C1-tetrahydrofolate Synthase Research Articles

Descriptive analyses of differentially expressed proteins during intrapuparial stage based on the label-free proteomics technique between Chrysomya megacephala and Synthesiomyia nudiseta

The lack of rapid and accurate species identification methods on pupae restricts the practical application of forensic entomology. It is a new idea to construct portable and rapid identification kits based on the principle of antigen/antibody interaction. Screening differentially expressed proteins (DEPs) of fly pupae is a basis of solving the problem. Here, we used the label-free proteomics technique to discover the DEPs and further validate using the parallel reaction monitoring technique (PRM) in the common flies. In this study, we reared the Chrysomya megacephala and Synthesiomyia nudiseta at constanttemperature, and then we sampled at least four pupae at 24 h intervals until the end of the intrapuparial stage. We found 132 DEPs between Ch. megacephala, and S. nudiseta groups, with 68 and 64 proteins being up-regulated and down-regulated between the two groups. Among the 132 DEPs, we selected five proteins having potential for further development and utilization, such as C1-tetrahydrofolate synthase, Malate dehydrogenase, Transferrin, Protein disulfide-isomerase, and Fructose-bisphosphate aldolase, for further validation using PRM-targeted proteomics, with the trends of PRM results being consistent with the label-free data for corresponding proteins. The present study investigated DEPs via the label-free technique during the pupal development in the Ch. megacephala, and S. nudiseta and provided reference data for development of rapid and accurate identification kits.

Orthogonal genome-wide screens of bat cells identify MTHFD1 as a target of broad antiviral therapy

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.

Molecular cloning and characterization of C1 tetrahydrofolate (C1-THF) synthase in Bombyx mori, silkworm

Folate metabolism pathway is mainly associated with syntheses of nucleic acids and proteins as well as DNA methylation and repair. Cytoplasmic C1 tetrahydrofolate (C1-THF) synthase is a central enzyme in folate metabolism and plays an important role in C1-THF conversion. The full-length sequence of silkworm (Bombyx mori) cytoplasmic C1-THF synthase (BmC1-THF synthase) gene including an open reading frame (ORF) of 932 amino acid residues was cloned. Phylogenetic analysis indicated that BmC1-THF synthase shares 84% and 81% identity with C1-THF synthase of Papilio Xuthus and Danaus plexippus, respectively. Conserved sequence analysis showed that the 330 to 710 amino acid residues of BmC1-THF synthase were highly conserved among various organisms. Recombinant BmC1-THF synthase protein with a molecular weight of about 105.5 kDa was expressed in E. coli cells and identified using SDS-PAGE and western blot analysis. The expression of BmC1-THF synthase gene in eight silkworm tissues revealed that BmC1-THF synthase gene is widely expressed in various tissues of silkworm and highly expressed in the fat body. The expression characteristics of BmC1-THF synthase gene in response to oxidative stress were conducted under phoxim and high temperature (30 °C) exposure. The results showed that the transcriptional level of BmC1-THF synthase gene was upregulated by 3.435-fold and 6.845-fold after phoxim and high temperature (30 °C) exposure, respectively, indicating that BmC1-THF synthase gene may play a significant role in response of silkworm to oxidative stresses.

Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs.

Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice

MTHFD1 encodes C1-tetrahydrofolate synthase, which is a folate-dependent enzyme that catalyzes the formation and interconversion of folate-activated one-carbon groups for nucleotide biosynthesis and cellular methylation. A polymorphism in MTHFD1 (1958G→A) impairs enzymatic activity and is associated with increased risk of adverse pregnancy outcomes, but the mechanisms are unknown. The objective of this study was to determine whether disruption of the embryonic or maternal Mthfd1 gene or both interacts with impaired folate and choline status to affect neural tube closure, fetal growth, and fertility in mice and to investigate the underlying metabolic disruptions. Dams with a gene-trapped (gt) allele in Mthfd1 and wild-type dams were fed a control or folate- and choline-deficient AIN93G diet (Dyets Inc). Litters were examined for gross morphologic defects, crown-rump length, and resorptions. Folate status and amounts of folate-related metabolites were determined in pregnant dams. Reduced folate and choline status resulted in severe fetal growth restriction (FGR) and impaired fertility in litters harvested from Mthfd1(gt/+) dams, but embryonic Mthfd1(gt/+) genotype did not affect fetal growth. Gestational supplementation of Mthfd1(gt/+) dams with hypoxanthine increased FGR frequency and caused occasional neural tube defects (NTDs) in Mthfd1(gt/+) embryos. Mthfd1(gt/+) dams exhibited lower red blood cell folate and plasma methionine concentrations than did wild-type dams. Maternal Mthfd1(gt/+) genotype impairs fetal growth but does not cause NTDs when dams are maintained on a folate- and choline-deficient diet. Mthfd1(gt/+) mice exhibit a spectrum of adverse reproductive outcomes previously attributed to the human MTHFD1 1958G→A polymorphism. Mthfd1 heterozygosity impairs folate status in pregnant mice but does not significantly affect homocysteine metabolism.

Mthfd1 is a modifier of chemically induced intestinal carcinogenesis.

The causal metabolic pathways underlying associations between folate and risk for colorectal cancer (CRC) have yet to be established. Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate and methionine. Methionine is converted to S-adenosylmethionine (AdoMet), the major one-carbon donor for cellular methylation reactions. Impairments in folate metabolism can modify DNA synthesis, genomic stability and gene expression, characteristics associated with tumorigenesis. The Mthfd1 gene product, C1-tetrahydrofolate synthase, is a trifunctional enzyme that generates one-carbon substituted tetrahydrofolate cofactors for one-carbon metabolism. In this study, we use Mthfd1(gt/+) mice, which demonstrate a 50% reduction in C1-tetrahydrofolate synthase, to determine its influence on tumor development in two mouse models of intestinal cancer, crosses between Mthfd1(gt/+) and Apc(min)(/+) mice and azoxymethane (AOM)-induced colon cancer in Mthfd1(gt/+) mice. Mthfd1 hemizygosity did not affect colon tumor incidence, number or load in Apc(min/+) mice. However, Mthfd1 deficiency increased tumor incidence 2.5-fold, tumor number 3.5-fold and tumor load 2-fold in AOM-treated mice. DNA uracil content in the colon was lower in Mthfd1(gt/+) mice, indicating that thymidylate biosynthesis capacity does not play a significant role in AOM-induced colon tumorigenesis. Mthfd1 deficiency-modified cellular methylation potential, as indicated by the AdoMet: S-adenosylhomocysteine ratio and gene expression profiles, suggesting that changes in the transcriptome and/or decreased de novo purine biosynthesis and associated mutability cause cellular transformation in the AOM CRC model. This study emphasizes the impact and complexity of gene-nutrient interactions with respect to the relationships among folate metabolism and colon cancer initiation and progression.

Mitochondrial C1-Tetrahydrofolate Synthase (MTHFD1L) Supports the Flow of Mitochondrial One-carbon Units into the Methyl Cycle in Embryos

Mitochondrial folate-dependent one-carbon (1-C) metabolism converts 1-C donors such as serine and glycine to formate, which is exported and incorporated into the cytoplasmic tetrahydrofolate (THF) 1-C pool. Developing embryos depend on this mitochondrial pathway to provide 1-C units for cytoplasmic process such as de novo purine biosynthesis and the methyl cycle. This pathway is composed of sequential methylene-THF dehydrogenase, methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase activities. In embryonic mitochondria, the bifunctional MTHFD2 enzyme catalyzes the dehydrogenase and cyclohydrolase reactions, but the enzyme responsible for the mitochondrial synthetase reaction has not been identified in embryos. A monofunctional 10-formyl-THF synthetase (MTHFD1L gene product) functions in adult mitochondria and is a likely candidate for the embryonic activity. Here we show that the MTHFD1L enzyme is present in mitochondria from normal embryonic tissues and embryonic fibroblast cell lines, and embryonic mitochondria possess the ability to synthesize formate from glycine. The MTHFD1L transcript was detected at all stages of mouse embryogenesis examined. In situ hybridizations showed that MTHFD1L was expressed ubiquitously throughout the embryo but with localized regions of higher expression. The spatial pattern of MTHFD1L expression was virtually indistinguishable from that of MTHFD2 and MTHFD1 (cytoplasmic C(1)-THF synthase) in embryonic day 9.5 mouse embryos, suggesting coordinated regulation. Finally, we show using stable isotope labeling that in an embryonic mouse cell line, greater than 75% of 1-C units entering the cytoplasmic methyl cycle are mitochondrially derived. Thus, a complete pathway of enzymes for supplying 1-C units from the mitochondria to the methyl cycle in embryonic tissues is established.

