Journal articles on the topic 'Tetrahydrofolate dehydrogenase'
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Invernizzi, R., R. Nano, O. Perugini, P. Fazio, L. Nespoli, G. Gerzeli, and E. Ascari. "Tetrahydrofolate dehydrogenase cytochemistry in acute lymphoblastic leukemia." European Journal of Haematology 41, no. 2 (April 24, 2009): 109–14. http://dx.doi.org/10.1111/j.1600-0609.1988.tb00879.x.
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Brosnan, Margaret E., Garrett Tingley, Luke MacMillan, Brian Harnett, Theerawat Pongnopparat, Jenika D. Marshall, and John T. Brosnan. "Plasma Formate Is Greater in Fetal and Neonatal Rats Compared with Their Mothers." Journal of Nutrition 150, no. 5 (January 7, 2020): 1068–75. http://dx.doi.org/10.1093/jn/nxz329.
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ABSTRACT Background Formate can be incorporated into 10-formyl-tetrahydrofolate (10-formyl-THF), which is a substrate for purine synthesis, and after further reduction of the one-carbon group, may be used as a substrate for thymidylate synthesis and for homocysteine remethylation. Objective We examined plasma formate concentrations and the expression of genes involved in the production and utilization of formate in fetal and neonatal rats and in pregnant and virgin female rats. Methods In 1 experiment, plasma formate was measured by GC-MS in rats aged 1–56 d. In a second experiment, virgin female (adult) rats, 19-d pregnant rats (P) and their male and female fetuses (F), and 3-d-old (N) and 7-d-old (J) offspring had plasma and amniotic fluid analyzed for formate by GC-MS, mRNA abundance in liver and placenta by qPCR, and several plasma amino acids by HPLC. Results The plasma formate concentration was significantly higher in fetuses at embryonic day 19 than in the mothers. It was also significantly higher in neonatal rats but slowly returned to adult concentrations by ∼3 wk. The abundance of mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase (Mthfd1l) mRNA was significantly higher in placenta (PP) and F liver than in liver of N or J. Expression of mitochondrial bifunctional NAD-dependent methylene-tetrahydrofolate dehydrogenase/methenyl-tetrahydrofolate cyclohydrolase (Mthfd2) was significantly enriched in PP and liver of P, intermediate in F liver, and much lower in liver of N and J, relative to PP. Serine hydroxymethyltransferase 2 (Shmt2), methylenetetrahydrofolate dehydrogenase 1 (Mthfd1), and glycine decarboxylase protein of the glycine cleavage system (Gldc) mRNA expression was significantly lower in PP compared with other groups. Cytoplasmic NAD(P)-dependent 10-formyl-tetrahydrofolate dehydrogenase (Aldh1/1) and mitochondrial NAD(P)-dependent 10-formyl-tetrahydrofolate dehydrogenase (Aldh1/2) , genes responsible for the catabolism of 10-formylTHF, were very weakly expressed in PP, low in livers of F and N, and reached the significantly higher adult levels in J. Serine, glycine, and methionine concentrations in plasma of F were significantly higher than in plasma of P. Conclusions Formate metabolism is highly active in fetuses and in placenta of pregnant rats.
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Krupenko, S. A., C. Wagner, and R. J. Cook. "Recombinant 10-formyltetrahydrofolate dehydrogenase catalyses both dehydrogenase and hydrolase reactions utilizing the synthetic substrate 10-formyl-5,8-dideazafolate." Biochemical Journal 306, no. 3 (March 15, 1995): 651–55. http://dx.doi.org/10.1042/bj3060651.
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10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) is a bifunctional enzyme, displaying both NADP(+)-dependent dehydrogenase activity for the formation of tetrahydrofolate and CO2, and NADP(+)-independent hydrolase activity for the formation of tetrahydrofolate and formate. A previous report [Case, Kaisaki and Steele (1988) J. Biol. Chem. 263, 1024-1027] claimed that dehydrogenase and hydrolase activities were products of separate cytosolic and mitochondrial forms of this enzyme. Here we report that recombinant 10-formyltetrahydrofolate dehydrogenase carries out both enzymic reactions, proving that a product of a single gene, i.e. one protein, not two, has both activities. The stable synthetic analogue 10-formyl-5,8-dideazafolate can substitute for the labile natural substrate, 10-formyltetrahydrofolate, in both reactions. This was shown with both native and recombinant rat liver enzyme. The Km values for 10-formyl-5,8-dideazafolate were half of those for 10-formyltetrahydrofolate in both the dehydrogenase and hydrolytic reactions. The Vmax, values were similar for both substrates. Both dehydrogenase and hydrolase reactions were dependent on the presence of 2-mercaptoethanol. The pH optima were 7.8 and 5.6 for the dehydrogenase and hydrolase reactions respectively, consistent with the presence of two active sites in the enzyme.
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Hattori, Satoshi, Alexander S. Galushko, Yoichi Kamagata, and Bernhard Schink. "Operation of the CO Dehydrogenase/Acetyl Coenzyme A Pathway in both Acetate Oxidation and Acetate Formation by the Syntrophically Acetate-Oxidizing Bacterium Thermacetogenium phaeum." Journal of Bacteriology 187, no. 10 (May 15, 2005): 3471–76. http://dx.doi.org/10.1128/jb.187.10.3471-3476.2005.
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ABSTRACT Thermacetogenium phaeum is a homoacetogenic bacterium that can grow on various substrates, such as pyruvate, methanol, or H2/CO2. It can also grow on acetate if cocultured with the hydrogen-consuming methanogenic partner Methanothermobacter thermautotrophicus. Enzyme activities of the CO dehydrogenase/acetyl coenzyme A (CoA) pathway (CO dehydrogenase, formate dehydrogenase, formyl tetrahydrofolate synthase, methylene tetrahydrofolate dehydrogenase) were detected in cell extracts of pure cultures and of syntrophic cocultures. Mixed cell suspensions of T. phaeum and M. thermautotrophicus oxidized acetate rapidly and produced acetate after addition of H2/CO2 after a short time lag. CO dehydrogenase activity staining after native polyacrylamide gel electrophoresis exhibited three oxygen-labile bands which were identical in pure culture and coculture. Protein profiles of T. phaeum cells after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the strain exhibited basically the same protein patterns in both pure and syntrophic culture. These results indicate that T. phaeum operates the CO dehydrogenase/acetyl-CoA pathway reversibly both in acetate oxidation and in reductive acetogenesis by using the same biochemical apparatus, although it has to couple this pathway to ATP synthesis in different ways.
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Vorholt, Julia A., Marina G. Kalyuzhnaya, Christoph H. Hagemeier, Mary E. Lidstrom, and Ludmila Chistoserdova. "MtdC, a Novel Class of Methylene Tetrahydromethanopterin Dehydrogenases." Journal of Bacteriology 187, no. 17 (September 1, 2005): 6069–74. http://dx.doi.org/10.1128/jb.187.17.6069-6074.2005.
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ABSTRACT Novel methylene tetrahydromethanopterin (H4MPT) dehydrogenase enzymes, named MtdC, were purified after expressing in Escherichia coli genes from, respectively, Gemmata sp. strain Wa1-1 and environmental DNA originating from unidentified microbial species. The MtdC enzymes were shown to possess high affinities for methylene-H4MPT and NADP but low affinities for methylene tetrahydrofolate or NAD. The substrate range and the kinetic properties revealed by MtdC enzymes distinguish them from the previously characterized bacterial methylene-H4MPT dehydrogenases, MtdA and MtdB. While revealing higher sequence similarity to MtdA enzymes, MtdC enzymes appear to fulfill a function homologous to the function of MtdB, as part of the H4MPT-linked pathway for formaldehyde oxidation/detoxification.
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Vorholt, Julia A., Ludmila Chistoserdova, Mary E. Lidstrom, and Rudolf K. Thauer. "The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in Methylobacterium extorquens AM1." Journal of Bacteriology 180, no. 20 (October 15, 1998): 5351–56. http://dx.doi.org/10.1128/jb.180.20.5351-5356.1998.
