Methionine and threonine synthesis are limited by homoserine availability and not the activity of homoserine kinase in Arabidopsis thaliana

Homoserine kinase (HSK) produces O-phospho-l-homoserine (HserP) used by cystathionine gamma-synthase (CGS) for Met synthesis and threonine synthase (TS) for Thr synthesis. The effects of overexpressing Arabidopsis thaliana HSK, CGS, and Escherichia coli TS (eTS), each controlled by the 35S promoter, were compared. The results indicate that in Arabidopsis Hser supply is the major factor limiting the synthesis of HserP, Met and Thr. HSK is not limiting and CGS or TS control the partitioning of HserP. HSK overexpression had no effect on the level of soluble HserP, Met or Thr, however, when treated with Hser these plants produced far more HserP than wild type. Met and Thr also accumulated markedly after Hser treatment but the increase was similar in HSK overexpressing and wild-type plants. CGS overexpression was previously shown to increase Met content, but had no effect on Thr. After Hser treatment Met accumulation increased in CGS-overexpressing plants compared with wild type, whereas HserP declined and Thr was unaffected. Arabidopsis responded differentially to eTS expression depending on the level of the enzyme. At the highest eTS level the Thr content was not increased, but the phenotype was negatively affected and the T1 plants died before reproducing. Comparatively low eTS did not affect phenotype or Thr/Met level, however after Hser treatment HserP and Met accumulation were reduced compared with wild type and Thr was increased slightly. At intermediate eTS activity seedling growth was retarded unless Met was supplied and CGS expression was induced, indicating that eTS limited HserP availability for Met synthesis.     

The N-terminal region of Arabidopsis cystathionine gamma-synthase plays an important regulatory role in methionine metabolism

Cystathionine gamma-synthase (CGS) is a key enzyme of Met biosynthesis in bacteria and plants. Aligning the amino acid sequences revealed that the plant enzyme has an extended N-terminal region that is not found in the bacterial enzyme. However, this region is not essential for the catalytic activity of this enzyme, as deduced from the complementation test of an Escherichia coli CGS mutant. To determine the function of this N-terminal region, we overexpressed full-length Arabidopsis CGS and its truncated version that lacks the N-terminal region in transgenic tobacco (Nicotiana tabacum) plants. Transgenic plants expressing both types of CGS had a significant higher level of Met, S-methyl-Met, and Met content in their proteins. However, although plants expressing full-length CGS showed the same phenotype and developmental pattern as wild-type plants, those expressing the truncated CGS showed a severely abnormal phenotype. These abnormal plants also emitted high levels of Met catabolic products, dimethyl sulfide and carbon disulfide. The level of ethylene, the Met-derived hormone, was 40 times higher than in wild-type plants. Since the alien CGS was expressed at comparable levels in both types of transgenic plants, we further suggest that post-translational modification(s) occurs in this N-terminal region, which regulate CGS and/or Met metabolism. More specifically, since the absence of the N-terminal region leads to an impaired Met metabolism, the results further suggest that this region plays a role in protecting plants from a high level of Met catabolic products such as ethylene.     

Insertional inactivation of the methionine s-methyltransferase gene eliminates the s-methylmethionine cycle and increases the methylation ratio

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met and S-adenosyl-Met (Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met to S-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lower S-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%-60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.     

Folates in plants: biosynthesis, distribution, and enhancement

Folates are crucial intermediates for a set of reactions that involve the transfer of single-carbon units (C1 metabolism). They are directly involved in the synthesis of nucleic acids, methionine, pantothenate, glycine and serine, and indirectly, through S-adenosyl methionine, in all methylation reactions. Humans cannot synthesize folates de novo. In these organisms, folate deficiency has severe effects on health and affects large population groups around the world. Because plants are the main source of dietary folates, there are great concerns to select plant food having high concentrations of folates or to engineer their folate metabolism to increase the initial amount. All these attempts rely on what we know about the metabolism of folates. During these last 10 years, the complex pathway leading to the synthesis of folates has been deciphered. Our knowledge about folate synthesis and distribution during plant growth and development also increased substantially. However, important aspects of folate metabolism remain unclear, such as catabolism, transport and regulation of the homeostasis. The aim of this review was to summarize our recent findings, to describe the few attempts reported in the literature to engineer folate level in plants, and to discuss potential strategies that could be used for enhancement.     

Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine gamma-synthase cDNA

To investigate the role of cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of cystathionine beta-lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.     

The folylpolyglutamate synthetase plastidial isoform is required for postembryonic root development in Arabidopsis

A recessive Arabidopsis (Arabidopsis thaliana) mutant with short primary roots and root hairs was identified from a forward genetic screen. The disrupted gene in the mutant encoded the plastidial isoform of folylpolyglutamate synthetase (FPGS), previously designated as AtDFB, an enzyme that catalyzes the addition of glutamate residues to the folate molecule to form folylpolyglutamates. The short primary root of atdfb was associated with a disorganized quiescent center, dissipated auxin gradient in the root cap, bundled actin cytoskeleton, and reduced cell division and expansion. The accumulation of monoglutamylated forms of some folate classes in atdfb was consistent with impaired FPGS function. The observed cellular defects in roots of atdfb underscore the essential role of folylpolyglutamates in the highly compartmentalized one-carbon transfer reactions (C1 metabolism) that lead to the biosynthesis of compounds required for metabolically active cells found in the growing root apex. Indeed, metabolic profiling uncovered a depletion of several amino acids and nucleotides in atdfb indicative of broad alterations in metabolism. Methionine and purines, which are synthesized de novo in plastids via C1 enzymatic reactions, were particularly depleted. The root growth and quiescent center defects of atdfb were rescued by exogenous application of 5-formyl-tetrahydrofolate, a stable folate that was readily converted to metabolically active folates. Collectively, our results indicate that AtDFB is the predominant FPGS isoform that generates polyglutamylated folate cofactors to support C1 metabolism required for meristem maintenance and cell expansion during postembryonic root development in Arabidopsis.     

The effect of methotrexate on intracellular folate pools in human MCF-7 breast cancer cells. Evidence for direct inhibition of purine synthesis

This report details the effects of methotrexate on the intracellular folate pools of the MCF-7 human breast cancer cell line. To achieve this goal, we designed a high-pressure liquid chromatography system capable of separating the physiologic folates. The folate pools were quantitated following growth and equilibration in 2.25 microM radiolabeled folic acid. Each of the intracellular folates was identified by coelution with standard folates and by chemical/biochemical tests unique to each of the various folates. The 10-formyl-H4PteGlu (where H4PteGlu represents dl-tetrahydrofolic acid) pool accounted for 20.5% of the total intracellular folate pool in untreated cells, whereas 5-formyl-H4PteGlu and H4PteGlu accounted for 6.5 and 10.6%, respectively. The levels of these three folates remained stable throughout cell growth. The 5-methyl-H4PteGlu pool accounted for less than 10% in early growth phase cells but assumed greater than 60% of the total pool by the mid- and late-log phases of cell growth. When the MCF-7 cells were exposed to 1 microM methotrexate, de novo purine synthesis and de novo thymidylate synthesis were rapidly inhibited to less than 20% of control within 3 h. During this time period, rapid alterations in the folate pools also occurred such that dihydrofolic acid levels rose from less than 1% in untreated cells to greater than 30% of the total pool. This rise was accompanied by a parallel fall in 5-methyl-H4PteGlu. H4PteGlu and 5-formyl-H4PteGlu were undetectable following 2 h of methotrexate exposure, but 10-formyl-H4PteGlu, the required cosubstrate for de novo purine synthesis, was preserved at greater than 80% of pretreatment values following a 1 microM methotrexate exposure of up to 21 h. The rapid inhibition of de novo purine synthesis in these cells following methotrexate exposure coupled with a relatively preserved 10-formyl-H4PteGlu pool suggests direct inhibition of this synthetic pathway by the temporally coincident accumulation of dihydrofolic acid and/or methotrexate polyglutamates. This inhibition cannot be ascribed to depletion of the folate cofactor 10-formyl-H4PteGlu.     

