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Dean R Appling - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial C1-Tetrahydrofolate Synthase (MTHFD1L) supports the flow of mitochondrial one-carbon units into the methyl cycle in embryos
Journal of Biological Chemistry, 2010Co-Authors: Schuyler T. Pike, Rashmi Rajendra, Karen Artzt, Dean R ApplingAbstract: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.
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Yeast AEP3p is an accessory factor in initiation of mitochondrial translation.
The Journal of biological chemistry, 2009Co-Authors: Changkeun Lee, Anne S. Tibbetts, Gisela Kramer, Dean R ApplingAbstract:Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNAfMet). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNAfMet, as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNAfMet to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNAfMet. This accessory factor might be unnecessary when formylated Met-tRNAfMet is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Δfmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNAfMet due to loss of the MIS1 gene that encodes the mitochondrial C1-Tetrahydrofolate Synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.
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human mitochondrial c1 Tetrahydrofolate Synthase submitochondrial localization of the full length enzyme and characterization of a short isoform
Archives of Biochemistry and Biophysics, 2009Co-Authors: Priya Prasannan, Dean R ApplingAbstract:Mammalian mitochondrial C1-Tetrahydrofolate (THF) Synthase (MTHFDIL gene product) is a monofunctional 10-formyl-THF synthetase, lacking the 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities typically found in the trifunctional cytoplasmic proteins. Here, we report the submitochondrial localization of epitope-tagged human mitochondrial C1-THF Synthase expressed in Chinese hamster ovary cells. Mitochondrial fractionation experiments show that human mitochondrial C1-THF Synthase behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. Inner mitochondrial membrane association was also observed for the endogenous mitochondrial C1-THF Synthase in adult rat spleen. We also purified and characterized the recombinant protein product (short isoform) of the alternatively spliced short transcript of the mitochondrial isozyme. Methylene-THF dehydrogenase assays confirmed that the short isoform is not enzymatically active. The purified short isoform was used in the production of polyclonal antibodies specific for the mitochondrial isozyme. These antibodies detected endogenous full-length mitochondrial C1-THF Synthase in mitochondria from adult rat spleen and human placenta, confirming the expression of the mitochondrial isozyme in adult mammalian tissues.
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enzymatic characterization of human mitochondrial c1 Tetrahydrofolate Synthase
Archives of Biochemistry and Biophysics, 2005Co-Authors: Addie S Walkup, Dean R ApplingAbstract:Abstract A human mitochondrial isozyme of C 1 -Tetrahydrofolate (THF) Synthase was previously identified by its similarity to the human cytoplasmic C 1 -THF Synthase. All C 1 -THF Synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C 1 -THF Synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C 1 -THF Synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6 R , S )-H 4 -PteGlu 1 , (6 R , S )-H 4 -PteGlu 3 , and (6 R , S )-H 4 -PteGlu 5 substrates. The (6 R , S )-H 4 -PteGlu n K m dropped from greater than 500 μM for the monoglutamate to 15 μM and 3.6 μM for the tri- and pentaglutamates, respectively. The K m values for formate and ATP also are lowered when THF polyglutamates are used. The formate K m dropped 79-fold and the ATP K m dropped more than 5-fold when (6 R , S )-H 4 -PteGlu 5 was used as the substrate in place of (6 R , S )-H 4 -PteGlu 1 .
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Characterization of the rat cytoplasmic C1-Tetrahydrofolate Synthase gene and analysis of its expression in liver regeneration and fetal development
Gene, 2003Co-Authors: Katherine M Howard, Stephanie J. Muga, Liwen Zhang, Anice E. Thigpen, Dean R ApplingAbstract:Abstract The eukaryotic trifunctional enzyme, C1-Tetrahydrofolate (THF) Synthase, interconverts folic acid derivatives between various oxidation states and is critical for normal cellular function, growth, and differentiation. Using a rat C1-THF Synthase cDNA and synthetic oligonucleotides, the rat C1-THF Synthase gene was isolated and characterized. The gene consists of 28 exons and spans 67.5 kbp. Primer extension, RNase protection, and rapid amplification of cDNA ends (RACE) experiments indicate the presence of multiple transcription start points (tsp) within a 250-bp window located between 50 and 300 bp upstream from the start codon. The 5′ flanking region is devoid of a TATA consensus sequence motif, but putative regulatory elements, including NF-κβ, HNF-4α1, RARα1, C/EBP, and PPAR are present in the promoter region. The 5′ flanking region also contains two sets of tetranucleotide repeats and two short interspersed nuclear elements (SINES). The initial 2500 bp of 5′ flanking sequences of the rat and mouse cytoplasmic C1-THF Synthase genes share 70% identity. However, comparison with the human gene from the Human Genome Data Bank revealed no significant homology in the 5′ flanking region. The gene structure characterization led to the identification of a pseudogene that is 94% identical to the C1-THF Synthase gene and probably diverged 10–12 million years ago. In addition, the gene expression patterns of C1-THF Synthase were investigated during liver regeneration and liver and kidney organogenesis, two highly regulated events. In both processes, C1-THF Synthase expression correlated with increased nucleotide metabolism. This pattern suggests that the gene is regulated in response to changes in the demand for folate-dependent one-carbon units.
