Folate derivatives, 5-methyltetrahydrofolate and 10-formyltetrahydrofolate, protect BEAS-2B cells from high glucose-induced oxidative stress and inflammation.

Folate (vitamin B9) and its biologically active derivatives are well-known antioxidant molecules protecting cells from oxidative degradation. The presence of high glucose, often found in diabetic patients, causes oxidative stress resulting in cellular stress and inflammatory injury. Cells in organs such as the lung are highly prone to inflammation, and various protective mechanisms exist to prevent the progressive disorders arising from inflammation. In the present study, the synthetic form of folate, i.e. folic acid, and active forms of folate, i.e. 5-methyltetrahydrofolate and 10-formyltetrahydrofolate, were evaluated for their antioxidant and antiinflammatory potential against high glucose (50 mM)-mediated oxidative stress and inflammation in BEAS-2B cells, an immortalised bronchial epithelial cell line. High glucose treatment showed a 67% reduction in the viability of BEAS-2B cells, which was restored to the viability levels seen in control cultures by the addition of active folate derivatives to the culture media. The DCFH-DA fluorometric assay was performed for oxidative stress detection. The high glucose-treated cells showed a significantly higher fluorescence intensity (1.81- and 3.8-fold for microplate assay and microscopic observation, respectively), which was normalised to control levels on supplementation with active folate derivatives. The proinflammatory NF-κB p50 protein expression in the active folate derivative-supplemented high glucose-treated cells was significantly lower compared to the folic acid treatment. In support of these findings, in silico microarray GENVESTIGATOR database analysis showed that in bronchiolar small airway epithelial cells exposed to inflammatory condition, folate utilization pathway genes are largely downregulated. However, the folate-binding protein gene, which encodes to the folate receptor 1 (FOLR1), is significantly upregulated, suggesting a high demand for folate by these cells  in inflammatory situations. Supplementation of the active folate derivatives 5-methyltetrahydrofolate and 10-formyltetrahydrofolate resulted in significantly higher protection over the folic acid from high glucose-induced oxidative stress and inflammation. Therefore, the biologically active folate derivatives could be a suitable alternative over the folic acid for alleviating inflammatory injury-causing oxidative stress.

Determination of the effect of germination on the folate content of the seeds of some legumes using HPTLC-mass spectrometry-multivariate image analysis.

Legumes are the main sources of folates which are not synthesized in the human body. The five folate species: 5-methyl tetrahydrofolate, tetrahydrofolate, pteroyl glutamate, 5-formyl tetrahydrofolate and 10-formyl tetrahydrofolate were quantitatively determined in legumes seeds and sprouts by a newly developed and validated high performance thin layer chromatography method. High resolution plate imaging hyphenated to mass spectrometry was exploited for fingerprint analysis of tested samples. Results indicated that germination of all seeds resulted in a 2.5-4 fold increase in the content of total folates as well as the individual vitamers. The total amount of folate reached a maximum on the fifth day in the case of black-eyed peas (861 µg/100 g Fresh Weight), white beans (755 µg/100 g FW) and brown lentils (681 µg/100 g FW). 5-CH3-H4 folate was found to be the most dominating folate species reaching its maximum content in day 5 sprouts of black-eyed peas (490 µg/100 g FW).

Aldh1l2 knockout mouse metabolomics links the loss of the mitochondrial folate enzyme to deregulation of a lipid metabolism observed in rare human disorder.