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ABSTRACT An NADP-dependent methylene tetrahydromethanopterin (H4MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO2 inMethylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be themtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H4MPT with NADP+rather than with NAD+, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H4F) with NADP+. With methylene H4F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (V max/Km ) were approximately 20-fold lower than with methylene H4MPT. Whereas the dehydrogenation of methylene H4MPT (E0 = −390 mV) with NADP+ (E0 = −320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H4F (E0 = −300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase fromM. extorquens AM1 with those of methylene H4F dehydrogenases from other bacteria and eucarya and with those of methylene H4MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).
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Dimri, G. P., G. F. Ames, L. D'Ari, and J. C. Rabinowitz. "Physical map location of the Escherichia coli gene encoding the bifunctional enzyme 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methenyl-tetrahydrofolate cyclohydrolase." Journal of Bacteriology 173, no. 17 (1991): 5251. http://dx.doi.org/10.1128/jb.173.17.5251-.1991.
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Dimri, G. P., G. F. Ames, L. D'Ari, and J. C. Rabinowitz. "Physical map location of the Escherichia coli gene encoding the bifunctional enzyme 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methenyl-tetrahydrofolate cyclohydrolase.:." Journal of Bacteriology 173, no. 17 (September 1991): 5251. http://dx.doi.org/10.1128/jb.173.17.5251.1991.
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Wilquet, Valérie, Mark Van de Casteele, Daniel Gigot, Christianne Legrain, and Nicolas Glansdorff. "Dihydropteridine Reductase as an Alternative to Dihydrofolate Reductase for Synthesis of Tetrahydrofolate in Thermus thermophilus." Journal of Bacteriology 186, no. 2 (January 15, 2004): 351–55. http://dx.doi.org/10.1128/jb.186.2.351-355.2004.
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ABSTRACT A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH Tt ] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH Tt appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.
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Wang, Hongchao, Qizai Wang, Chen Zhang, Haiqin Chen, Wenwei Lu, Zhennan Gu, Jianxin Zhao, Hao Zhang, Yong Q. Chen, and Wei Chen. "The role of MTHFDL in mediating intracellular lipogenesis in oleaginous Mortierella alpina." Microbiology 166, no. 7 (July 1, 2020): 617–23. http://dx.doi.org/10.1099/mic.0.000897.
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The oleaginous fungus Mortierella alpina can synthesize a variety of polyunsaturated fatty acids, which are used extensively in industry for the production of arachidonic acid (AA). NADPH is the limiting factor and critical reducing agent in lipid biosynthesis. In the folate cycle, methylenetetrahydrofolate dehydrogenase (MTHFDL) catalyzes the conversion of methylene tetrahydrofolate into 10-formyl-tetrahydrofolate with the reduction of NADP+ to NADPH. MTHFDL RNAi was used to investigate the role of the folate cycle in lipogenesis. Gene knockdown decreased the transcript levels of MTHFDL by about 50 % and attenuated cell fatty acid synthesis. The observation of decreased NADPH levels and downregulated NADPH-producing genes in response to MTHFDL RNAi indicates a novel aspect of the NADPH regulatory mechanism. Thus, our study demonstrates that MTHFDL plays key role in the mediation of NADPH in lipogenesis in M. alpina.
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Studer, Alex, Craig McAnulla, Rainer Büchele, Thomas Leisinger, and Stéphane Vuilleumier. "Chloromethane-Induced Genes Define a Third C1 Utilization Pathway in Methylobacterium chloromethanicum CM4." Journal of Bacteriology 184, no. 13 (July 1, 2002): 3476–84. http://dx.doi.org/10.1128/jb.184.13.3476-3484.2002.
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ABSTRACT Methylobacterium chloromethanicum CM4 is an aerobic α-proteobacterium capable of growth with chloromethane as the sole carbon and energy source. Two proteins, CmuA and CmuB, were previously purified and shown to catalyze the dehalogenation of chloromethane and the vitamin B12-mediated transfer of the methyl group of chloromethane to tetrahydrofolate. Three genes located near cmuA and cmuB, designated metF, folD and purU and encoding homologs of methylene tetrahydrofolate (methylene-H4folate) reductase, methylene-H4folate dehydrogenase-methenyl-H4folate cyclohydrolase and formyl-H4folate hydrolase, respectively, suggested the existence of a chloromethane-specific oxidation pathway from methyl-tetrahydrofolate to formate in strain CM4. Hybridization and PCR analysis indicated that these genes were absent in Methylobacterium extorquens AM1, which is unable to grow with chloromethane. Studies with transcriptional xylE fusions demonstrated the chloromethane-dependent expression of these genes. Transcriptional start sites were mapped by primer extension and allowed to define three transcriptional units, each likely comprising several genes, that were specifically expressed during growth of strain CM4 with chloromethane. The DNA sequences of the deduced promoters display a high degree of sequence conservation but differ from the Methylobacterium promoters described thus far. As shown previously for purU, inactivation of the metF gene resulted in a CM4 mutant unable to grow with chloromethane. Methylene-H4folate reductase activity was detected in a cell extract of strain CM4 only in the presence of chloromethane but not in the metF mutant. Taken together, these data provide evidence that M. chloromethanicum CM4 requires a specific set of tetrahydrofolate-dependent enzymes for growth with chloromethane.
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Shen, Betty W., David H. Dyer, Jie-Yu Huang, Linda D'Ari, Jesse Rabinowitz, and Barry L. Stoddard. "The crystal structure of a bacterial, bifunctional 5, 10 methylene-tetrahydrofolate dehydrogenase/cyclohydrolase." Protein Science 8, no. 6 (1999): 1342–49. http://dx.doi.org/10.1110/ps.8.6.1342.
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Goenrich, Meike, Jan Bursy, Eva Hübner, Dietmar Linder, Arnold C. Schwartz, and Julia A. Vorholt. "Purification and characterization of the methylene tetrahydromethanopterin dehydrogenase MtdB and the methylene tetrahydrofolate dehydrogenase FolD from Hyphomicrobium zavarzinii ZV580." Archives of Microbiology 177, no. 4 (January 31, 2002): 299–303. http://dx.doi.org/10.1007/s00203-001-0394-y.
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Luka, Zigmund, Svetlana Pakhomova, Lioudmila V. Loukachevitch, Marcia E. Newcomer, and Conrad Wagner. "Folate in demethylation: The crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate." Biochemical and Biophysical Research Communications 449, no. 4 (July 2014): 392–98. http://dx.doi.org/10.1016/j.bbrc.2014.05.064.
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Fu, Tzu-Fun, Bruno Maras, Donatella Barra, and Verne Schirch. "A Noncatalytic Tetrahydrofolate Tight Binding Site Is on the Small Domain of 10-Formyltetrahydrofolate Dehydrogenase." Archives of Biochemistry and Biophysics 367, no. 2 (July 1999): 161–66. http://dx.doi.org/10.1006/abbi.1999.1262.
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Murta, Silvane M. F., Tim J. Vickers, David A. Scott, and Stephen M. Beverley. "Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential inLeishmania major." Molecular Microbiology 71, no. 6 (March 2009): 1386–401. http://dx.doi.org/10.1111/j.1365-2958.2009.06610.x.
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Field, Martha S., Elena Kamynina, David Watkins, David S. Rosenblatt, and Patrick J. Stover. "Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis." Proceedings of the National Academy of Sciences 112, no. 2 (December 29, 2014): 400–405. http://dx.doi.org/10.1073/pnas.1414555112.
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An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.
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Cheung, Edwin, Linda D'Ari, Jesse C. Rabinowitz, David H. Dyer, Jie-Yu Huang, and Barry L. Stoddard. "Purification, crystallization, and preliminary X-ray studies of a bifunctional 5,10-methenyl/methylene tetrahydrofolate cyclohydrolase/dehydrogenase fromEscherichia coli." Proteins: Structure, Function, and Genetics 27, no. 2 (February 1997): 322–24. http://dx.doi.org/10.1002/(sici)1097-0134(199702)27:2<322::aid-prot19>3.0.co;2-o.