Jesse C. Rabinowitz - One of the best experts on this subject based on the ideXlab platform.
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Function of yeast cytoplasmic Cl-Tetrahydrofolate Synthase (Saccharomyces cerevisiae/heterologous gene expression/ADE3/purine biosynthesis)
2016Co-Authors: Jae Mahn Song, Jesse C. RabinowitzAbstract:The protein product of the ADE3 gene of the yeast Saccharomyces cerevisiae has been identified as the cy- toplasmic trifunctional Cl-Tetrahydrofolate (THF) Synthase, which possesses 10-formyl-THF synthetase (EC 6.3.4.3), 5,10- methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10- methylene-THF dehydrogenase (EC 1.5.1.5) activities. How- ever, it has been suggested that the ADE3-encoded C1-THF Synthase does not play a role in providing the enzymes involved in the generation of one-carbon intermediates in the biosyn- thesis of the purine bases but functions in maintaining the structural integrity of the enzyme complex involved in purine biosynthesis (Barlowe, C. K. & Appling, D. A. (1990) Mol. Cell. Biol. 10, 5679-5687). This hypothesis is based on their finding that the presence of the full-length ADE3 C1-THF Synthase, whether catalytically active or not, is correlated with the Ade+ phenotype. In contrast to their results, our deletion analysis of the ADE3 gene indicates that the presence of either the synthetase or dehydrogenase/cyclohydrolase domains of C1-THF Synthase is enough to complement the adenine require- ment in ade3 strains. These results are also consistent with those obtained in heterologous expression of spinach and Clostridium acidiurici monofunctional synthetases in ade3 strains. Heterologous expression studies show that the high synthetase activity may be correlated with the increased growth in medium lacking adenine. These results suggest that the catalytic activity of the CI-THF Synthase is involved in purinr biosynthesis.
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function of yeast cytoplasmic cl Tetrahydrofolate Synthase saccharomyces cerevisiae heterologous gene expression ade3 purine biosynthesis
2016Co-Authors: Jae Mahn Song, Jesse C. RabinowitzAbstract:The protein product of the ADE3 gene of the yeast Saccharomyces cerevisiae has been identified as the cy- toplasmic trifunctional Cl-Tetrahydrofolate (THF) Synthase, which possesses 10-formyl-THF synthetase (EC 6.3.4.3), 5,10- methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10- methylene-THF dehydrogenase (EC 1.5.1.5) activities. How- ever, it has been suggested that the ADE3-encoded C1-THF Synthase does not play a role in providing the enzymes involved in the generation of one-carbon intermediates in the biosyn- thesis of the purine bases but functions in maintaining the structural integrity of the enzyme complex involved in purine biosynthesis (Barlowe, C. K. & Appling, D. A. (1990) Mol. Cell. Biol. 10, 5679-5687). This hypothesis is based on their finding that the presence of the full-length ADE3 C1-THF Synthase, whether catalytically active or not, is correlated with the Ade+ phenotype. In contrast to their results, our deletion analysis of the ADE3 gene indicates that the presence of either the synthetase or dehydrogenase/cyclohydrolase domains of C1-THF Synthase is enough to complement the adenine require- ment in ade3 strains. These results are also consistent with those obtained in heterologous expression of spinach and Clostridium acidiurici monofunctional synthetases in ade3 strains. Heterologous expression studies show that the high synthetase activity may be correlated with the increased growth in medium lacking adenine. These results suggest that the catalytic activity of the CI-THF Synthase is involved in purinr biosynthesis.
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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.
FEBS letters, 1995Co-Authors: Jae Mahn Song, Jesse C. RabinowitzAbstract:Abstract 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.
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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
FEBS Letters, 1995Co-Authors: Jae Mahn Song, Jesse C. RabinowitzAbstract:Abstract 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.
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Nucleotide sequence and characterization of the Saccharomyces cerevisiae RPL19A gene encoding a homolog of the mammalian ribosomal protein l19
Yeast (Chichester England), 1995Co-Authors: Jae Mahn Song, Edwin Cheung, Jesse C. RabinowitzAbstract: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% identity 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. The nucleotide sequence(s) reported in this paper has been submitted to the GenBanktm/EMBL data bank with the accession number Z36751.