BACKGROUND: Mitochondrial folate enzyme ALDH1L2 (aldehyde dehydrogenase 1 family member L2) converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2 simultaneously producing NADPH. We have recently reported that the lack of the enzyme due to compound heterozygous mutations was associated with neuro-ichthyotic syndrome in a male patient. Here, we address the role of ALDH1L2 in cellular metabolism and highlight the mechanism by which the enzyme regulates lipid oxidation. METHODS: We generated Aldh1l2 knockout (KO) mouse model, characterized its phenotype, tissue histology, and levels of reduced folate pools and applied untargeted metabolomics to determine metabolic changes in the liver, pancreas, and plasma caused by the enzyme loss. We have also used NanoString Mouse Inflammation V2 Code Set to analyze inflammatory gene expression and evaluate the role of ALDH1L2 in the regulation of inflammatory pathways. RESULTS: Both male and female Aldh1l2 KO mice were viable and did not show an apparent phenotype. However, H&E and Oil Red O staining revealed the accumulation of lipid vesicles localized between the central veins and portal triads in the liver of Aldh1l2-/- male mice indicating abnormal lipid metabolism. The metabolomic analysis showed vastly changed metabotypes in the liver and plasma in these mice suggesting channeling of fatty acids away from ß-oxidation. Specifically, drastically increased plasma acylcarnitine and acylglycine conjugates were indicative of impaired ß-oxidation in the liver. Our metabolomics data further showed that mechanistically, the regulation of lipid metabolism by ALDH1L2 is linked to coenzyme A biosynthesis through the following steps. ALDH1L2 enables sufficient NADPH production in mitochondria to maintain high levels of glutathione, which in turn is required to support high levels of cysteine, the coenzyme A precursor. As the final outcome, the deregulation of lipid metabolism due to ALDH1L2 loss led to decreased ATP levels in mitochondria. CONCLUSIONS: The ALDH1L2 function is important for CoA-dependent pathways including ß-oxidation, TCA cycle, and bile acid biosynthesis. The role of ALDH1L2 in the lipid metabolism explains why the loss of this enzyme is associated with neuro-cutaneous diseases. On a broader scale, our study links folate metabolism to the regulation of lipid homeostasis and the energy balance in the cell.

Cellular Pharmacodynamics of a Novel Pyrrolo[3,2-d]pyrimidine Inhibitor Targeting Mitochondrial and Cytosolic One-Carbon Metabolism.

Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-d]pyrimidine inhibitors of SHMT2 and de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase with potent in vitro and in vivo antitumor efficacy toward pancreatic adenocarcinoma cells. In this report, we extend our findings to an expanded panel of pancreatic cancer models. We used our lead analog AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-l-glutamic acid] to characterize pharmacodynamic determinants of antitumor efficacy for this series and demonstrated plasma membrane transport into the cytosol, uptake from cytosol into mitochondria, and metabolism to AGF347 polyglutamates in both cytosol and mitochondria. Antitumor effects of AGF347 downstream of SHMT2 and purine biosynthesis included suppression of mammalian target of rapamycin signaling, and glutathione depletion with increased levels of reactive oxygen species. Our results provide important insights into the cellular pharmacology of novel pyrrolo[3,2-d]pyrimidine inhibitors as antitumor compounds and establish AGF347 as a unique agent for potential clinical application for pancreatic cancer, as well as other malignancies. SIGNIFICANCE STATEMENT: This study establishes the antitumor efficacies of novel inhibitors of serine hydroxymethyltransferase 2 and of cytosolic targets toward a panel of clinically relevant pancreatic cancer cells and demonstrates the important roles of plasma membrane transport, mitochondrial accumulation, and metabolism to polyglutamates of the lead compound AGF347 to drug activity. We also establish that loss of serine catabolism and purine biosynthesis resulting from AGF347 treatment impacts mammalian target of rapamycin signaling, glutathione pools, and reactive oxygen species, contributing to antitumor efficacy.

Cytosolic 10-formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver.

ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1-/- mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1-/- mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1-/- mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1-/- mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.

Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor.

To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD+ and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. Cancer Res; 77(4); 937-48. ©2017 AACR.

Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway.

One-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here, we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-tetrahydrofolate (10-formyl-THF), and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis, with the mitochondrial pathway required in nutrient-poor conditions.

Folate-Dependent Purine Nucleotide Biosynthesis in Humans.

