Tetrahydrofolate Metabolism Research Articles

Insights into physiological roles of flavonoids in plant coldacclimation.

Flavonoids represent a diverse group of plant specialised metabolites which are also discussed in the context of dietary health and inflammatory response. Numerous studies have revealed that flavonoids play a central role in plant acclimation to abiotic factors like low temperature or high light, but their structural and functional diversity frequently prevents a detailed mechanistic understanding. Further complexity in analysing flavonoid metabolism arises from the different subcellular compartments which are involved in biosynthesis and storage. In the present study, non-aqueous fractionation of Arabidopsis leaf tissue was combined with metabolomics and proteomics analysis to reveal the effects of flavonoid deficiencies on subcellular metabolism during cold acclimation. During the first 3 days of a 2-week cold acclimation period, flavonoid deficiency was observed to affect pyruvate, citrate and glutamate metabolism which indicated a role in stabilising C/N metabolism and photosynthesis. Also, tetrahydrofolate metabolism was found to be affected, which had significant effects on the proteome of the photorespiratory pathway. In the late stage of cold acclimation, flavonoid deficiency was found to affect protein stability, folding and proteasomal degradation, which resulted in a significant decrease in total protein amounts in both mutants. In summary, these findings suggest that flavonoid metabolism plays different roles in the early and late stages of plant cold acclimation and significantly contributes to establishing a new protein homeostasis in a changing environment.

Proteomic Analysis of Arsenic Resistance during Cyanide Assimilation by Pseudomonas pseudoalcaligenes CECT 5344.

Wastewater from mining and other industries usually contains arsenic and cyanide, two highly toxic pollutants, thereby creating the need to develop bioremediation strategies. Here, molecular mechanisms triggered by the simultaneous presence of cyanide and arsenite were analyzed by quantitative proteomics, complemented with qRT-PCR analysis and determination of analytes in the cyanide-assimilating bacterium Pseudomonas pseudoalcaligenes CECT 5344. Several proteins encoded by two ars gene clusters and other Ars-related proteins were up-regulated by arsenite, even during cyanide assimilation. Although some proteins encoded by the cio gene cluster responsible for cyanide-insensitive respiration decreased in the presence of arsenite, the nitrilase NitC required for cyanide assimilation was unaffected, thus allowing bacterial growth with cyanide and arsenic. Two complementary As-resistance mechanisms were developed in this bacterium, the extrusion of As(III) and its extracellular sequestration in biofilm, whose synthesis increased in the presence of arsenite, and the formation of organoarsenicals such as arseno-phosphoglycerate and methyl-As. Tetrahydrofolate metabolism was also stimulated by arsenite. In addition, the ArsH2 protein increased in the presence of arsenite or cyanide, suggesting its role in the protection from oxidative stress caused by both toxics. These results could be useful for the development of bioremediation strategies for industrial wastes co-contaminated with cyanide and arsenic.

Down regulation of the expression of mitochondrial phosphopantetheinyl-proteins in pantothenate kinase-associated neurodegeneration: pathophysiological consequences and therapeutic perspectives

BackgroundNeurodegeneration with brain iron accumulation (NBIA) is a group of genetic neurological disorders frequently associated with iron accumulation in the basal nuclei of the brain characterized by progressive spasticity, dystonia, muscle rigidity, neuropsychiatric symptoms, and retinal degeneration or optic nerve atrophy. Pantothenate kinase-associated neurodegeneration (PKAN) is the most widespread NBIA disorder. It is caused by mutations in the gene of pantothenate kinase 2 (PANK2) which catalyzes the first reaction of coenzyme A (CoA) biosynthesis. Thus, altered PANK2 activity is expected to induce CoA deficiency as well as low levels of essential metabolic intermediates such as 4′-phosphopantetheine which is a necessary cofactor for critical proteins involved in cytosolic and mitochondrial pathways such as fatty acid biosynthesis, mitochondrial respiratory complex I assembly and lysine and tetrahydrofolate metabolism, among other metabolic processes.MethodsIn this manuscript, we examined the effect of PANK2 mutations on the expression levels of proteins with phosphopantetheine cofactors in fibroblast derived from PKAN patients. These proteins include cytosolic acyl carrier protein (ACP), which is integrated within the multifunctional polypeptide chain of the fatty acid synthase involved in cytosolic fatty acid biosynthesis type I (FASI); mitochondrial ACP (mtACP) associated with mitocondrial fatty acid biosynthesis type II (FASII); mitochondrial alpha-aminoadipic semialdehyde synthase (AASS); and 10-formyltetrahydrofolate dehydrogenases (cytosolic, ALD1L1, and mitochondrial, ALD1L2).ResultsIn PKAN fibroblasts the expression levels of cytosolic FAS and ALD1L1 were not affected while the expression levels of mtACP, AASS and ALD1L2 were markedly reduced, suggesting that 4′-phosphopantetheinylation of mitochondrial but no cytosolic proteins were markedly affected in PKAN patients. Furthermore, the correction of PANK2 expression levels by treatment with pantothenate in selected mutations with residual enzyme content was able to correct the expression levels of mitochondrial phosphopantetheinyl-proteins and restore the affected pathways. The positive effects of pantothenate in particular mutations were also corroborated in induced neurons obtained by direct reprograming of mutant PANK2 fibroblasts.ConclusionsOur results suggest that the expression levels of mitochondrial phosphopantetheinyl-proteins are severely reduced in PKAN cells and that in selected mutations pantothenate increases the expression levels of both PANK2 and mitochondrial phosphopantetheinyl-proteins associated with remarkable improvement of cell pathophysiology.