Jae Mahn Song - One of the best experts on this subject based on the ideXlab platform.
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Function of yeast cytoplasmic Cl-Tetrahydrofolate Synthase (Saccharomyces cerevisiae/heterologous gene expression/ADE3/purine biosynthesis)
2016Co-Authors: Jae Mahn Song, Jesse C. RabinowitzAbstract:The protein product of the ADE3 gene of the yeast Saccharomyces cerevisiae has been identified as the cy- toplasmic trifunctional Cl-Tetrahydrofolate (THF) Synthase, which possesses 10-formyl-THF synthetase (EC 6.3.4.3), 5,10- methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10- methylene-THF dehydrogenase (EC 1.5.1.5) activities. How- ever, it has been suggested that the ADE3-encoded C1-THF Synthase does not play a role in providing the enzymes involved in the generation of one-carbon intermediates in the biosyn- thesis of the purine bases but functions in maintaining the structural integrity of the enzyme complex involved in purine biosynthesis (Barlowe, C. K. & Appling, D. A. (1990) Mol. Cell. Biol. 10, 5679-5687). This hypothesis is based on their finding that the presence of the full-length ADE3 C1-THF Synthase, whether catalytically active or not, is correlated with the Ade+ phenotype. In contrast to their results, our deletion analysis of the ADE3 gene indicates that the presence of either the synthetase or dehydrogenase/cyclohydrolase domains of C1-THF Synthase is enough to complement the adenine require- ment in ade3 strains. These results are also consistent with those obtained in heterologous expression of spinach and Clostridium acidiurici monofunctional synthetases in ade3 strains. Heterologous expression studies show that the high synthetase activity may be correlated with the increased growth in medium lacking adenine. These results suggest that the catalytic activity of the CI-THF Synthase is involved in purinr biosynthesis.
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function of yeast cytoplasmic cl Tetrahydrofolate Synthase saccharomyces cerevisiae heterologous gene expression ade3 purine biosynthesis
2016Co-Authors: Jae Mahn Song, Jesse C. RabinowitzAbstract:The protein product of the ADE3 gene of the yeast Saccharomyces cerevisiae has been identified as the cy- toplasmic trifunctional Cl-Tetrahydrofolate (THF) Synthase, which possesses 10-formyl-THF synthetase (EC 6.3.4.3), 5,10- methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10- methylene-THF dehydrogenase (EC 1.5.1.5) activities. How- ever, it has been suggested that the ADE3-encoded C1-THF Synthase does not play a role in providing the enzymes involved in the generation of one-carbon intermediates in the biosyn- thesis of the purine bases but functions in maintaining the structural integrity of the enzyme complex involved in purine biosynthesis (Barlowe, C. K. & Appling, D. A. (1990) Mol. Cell. Biol. 10, 5679-5687). This hypothesis is based on their finding that the presence of the full-length ADE3 C1-THF Synthase, whether catalytically active or not, is correlated with the Ade+ phenotype. In contrast to their results, our deletion analysis of the ADE3 gene indicates that the presence of either the synthetase or dehydrogenase/cyclohydrolase domains of C1-THF Synthase is enough to complement the adenine require- ment in ade3 strains. These results are also consistent with those obtained in heterologous expression of spinach and Clostridium acidiurici monofunctional synthetases in ade3 strains. Heterologous expression studies show that the high synthetase activity may be correlated with the increased growth in medium lacking adenine. These results suggest that the catalytic activity of the CI-THF Synthase is involved in purinr biosynthesis.
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Cooperative regulation of ADE3 transcription by Gcn4p and Bas1p in Saccharomyces cerevisiae
Eukaryotic cell, 2009Co-Authors: Yoo Jin Joo, Jae Mahn Song, Jung Ae Kim, Joung Hee Baek, Ki Moon Seong, Kyung Duk Han, Jin Young Choi, Joon KimAbstract:The one-carbon response regulon is essential for the biosynthesis of nucleic acids as well as several amino acids. The ADE3 gene is known to encode a crucial one-carbon regulon enzyme, Tetrahydrofolate Synthase, which is involved in the biosynthesis of purine and the amino acids methionine and glycine. Therefore, the mechanism through which ADE3 transcription is regulated appears to be critical for the cross-talk among these metabolic pathways. Even so, the direct involvement of ADE3 transcription through gene-specific transcription factors has not been shown clearly. In this study, the promoter structure of the ADE3 gene was investigated in detail, and a genuine Gcn4p responsive element (GCRE) was confirmed among three putative GCRE elements in vivo and in vitro. Through gene deletion studies of Gcn4p and Bas1p, it was established that both factors are involved in the transcriptional regulation of the ADE3 gene. Direct binding to this GCRE and the occupancy of the ADE3 promoter by these factors were also confirmed. Taking these results together, we concluded that Gcn4p is responsible for the basal and inducible expression of the ADE3 gene, while Bas1p is required for its basal expression.