Purine nucleotide biosynthesis de novo (PNB) requires 2 folate-dependent transformylases-5'-phosphoribosyl-glycinamide (GAR) and 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylases-to introduce carbon 8 (C8) and carbon 2 (C2) into the purine ring. Both transformylases utilize 10-formyltetrahydrofolate (10-formyl-H4folate), where the formyl-carbon sources include ring-2-C of histidine, 3-C of serine, 2-C of glycine, and formate. Our findings in human studies indicate that glycine provides the carbon for GAR transformylase (exclusively C8), whereas histidine and formate are the predominant carbon sources for AICAR transformylase (C2). Contrary to the previous notion, these carbon sources may not supply a general 10-formyl-H4folate pool, which was believed to equally provide carbons to C8 and C2. To explain these phenomena, we postulate that GAR transformylase is in a complex with the trifunctional folate-metabolizing enzyme (TFM) and serine hydroxymethyltransferase to channel carbons of glycine and serine to C8. There is no evidence for channeling carbons of histidine and formate to AICAR transformylase (C2). GAR transformylase may require the TFM to furnish 10-formyl-H4folate immediately after its production from serine to protect its oxidation to 10-formyldihydrofolate (10-formyl-H2folate), whereas AICAR transformylase can utilize both 10-formyl-H2folate and 10-formyl-H4folate. Human liver may supply AICAR to AICAR transformylase in erythrocytes/erythroblasts. Incorporation of ring-2-C of histidine and formate into C2 of urinary uric acid presented a circadian rhythm with a peak in the morning, which corresponds to the maximum DNA synthesis in the bone marrow, and it may be useful in the timing of the administration of drugs that block PNB for the treatment of cancer and autoimmune disease.

Differential behavior of serum and red blood cell folate during a treatment with levofolinic acid. Gender differences.

BACKGROUND: Folates are essential nutrients that maintain nucleotide synthesis and methylation reactions. Folate levels depend essentially on the diet. In the present work, the changes in the folate-homocysteine (Hcy) metabolic axis were studied in response to treatment with levofolinic acid. METHODS: 49 college students (23 men and 26 women) underwent a treatment voluntarily with 5 mg/day levofolinic acid for one month. Serum and red blood cell folate, vitamin B12, and Hcy levels were determined on days 2, 5, 10, and 30 during treatment and 30 days after completion of treatment. RESULTS: Serum folate and Hcy levels showed a plateau beginning on day 10, while red blood cell folate increased towards treatment completion. Gender differences were found in basal levels of Hcy, these differences remaining until the 10th day of treatment and reappearing 30 days after the treatment was finished. Between gender differences in treatment evolution were found only in percentage changes in red blood cell folate in women and men at day 30 of treatment. CONCLUSIONS: There is a compartmentalization of folates in the body that presents a plateau in serum and an erythrocyte reservoir. Folate metabolism presents differential features between genders. The greater physiological need for folate in women of childbearing age could be the determining factor in this difference.

Quantitative flux analysis reveals folate-dependent NADPH production.

ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP(+) to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP(+) and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

A novel mouse model for genetic variation in 10-formyltetrahydrofolate synthetase exhibits disturbed purine synthesis with impacts on pregnancy and embryonic development.

Genetic variants in one-carbon folate metabolism have been identified as risk factors for disease because they may impair the production or use of one-carbon folates required for nucleotide synthesis and methylation. p.R653Q (1958G>A) is a single-nucleotide polymorphism (SNP) in the 10-formyltetrahydrofolate (formylTHF) synthetase domain of the trifunctional enzyme MTHFD1; this domain produces the formylTHF which is required for the de novo synthesis of purines. Approximately 20% of Caucasians are homozygous for the Q allele. MTHFD1 p.R653Q has been proposed as a risk factor for neural tube defects (NTDs), congenital heart defects (CHDs) and pregnancy losses. We have generated a novel mouse model in which the MTHFD1 synthetase activity is inactivated without affecting protein expression or the other activities of this enzyme. Complete loss of synthetase activity (Mthfd1S(-/-)) is incompatible with life; embryos die shortly after 10.5 days gestation, and are developmentally delayed or abnormal. The proportion of 10-formylTHF in the plasma and liver of Mthfd1S(+/-) mice is reduced (P < 0.05), and de novo purine synthesis is impaired in Mthfd1S(+/-) mouse embryonic fibroblasts (MEFs, P < 0.005). Female Mthfd1S(+/-) mice had decreased neutrophil counts (P < 0.05) during pregnancy and increased incidence of developmental defects in embryos (P = 0.052). These findings suggest that synthetase deficiency may lead to pregnancy complications through decreased purine synthesis and reduced cellular proliferation. Additional investigation of the impact of synthetase polymorphisms on human pregnancy is warranted.

Zebrafish 10-formyltetrahydrofolate dehydrogenase is similar to its mammalian isozymes for its structural and catalytic properties.

