In Vivo Analysis of Folate Coenzymes and Their Compartmentation in Saccharomyces Cerevisiae

In eukaryotes, enzymes responsible for the interconversion of one-carbon units exist in parallel in both mitochondria and the cytoplasm. Strains of Saccharomyces cerevisiae were constructed that possess combinations of gene disruptions at the SHM1 [mitochondrial serine hydroxymethyltransferase (SHMTm)], SHM2 [cytoplasmic SHMT (SHMTc)], MIS1 [mitochondrial C(1)-tetrahydrofolate synthase (C(1)-THFSm)], ADE3 [cytoplasmic C(1)-THF synthase (C(1)-THFSc)], GCV1 [glycine cleavage system (GCV) protein T], and the GLY1 (involved in glycine synthesis) loci. Analysis of the in vivo growth characteristics and phenotypes was used to determine the contribution to cytoplasmic nucleic acid and amino acid anabolism by the mitochondrial enzymes involved in the interconversion of folate coenzymes. The data indicate that mitochondria transport formate to the cytoplasmic compartment and mitochondrial synthesis of formate appears to rely primarily on SHMTm rather than the glycine cleavage system. The glycine cleavage system and SHMTm cooperate to specifically synthesize serine. With the inactivation of SHM1, however, the glycine cleavage system can make an observable contribution to the level of mitochondrial formate. Inactivation of SHM1, SHM2 and ADE3 is required to render yeast auxotrophic for TMP and methionine, suggesting that TMP synthesized in mitochondria may be available to the cytoplasmic compartment.     

Investigating the regulation of one-carbon metabolism in Arabidopsis thaliana

Serine (Ser) biosynthesis in C(3) plants can occur via several pathways. One major route involves the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC, EC 2.1.1.10) and serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) with glycine (Gly) as one-carbon (1-C) source. An alternative THF-dependent pathway involves the C1-THF synthase/SHMT activities with formate as 1-C source. Here, we have investigated aspects of the regulation of these two folate-mediated pathways in Arabidopsis thaliana (L.) Heynh. Columbia using two approaches. Firstly, transgenic plants overexpressing formate dehydrogenase (FDH, EC 1.2.1.2) were used to continue our previous studies on the function of FDH in formate metabolism. The formate pool size was approximately 73 nmol (g FW)(-1) in wild type (WT) Arabidopsis plants; three independent transgenic lines had similar-sized pools of formate. Transgenic plants produced more (13)CO(2) from supplied [(13)C]formate than did WT plants but were not significantly different from WT plants in their synthesis of Ser. We concluded that FDH has no direct role in the regulation of the above two pathways of Ser synthesis; the breakdown of formate to CO(2) by the FDH reaction is the primary and preferred fate of the organic acid in Arabidopsis. The ratio between the GDC/SHMT and C1-THF synthase/SHMT pathways of Ser synthesis from [alpha-(13)C]Gly and [(13)C]formate, respectively, in Arabidopsis shoots was 21 : 1; in roots, 9 : 1. In shoots, therefore, the pathway from formate plays only a small role in Ser synthesis; in the case of roots, results indicated that the 9 : 1 ratio was as a result of greater fluxes of (13)C through both pathways together with a relatively higher contribution from the C1-THF synthase/SHMT route than in shoots. We also examined the synthesis of Ser in a GDC-deficient mutant of Arabidopsis (glyD) where the GDC/SHMT pathway was impaired. Compared with WT, glyD plants accumulated 5-fold more Gly than WT after supplying [alpha-(13)C]Gly for 24 h; the accumulation of Ser from [alpha-(13)C]Gly was reduced by 25% in the same time period. On the other hand, the accumulation of Ser through the C1-THF synthase/SHMT pathway in glyD plants was 2.5-fold greater than that in WT plants. Our experiments confirmed that the GDC/SHMT and C1-THF synthase/SHMT pathways normally operate independently in Arabidopsis plants but that when the primary GDC/SHMT pathway is impaired the alternative C1-THF synthase/SHMT pathway can partially compensate for deficiencies in the synthesis of Ser.     

13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.

In C3 plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1-THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways in Arabidopsis thaliana (L.) Heynh. Columbia wild type. We confirmed the tight coupling of the GDC/ SHMT system and observed directly in a higher plant the flux of formate through the C1-THF synthase/SHMT system. The accumulation of 13C-enriched serine over 24 h from the GDC/SHMT activities was 4-fold greater than that from C1-THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. The results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment.     

Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase

A human mitochondrial isozyme of C1-tetrahydrofolate (THF) synthase was previously identified by its similarity to the human cytoplasmic C1-THF synthase. All C1-THF synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C1-THF synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C1-THF synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6R,S)-H4-PteGlu1, (6R,S)-H4-PteGlu3, and (6R,S)-H4-PteGlu5 substrates. The (6R,S)-H4-PteGlun Km dropped from greater than 500 microM for the monoglutamate to 15 microM and 3.6 microM for the tri- and pentaglutamates, respectively. The Km values for formate and ATP also are lowered when THF polyglutamates are used. The formate Km dropped 79-fold and the ATP Km dropped more than 5-fold when (6R,S)-H4-PteGlu5 was used as the substrate in place of (6R,S)-H4-PteGlu1.     