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Brosnan, Margaret E., Luke MacMillan, Jennifer R. Stevens, and John T. Brosnan. "Division of labour: how does folate metabolism partition between one-carbon metabolism and amino acid oxidation?" Biochemical Journal 472, no. 2 (November 13, 2015): 135–46. http://dx.doi.org/10.1042/bj20150837.
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One-carbon metabolism is usually represented as having three canonical functions: purine synthesis, thymidylate synthesis and methylation reactions. There is however a fourth major function: the metabolism of some amino acids (serine, glycine, tryptophan and histidine), as well as choline. These substrates can provide cells with more one-carbon groups than they need for these three canonical functions. Therefore, there must be mechanisms for the disposal of these one-carbon groups (when in excess) which maintain the complement of these groups required for the canonical functions. The key enzyme for these mechanisms is 10-formyl-THF (tetrahydrofolate) dehydrogenase (both mitochondrial and cytoplasmic isoforms) which oxidizes the formyl group to CO2 with the attendant reduction of NADP+ to NADPH and release of THF. In addition to oxidizing the excess of these compounds, this process can reduce substantial quantities of NADP+ to NADPH.
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Barlowe, C. K., and D. R. Appling. "Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme." Molecular and Cellular Biology 10, no. 11 (November 1990): 5679–87. http://dx.doi.org/10.1128/mcb.10.11.5679.
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In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.
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Barlowe, C. K., and D. R. Appling. "Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme." Molecular and Cellular Biology 10, no. 11 (November 1990): 5679–87. http://dx.doi.org/10.1128/mcb.10.11.5679-5687.1990.
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In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.
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Bang, Junho, and Sang Yup Lee. "Assimilation of formic acid and CO2by engineeredEscherichia coliequipped with reconstructed one-carbon assimilation pathways." Proceedings of the National Academy of Sciences 115, no. 40 (September 17, 2018): E9271—E9279. http://dx.doi.org/10.1073/pnas.1810386115.
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Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO2can be an attractive raw material for bio-based chemicals. Here, we report the development ofEscherichia colistrains assimilating FA and CO2through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. TheMethylobacterium extorquensformate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA assimilation. The gcv reaction was reversed by knocking out the repressor gene (gcvR) and overexpressing thegcvTHPgenes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO2in a glucose-containing medium. Native serine deaminase converted serine to pyruvate, showing 4.5% of pyruvate-forming flux comes from FA and CO2. The pyruvate-forming flux from FA and CO2could be increased to 14.9% by knocking outgcvR,pflB, andserA, chromosomally expressinggcvTHPundertrc, and overexpressing the reconstructed THF cycle,gcvTHP, andlpdgenes in one vector. To reduce glucose usage required for energy and redox generation, theCandida boidiniiformate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO2consumption rates of 370.2, 145.6, and 14.9 mg⋅g dry cell weight (DCW)−1⋅h−1, respectively. The C1 assimilation pathway consumed 21.3 wt% of FA. Furthermore, cells sustained slight growth using only FA and CO2after glucose depletion, suggesting that combined use of the C1 assimilation pathway andC. boidiniiFdh will be useful for eventually developing a strain capable of utilizing FA and CO2without an additional carbon source such as glucose.
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Okano, Kenji, Yu Sato, Shota Inoue, Shizuka Kawakami, Shigeru Kitani, and Kohsuke Honda. "Enhancement of S-Adenosylmethionine-Dependent Methylation by Integrating Methanol Metabolism with 5-Methyl-Tetrahydrofolate Formation in Escherichia coli." Catalysts 10, no. 9 (September 2, 2020): 1001. http://dx.doi.org/10.3390/catal10091001.
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S-Adenosylmethionine (SAM)-dependent methyltransferases are important tools for the biocatalytic methylation of diverse biomolecules. Methylation by a whole-cell biocatalyst allows the utilization of intrinsic SAM and its regeneration system, which consists of a cyclic and multi-step enzymatic cascade. However, low intracellular availability of 5-methyl-tetrahydrofolate (5-methyl-THF), which functions as a methyl group donor, limits SAM regeneration. Here, we integrated methanol metabolism with 5-methyl-THF formation into SAM-dependent methylation system in Escherichia coli, driven by heterologously expressed methanol dehydrogenase (MDH). The coupling of MDH-catalyzed methanol oxidation with the E. coli endogenous reactions enhances the formation of 5-methyl-THF using methanol as a source of methyl group, thereby promoting both the SAM regeneration and methylation reactions. Co-expression of the mutant MDH2 from Cupriavidus necator N-1 with the O-methyltransferase 5 from Streptomyces avermitilis MA-4680 enhanced O-methylation of esculetin 1.4-fold. Additional overexpression of the E. coli endogenous 5,10-methylene-THF reductase, which catalyzes the last step of 5-methyl-THF formation, further enhanced the methylation reaction by 1.9-fold. Together with deregulation of SAM biosynthesis, the titer of methylated compounds was increased about 20-fold (from 0.023 mM to 0.44 mM). The engineered E. coli strain with enhanced 5-methyl-THF formation is now available as a chassis strain for the production of a variety of methylated compounds.
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Salmassi, Tina M., and Jared R. Leadbetter. "Analysis of genes of tetrahydrofolate-dependent metabolism from cultivated spirochaetes and the gut community of the termite Zootermopsis angusticollis." Microbiology 149, no. 9 (September 1, 2003): 2529–37. http://dx.doi.org/10.1099/mic.0.26351-0.
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The hindguts of wood-feeding termites are the sites of intense, CO2-reductive acetogenesis. This activity profoundly influences host nutrition and methane emissions. Homoacetogens previously isolated from diverse termites comprised novel taxa belonging to two distinct bacterial phyla, Firmicutes and Spirochaetes. Little else is known about either the diversity or abundance of homoacetogenic species present in any given termite or the genetic details underlying CO2-reductive acetogenesis by Spirochaetes. A key enzyme of CO2-reductive acetogenesis is formyltetrahydrofolate synthetase (FTHFS). A previously designed primer set was used to amplify FTHFS genes from three isolated termite-gut spirochaetes. Sequencing DNA flanking the FTHFS gene of Treponema strain ZAS-2 revealed genes encoding two acetogenesis-related enzymes, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase. Although termite-gut spirochaetes are only distantly related to clostridia at the ribosomal level, their tetrahydrofolate-dependent enzymes appear to be closely related. In contrast, homologous proteins identified in the non-homoacetogenic oral spirochaete Treponema denticola were only distantly related to those from clostridia and the termite-gut treponemes. Having demonstrated their utility with spirochaete pure cultures, the FTHFS primers were used to construct a 91-clone library from the termite-gut community DNA. From this, 19 DNA and eight amino acid FTHFS types were identified. Over 75 % of the retrieved clones formed a novel, coherent cluster with the FTHFS homologues obtained from the termite-gut treponemes. Thus, FTHFS gene diversity in the gut of the termite Zootermopsis angusticollis appears to be dominated by spirochaetes. The homoacetogenic capacity of termite-gut spirochaetes may have been acquired via lateral gene transfer from clostridia.
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Di Mauro, Debora, Monica Currò, Fabio Trimarchi, Mercurio Vecchio, Giuseppina Rizzo, Davide Barreca, Giuseppa Visalli, Riccardo Ientile, and Daniela Caccamo. "Role of Genetic Background in Cardiovascular Risk Markers Changes in Water Polo Players." International Journal of Sports Medicine 39, no. 05 (March 21, 2018): 390–96. http://dx.doi.org/10.1055/s-0044-101459.