Identification of metastasis-associated proteins involved in gallbladder carcinoma metastasis by proteomic analysis and functional exploration of chloride intracellular channel 1

Advanced gallbladder cancer has an extremely poor prognosis because of metastasis. Identification of metastasis-related biomarkers is essential to improve patient survival. In the present study, metastasis-associated proteins were identified by comparative proteomic analysis and the metastasis-related function of the candidate protein, chloride intracellular channel 1 (CLIC1), was further elucidated. Two cell lines with high or low metastatic potential (termed GBC-SD18H and GBC-SD18L, respectively), originating from the same parental gallbladder carcinoma GBC-SD cell line, were identified by spontaneous metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised of two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between GBC-SD18L and GBC-SD18H. Twenty-six proteins were identified and further verified by one-dimensional Western blotting and semiquantitative reverse transcriptase polymerase chain reaction analysis. It was determined that CLIC1, ezrin, vimentin, annexin A3, WD repeat domain 1, triosephosphate isomerase, C1-tetrahydrofolate synthase, Rho GDP-dissociation inhibitor 1, T-complex protein 1, heterogeneous nuclear ribonucleoprotein K, glutamate dehydrogenase 1, proteasome activator complex subunit 3 and Rab GDP-dissociation inhibitor beta were significantly up-regulated in the highly metastatic GBC-SD18H cell line compared to the poorly metastatic GBC-SD18L cell line. However, phosphoglycerate kinase 1 and programmed cell death protein 8 were significantly down-regulated in the highly metastatic GBC-SD18H cell line compared to GBC-SD18L. Considering that CLIC1 was profuse in highly metastatic GBC-SD18H but scarce in poorly metastatic GBC-SD18L, the association of CLIC1 with metastasis was further elucidated by the overexpression and RNA interference of CLIC1 in GBC-SD18L cells and GBC-SD18H cells, respectively. The results demonstrated that the overexpression of CLIC1 promoted cell motility and invasion of GBC-SD18L in vitro, while RNA interference of CLIC1 remarkably decreased cell motility and invasive potency of GBC-SD18H in vitro, indicating that CLIC1 might play an important role in metastasis of gallbladder carcinoma.

Mthfd1 Is an Essential Gene in Mice and Alters Biomarkers of Impaired One-carbon Metabolism

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.

Regulation of Folate-mediated One-carbon Metabolism by 10-Formyltetrahydrofolate Dehydrogenase

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.

Human Mitochondrial C1-Tetrahydrofolate Synthase: GENE STRUCTURE, TISSUE DISTRIBUTION OF THE mRNA, AND IMMUNOLOCALIZATION IN CHINESE HAMSTER OVARY CELLS