10-Formyltetrahydrofolate dehydrogenase from zebrafish has been cloned and expressed in both Escherichia coli and yeast. In addition, the N-terminal and C-terminal domains have also been cloned and expressed. Each expressed protein was purified to homogeneity and structural and kinetic properties determined. These studies show that the zebrafish enzyme is structurally and catalytically very similar to the enzymes from mammalian sources, suggesting that zebrafish can be used to study the in vivo function of 10-formyltetrahydrofolate dehydrogenase.

Resection of hepatic metastasis after 5-fluorouracil and cofactor for colon cancer.

Hepatic metastases are common in colorectal cancer. However, only a small percentage of patients are candidates for resection. Neoadjuvant chemotherapy is used to downstage tumors so surgical resection becomes a viable option. We present a case of resection of hepatic metastasis from an 85-year-old patient with metastatic colorectal cancer after treatment with 5-Fluorouracil and 5,10-methylenetetrafolate (CoFactor), an analog of leucovorin, in a Phase II Clinical Trial. CoFactor was developed as a more active replacement of leucovorin to potentially allow reduced dosing of 5-FU. This could potentially be associated with diminished side effects. 5-Fluorouracil with leucovorin or CoFactor could represent another alternative for neoadjuvant chemotherapy prior to resection in metastatic colorectal cancer and warrants further studies, especially in elderly patients.

The enzymes of the 10-formyl-tetrahydrofolate synthetic pathway are found exclusively in the cytosol of the trypanosomatid parasite Leishmania major.

In most organisms 10-formyl-tetrahydrofolate (10-CHO-THF) participates in the synthesis of purines in the cytosol and formylation of mitochondrial initiator methionyl-tRNA(Met). Here we studied 10-CHO-THF biosynthesis in the protozoan parasite Leishmania major, a purine auxotroph. Two distinct synthetic enzymes are known, a bifunctional methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (DHCH) or formyl-tetrahydrofolate ligase (FTL), and phylogenomic profiling revealed considerable diversity for these in trypanosomatids. All species surveyed contain a DHCH1, which was shown recently to be essential in L. major. A second DHCH2 occurred only in L. infantum, L. mexicana and T. cruzi, and as a pseudogene in L. major. DHCH2s bear N-terminal extensions and we showed a LiDHCH2-GFP fusion was targeted to the mitochondrion. FTLs were found in all species except Trypanosoma brucei. L. major ftl(-) null mutants were phenotypically normal in growth, differentiation, animal infectivity and sensitivity to a panel of pteridine analogs, but grew more slowly when starved for serine or glycine, as expected for amino acids that are substrates in C1-folate metabolism. Cell fractionation and western blotting showed that both L. major DHCH1 and FTL were localized to the cytosol and not the mitochondrion. These localization data predict that in L. major cytosolic 10-formyl-tetrahydrofolate must be transported into the mitochondrion to support methionyl-tRNA(Met) formylation. The retention in all the trypanosomatids of at least one enzyme involved in 10-formyl-tetrahydrofolate biosynthesis, and the essentiality of this metabolite in L. major, suggests that this pathway represents a promising new area for chemotherapeutic attack in these parasites.

Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in Leishmania major.

10-Formyl tetrahydrofolate (10-CHO-THF) is a key metabolite in C1 carbon metabolism, arising through the action of formate-tetrahydrofolate ligase (FTL) and/or 5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP(+)-dependent methylene tetrahydrofolate DH and 5,10-methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analogue 5,10-CO-THF (K(i) 105 nM), as was Leishmania growth (EC(50) 1.1 microM). Previous studies showed null ftl(-) mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10-CHO-THF dispensable in evolution. We were unable to generate dhch1(-) null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania, based on segregational loss of episomal complementing genes rather than transfection; analysis of approximately 1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed 'genetic metabolite complementation' using ectopically expressed FTL as an alternative source of 10-CHO-THF; now dhch1(-) null parasites were readily obtained. These data establish a requirement for 10-CHO-THF metabolism in L. major, and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites.

10-formyltetrahydrofolate dehydrogenase requires a 4'-phosphopantetheine prosthetic group for catalysis.