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AbstractMethylene-tetrahydrofolate reductase (MTHFR) and paraoxonase 1 (PON1) gene polymorphisms have been associated with hyperhomocysteinemia and oxidative stress increase, that are established cardiovascular risk factors. Given that intense physical activity may increase the susceptibility to adverse cardiovascular outcomes, here we investigated the effects of MTHFR C677T and A1298C as well as PON1 Q192R gene polymorphisms on cardiovascular risk markers in twenty-eight male water polo elite players. The mean plasma levels of homocysteine (Hcy) and advanced oxidation protein products (AOPP) were above reference limits in resting conditions, and increased after competition. Moreover, a positive correlation was found between Hcy and AOPP concentrations, and also between their variations (ratio post-exercise/pre-exercise values) and the variations of lactic dehydrogenase (LDH) and creatine kinase (CK) activities, known as muscle damage markers. The highest Hcy and AOPP values were found in subjects having either MTHFR CT/AC or TT/AA, and PON1 QR192 genotype, respectively. After exercise, Hcy concentrations significantly increased in CT/AC or TT/AA subjects than in athletes having other MTHFR genotypes. A training-induced increase in plasma levels of LDH and CK activities, as well as myoglobin concentrations, was also observed, even if significant differences were found only for CK activity in athletes with MTHFR CT/AC or TT/AA athletes.
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Jimenez, Alicia Requena, Naila Naz, and Jaleel A. Miyan. "Altered folate binding protein expression and folate delivery are associated with congenital hydrocephalus in the hydrocephalic Texas rat." Journal of Cerebral Blood Flow & Metabolism 39, no. 10 (May 25, 2018): 2061–73. http://dx.doi.org/10.1177/0271678x18776226.
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Hydrocephalus (HC) is an imbalance in cerebrospinal fluid (CSF) secretion/absorption resulting in fluid accumulation within the brain with consequential pathophysiology. Our research has identified a unique cerebral folate system in which depletion of CSF 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is associated with cortical progenitor cell-cycle arrest in hydrocephalic Texas (H-Tx) rats. We used tissue culture, immunohistochemistry, in-situ PCR and RT-PCR and found that the in-vitro proliferation of arachnoid cells is highly folate-dependent with exacerbated proliferation occurring in hydrocephalic CSF that has low FDH but high folate-receptor-alpha (FRα) and folate. Adding FDH to this CSF prevented aberrant proliferation indicating a regulatory function of FDH on CSF folate concentration. Arachnoid cells have no detectable mRNA for FRα or FDH, but FDH mRNA is found in the choroid plexus (CP) and CSF microvesicles. Co-localization of FDH, FRα and folate suggests important functions of FDH in cerebral folate transport, buffering and function. In conclusion, abnormal CSF levels of FDH, FRα and folate inhibit cortical cell proliferation but allow uncontrolled arachnoid cell division that should increase fluid absorption by increasing the arachnoid although this fails in the hydrocephalic brain. FDH appears to buffer available folate to control arachnoid proliferation and function.
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Song, Jae Mahn, and Jesse C. Rabinowitz. "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 376, no. 3 (December 4, 1995): 229–32. http://dx.doi.org/10.1016/0014-5793(95)01288-9.
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Sah, Shivjee, Srinivas Aluri, Kervin Rex, and Umesh Varshney. "One-Carbon Metabolic Pathway Rewiring in Escherichia coli Reveals an Evolutionary Advantage of 10-Formyltetrahydrofolate Synthetase (Fhs) in Survival under Hypoxia." Journal of Bacteriology 197, no. 4 (December 1, 2014): 717–26. http://dx.doi.org/10.1128/jb.02365-14.
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In cells,N10-formyltetrahydrofolate (N10-fTHF) is required for formylation of eubacterial/organellar initiator tRNA and purine nucleotide biosynthesis. Biosynthesis ofN10-fTHF is catalyzed by 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). All eubacteria possess FolD, but some possess both FolD and Fhs. However, the reasons for possessing Fhs in addition to FolD have remained unclear. We usedEscherichia coli, which naturally lacksfhs, as our model. We show that inE. coli, the essential function offolDcould be replaced byClostridium perfringensfhswhen it was provided on a medium-copy-number plasmid or integrated as a single-copy gene in the chromosome. Thefhs-supportedfolDdeletion (ΔfolD) strains grow well in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. Thein vivolevels ofN10-fTHF in the ΔfolDstrain (supported by plasmid-bornefhs) were limiting despite the high capacity of the available Fhs to synthesizeN10-fTHFin vitro. Auxotrophy for purines could be alleviated by supplementing formate to the medium, and that for glycine was alleviated by engineering THF import into the cells. The ΔfolDstrain (harboringfhson the chromosome) showed a high NADP+-to-NADPH ratio and hypersensitivity to trimethoprim. The presence offhsinE. coliwas disadvantageous for its aerobic growth. However, under hypoxia,E. colistrains harboringfhsoutcompeted those lacking it. The computational analysis revealed a predominant natural occurrence offhsin anaerobic and facultative anaerobic bacteria.
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Lin, Chien-Chih, Phimonphan Chuankhayan, Wen-Ni Chang, Tseng-Ting Kao, Hong-Hsiang Guan, Hoong-Kun Fun, Atsushi Nakagawa, Tzu-Fun Fu, and Chun-Jung Chen. "Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition." Acta Crystallographica Section D Biological Crystallography 71, no. 4 (March 27, 2015): 1006–21. http://dx.doi.org/10.1107/s1399004715002928.
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10-Formyltetrahydrofolate dehydrogenase (FDH), which is composed of a small N-terminal domain (Nt-FDH) and a large C-terminal domain, is an abundant folate enzyme in the liver and converts 10-formyltetrahydrofolate (10-FTHF) to tetrahydrofolate (THF) and CO2. Nt-FDH alone possesses a hydrolase activity, which converts 10-FTHF to THF and formate in the presence of β-mercaptoethanol. To elucidate the catalytic mechanism of Nt-FDH, crystal structures of apo-form zNt-FDH from zebrafish and its complexes with the substrate analogue 10-formyl-5,8-dideazafolate (10-FDDF) and with the products THF and formate have been determined. The structures reveal that the conformations of three loops (residues 86–90, 135–143 and 200–203) are altered upon ligand (10-FDDF or THF) binding in the active site. The orientations and geometries of key residues, including Phe89, His106, Arg114, Asp142 and Tyr200, are adjusted for substrate binding and product release during catalysis. Among them, Tyr200 is especially crucial for product release. An additional potential THF binding site is identified in the cavity between two zNt-FDH molecules, which might contribute to the properties of product inhibition and THF storage reported for FDH. Together with mutagenesis studies and activity assays, the structures of zNt-FDH and its complexes provide a coherent picture of the active site and a potential THF binding site of zNt-FDH along with the substrate and product specificity, lending new insights into the molecular mechanism underlying the enzymatic properties of Nt-FDH.
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Schilhabel, Anke, Sandra Studenik, Martin Vödisch, Sandra Kreher, Bernhard Schlott, Antonio Y. Pierik, and Gabriele Diekert. "The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes." Journal of Bacteriology 191, no. 2 (November 14, 2008): 588–99. http://dx.doi.org/10.1128/jb.01104-08.
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ABSTRACT Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases.
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de Jonge, Robert, Wim J. E. Tissing, Jan Hendrik Hooijberg, Gerrit Jansen, Gertjan J. L. Kaspers, Jan Lindemans, Godefridus J. Peters, and Rob Pieters. "Polymorphisms in folate-related genes and risk of pediatric acute lymphoblastic leukemia." Blood 113, no. 10 (March 5, 2009): 2284–89. http://dx.doi.org/10.1182/blood-2008-07-165928.