C1-tetrahydrofolate (THF) synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The cytoplasmic isozyme of C1-THF synthase is well characterized in a number of mammals, including humans; but a mitochondrial isozyme has been previously identified only in the yeast Saccharomyces. Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase. The gene spans 236 kilobase pairs on chromosome 6 and consists of 28 exons plus one alternative exon. The gene encodes a protein of 978 amino acids, including an N-terminal mitochondrial targeting sequence. The mitochondrial isozyme is 61% identical to the human cytoplasmic isozyme. Expression of the gene was detected in most human tissues, but transcripts were highest in placenta, thymus, and brain. Two mRNAs were detected, a 3.6-kb transcript and a 1.1-kb transcript, and both transcripts were observed in varying ratios in each tissue. The shorter transcript results from an alternative splicing event, where exon 7 is spliced to exon 8a instead of exon 8. Exon 8a is derived from an exonized Alu sequence, sharing no homology with exon 8 of the long transcript, and encodes just 15 amino acids followed by a stop codon and a polyadenylation signal. This short transcript potentially encodes a bifunctional enzyme lacking 10-formyl-THF synthetase activity. Both transcripts initiate at the same 5'-site, 107 nucleotides up-stream of the ATG start codon. The full-length (2934 bp) cDNA fused to a C-terminal V5 epitope tag was expressed in Chinese hamster ovary cells. Immunoblots of subfractionated cells revealed a 107-kDa protein only in the mitochondrial fractions of these cells, confirming the mitochondrial localization of the protein. Yeast cells expressing the full-length human cDNA exhibited elevated 10-formyl-THF synthetase activity, confirming its identification as the human mitochondrial C1-THF synthase.

Photorespiratory metabolism of glyoxylate and formate in glycine-accumulating mutants of barley and Amaranthus edulis

Glycine-accumulating mutants of barley (Hordeum vulgare L.) and Amaranthus edulis (Speg.), which lack the ability to decarboxylate glycine by glycine decarboxylase (GDC; EC 2.1.2.10), were used to study the significance of an alternative photorespiratory pathway of serine formation. In the normal photorespiratory pathway, 5,10-methylenetetrahydrofolate is formed in the reaction catalysed by GDC and transferred to serine by serine hydroxymethyltransferase. In an alternative pathway, glyoxylate could be decarboxylated to formate and formate could be converted into 5,10-methylenetetrahydrofolate in the C1-tetrahydrofolate synthase pathway. In contrast to wild-type plants, the mutants showed a light-dependent accumulation of glyoxylate and formate, which was suppressed by elevated (0.7%) CO2 concentrations. After growth in air, the activity and amount of 10-formyltetrahydrofolate synthetase (FTHF synthetase; EC 6.3.4.4), the first enzyme of the conversion of formate into 5,10-methylenetetrahydrofolate, were increased in the mutants compared to the wild types. A similar increase in FTHF synthetase could be induced by incubating leaves of wild-type plants with glycine under illumination, but not in the dark. Experiments with 14C showed that the barley mutants incorporated [14C]formate and [2-14C]glycollate into serine. Together, the accumulation of glyoxylate and formate under photorespiratory conditions, the increase in FTHF synthetase and the ability to utilise formate and glycollate for the formation of serine indicate that the mutants are able partially to compensate for the lack of GDC activity by bypassing the normal photorespiratory pathway.

Multiple folate enzyme inhibition: mechanism of a novel pyrrolopyrimidine-based antifolate LY231514 (MTA).

Extensive biochemical and pharmacological evidence indicates that LY231514 is a novel antifolate antimetabolite. LY231514 is transported into cells mainly through the reduced folate carrier system and extensively metabolized to polyglutamated forms. The polyglutamates of LY231514 inhibit at least three key folate enzymes: TS, DHFR, and GARFT, and to a lesser extent AICARFT and C1-tetrahydrofolate synthase. The combined effects of the inhibition exerted by LY231514 at each target give rise to an unusual end-product reversal pattern at the cellular level that is distinct from those of other inhibitors such as methotrexate and the quinazoline antifolates. The metabolic effects exerted by LY231514 on the folate and nucleotide pools are also quite distinct from those of MTX and LY309887. The efficient polyglutamation, longer cellular retention and the multiple folate enzyme inhibition mechanism may all have contributed directly to the exciting antitumor responses now observed in Phase I and II studies. The multitargeted inhibition mechanism of LY231514 is particularly intriguing. This new level of mechanistic insight, which has evolved from the study of LY231514, challenges the traditional concept and paradigm of antifolate drug discovery and development which focused on developing very potent and selective inhibitors of single folate enzyme targets, such as DHFR, TS or one of the enzymes along the de novo purine biosynthetic pathway. Given the complex nature of folate metabolism and the critical role of folates in maintaining the physiological functions of living systems, it is completely reasonable to suspect that agents which can interfere at multiple sites in the folate pathway may trigger and cause more biochemical imbalance in the cellular DNA and RNA synthesis of malignant cells than agents that act on a single point (Fig. 5). In conclusion, LY231514 (MTA) is a new generation antifolate antimetabolite demonstrating inhibitory activity against multiple folate enzymes including TS, DHFR and GARFT. In current phase II studies, MTA is broadly active as a single agent and is showing very encouraging antitumor activity in multiple solid tumors including colorectal, breast and non-small cell lung cancers (38-43). The every three week dosing schedule has proven to be convenient and easy to administer and the clinical toxicities of LY231514 seem to be well tolerated. More advanced and extensive clinical trials of LY231514 are currently in progress.