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.     

Glycine and serine catabolism in non-photosynthetic higher plant cells: their role in C1 metabolism

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C-labelled substrates, we studied the catabolism of each of these amino acids in non-photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra- hydrofolate (CH2-THF) produced during this reaction did not equilibrate with the overall CH2-THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two-thirds) involved serine-to-glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC-SHMT enzymatic system, which is therefore required in all plant tissues.     

Cytoplasmic serine hydroxymethyltransferase regulates the metabolic partitioning of methylenetetrahydrofolate but is not essential in mice

The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMT-deficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm.     

A general method for generation and analysis of defined mutations in enzymes involved in a tetrahydrofolate-interconversion pathway

In eukaryotes, 10-formyltetrahydrofolate (THF) synthetase, 5,10-methenyl-THF cyclohydrolase and 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. These reactions are generally catalyzed by three separate monofunctional enzymes in prokaryotic cells. In this report a general method for the generation, detection and analysis of specific mutations affecting the catalytic activity of any of the reactions catalyzed by C1-THF synthase or its monofunctional counterparts is described. The method relies on plasmid-borne expression of genes in strains of the yeast Saccharomyces cerevisiae that are missing one or more of the activities of C1-THF synthase. Specific segments of the gene are subjected in vitro to random mutagenesis, the mutant genes expressed in yeast and screened by phenotype for inactivating mutations. Plasmids encoding mutant enzymes are recovered for sequence analysis. One-step purification of C1-THF synthase from the yeast expression system is demonstrated. The feasibility and versatility of the method is shown with the yeast ADE3 gene encoding the cytoplasmic C1-THF synthase and the gene encoding the monofunctional 10-formyl-THF synthetase from Clostridium acidiurici.     

Nitrous oxide exposure reduces hepatic C1-tetrahydrofolate synthase expression in rats

C1-tetrahydrofolate synthase (C1-THF synthase) is a trifunctional enzyme which catalyzes the interconversion of one-carbon units attached to the coenzyme THF. Nitrous oxide (N2O) inhalation is known to inactivate hepatic cobalamin-dependent methionine synthase leading to methionine deficiency and trapping of THF in the methyl-THF form. Liver tissue from rats exposed to N2O for 48 hours exhibited a coordinate decrease in all three activities of C1-THF synthase of approximately 25%. A corresponding 25% decrease in immunoreactive C1-THF synthase was also observed after 48 hours. Thus, the decrease in the concentration of C1-THF synthase accounted entirely for the decreases observed in the three activities. These results suggest that perturbations of hepatic THF pools by N2O affect the level of C1-THF synthase expression at a translational or pretranslational level.     

Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae

Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.     

Purine biosynthesis de novo in rat skeletal muscle.

Purine biosynthesis by the 'de novo' pathway was demonstrated in isolated rat extensor digitorum longus muscle with [1-14C]glycine, [3-14C]serine and sodium [14C]formate as nucleotide precursors. Evidence is presented which suggests that the source of glycine and serine for purine biosynthesis is extracellular rather than intracellular. The relative incorporation rates of the three precursors were formate greater than glycine greater than serine. Over 85% of the label from formate and glycine was recovered in the adenine nucleotides, principally ATP. Azaserine markedly inhibited purine biosynthesis from both formate and glycine. Cycloserine inhibited synthesis from serine, but not from formate. Adenine, hypoxanthine and adenosine markedly inhibited purine synthesis from sodium [14C]formate.     

Methylthioadenosine serves as a single carbon source to the folate co-enzyme pool in rat bone marrow cells

[ribose-U-14C]Methylthioadenosine (MTA) was prepared by incubating methionine with [14C-U]ATP in the presence of methionine adenosyltransferase and the resulting S-adenosylmethionine was heated to release MTA. Labelled [14C]MTA, when incubated with rat bone marrow cells, yielded [14C]formate which was used in the synthesis of adenine and guanine. Unlike 14C from sodium, formate, serine and glycine, there was no decline in 14C utilization from MTA with bone marrow cells from rats in which cobalamin had been inactivated by exposure to nitrous oxide. It was concluded that methionine via MTA is a significant contributor of single-carbon units at the formate level of oxidation and that this pathway is maintained in cobalamin 'deficiency'.     

Effect of glycine on the cell yield and growth rate of Escherichia coli: evidence for cell-density-dependent glycine degradation as determined by (13)C NMR spectroscopy.