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Abstract Polymorphisms in folate pathway genes may influence the susceptibility to acute lymphoblastic leukemia (ALL). DNA was isolated from 245 pediatric ALL patients (cases) and from 500 blood bank donors (controls). Polymorphisms in methylene-tetrahydrofolate reductase (MTHFR 677C>T, 1298A>C), methionine synthase (MTR 2756A>G), methionine synthase reductase (MTRR 66A>G), methylenetetrahydrofolate dehydrogenase (MTHFD1 1958G>A), nicotinamide N-methyltransferase (NNMT IVS −151C>T), serine hydroxymethyl transferase (SHMT1 1420C>T), thymidylate synthase (TS 2R3R), and the reduced folate carrier (RFC1 80G>A) were detected. In ALL patients, an increased occurrence was observed of the RFC1 80AA variant (odds ratio [OR] = 2.1; 95% confidence interval [CI] = 1.3-3.2; P = .002) and the RFC1 80A allele (OR = 1.5; 95% CI, 1.1-2.1; P = .02). Likewise, the NNMT IVS −151TT genotype showed a 2.2-fold increased ALL risk (OR = 2.2; 95% CI, 1.1-4.6; P = .04). A 1.4-fold reduction in ALL risk was observed for (heterozygous or homozygous) carriers of the TS 2R allele and the MTHFR 677T allele (OR = 0.7; 95% CI, 0.5-1.0; P < .05). Furthermore, interactions between NNMT and MTHFR 677C>T and RFC1 were observed. NNMT IVS −151CC/MTHFR 677CT + TT patients exhibited a 2-fold reduction in ALL risk whereas RFC1 80AA/NNMT IVS −151CT + TT subjects had a 4.2-fold increase in ALL risk (P = .001). For the first time, we associate the RFC1 80G>A and NNMT IVS −151C>T variants to an increased ALL susceptibility.
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Schwab, Matthias, Ulrich M. Zanger, Claudia Marx, Elke Schaeffeler, Kathrin Klein, Jürgen Dippon, Reinhold Kerb, et al. "Role of Genetic and Nongenetic Factors for Fluorouracil Treatment-Related Severe Toxicity: A Prospective Clinical Trial by the German 5-FU Toxicity Study Group." Journal of Clinical Oncology 26, no. 13 (May 1, 2008): 2131–38. http://dx.doi.org/10.1200/jco.2006.10.4182.
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Purpose To assess the predictive value of polymorphisms in dihydropyrimidine dehydrogenase (DPYD ), thymidylate synthase (TYMS ), and methylene tetrahydrofolate reductase (MTHFR ) and of nongenetic factors for severe leukopenia, diarrhea, and mucositis related to fluorouracil (FU) treatment. Patients and Methods A multicenter prospective clinical trial included 683 patients with cancer treated with FU monotherapy. Toxicity was documented according to World Health Organization grades. DPYD, TYMS, and MTHFR genotypes were determined, and DPYD was resequenced in patients with severe toxicity. Results Grade 3 to 4 toxicity occurred in 16.1% of patients. The sensitivity of DPYD*2A genotyping for overall toxicity was 5.5% (95%CI, 0.02 to 0.11), with a positive predictive value of 0.46 (95% CI, 0.19 to 0.75; P = .01). Inclusion of additional DPYD variants improved prediction only marginally. Analysis according to toxicity type revealed significant association of DPYD with mucositis and leukopenia, whereas TYMS was associated with diarrhea. Genotype, female sex, mode of FU administration, and modulation by folinic acid were identified as independent risk factors by multivariable analysis. A previously unrecognized significant interaction was found between sex and DPYD, which resulted in an odds ratio for toxicity of 41.8 for male patients (95% CI, 9.2 to 190; P < .0001) but only 1.33 (95% CI, 0.34 to 5.2) in female patients. Homozygosity for the TYMS enhancer region double repeat allele increased risk for toxicity 1.6-fold (95% CI, 1.08 to 2.22; P = .02). Conclusion DPYD, TYMS, and MTHFR play a limited role for FU related toxicity but a pronounced DPYD gene/sex-interaction increases prediction rate for male patients. Toxicity risk assessment should include sex, mode of administration, and folinic acid as additional predictive factors.
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Vokes, E. E., R. Mick, M. S. Kies, M. E. Dolan, D. Malone, I. Athanasiadis, D. J. Haraf, M. Kozloff, R. R. Weichselbaum, and M. J. Ratain. "Pharmacodynamics of fluorouracil-based induction chemotherapy in advanced head and neck cancer." Journal of Clinical Oncology 14, no. 5 (May 1996): 1663–71. http://dx.doi.org/10.1200/jco.1996.14.5.1663.
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PURPOSE AND METHODS To optimize the biochemical modulation of fluorouracil (5-FU), we administered the pure I-stereoisomer of leucovorin (LV) as a 132-hour continuous intravenous infusion (CIV) with cisplatin 100 mg/m2, 5-FU 640 mg/m2/d as a 120-hour CIV, and interferon alfa-2b (IFN) at 2 MU/m2/d for 6 days for three cycles (I-PFL-IFN). Pharmacologic parameters included morning (AM) and afternoon (PM) plasma concentrations of 5-FU, LV and its active metabolite 5-methyl tetrahydrofolate (MTHF), and dihydropyrimidine dehydrogenase (DPD) activity in peripheral mononuclear cells. RESULTS Eighty-nine patients were treated (86 stage IV). Neutropenia and mucositis were the most common toxicities. Sixty-six percent achieved a complete remission (CR). There was a trend for higher PM versus AM 5-FU concentrations (median, 1.64 v 1.51 mumoles/L; P = .08), but not for LV plus MTHF (P = .66). The mean +/- SD DPD activity was 0.21 +/- 0.14 nmol/min/mg and did not correlate with plasma concentrations of 5-FU or LV plus MTHF or clinical toxicities. Higher PM 5-FU concentrations correlated with worse leukopenia (P = .04) and severity of mucositis (P = .04). PM 5-FU concentration was higher in women than in men (P = .07), with no apparent difference in severity of toxicities. The maximum 5-FU concentration was higher in CR than non-CR patients (median, 2.01 v 1.54 mumoles/L; P = .02) and higher in women than men who achieved a CR (median, 2.77 v 1.91 mumoles/L; P = .03). No correlation of CR with dose-intensity was found. CONCLUSION L-PFL-IFN is active in stage IV head and neck cancer. 5-FU concentration is a significant predictor of toxicity. In women, optimization of response outcome requires a higher 5-FU concentration. Individualized 5-FU dosing to obtain higher 5-FU plasma concentrations may be indicated.
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Pelletier, Joelle N., and Robert E. MacKenzie. "Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase." Biochemistry 34, no. 39 (October 1995): 12673–80. http://dx.doi.org/10.1021/bi00039a025.
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Chen, Liangfu, Frank E. Nargang, and Edwin A. Cossin. "Isolation and Sequencing of a Plant cDNA Encoding a Bifunctional Methylenetetrahydrofolate Dehydrogenase : Methenyltetrahydrofolate Cyclohydrolase Protein." Pteridines 10, no. 4 (November 1999): 171–77. http://dx.doi.org/10.1515/pteridines.1999.10.4.171.
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Summary In plant cells, the interconversion of formyl- and methylene-tetrahydrofolates is catalyzed by a bifunctional protein possessing methenyltetrallydrofolate cydohydrolase (EC 3.5.4.9) and methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. The present work reports the isolation and sequencing of a cDNA that encodes this protein. Polydonal antibodies, raised against purified pea cytosolic dehydrogenase:cyclohydrolase, were used to screen a λgt 11 cDNA expression library, constructed from leaf extracts of this species. The screen identified a phage containing a cDNA insert with an open reading frame encoding a 294 amino acid protein (Mr 31,344). The deduced primary structure of this protein contained most of the conserved regions found in other dehydrogenase:cyclohydrolase proteins including the corresponding domains of the trifunctional C1-THF synthases of mammalian and yeast origins.
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Stumpf, Anita N., Edith D. van der Meijden, Cornelis A. M. van Bergen, Roelof Willemze, J. H. Frederik Falkenburg, and Marieke Griffioen. "Identification of Four New HLA Class II Restricted Minor Histocompatibility Antigens Contributing to Graft Versus Leukemia Reactivity." Blood 112, no. 11 (November 16, 2008): 3247. http://dx.doi.org/10.1182/blood.v112.11.3247.3247.
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Abstract Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+ and CD4+ T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+ T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+ T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+ T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+ CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+ T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+ T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+ T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.