Site-Directed Mutagenesis of a Highly Conserved Aspartate in the Putative 10-Formyl-Tetrahydrofolate Binding Site of Yeast C1-Tetrahydrofolate Synthase

C1-tetrahydrofolate (THF) synthase is a eukaryotic trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. Although the 10-formyl-THF synthetase reaction (a reversible ATP-dependent formylation of THF) has been studied extensively, little is known about specific residues involved in the catalytic mechanism. In this study, we have examined the role of a highly conserved aspartate residue, Asp449of yeast cytoplasmic C1-THF synthase. Asp449is part of a putative folate binding site found in many proteins that bind 10-formyl-THF. The corresponding aspartate has been identified as a critical catalytic residue inEscherichia coliand human GAR transformylase, which catalyzes a 10-formyl-THF-dependent formyl transfer. In order to determine if Asp449has a similar catalytic role in the 10-formyl-THF synthetase reaction, three mutant proteins were produced by site-directed mutagenesis in which Asp449of yeast cytoplasmic C1-THF synthase was changed to Asn, Glu, or Ala. The mutant proteins were expressed in yeast, purified, and characterized with respect to kinetic properties and enzyme stability. All three of the mutant enzymes retained substantial 10-formyl-THF synthetase activity, indicating that Asp449is not a critical catalytic residue. However, our data suggest that it does play a role in folate binding, probably by contributing to the proper conformation of the active site. Thus, these results suggest that the 10-formyl-THF binding site differs significantly between the GAR transformylase and 10-formyl-THF synthetase families, and that the conserved aspartate plays different roles in the two enzymes.

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.

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 C1-tetrahydrofolate synthase (C1-THFSm)], ADE3 [cytoplasmic C1-THF synthase (C1-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.

Properties of tetrahydropteroylpentaglutamate bound to 10-formyltetrahydrofolate dehydrogenase.

A new rapid procedure for purifying 10-formyltetrahydrofolate dehydrogenase results in 90 mg of pure enzyme from two rabbit livers. This abundant liver enzyme is known to bind its product tetrahydropteroylpentaglutamate (H4PteGlu5) so tightly that it does not dissociate during size exclusion chromatography. 10-Formyltetrahydrofolate dehydrogenase is also known to exhibit strong product inhibition by H4PteGlu5. There is a several-fold excess of 10-formyltetrahydrofolate dehydrogenase subunits in liver relative to the concentration of H4PteGlun, suggesting that in vivo this enzyme may bind significant amounts of this coenzyme in a nearly irreversible enzyme. H4PteGlu5 complex. How this tightly bound H4PteGlun is transferred to the other two enzymes in the cytosol, serine hydroxymethyltransferase and C1-tetrahydrofolate synthase, which use H4PteGlu5 as a substrate, is the subject of this investigation. Analysis of the product inhibition curve for 10-formyltetrahydrofolate dehydrogenase shows that H4-PteGlu5 has a dissociation constant near 15 nM which is 60-fold lower than the Ks for 10-formyl-H4PteGlu5. Fluorescence titration studies also yield a Kd of about 20 nM for H4PteGlu5. Coupling the 10-formyltetrahydrofolate dehydrogenase reaction to an excess of either serine hydroxymethyltransferase or C1-tetrahydrofolate synthase not only abolishes product inhibition but also increases the initial rate of its activity by about 2-fold. Passage of a reaction mixture of 10-formyltetrahydrofolate dehydrogenase down a size exclusion column results in enzyme with 1 equiv of H4PteGlu5 bound per subunit. However, addition of either serine hydroxymethyltransferase or C1-tetrahydrofolate synthase results in a rapid transfer of this bound folate to these enzymes. Evidence is also presented that the tightly bound folate is in equilibrium with solvent H4PteGlu5.