Regulation of methotrexate polyglutaminate formation in Ehrlich ascites carcinoma cells by endogenous folate pool

The conversion of methotrexate to poly-gamma-glutamyl derivatives in Ehrlich ascites carcinoma cells which are characterized by different pools of endogenous folates is described. The cells in which folate pool was high (the 5-fluorodeoxy-uridine-resistant cell line) the ability to convert methotrexate to its polyglutamate derivatives was much lower than in the cells in which folate pool was smaller (the parental cell line). When the cellular folate pool was reduced by treatment of the cells with lysolecithin, a similar methotrexate polyglutamate concentration in both cell lines was observed. These data suggest that cellular folate pool has a regulatory effect on methotrexate polyglutamate synthesis.     

Synthesis and turnover of folates in plants

Folates are essential cofactors for one-carbon transfer reactions, which are central to plant metabolism. Plants synthesize folates de novo, and are key sources of dietary folate for humans. Research into plant folates therefore impacts human nutrition. Biochemical progress, the sequencing of the Arabidopsis genome, and EST databases are now painting a clear picture of the folate synthesis pathway in plants and its surprising compartmentation. Moreover, new analytical advances will help to elucidate plant folate turnover and transport, which are practically unexplored.     

Folate metabolism in plants: an Arabidopsis homolog of the mammalian mitochondrial folate transporter mediates folate import into chloroplasts

The distribution of folates in plant cells suggests a complex traffic of the vitamin between the organelles and the cytosol. The Arabidopsis thaliana protein AtFOLT1 encoded by the At5g66380 gene is the closest homolog of the mitochondrial folate transporters (MFTs) characterized in mammalian cells. AtFOLT1 belongs to the mitochondrial carrier family, but GFP-tagging experiments and Western blot analyses indicated that it is targeted to the envelope of chloroplasts. By using the glycine auxotroph Chinese hamster ovary glyB cell line, which lacks a functional MFT and is deficient in folates transport into mitochondria, we showed by complementation that AtFOLT1 functions as a folate transporter in a hamster background. Indeed, stable transfectants bearing the AtFOLT1 cDNA have enhanced levels of folates in mitochondria and can support growth in glycine-free medium. Also, the expression of AtFOLT1 in Escherichia coli allows bacterial cells to uptake exogenous folate. Disruption of the AtFOLT1 gene in Arabidopsis does not lead to phenotypic alterations in folate-sufficient or folate-deficient plants. Also, the atfolt1 null mutant contains wild-type levels of folates in chloroplasts and preserves the enzymatic capacity to catalyze folate-dependent reactions in this subcellular compartment. These findings suggest strongly that, despite many common features shared by chloroplasts and mitochondria from mammals regarding folate metabolism, the folate import mechanisms in these organelles are not equivalent: folate uptake by mammalian mitochondria is mediated by a unique transporter, whereas there are alternative routes for folate import into chloroplasts.     

Pterin and folate salvage. Plants and Escherichia coli lack capacity to reduce oxidized pterins

Dihydropterins are intermediates of folate synthesis and products of folate breakdown that are readily oxidized to their aromatic forms. In trypanosomatid parasites, reduction of such oxidized pterins is crucial for pterin and folate salvage. We therefore sought evidence for this reaction in plants. Three lines of evidence indicated its absence. First, when pterin-6-aldehyde or 6-hydroxymethylpterin was supplied to Arabidopsis (Arabidopsis thaliana), pea (Pisum sativum), or tomato (Lycopersicon esculentum) tissues, no reduction of the pterin ring was seen after 15 h, although reduction and oxidation of the side chain of pterin-6-aldehyde were readily detected. Second, no label was incorporated into folates when 6-[(3)H]hydroxymethylpterin was fed to cultured Arabidopsis plantlets for 7 d, whereas [(3)H]folate synthesis from p-[(3)H]aminobenzoate was extensive. Third, no NAD(P)H-dependent pterin ring reduction was found in tissue extracts. Genetic evidence showed a similar situation in Escherichia coli: a GTP cyclohydrolase I (folE) mutant, deficient in pterin synthesis, was rescued by dihydropterins but not by the corresponding oxidized forms. Expression of a trypanosomatid pterin reductase (PTR1) enabled rescue of the mutant by oxidized pterins, establishing that E. coli can take up oxidized pterins but cannot reduce them. Similarly, a GTP cyclohydrolase I (fol2) mutant of yeast (Saccharomyces cerevisiae) was rescued by dihydropterins but not by most oxidized pterins, 6-hydroxymethylpterin being an exception. These results show that the capacity to reduce oxidized pterins is not ubiquitous in folate-synthesizing organisms. If it is lacking, folate precursors or breakdown products that become oxidized will permanently exit the metabolically active pterin pool.     