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Lucena, Maylla Rodrigues, Clovis Paniz, Juliano Felix Bertinato, Patricia Mendonça da Silva Amorim, Tamara Ramos Jorge, Maria Stella Figueiredo, Rodolfo Cancado, et al. "No Association Was Found Between MTHFR and MTHFD1 SNPs and Vitamin Levels in People with Increased and Normal Erythropoiesis, after Compulsory Flour Fortification with Folic Acid." Blood 124, no. 21 (December 6, 2014): 4877. http://dx.doi.org/10.1182/blood.v124.21.4877.4877.
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Abstract Background The methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase (MTHFD1) enzymes play important roles in the metabolism of folate. MTHFR catalyses the reduction of 5,10-methylene-tetrahydrofolate (5,10-methylene-THF) to 5-methyl-THF and present a crucial role in the regulation of available folate to homocysteine remethylation, in a cobalamin (Cbl) dependent reaction. MTHFD1 has three distinct enzymatic activities in folate metabolism: the conversion of THF to 10-formyl-THF; the reversible conversion of 10-formyl-THF to 5,10-methenyl-THF; and the conversion of 5,10-methenyl-THF to 5,10-methylene-THF. The two enzymes were related with DNA synthesis and single nucleotides polymorphisms (SNPs) in their genes have been associated with lower folate levels. People who have increased cellular duplication could be affected by low folate levels. Hereditary spherocytosis (HS) patients had an increased erythropoiesis and need more amounts of acid folic. In Brazil, HS patients are usually treated with 5mg/day of folic acid, the only formulation available. The use of folic acid by β-thalassemia heterozygotes (β-TH) subjects is not common, and depends of the individual clinical feature. Furthermore, in Brazil, HS patients and β-TH people have been exposed to food fortification (wheat and corn flours) with 150 µg of FA since 2004. The influences of variants of MTHFR and MTHFD1 SNPs in people with increased erythropoiesis, especially in HS patients, are not known. Objective The aim of the study was to evaluate the interaction between MTHFR c. 677C>T and c. 1298A>C and MTHFD1 c. 1958G>A SNPs and the use or not of FA supplementation in HS patients. It was also our objective to compare the folate and Cbl levels according to genotypes for MTHFR and MTHFD1 SNPs in β-TH subjects and their controls. Methods Twenty-five patients with HS, 49 β-TH subjects and 98 healthy people exposed to mandatory fortification of wheat and corn flours with FA were included in this study. Serum folate and Cbl were determined by microbiologic assays. Genomic DNA was extracted from whole blood using commercial kit. The genotypes for MTHFR c. 1298A>C and MTHFD1 c.1958G>A SNPs were determined by Real Time PCR using TaqMan assays. PCR-RFLP was used for genotyping MTHFR c. 677C>T SNP. Haplotype frequencies (MTHFR c. 677C>T and c. 1298 A>G) and the standardized disequilibrium coefficient (D’) for pair-wise linkage disequilibrium were assessed by the Expectation-Maximization (EM) algorism using the Haploview Software. We considered linkage disequilibrium when D’ ≥0.50. The study of linkage disequilibrium was performed by calculating the coefficient D' of Lewontin. Results The frequencies of genotypes for MTHFR (c. 677C>T and c. 1298A>C) and MTHFD1 c. 1958G>A SNPs were similar in HS, β-TH and control groups (p>0.05). When genotypes were grouped according to the presence of the mutated allele (CC vs. CT+TT for MTHFR c. 677C>T, AA vs. AC+CC for MTHFR c. 1298A>C and GG vs. GA+AA for MTHFD1 c. 1958G>A), no differences were also found (p>0.05). The MTHFR c.677C>T and c. 1298A>C SNPs are in complete linkage disequilibrium (D'=1.0). Regarding the correlation coefficient between the two loci (r2), the value found in this analysis was equal to 0.169 in HS versus control group and 0.205 in β-TH versus control group. The haplotype CA, which is formed by the ancestral allele of MTHFR c. 677C>T and c. 1298A>C SNPs respectively, was more frequent in controls (41.9%) than in the β-TH group (28.1%). However, this finding was not observed in HS group comared with control group. No association was found between genotypes for MTHFR c. 677C>T and c. 1298A>C and MTHFD1 c. 1958G>A and alterations in folate and Cbl levels in three groups. ANCOVA was used for evaluating the interaction between the use of FA supplementation and MTHFR c.677C>T SNP on folate levels in HS patients. Only FA supplementation was associated with increased serum folate, and no associations were observed with MTHFR SNP or interaction between both. Conclusion The SNPs were not associated with reduced serum folate levels in the three groups studied. This finding suggests that FA flour fortification in β-TH and controls, as well as, the supplementation plus fortification in HS group, could offset the effect of variants of SNPs, especially for MTHFR c.677C>T. Disclosures No relevant conflicts of interest to declare.
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Paniz, Clovis, Maylla Rodrigues Lucena, Juliano Felix Bertinato, Patricia Mendonça da Silva Amorim, Guilherme Wataru Gomes, Maria Stella Figueiredo, Ralph Green, et al. "Elevated Serum Folic Acid Concentrations Were Associated with Higher mRNA Expression of DHFR Gene in Patients with Hereditary Spherocytosis." Blood 124, no. 21 (December 6, 2014): 4005. http://dx.doi.org/10.1182/blood.v124.21.4005.4005.
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Abstract Background Folic acid (FA) is the synthetic form of folate, a B complex vitamin which plays an important role in several reactions in the body. Folate deficiency produces several clinical complications including anemia and is associated with an increased risk of cancer, cardiovascular disease and neural tube defects. With the advent of FA and iron fortification of wheat and corn flour (150 µg of folic acid and 4.2 mg of iron/100 grams) in Brazil since 2004, the population was exposed to amounts of folate beyond that normally present in foods. The form present in fortified food is taken up by cells and reduced by the enzyme dihydrofolate reductase (DHFR) first to dihydrofolate (DHF) and then tetrahydrofolate (THF). DHF is the preferred natural substrate. Amounts of FA higher than the defined tolerable upper intake of 1 mg/day could impair the ability of DHFR to convert DHF to THF. Some patients with hemolytic anemia, such as hereditary spherocytosis (HS), need larger amounts of FA to compensate for their increase in erythropoiesis and have been receiving 5mg/day of supplemental FA, the only formulation available in Brazil, in addition to being exposed to compulsory food fortification with FA. Objective The aim of this study was to compare the effects of 5mg/day FA on serum folate levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes; and cytotoxicity of lymphocytes and NK cells in patients with HS and healthy individuals not receiving supplemental FA. Material and Methods Twenty-five patients with HS exposed to mandatory fortification and in use or not of 5 mg/day of FA were included in this study. Forty-five healthy people were recruited as a control group, and matched with HS patients according to age, gender, body mass index and self-reported skin color. Blood count, including reticulocytes, C-reative protein and lactic dehydrogenase (LDH) were performed. Serum folate (SF) and vitamin B12 were determined by a microbiological method. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed in duplicate, using Real Time PCR. Cytotoxicity of lymphocytes and NK cells were also carried out using a flow cytometric assay. Results Eight HS patients did not use FA 5 mg everyday (4 declared no use anytime and 4 reported intermittent use) and none of control group used FA supplementation. Reticulocytes and LDH were higher in HS group (P<0.05), however no difference was found between levels of C-reative protein (P=0.173) and vitamin B12 (P= 0.699) when compared with control group. The HS group had higher SF levels and elevated mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 when compared with controls (P<0.05, Figure 1). It is not clear whether FA use or underlying disease was be responsible for increasing of mRNA expression of these genes. To verify the effect of SF levels on HS patients, this group was classified into two subgroups according to median SF (< 46.6 and ≥ 46.6 nmol/L). Interestingly, the subgroup with higher SF levels showed significantly elevated DHFR mRNA expression but no difference was found in the mRNA expression of the other genes studied. The two subgroups were similar according to WBC, RBC, hemoglobin, MCV, reticulocytes, LDH, C-reative protein, vitamin B12 and cytotoxic capacity of lymphocytes and NK cells. Conclusions Elevated SF concentrations were associated with higher mRNA expression of DHFR gene in HS patients, suggesting that the use of higher amounts of FA might influence the expression and activity of DHFR and thus affect folate metabolism in these patients. It is not known whether normal subjects receiving similar high doses of FA show the same effects. Financing: FAPESP 2012/12912-1 and CNPq 4826412012-6 Figure 1 – Serum folate levels (A) and DHFR mRNA (B) in hereditary spherocytosis and control group. The DHFR mRNA expressions in HS patients according to serum folate levels were shown (C). Figure 1 – Serum folate levels (A) and DHFR mRNA (B) in hereditary spherocytosis and control group. The DHFR mRNA expressions in HS patients according to serum folate levels were shown (C). The line in each graphic is the median. The median and percentiles 25 and 75 (P25 – P75) for control and hereditary spherocytosis groups were, respectively: A- serum folate: 22.1 (14.5 – 36.6 nmolL) and 51.5 (19.5 – 95.2 nmolL), B- DHFR mRNA expression: 1.59 (1.00 – 2.48) and 35.8 (19.0 – 52.7). In Figure C, the median and percentiles 25 and 75 (P25 – P75) for DHFR mRNA expression, according to serum folate levels (SF <46.6 and SF ≥ 46.6 nmol/L), were, respectively: 19.6 (1.4 – 38.6) and 50.3 (23 – 66.8). Disclosures No relevant conflicts of interest to declare.