The N-terminal, dehydrogenase/cyclohydrolase domain of yeast cytoplasmic trifunctional C1-tetrahydrofolate synthase requires the C-terminal, synthetase domain for the catalytic activity in vitro

The yeast ADE3(1–333) gene which encodes a truncated protein containing the N-terminal 5,10-methylene-tetrahydrofolate (THF) dehydrogenase (D)/5,10-methynyl-THF cyclohydrolase (C) domain of cytoplasmic trifunctional C1-THF synthase is able to complement all the phenotypes associated with ade3 mutations in vivo. However, expression of the ADE3(1–333) gene in an ade3 strain does not retain any D activity in vitro. Expression in a yeast ade3 strain of the ADE3(1–333) fused to the Escherichia coli lacZ gene or to the yeast SER2 gene allows detection of D and C activities in vitro. These results indicate that the N-terminal D/C domain of C1-THF synthase requires the C-terminal 10-formyl-THF synthetase domain for stable catalytic activity in vitro.

Nucleotide sequence and characterization of the Saccharomyces cerevisiae RPL19A gene encoding a homolog of the mammalian ribosomal protein L19.

A gene designated RPL19A has been identified in the region downstream from the 3'-end of the Saccharomyces cerevisiae MIS1 gene encoding the mitochondrial C1-tetrahydrofolate synthase. The gene codes for the yeast ribosomal protein YL19 which exhibits 57.5% identify with the mammalian ribosomal protein L19. RPL19A is one of two functional copies of the YL19 gene located on chromosome II. The disruption of RPL19A has no effect on the growth of the yeast. The RPL19A gene contains an intron located near the 5'-end. The 5'-flanking region contains one similar and one complete UASrpg upstream activating sequence. RPL19A was also found to be adjacent to the chromosome II AAC3 gene, encoding the mitochondrial ADP/ATP carrier protein.

Whole-cell detection by 13C NMR of metabolic flux through the C1-tetrahydrofolate synthase/serine hydroxymethyltransferase enzyme system and effect of antifolate exposure in Saccharomyces cerevisiae.

Folate-mediated one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth. A potential source of one-carbon units is formate. This one-carbon unit is activated to 10-formyltetrahydrofolate via the synthetase activity of the trifunctional enzyme C1-tetrahydrofolate (THF) synthase for use in purine synthesis or can be further reduced to 5,10-methylene-THF by the dehydrogenase activity of the same enzyme. 5,10-Methylene-THF is used by serine hydroxymethyltransferase (SHMT) in the synthesis of serine. Recently, 13C NMR has been used to establish that the C1-THF synthase/SHMT enzyme system is the only route from formate to serine in vivo in the yeast Saccharomyces cerevisiae [Pasternack et al. (1992) Biochemistry 31, 8713-8719]. In vitro studies have considered the kinetics of the C1-THF synthase/SHMT enzyme system in the catalytic conversion of formate to serine [Strong et al. (1987) J. Biol. Chem. 262, 12519-12525]. In the present work, we begin to study the kinetics of this two-enzyme system in its natural environment. Provision of [13C]formate and direct detection of an intracellular accumulating pool of [3-13C]serine by 13C NMR of whole cells allow us to monitor the rate of flux through this enzyme system in vivo. The rate of accumulation of soluble [3-13C]serine under [13C]formate-saturating conditions is 13.0 +/- 1.2 microM/min relative to an external standard of serine in D2O. The extracellular formate concentration at half-maximal flux was determined to be 900 microM.(ABSTRACT TRUNCATED AT 250 WORDS)