The effect of folic acid and/or methionine deficiency on deoxyribonucleotide pools and cell cycle distribution in mitogen-stimulated rat lymphocytes

Abstract. Folate deficiency will induce abnormal deoxynucleoside triphosphate (dNTP) metabolism because folate-derived one-carbon groups are essential for de novo synthesis of purines and the pyrimidine, thymidylate. Under conditions of methionine deprivation, a functional folate deficiency for deoxynucleoside triphosphate synthesis is induced as a result of the irreversible diversion of available folates toward endogenous methionine resynthesis from homocysteine. The purpose of the present study was to examine the effect of nutritional folate and/or methionine deprivation in vitro on intracellular dNTP pools as related to DNA synthesis activity and cell cycle progression. Primary cultures of mitogen-stimulated rat splenic T-cells were incubated in complete RPMI 1640 medium or in custom-prepared RPMI 1640 medium lacking in folic acid and/or methionine. Parallel cultures, initiated from the same cell suspension, were analysed for deoxyribonucleotide pool levels and for cell proliferation. The distribution of cells within the cell cycle was quantified by dual parameter flow cytometric bromodeoxyuridine/propidium iodide DNA analysis which allows more accurate definition of DNA synthesizing S-phase cells than the traditional DNA-specific staining with propidium iodide alone. Relative to cells cultured in complete RPMI 1640 media, the cells cultured in media deficient in folate, methionine or in both nutrients manifested increases in the deoxythymidylate pool and an apparent depletion of the deoxyguanosine triphosphate pool. Both adenosine triphosphate and nicotinamide adenine diphosphate levels were significantly reduced with single or combined deficiencies of folate and methionine. These nucleotide pool alterations were associated with a decrease in the proportion of cells actively synthesizing DNA and an increase in cells in G₂+ M phase of the cell cycle. Folate deprivation in the presence of adequate methionine produced a moderate decrease in DNA synthesizing cells over the 68 h incubation. However, methionine deprivation, in the presence or absence of folate, severely compromised DNA synthesis activity. These results are consistent with the established ‘methyl trap’ diversion of available folates towards the resynthesis of methionine from homocysteine and away from nucleotide synthesis. The data confirm the metabolic interdependence of folic acid and methionine and emphasize the pivotal role of methionine on the availability of folate one-carbon groups for deoxynucleotide synthesis. The decrease in DNA synthesis activity under nutrient conditions that negatively affect nucleotide biosynthesis suggest a possible role for abnormal dNTP metabolism in the regulation of cell cycle progression and DNA synthesis.     

Synthesis of folate derivatives in aerated storage tissue disks

Microbiological assay of extracts prepared from carrot, potato, turnip and beet storage tissue disks revealed that folate derivatives were synthesized during a 48 hr aeration period in sterile distilled water. The composition of the folate pool in carrot was examined by DEAE—cellulose column chromatography, γ-glutamylcarboxypeptidase treatment and differential assay of individual derivatives using Lactobacillus casei and Streptococcus faecalis. The principal folates were polyglutamates of formyl and methyl tetrahydrofolate. Smaller quantities of the corresponding mono- and di-glutamates were also detected. The latter derivatives occurred in pools having a high degree of metabolic turnover. The specific activities of three enzymes catalyzing production of these derivatives from tetrahydrofolate increased during the first 12 hr of aeration. Amino acid analyses revealed that folate synthesis in carrot disks was accompanied by depletion of free serine and by net synthesis of free and protein methionine.     

A central role for gamma-glutamyl hydrolases in plant folate homeostasis

Most cellular folates carry a short poly-γ-glutamate tail, and this tail is believed to affect their efficacy and stability. The tail can be removed by γ-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2) alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates, accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.     