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DiRisio, Aislyn, Elena Kamynina, and Patrick Stover. "The Role of Serine Hydroxymethyltransferase and Methylene‐Tetrahydrofolate Dehydrogenase in Genome Stability." FASEB Journal 29, S1 (April 2015). http://dx.doi.org/10.1096/fasebj.29.1_supplement.919.1.
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Krupenko, Natalia I., Jaspreet Sharma, Peter Pediaditakis, Baharan Fekry, Kristi L. Helke, Xiuxia Du, Susan Sumner, and Sergey A. Krupenko. "Cytosolic 10-formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver." Scientific Reports 9, no. 1 (October 17, 2019). http://dx.doi.org/10.1038/s41598-019-51397-1.
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Abstract ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1−/− mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1−/− mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1−/− mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1−/− mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.
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Yao, Shigang, Le Chen, Zhou Yang, Li Yao, Jianchun Zhu, Jiguo Qiu, Guoxiang Wang, and Jian He. "The Properties of 5-Methyltetrahydrofolate Dehydrogenase (MetF1) and Its Role in the Tetrahydrofolate-Dependent Dicamba Demethylation System inRhizorhabdus dicambivoransNdbn-20." Journal of Bacteriology 201, no. 17 (June 17, 2019). http://dx.doi.org/10.1128/jb.00096-19.
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ABSTRACTThe herbicide dicamba is initially degraded via the tetrahydrofolate (THF)-dependent demethylation system inRhizorhabdus dicambivoransNdbn-20. Two THF-dependent dicamba methyltransferase gene clusters, scaffold 50 and scaffold 66, were found in the genome of strain Ndbn-20. Each cluster contains a dicamba methyltransferase gene and three THF metabolism-related genes, namely,metF(coding for 5,10-CH2-THF reductase),folD(coding for 5,10-CH2-THF dehydrogenase–5,10-methenyl-THF cyclohydrolase), andpurU(coding for 10-formyl-THF deformylase). In this study, reverse transcription-PCR (RT-PCR) results showed that only genes in scaffold 66, not those in scaffold 50, were transcribed in dicamba-cultured cells. ThemetFgene of scaffold 66 (metF1) was expressed inEscherichia coliBL21(DE3), and the product was purified as a His6-tagged protein. Purified MetF1 was found to be a monomer and exhibited 5-CH3-THF dehydrogenase activityin vitro. ThekcatandKmfor 5-CH3-THF were 0.23 s−1and 16.48 μM, respectively. However, 5,10-CH2-THF reductase activity was not detected for MetF1 under the conditions tested. Gene disruption results showed thatmetF1is essential for dicamba degradation, whereasfolD1is dispensable.IMPORTANCEThere are several THF-dependent methyltransferase genes and THF-metabolic genes in the genome ofR. dicambivoransNdbn-20; however, which genes are involved in dicamba demethylation and the mechanism underlying THF regeneration remain unknown. This study revealed that scaffold 66 is responsible for dicamba demethylation and that MetF1 physiologically catalyzes the dehydrogenation of 5-CH3-THF to 5,10-CH2-THF in the THF-dependent dicamba demethylation system inR. dicambivoransNdbn-20. Furthermore, the results showed that MetF1 differs from previously characterized MetF in phylogenesis, biochemical properties, and catalytic activity; e.g., MetF1in vitrodid not show 5,10-CH2-THF reductase activity, which is the physiological function ofEscherichia coliMetF. This study provides new insights into the mechanism of the THF-dependent methyltransferase system.
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Wegner, Carl-Eric, Linda Gorniak, Stefan Riedel, Martin Westermann, and Kirsten Küsel. "Lanthanide-Dependent Methylotrophs of the Family Beijerinckiaceae: Physiological and Genomic Insights." Applied and Environmental Microbiology 86, no. 1 (October 11, 2019). http://dx.doi.org/10.1128/aem.01830-19.
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ABSTRACT Methylotrophic bacteria use methanol and related C1 compounds as carbon and energy sources. Methanol dehydrogenases are essential for methanol oxidation, while lanthanides are important cofactors of many pyrroloquinoline quinone-dependent methanol dehydrogenases and related alcohol dehydrogenases. We describe here the physiological and genomic characterization of newly isolated Beijerinckiaceae bacteria that rely on lanthanides for methanol oxidation. A broad physiological diversity was indicated by the ability to metabolize a wide range of multicarbon substrates, including various sugars, and organic acids, as well as diverse C1 substrates such as methylated amines and methylated sulfur compounds. Methanol oxidation was possible only in the presence of low-mass lanthanides (La, Ce, and Nd) at submicromolar concentrations (>100 nM). In a comparison with other Beijerinckiaceae, genomic and transcriptomic analyses revealed the usage of a glutathione- and tetrahydrofolate-dependent pathway for formaldehyde oxidation and channeling methyl groups into the serine cycle for carbon assimilation. Besides a single xoxF gene, we identified two additional genes for lanthanide-dependent alcohol dehydrogenases, including one coding for an ExaF-type alcohol dehydrogenase, which was so far not known in Beijerinckiaceae. Homologs for most of the gene products of the recently postulated gene cluster linked to lanthanide utilization and transport could be detected, but for now it remains unanswered how lanthanides are sensed and taken up by our strains. Studying physiological responses to lanthanides under nonmethylotrophic conditions in these isolates as well as other organisms is necessary to gain a more complete understanding of lanthanide-dependent metabolism as a whole. IMPORTANCE We supplemented knowledge of the broad metabolic diversity of the Beijerinckiaceae by characterizing new members of this family that rely on lanthanides for methanol oxidation and that possess additional lanthanide-dependent enzymes. Considering that lanthanides are critical resources for many modern applications and that recovering them is expensive and puts a heavy burden on the environment, lanthanide-dependent metabolism in microorganisms is an exploding field of research. Further research into how isolated Beijerinckiaceae and other microbes utilize lanthanides is needed to increase our understanding of lanthanide-dependent metabolism. The diversity and widespread occurrence of lanthanide-dependent enzymes make it likely that lanthanide utilization varies in different taxonomic groups and is dependent on the habitat of the microbes.
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Picone, Nunzia, Pieter Blom, Carmen Hogendoorn, Jeroen Frank, Theo van Alen, Arjan Pol, Antonina L. Gagliano, et al. "Metagenome Assembled Genome of a Novel Verrucomicrobial Methanotroph From Pantelleria Island." Frontiers in Microbiology 12 (May 19, 2021). http://dx.doi.org/10.3389/fmicb.2021.666929.
