homocysteine methyltransferases simultaneously impaired.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: induction of 5-methyltetrahydrofolate homocysteine cobalamin methyltransferase by folate and methionine.

The effects of media vitamin B₁₂(CNB₁₂), l-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH₄folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mm methionine elevates the amount of B₁₂ methyltransferase 1.8 – 2.3-fold at media folate concentrations of 0.2 – 2.0 μm. Conversely, excess 100 μm folic acid increases the amount of B₁₂ holoenzyme by 2.4 – 3.0-fold when the medium contains 0.01 – 0.1 mm methionine. These increases in B₁₂ methyltransferase promoted by 100 μm media folate and 10 mm methionine are inhibited by cycloheximide. 5-MeH₄folate will support growth and induce methyltransferase synthesis more efficiently than folic acid.Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 μm CNB₁₂, 0.1 mm homocysteine, and 100 μm folic acid or 10 μm dl-5-MeH₄folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB₁₂ does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mm methionine-containing to methionine-free media is dependent upon the B₁₂ holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 μm CNB₁₂ to stabilize and convert apo- to holomethyltransferase, and 100 μm folate (or 10 μm dl-5-MeH₄folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20–25% increase in the cellular B₁₂ enzyme content over the level already induced by 100 μm folate in 0.1 mm methionine-supplemented media. A mutant auxotroph CHO AUXB1 with a triple growth requirement for glycine + adenosine + thymidine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) cannot survive in media lacking exogenous methionine. High concentrations of media folic acid or dl-5-MeH₄folate fail to induce elevated amounts of B₁₂ methyltransferase in this mutant. Excess 10 mm medium methionine does, however, elevate its B₁₂ enzyme as in the parent CHO cells. An additional mutant AUXB3 that requires glycine + adenosine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) barely survives in methionine-deficient media. It has a folate-induced B₁₂ enzyme level intermediate between wild-type CHO cells and AUXB1. The level of B₁₂ methyltransferase induced by high media folate concentrations is a critical determinant of CHO cell survival in methionine-free media.