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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.
FEBS letters, 1995Co-Authors: Jae Mahn Song, Jesse C. RabinowitzAbstract:Abstract 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.
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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
FEBS Letters, 1995Co-Authors: Jae Mahn Song, Jesse C. RabinowitzAbstract:Abstract 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.
Laura B. Pasternack - One of the best experts on this subject based on the ideXlab platform.
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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
Biochemistry, 1994Co-Authors: Laura B. Pasternack, David A. Laude, Dean R ApplingAbstract: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)
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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.
Biochemistry, 1994Co-Authors: Laura B. Pasternack, David A. Laude, Dean R ApplingAbstract: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)
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13C NMR analysis of intercompartmental flow of one-carbon units into choline and purines in Saccharomyces cerevisiae.
Biochemistry, 1994Co-Authors: Laura B. Pasternack, David A. Laude, Dean R ApplingAbstract:In Saccharomyces cerevisiae, the three-carbon of serine is normally the major one-carbon donor, although glycine and formate can substitute for serine. The second carbon of glycine enters via the glycine cleavage system in the mitochondria and can satisfy all cellular one-carbon requirements. It remains unresolved, however, as to the route by which these mitochondrial one-carbon units supply cytosolic anabolic processes. In the present work, we have used yeast mutants blocked at selected sites and 13C NMR to trace the incorporation of glycine-derived mitochondrial 5,10-methyleneTetrahydrofolate into nonmitochondrial synthesis of choline and purines. Label incorporation into choline traces the methylation pathway of choline synthesis from production of serine to methylation of phosphatidylethanolamine. The active one-carbon unit of S-adenosylmethionine involved in methylation reactions originates almost solely from C3 of serine. On the other hand, flow of mitochondrial one-carbon units to 10-formylTetrahydrofolate for purine synthesis is shown to occur via both serine and formate. Formate transport accounts for at least 25% of the total, even during growth with sufficient serine to provide for the one-carbon requirements of the cell. This work shows that the synthetase function of the cytosolic C1-Tetrahydrofolate Synthase plays a critical role in the processing of mitochondrial one-carbon units to 10-formylTetrahydrofolate pools. In addition, this study provides evidence of two pools of glycine within the mitochondria and establishes a system of analyzing flux into the different folate derivatives.
David A. Laude - One of the best experts on this subject based on the ideXlab platform.
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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
Biochemistry, 1994Co-Authors: Laura B. Pasternack, David A. Laude, Dean R ApplingAbstract: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)
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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.
Biochemistry, 1994Co-Authors: Laura B. Pasternack, David A. Laude, Dean R ApplingAbstract: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)
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13C NMR analysis of intercompartmental flow of one-carbon units into choline and purines in Saccharomyces cerevisiae.
Biochemistry, 1994Co-Authors: Laura B. Pasternack, David A. Laude, Dean R ApplingAbstract:In Saccharomyces cerevisiae, the three-carbon of serine is normally the major one-carbon donor, although glycine and formate can substitute for serine. The second carbon of glycine enters via the glycine cleavage system in the mitochondria and can satisfy all cellular one-carbon requirements. It remains unresolved, however, as to the route by which these mitochondrial one-carbon units supply cytosolic anabolic processes. In the present work, we have used yeast mutants blocked at selected sites and 13C NMR to trace the incorporation of glycine-derived mitochondrial 5,10-methyleneTetrahydrofolate into nonmitochondrial synthesis of choline and purines. Label incorporation into choline traces the methylation pathway of choline synthesis from production of serine to methylation of phosphatidylethanolamine. The active one-carbon unit of S-adenosylmethionine involved in methylation reactions originates almost solely from C3 of serine. On the other hand, flow of mitochondrial one-carbon units to 10-formylTetrahydrofolate for purine synthesis is shown to occur via both serine and formate. Formate transport accounts for at least 25% of the total, even during growth with sufficient serine to provide for the one-carbon requirements of the cell. This work shows that the synthetase function of the cytosolic C1-Tetrahydrofolate Synthase plays a critical role in the processing of mitochondrial one-carbon units to 10-formylTetrahydrofolate pools. In addition, this study provides evidence of two pools of glycine within the mitochondria and establishes a system of analyzing flux into the different folate derivatives.