Addition of selected amino acids could be a means to improve production of recombinant proteins in industrial processes. We found that glycine increased the maximum specific growth rate of Escherichia coli from 0.67 to 0.78 h(-1), and the cell yield from 0.57 to 0.98 g dry weight per g substrate, when supplemented to batch cultures in a glucose-mineral medium. Maximum effect occurred at pH 6.8, at a glycine concentration of 6-12 mmol l(-1), and at cell densities below 1.15 g dry weight l(-1) (0D(610).3). When glycine was added to a culture at a cell density of 1.15 g l(-1) or above, no growth promoting effect of glycine was seen. The 'glycine effect' was not due to CO(2) produced by the glycine cleavage system (GCV), and the lack of effect at higher cell densities was not masked by acetate accumulation, but coincided with increased acetate production. The metabolism of glycine was further investigated in cultures supplied with [2-(13)C] labelled glycine, and the redistribution of label in the [1-(13)C], [2-(13)C], and [1,2-(13)C] isotopomeres of excreted acetate was analysed by 13C NMR. The NMR data revealed that very little degradation of glycine occurred at cell densities below 1.15 g l(-1). Simultaneously the biosynthesis of serine and glycine was repressed as judged by the absence of [2-(13)C] acetate, implying that added glycine was used as a source of glycine, serine, one-carbon units, and threonine. At cell densities above 1.15 g l(-1), 53% of the consumed glycine carbon was excreted as acetate. Degradation of glycine was associated with an increased uptake rate, cleavage by GCV, and degradation of both glycine-derived serine, and glucose-derived serine to pyruvate. This switch in metabolism appears to be regulated by quorum sensing.     

The role of the mitochondrial glycine cleavage complex in the metabolism and virulence of the protozoan parasite Leishmania major

For the human pathogen Leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-CH(2)-THF). 5,10-CH(2)-THF can be synthesized from glycine by the mitochondrial glycine cleavage complex (GCC). Bioinformatic analysis revealed the four subunits of the GCC in the L. major genome, and the role of the GCC in parasite metabolism and virulence was assessed through studies of the P subunit (glycine decarboxylase (GCVP)). First, a tagged GCVP protein was expressed and localized to the parasite mitochondrion. Second, a gcvP(-) mutant was generated and shown to lack significant GCC activity using an indirect in vivo assay after incorporation of label from [2-(14)C]glycine into DNA. The gcvP(-) mutant grew poorly in the presence of excess glycine or minimal serine; these studies also established that L. major promastigotes require serine for optimal growth. Although gcvP(-) promastigotes and amastigotes showed normal virulence in macrophage infections in vitro, both forms of the parasite showed substantially delayed replication and lesion pathology in infections of both genetically susceptible or resistant mice. These data suggest that, as the physiology of the infection site changes during the course of infection, so do the metabolic constraints on parasite replication. This conclusion has great significance to the interpretation of metabolic requirements for virulence. Last, these studies call attention in trypanosomatid protozoa to the key metabolic intermediate 5,10-CH(2)-THF, situated at the junction of serine, glycine, and thymidylate metabolism. Notably, genome-based predictions suggest the related parasite Trypanosoma brucei is totally dependent on the GCC for 5,10-CH(2)-THF synthesis.     

In Vivo Kinetics of Formate Metabolism in Folate-deficient and Folate-replete Rats

It is now established that the mitochondrial production of formate is a major process in the endogenous generation of folate-linked one-carbon groups. We have developed an in vivo approach involving the constant infusion of [¹³C]formate until isotopic steady state is attained to measure the rate of endogenous formate production in rats fed on either a folate-replete or folate-deficient diet. Formate was produced at a rate of 76 μmol·h⁻¹·100 g of body weight⁻¹ in the folate-replete rats, and this was decreased by 44% in folate-deficient rats. This decreased formate production was confirmed in isolated rat liver mitochondria where formate production from serine, the principal precursor of one-carbon groups, was decreased by 85%, although formate production from sarcosine and dimethylglycine (choline metabolites) was significantly increased. We attribute this unexpected result to the demonstrated production of formaldehyde by sarcosine dehydrogenase and dimethylglycine dehydrogenase from their respective substrates in the absence of tetrahydrofolate and subsequent formation of formate by formaldehyde dehydrogenase. Comparison of formate production with the ingestion of dietary formate precursors (serine, glycine, tryptophan, histidine, methionine, and choline) showed that ∼75% of these precursors were converted to formate, indicating that formate is a significant, although underappreciated end product of choline and amino acid oxidation. Ingestion of a high protein diet did not result in increased production of formate, suggesting a regulation of the conversion of these precursors at the mitochondrial level to formate.     

Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals

In eukaryotes, folate metabolism is compartmentalized between the cytoplasm and organelles. The folate pathways of mitochondria are adapted to serve the metabolism of the organism. In yeast, mitochondria support cytoplasmic purine synthesis through the generation of formate. This pathway is important but not essential for survival, consistent with the flexibility of yeast metabolism. In plants, the mitochondrial pathways support photorespiration by generating serine from glycine. This pathway is essential under photosynthetic conditions and the enzyme expression varies with photosynthetic activity. In mammals, the expression of the mitochondrial enzymes varies in tissues and during development. In embryos, mitochondria supply formate and glycine for purine synthesis, a process essential for survival; in adult tissues, flux through mitochondria can favor serine production. The differences in the folate pathways of mitochondria depending on species, tissues and developmental stages, profoundly alter the nature of their metabolic contribution.