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Verrucomicrobial methanotrophs are a group of aerobic bacteria isolated from volcanic environments. They are acidophiles, characterized by the presence of a particulate methane monooxygenase (pMMO) and a XoxF-type methanol dehydrogenase (MDH). Metagenomic analysis of DNA extracted from the soil of Favara Grande, a geothermal area on Pantelleria Island, Italy, revealed the presence of two verrucomicrobial Metagenome Assembled Genomes (MAGs). One of these MAGs did not phylogenetically classify within any existing genus. After extensive analysis of the MAG, we propose the name of “Candidatus Methylacidithermus pantelleriae” PQ17 gen. nov. sp. nov. The MAG consisted of 2,466,655 bp, 71 contigs and 3,127 predicted coding sequences. Completeness was found at 98.6% and contamination at 1.3%. Genes encoding the pMMO and XoxF-MDH were identified. Inorganic carbon fixation might use the Calvin-Benson-Bassham cycle since all genes were identified. The serine and ribulose monophosphate pathways were incomplete. The detoxification of formaldehyde could follow the tetrahydrofolate pathway. Furthermore, “Ca. Methylacidithermus pantelleriae” might be capable of nitric oxide reduction but genes for dissimilatory nitrate reduction and nitrogen fixation were not identified. Unlike other verrucomicrobial methanotrophs, genes encoding for enzymes involved in hydrogen oxidation could not be found. In conclusion, the discovery of this new MAG expands the diversity and metabolism of verrucomicrobial methanotrophs.
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Purwantini, Endang, Usha Loganathan, and Biswarup Mukhopadhyay. "Coenzyme F420-Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F420in Mycobacteria." Journal of Bacteriology 200, no. 23 (September 24, 2018). http://dx.doi.org/10.1128/jb.00375-18.
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ABSTRACTCoenzyme F420plays a key role in the redox metabolisms of various archaea and bacteria, includingMycobacterium tuberculosis. InM. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420carries multiple glutamate residues in the side chain, forming F420-nspecies (n, number of glutamate residues), and the length of this side chain impacts cellular physiology.M. tuberculosisstrains with F420species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420. However, purified FbiB ofM. tuberculosis(MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that,in vitro,MtbFbiB synthesized side chains containing up to seven glutamate residues if F420was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis inMycobacterium bovisBCG andMycobacterium smegmatisand an analysis of literature data onM. tuberculosisrevealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420to F420H2. We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate.IMPORTANCECoenzyme F420-dependent reactions ofMycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism ofM. tuberculosis. Mutations that eliminate the production of F420with longer tails makeM. tuberculosisresistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420requires concerted actions of two enzymes, one of which reduces the coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.
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Młodzik-Czyżewska, Monika A., Artur Szwengiel, Anna M. Malinowska, and Agata Chmurzynska. "Comparison of Associations between One-Carbon Metabolism, Lipid Metabolism, and Fatty Liver Markers in Normal-Weight and Overweight People Aged 20–40 Years." Annals of Nutrition and Metabolism, July 7, 2021, 1–10. http://dx.doi.org/10.1159/000517911.
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The aim of the present study was to compare biomarkers of one-carbon metabolism (OCM), lipid metabolism, and fatty liver in people with normal and increased body weight. The study was performed on 421 participants, aged 20–40 years, enrolled in Poznan, Poland, in 2016–2018. Choline and betaine intakes were assessed. DNA samples were genotyped for polymorphisms of phosphatidylethanolamine N-methyltransferase (<i>PEMT</i>; rs7946 and rs12325817), methylene tetrahydrofolate reductase (<i>MTHFR</i>; rs180113), methylenetetrahydrofolate dehydrogenase (<i>MTHFD1</i>; rs2236225), and dihydrofolate reductase (<i>DHFR</i>; rs70991108). To assess the associations between blood metabolites (choline, betaine, folate, L-carnitine, o-acetyl-L-carnitine, and trimethylamine N-oxide]), circulating lipids, and fatty liver indices, multiple logistic regression analyses were performed. Overweight/obese participants had 5.8% higher choline (<i>p</i> < 0.05) and 10% higher L-carnitine (<i>p</i> < 0.001) levels than normal-weight subjects. Serum folate and betaine levels were associated with lower total cholesterol (<i>p</i> < 0.001 and <i>p</i> < 0.05), low-density lipoprotein (LDL) cholesterol (<i>p</i> < 0.001 and <i>p</i> < 0.05, respectively), triacylglycerols (<i>p</i> < 0.01 and <i>p</i> < 0.001), and triglyceride glucose index (<i>p</i> < 0.001 and <i>p</i> < 0.01, respectively), though only in overweight/obese people. The <i>PEMT</i> rs12325817 CC genotype was associated with higher levels of high-density lipoprotein (HDL) cholesterol (<i>p</i> < 0.01) in overweight/obese people. The associations between OCM markers, fatty liver indices, and blood lipids differ in subjects with normal and excessive body weight.
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Krupenko, Natalia I., Jaspreet Sharma, Peter Pediaditakis, Kristi L. Helke, Madeline S. Hall, Xiuxia Du, Susan Sumner, and Sergey A. Krupenko. "Aldh1l2 knockout mouse metabolomics links the loss of the mitochondrial folate enzyme to deregulation of a lipid metabolism observed in rare human disorder." Human Genomics 14, no. 1 (November 9, 2020). http://dx.doi.org/10.1186/s40246-020-00291-3.
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Abstract Background Mitochondrial folate enzyme ALDH1L2 (aldehyde dehydrogenase 1 family member L2) converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2 simultaneously producing NADPH. We have recently reported that the lack of the enzyme due to compound heterozygous mutations was associated with neuro-ichthyotic syndrome in a male patient. Here, we address the role of ALDH1L2 in cellular metabolism and highlight the mechanism by which the enzyme regulates lipid oxidation. Methods We generated Aldh1l2 knockout (KO) mouse model, characterized its phenotype, tissue histology, and levels of reduced folate pools and applied untargeted metabolomics to determine metabolic changes in the liver, pancreas, and plasma caused by the enzyme loss. We have also used NanoString Mouse Inflammation V2 Code Set to analyze inflammatory gene expression and evaluate the role of ALDH1L2 in the regulation of inflammatory pathways. Results Both male and female Aldh1l2 KO mice were viable and did not show an apparent phenotype. However, H&E and Oil Red O staining revealed the accumulation of lipid vesicles localized between the central veins and portal triads in the liver of Aldh1l2-/- male mice indicating abnormal lipid metabolism. The metabolomic analysis showed vastly changed metabotypes in the liver and plasma in these mice suggesting channeling of fatty acids away from β-oxidation. Specifically, drastically increased plasma acylcarnitine and acylglycine conjugates were indicative of impaired β-oxidation in the liver. Our metabolomics data further showed that mechanistically, the regulation of lipid metabolism by ALDH1L2 is linked to coenzyme A biosynthesis through the following steps. ALDH1L2 enables sufficient NADPH production in mitochondria to maintain high levels of glutathione, which in turn is required to support high levels of cysteine, the coenzyme A precursor. As the final outcome, the deregulation of lipid metabolism due to ALDH1L2 loss led to decreased ATP levels in mitochondria. Conclusions The ALDH1L2 function is important for CoA-dependent pathways including β-oxidation, TCA cycle, and bile acid biosynthesis. The role of ALDH1L2 in the lipid metabolism explains why the loss of this enzyme is associated with neuro-cutaneous diseases. On a broader scale, our study links folate metabolism to the regulation of lipid homeostasis and the energy balance in the cell.
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Álvarez-Córdoba, Mónica, Marta Talaverón-Rey, Irene Villalón-García, Suleva Povea-Cabello, Juan M. Suárez-Rivero, Alejandra Suárez-Carrillo, Manuel Munuera-Cabeza, Joaquín J. Salas, and José A. Sánchez-Alcázar. "Down regulation of the expression of mitochondrial phosphopantetheinyl-proteins in pantothenate kinase-associated neurodegeneration: pathophysiological consequences and therapeutic perspectives." Orphanet Journal of Rare Diseases 16, no. 1 (May 5, 2021). http://dx.doi.org/10.1186/s13023-021-01823-3.
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Abstract Background Neurodegeneration 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. Methods In 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). Results In 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. Conclusions Our 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.