Chemoenzymatic Buta-1,3-diene Synthesis from Syngas Using Biological Decarboxylative Claisen Condensation and Zeolite-Based Dehydration.

A method for producing buta-1,3-diene (1,3-BD) by an amalgamation of chemical and biological approaches with syngas as the carbon source is proposed. Syngas is converted to the central intermediate, acetyl-CoA, by microorganisms through a tetrahydrofolate metabolism pathway. Acetyl-CoA is subsequently converted to malonyl-CoA using a carbonyl donor in the presence of a carboxylase enzyme. A decarboxylative Claisen condensation of malonyl-CoA and acetaldehyde ensues in the presence of acyltransferases to form 3-hydroxybutyryl-CoA, which is subsequently reduced by aldehyde reductase to give butane-1,3-diol (1,3-BDO). An ensuing dehydration step converts 1,3-BDO to 1,3-BD in the presence of a chemical dehydrating reagent.

Gene expression and molecular pathway activation signatures of MYCN-amplified neuroblastomas.

Neuroblastoma is a pediatric cancer arising from sympathetic nervous system. Remarkable heterogeneity in outcomes is one of its widely known features. One of the traits strongly associated with the unfavorable subtype is the amplification of oncogene MYCN. Here, we performed cross-platform biomarker detection by comparing gene expression and pathway activation patterns from the two literature reports and from our experimental dataset, combining profiles for the 761 neuroblastoma patients with known MYCN amplification status. We identified 109 / 25 gene expression / pathway activation biomarkers strongly linked with the MYCN amplification. The marker genes/pathways are involved in the processes of purine nucleotide biosynthesis, ATP-binding, tetrahydrofolate metabolism, building mitochondrial matrix, biosynthesis of amino acids, tRNA aminoacylation and NADP-linked oxidation-reduction processes, as well as in the tyrosine phosphatase activity, p53 signaling, cell cycle progression and the G1/S and G2/M checkpoints. To connect molecular functions of the genes involved in MYCN-amplified phenotype, we built a new molecular pathway using known intracellular protein interaction networks. The activation of this pathway was highly selective in discriminating MYCN-amplified neuroblastomas in all three datasets. Our data also suggest that the phosphoinositide 3-kinase (PI3K) inhibitors may provide new opportunities for the treatment of the MYCN-amplified neuroblastoma subtype.

A Tetrahydrofolate-Dependent Methyltransferase Catalyzing the Demethylation of Dicamba in Sphingomonas sp. Strain Ndbn-20