Lack of dihydrofolate reductase activity in brain tissue of mammalian species: possible implications

IT IS becoming increasingly clear that folates play a vital, yet until recently an unrecognized, role in the development and function of the brain. Thus several groups of patients have been found with severe maldevelopment of the brain and mental retardation associated with inborn errors of folate metabolism resulting from congenital deficiency in one or more enzymes involved in folate metabolism (ARAKAWA et al., 1965; 1966; 1967; MUDD, LEVY and ABELES, 1969; ARAKAWA, 1970). The presence of folate coenzymes in brain tissue has been reported by several investigators (ALLEN and KLIPSTEIN, 1970; MCCLAIN and BRIDGERS, 1969). MCCLAIN and BRIDGERS (1969) showed that much less of the folates in brain are in the form of the N5-methyl derivatives than is the case for folates in plasma, red blood cells and liver. Appreciable activity of several folate interconverting enzymes have been demonstrated in brain tissue; for example, N5-methyl tetrahydrofolate homocysteine methyl transferase has been found to exist in higher levels in brain than in liver or kidney (MANGUM, 1972); N5-methyl FH,N-dimethyl-dopamine methyl transferase (LADURON, 1972) and serine transhydroxymethylase (EC 2.1.1; L-Serine: tetrahydrofolate 5, 10-hydroxymethyl transferase) (BRIDGERS, 1968) have recently been detected in brain. The last enzyme is known to catalyse a reaction responsible for the generation of a major portion of one-carbon units. In mouse brain, the activity of this enzyme declines during the first 2 weeks of extra-uterine life (BRIDGERS, 1968). The aim of the present study was to determine the levels of dihydrofolate reductase(5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) in mammalian brain tissues in comparison to the levels in other tissues. This enzyme occupies the first and key position in folate metabolism, reducing the metabolically inert vitamin, folic acid, to tetrahydrofolate. This enzyme also functions in thymidylate synthesis to regenerate tetrahydrofolate from dihydrofolate, a product of the reaction (HWHREYS and GREENBERG, 1958). In this reduced state the molecule can accept one-carbon units from various sources to give rise to metabolically active coenzyme forms of folate. This communication reports the complete absence of dihydrofolate reductase in brain tissue of several mammalian species.     

Culture cycle dependence of folate interconversion and related enzymes in Euglena gracilis

Folates are involved in one-carbon metabolism in which one-carbon groups of increasing reduction state (formyl, methylene and methyl) are cyclically accepted and donated by the coenzyme form of folic acid, tetrahydrofolic acid. The latter originates by reduction of dihydrofolic acid, the coenzymatically inactive form. Euglena culture cycle dependence of folate distribution in oxidized, formyl and methyl forms and of enzyme activities for folate interconversion were studied. Distribution levels of all the components examined varied widely during the culture, and many of these changes occurred in the logarithmic phase of growth. In the phase of folate synthesis, there was an appreciable delay in the conversion of oxidized to reduced forms and of formyl to methyl forms. This delay appeared to be correlated with the level of corresponding enzymes. The methyl folate peak coincided with the highest level of total cell folates, at which point a severe repression of folate synthesis began. During the last phase of exponential growth, when cell folate content was reduced to one-fifth and folates had shorter glutamate chains, the level of coenzymatically inactive and inhibitory oxidized forms increased again. The reduced efficiency of the system and the change in growth rate are discussed. The activity patterns of dihydrofolate reductase and methylene tetrahydrofolate reductase were markedly different. The peak in methylene tetrahydrofolate reductase activity coincided with the absence of oxidized folates. A regulation of folate synthesis by the level of methyl folates and of methylene tetrahydrofolate reductase synthesis by the level of oxidized forms is proposed.     

Purine biosynthesis in the domain Archaea without folates or modified folates.

The established pathway for the last two steps in purine biosynthesis, the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) to IMP, is known to utilize 10-formyl-tetrahydrofolate as the required C1 donor cofactor. The biosynthetic conversion of ZMP to IMP in three members of the domain Archaea, Methanobacterium thermoautotrophicum deltaH, M. thermoautotrophicum Marburg, and Sulfolobus solfataricus, however, has been demonstrated to occur with only formate and ATP serving as cofactors. Thus, in these archaea, which use methanopterin (MPT) or another modified folate in place of folate as the C1 carrier coenzyme, neither folate nor a modified folate serves as a cofactor for this biosynthetic transformation. It is concluded that archaea, which function with modified folates such as MPT, are able to carry out purine biosynthesis without the involvement of folates or modified folates.