10-Formyltetrahydrofolate dehydrogenase (FDH) consists of two independent catalytic domains, N- and C-terminal, connected by a 100-amino acid residue linker (intermediate domain). Our previous studies on structural organization and enzymatic properties of rat FDH suggest that the overall enzyme reaction, i.e. NADP(+)-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO(2), consists of two steps: (i) hydrolytic cleavage of the formyl group in the N-terminal catalytic domain, followed by (ii) NADP(+)-dependent oxidation of the formyl group to CO(2) in the C-terminal aldehyde dehydrogenase domain. In this mechanism, it was not clear how the formyl group is transferred between the two catalytic domains after the first step. This study demonstrates that the intermediate domain functions similarly to an acyl carrier protein. A 4'-phosphopantetheine swinging arm bound through a phosphoester bond to Ser(354) of the intermediate domain transfers the formyl group between the catalytic domains of FDH. Thus, our study defines the intermediate domain of FDH as a novel carrier protein and provides the previously lacking component of the FDH catalytic mechanism.

Structures of the hydrolase domain of human 10-formyltetrahydrofolate dehydrogenase and its complex with a substrate analogue.

10-Formyltetrahydrofolate dehydrogenase is a ubiquitously expressed enzyme in the human body. It catalyses the formation of tetrahydrofolate and carbon dioxide from 10-formyltetrahydrofolate, thereby playing an important role in the human metabolism of one-carbon units. It is a two-domain protein in which the N-terminal domain hydrolyses 10-formyltetrahydrofolate into formate and tetrahydrofolate. The high-resolution crystal structure of the hydrolase domain from human 10-formyltetrahydrofolate dehydrogenase has been determined in the presence and absence of a substrate analogue. The structures reveal conformational changes of two loops upon ligand binding, while key active-site residues appear to be pre-organized for catalysis prior to substrate binding. Two water molecules in the structures mark the positions of key oxygen moieties in the catalytic reaction and reaction geometries are proposed based on the structural data.

NAD- and NADP-dependent mitochondrially targeted methylenetetrahydrofolate dehydrogenase-cyclohydrolases can rescue mthfd2 null fibroblasts.

Mouse fibroblasts in which the mthfd2 gene encoding mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) was previously inactivated were infected with retroviral expression constructs of dehydrogenase/cyclohydrolase cDNA. Cellular fractionation confirmed that the expressed proteins were properly targeted to the mitochondria. Expression of the NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase enzyme in mitochondria corrected the glycine auxotrophy of the null mutant cells. A construct in which the cyclohydrolase activity of NMDMC was inactivated by point mutation also rescued the glycine auxotrophy, although poorly. This suggests that the cyclohydrolase activity is also required to ensure optimal production of 10-formyltetrahydrofolate. The expression of the NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase in the mitochondria also reversed the glycine requirement of the null cells demonstrating that the use of the NAD cofactor is not absolutely essential to maintain the flux of one-carbon metabolites. All rescued cells demonstrated a decrease in the ratio of incorporation of exogenous formate to serine in standardized radiolabeling studies. This ratio, which is approximately 2.5 for nmdmc(-/-) cells and 0.3 for the wild type cells under the conditions used, is a qualitative indicator of the ability of the mitochondria of the cells to generate formate.

5-Formyltetrahydrofolate is an inhibitory but well tolerated metabolite in Arabidopsis leaves.

5-Formyltetrahydrofolate (5-CHO-THF) is formed via a second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 micromol of CO2 mol(-1)) and non-photorespiratory (3200 micromol of CO2 mol(-1)) conditions. The mutation had only mild visible effects at 370 micromol of CO2 mol(-1), reducing growth rate by approximately 20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 micromol of CO2 mol(-1), the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants.

Transport of 5-formyltetrahydrofolate into primary cultured cerebellar granule cells.

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured cerebellar granule cells (CGC) was studied. Uptake of 5-FTHF into CGC was saturable with K(m)=2.86 microM and V(max)=40.8 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF in the astrocytes has a similar style in the time curve. Uptake of 5-FTHF is characterized by countertransport because adding unlabeled 5-FTHF in the medium resulted in the efflux of labeled 5-FTHF. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methotrexate and folic acid (K(i)=6.64, 7.69, and 19.38 microM, respectively). Uptake was significantly decreased by high concentrations of sodium azide and sodium arsenate but not by sodium cyanide. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Acute exposure of the cells to ethanol (100 mM) did not affect the uptake. It is concluded that CGC have a carrier-mediated system for the uptake of 5-FTHF and other folates.