Sphingomonas sp. strain Ndbn-20 degrades and utilizes the herbicide dicamba as its sole carbon and energy source. In the present study, a tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt, was cloned from the strain, and three other genes, metF, dhc, and purU, which are involved in THF metabolism, were found to be located downstream of dmt A transcriptional study revealed that the four genes constituted one transcriptional unit that was constitutively transcribed. Lysates of cells grown with glucose or dicamba exhibited almost the same activities, which further suggested that the dmt gene is constitutively expressed in the strain. Dmt shared 46% and 45% identities with the methyltransferases DesA and LigM from Sphingomonas paucimobilis SYK-6, respectively. The purified Dmt catalyzed the transfer of methyl from dicamba to THF to form the herbicidally inactive metabolite 3,6-dichlorosalicylic acid (DCSA) and 5-methyl-THF. The activity of Dmt was inhibited by 5-methyl-THF but not by DCSA. The introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba. In conclusion, this study identified a THF-dependent dicamba methyltransferase, Dmt, with potential applications for the genetic engineering of dicamba-resistant crops. Dicamba is a very important herbicide that is widely used to control more than 200 types of broadleaf weeds and is a suitable target herbicide for the engineering of herbicide-resistant transgenic crops. A study of the mechanism of dicamba metabolism by soil microorganisms will benefit studies of its dissipation, transformation, and migration in the environment. This study identified a THF-dependent methyltransferase, Dmt, capable of catalyzing dicamba demethylation in Sphingomonas sp. Ndbn-20, and a preliminary study of its enzymatic characteristics was performed. Introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba, suggesting that the dmt gene has potential applications for the genetic engineering of herbicide-resistant crops.

Proteome- and transcriptome-driven reconstruction of the human myocyte metabolic network and its use for identification of markers for diabetes.

Skeletal myocytes are metabolically active and susceptible to insulin resistance and are thus implicated in type 2 diabetes (T2D). This complex disease involves systemic metabolic changes, and their elucidation at the systems level requires genome-wide data and biological networks. Genome-scale metabolic models (GEMs) provide a network context for the integration of high-throughput data. We generated myocyte-specific RNA-sequencing data and investigated their correlation with proteome data. These data were then used to reconstruct a comprehensive myocyte GEM. Next, we performed a meta-analysis of six studies comparing muscle transcription in T2D versus healthy subjects. Transcriptional changes were mapped on the myocyte GEM, revealing extensive transcriptional regulation in T2D, particularly around pyruvate oxidation, branched-chain amino acid catabolism, and tetrahydrofolate metabolism, connected through the downregulated dihydrolipoamide dehydrogenase. Strikingly, the gene signature underlying this metabolic regulation successfully classifies the disease state of individual samples, suggesting that regulation of these pathways is a ubiquitous feature of myocytes in response to T2D.

Genetic Polymorphism rs6922269 in the MTHFD1L Gene Is Associated with Survival and Baseline Active Vitamin B12 Levels in Post-Acute Coronary Syndromes Patients

Background and AimsThe methylene-tetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) gene is involved in mitochondrial tetrahydrofolate metabolism. Polymorphisms in MTHFD1L, including rs6922269, have been implicated in risk for coronary artery disease (CAD). We investigated the association between rs6922269 and known metabolic risk factors and survival in two independent cohorts of coronary heart disease patients.Methods and ResultsDNA and plasma from 1940 patients with acute coronary syndromes were collected a median of 32 days after index hospital admission (Coronary Disease Cohort Study, CDCS). Samples from a validation cohort of 842 patients post-myocardial infarction (PMI) were taken 24–96 hours after hospitalization. DNA samples were genotyped for rs6922269, using a TaqMan assay. Homocysteine and active vitamin B12 were measured by immunoassay in baseline CDCS plasma samples, but not PMI plasma. All cause mortality was documented over follow-up of 4.1 (CDCS) and 8.8 (PMI) years, respectively. rs6922269 genotype frequencies were AA n = 135, 7.0%; GA n = 785, 40.5% and GG n = 1020, 52.5% in the CDCS and similar in the PMI cohort. CDCS patients with AA genotype for rs6922269 had lower levels of co-variate adjusted baseline plasma active vitamin B12 (p = 0.017) and poorer survival than patients with GG or GA genotype (mortality: AA 19.6%, GA 12.0%, GG 11.6%; p = 0.007). In multivariate analysis, rs6922269 genotype predicted survival, independent of established covariate predictors (p = 0.03). However the association between genotype and survival was not validated in the PMI cohort.Conclusion MTHFD1L rs6922269 genotype is associated with active vitamin B12 levels at baseline and may be a marker of prognostic risk in patients with established coronary heart disease.

Crystal Structure of FolD Bifunctional Protein, a Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase from Campylobacter jejuni

Tetrahydrofolate (THF) plays a crucial role as a molecule for the production and utilization of monocarbon units in various oxidation states. Enzymes involved in THF metabolism are of pharmaceutical interest, as their function is crucial for amino acid and DNA synthesis. The interconversion of two major one‐carbon donors, methylene and methenyl groups, occurs through the sequential activities of NAD(P)‐dependent methylenetetrahydrofolate dehydrogenase (MTHFD) and methenyltetrahydrofolate cyclohydrolase (MTHFC). These activities frequently coexist as a part of a bifunctional enzyme in prokaryotes. In higher organisms, MTHFR and MTHFC are often linked together with another protein, formyl‐THF synthetase (FTHFS) and operate as a trifunctional enzyme. We have determined the crystal structure of a FolD protein, a predicted MTHFR/MTHFC from Campylobacter jejuni at 2.20 Å resolution. The protein exists as a dimer and each subunit is composed of two α/β domains that assemble to form a wide, largely hydrophobic cleft. Comparison with the structure of the homologous enzyme domain from a trifunctional human protein allowed the identification of a highly conserved cluster of basic residues that most likely participate in the binding of a THF substrate. Exploring the differences between the active sites of bacterial and human enzymes might allow the development of inhibitors specific to the bacterial target. This research was supported by the NIH contract No. HHSN272200700058C, and by the U.S. DOE, Office of Biological and Environmental Research, contract DE‐AC02‐06CH11357.

Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase.

Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.

Comparison of methotrexate polyglutamylation in l1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor: Lack of evidence for influx route-specific effects

We previously described a methotrexate-resistant L1210 cell line (MTX rA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTX rA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 μM, influx in the folate receptor transfectant (MTX rA-TF1) and in the reduced folate carrier transfectant (MTX rA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (~3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTX rA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.

Identification and characterization of human mitochondrial methenyltetrahydrofolate synthetase activity

We present evidence for the presence of the folate metabolism enzyme methenyltetrahydrofolate synthetase (MTHFS) in mitochondria. MTHFS activity was identified in the matrix of mitochondria purified from human liver biopsies. Mitochondrial and cytoplasmic MTHFS specific activities are similar, 85% of the total cellular MTHFS activity is in the cytoplasm and both native enzymes have similar molecular weights (approximately 25 kDa). Studies using purified mitochondrial MTHFS from CA46 human Burkitt lymphoma cells reveal that mitochondrial MTHFS behaves kinetically like the cytoplasmic enzyme with K m values of 4.7, 0.8 and 22 μM respectively for (6R,S)-5-formyltetrahydrofolate monoglutamate, (6S)-5-formyltetrahydrofolate pentaglutamate and ATP. This finding adds to previous observations that various folate-dependent enzymes reside in the mitochondria of eucaryotic cells. Intracellular tetrahydrofolate metabolism is highly compartmentalized and mitochondrial MTHFS activity is necessary for the entry of mitochondrial 5-formyltetrahydrofolate into the mitochondrial folate pool.

Characterization of cobalamin-dependent methionine synthase purified from the human malarial parasite, Plasmodium falciparum.

Methionine synthase, which catalyzes the reaction, 5-methyltetrahydrofolate (5-CH3-H4PteGlu) + homocysteine----methionine + tetrahydrofolate, was detected and partially purified from the human malarial parasite, Plasmodium falciparum (K1 isolate). Partial purification was achieved using high-performance size-exclusion and anion-exchange chromatography. The apparent relative molecular weight of the enzyme was estimated as 105,000 daltons, and the apparent Km for 5-CH3-H4PteGlu was 24.2 microM. The enzyme was dependent on adenosylcobalamin or methylcobalamin but not on cobalamin, cyanocobalamin, or hydroxocobalamin in either the absence or presence of S-adenosylmethionine. Preincubation with nitrous oxide markedly inhibited the enzyme. Methionine synthase in P. falciparum may play a role in the supply of methionine and in folate salvage using exogenous 5-CH3-H4PteGlu for tetrahydrofolate metabolism.

A qualitative study on vanadate effects in the tetrahydrofolate-dependent formate transfer in vitro and in vivo in mice.

Parenteral administration of sodium vanadate (NaVO3, 22 mg/kg b.w., i.p.) to mice depresses the oxidation rate of [14C]formate to [14C]CO2, as determined by radioactive breath analysis. The heavy metal-induced inhibition is relatively fast in onset, fairly intense (up to 80% inhibition), of short duration (about 2 h) and strongly correlated to the presence of vanadate (in the pentavalent state) in plasma. The [14C]CO2 exhalation rate from [14C]bicarbonate is less affected by vanadate in vivo, thereby suggesting a specific interference of vanadium in the intermediate step of formate oxidation to HCO3-. In vitro in mouse liver cytosolic fractions vanadate inhibits the enzymatic transfer of formate to tetrahydrofolic acid. The inhibition is accomplished by a vanadate-dependent oxidative degradation of tetrahydrofolate. In contrast, the concentrations of N5-methyltetrahydrofolate, dihydrofolate and folate remain unchanged upon in vitro-exposure to vanadate. The in vitro studies thus might explain the observed inhibition of formate oxidation to carbon dioxide in vivo by a vanadate-evoked depletion of its biological carrier tetrahydrofolic acid. Whether the interference in tetrahydrofolate metabolism also occurs under in vivo conditions, remains to be elucidated.

Stereochemistry associated with the conversion of C-3 of serine through methylenetetrahydrofolate and methenyltetrahydrofolate in the biosynthesis of tuberin.

Results of feeding experiments in Streptomyces amakusaensis with (2 S , 3 R )-[3- 2H 1]- and (2 S , 3 S )-[2,3- 2H 2]-serine show that the N -formyl group in tuberin (1) is formed from C-3 of serine via tetrahydrofolate-linked intermediates with retention of the 3- pro - S proton; retention is only partially stereospecific. The relevance to tetrahydrofolate metabolism is discussed.

The biosynthesis of tuberin from tyrosine and glycine; observations on the stereochemistry associated with the conversion of glycine through methylenetetrahydrofolate into methenyltetrahydrofolate

Tuberin (1) is shown to derive from glycine by way of tetrahydrofolate metabolism and from tyrosine. Glycine is incorporated into the N-formyl group of tuberin with (partial) stereospecific retention of the 2-pro-S proton; there is also some non-stereospecific loss of deuterium label from [2-13C, 2-2H2]glycine on incorporation into the C1 units present in tuberin. [2-14C]-threo-3-Hydroxytyrosine [as (7)] is only incorporated into tuberin after degradation; exclusive labelling of the two C1 units was observed. [2-3H]Octopamine [as (6)] is a poor precursor for tuberin.

Inhibition of glycine-serine interconversion in cultured human fibroblasts by products of isoleucine catabolism.

Extract: Preincubation of fibroblasts with isoleucine before freeze-thawing inhibited the interconversion of glycine and serine. Inhibition was not seen when isoleucine was added after the cells were broken, which suggests that isoleucine must be metabolized to form an inhibitor substance. Tiglic acid was shown to accumulate during this preincubation period and was demonstrated to inhibit interconversion. Methylene tetrahydrofolate production was also reduced by preincubation with isoleucine or incubation with tiglic acid but this decreased production could not be proven to be related to inhibition of serine-glycine interconversion. Speculation: The metabolic consequences of the three enzyme deficiencies associated with the “ketotic hyperglycinemia” syndrome are protean. The data presented in this paper suggest that some of the problems may be related to secondary inhibition of tetrahydrofolate metabolism. Folate therapy may be of some value in the clinical management of patients with this syndrome.

New mutant types at the ade3 locus of Saccharomyces cerevisiae.

Six mutants, allelic to ade3, were isolated after mutagenic treatment of a prototrophic strain of yeast. All six grow on medium supplemented with adenine alone and four respond to histidine. Supplementation with adenine plus histidine or methionine inhibits growth, but a mixture of these three is stimulatory. Heteroallelic diploids formed by the new mutants with the standard ade3 can resemble either parent or show an intermediate phenotype. The new mutants, unlike standard ade3, are not fully epistatic to ade2. The activities of three enzymes concerned in tetrahydrofolate metabolism have been assayed in the new and standard ade3 mutants and wild type. The only difference detected between the new and standard ade3 was in the levels of 10-formyltetrahydrofolate synthetase. Activity in the new mutants ranged from 36 to 109% of wild type compared with 10 to 12% in the standard ade3. Possible mechanisms to account for the varied phenotypes at the ade3 locus are discussed.

Biochemical deficiency associated with [formula omitted]3 mutations in saccharomyces cerevisiae I. Levels of three enzymes of tetrahydrofolate metabolism

In ad 3 mutants, the specific activities for formyltetrahydrofolate synthetase and methenyltetrahydrofolate cyclohydrolase, represent ca. 10 % of wild type activity and the activity for methylenetetrahydrofolate dehydrogenase can represent 50 % of wild type activity under appropriate assay conditions.