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Sample records for 10-formyl tetrahydrofolate deformylases

  1. Arabidopsis 10-Formyl Tetrahydrofolate Deformylases Are Essential for Photorespiration[W][OA

    PubMed Central

    Collakova, Eva; Goyer, Aymeric; Naponelli, Valeria; Krassovskaya, Inga; Gregory, Jesse F.; Hanson, Andrew D.; Shachar-Hill, Yair

    2008-01-01

    In prokaryotes, PurU (10-formyl tetrahydrofolate [THF] deformylase) metabolizes 10-formyl THF to formate and THF for purine and Gly biosyntheses. The Arabidopsis thaliana genome contains two putative purU genes, At4g17360 and At5g47435. Knocking out these genes simultaneously results in plants that are smaller and paler than the wild type. These double knockout (dKO) mutant plants show a 70-fold increase in Gly levels and accumulate elevated levels of 5- and 10-formyl THF. Embryo development in dKO mutants arrests between heart and early bent cotyledon stages. Mature seeds are shriveled, accumulate low amounts of lipids, and fail to germinate. However, the dKO mutant is only conditionally lethal and is rescued by growth under nonphotorespiratory conditions. In addition, culturing dKO siliques in the presence of sucrose restores normal embryo development and seed viability, suggesting that the seed and embryo development phenotypes are a result of a maternal effect. Our findings are consistent with the involvement of At4g17360 and At5g47435 proteins in photorespiration, which is to prevent excessive accumulation of 5-formyl THF, a potent inhibitor of the Gly decarboxylase/Ser hydroxymethyltransferase complex. Supporting this role, deletion of the At2g38660 gene that encodes the bifunctional 5,10-methylene THF dehydrogenase/5,10-methenyl THF cyclohydrolase that acts upstream of 5-formyl THF formation restored the wild-type phenotype in dKO plants. PMID:18628352

  2. Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation.

    PubMed Central

    Mazel, D; Pochet, S; Marlière, P

    1994-01-01

    Deformylase performs an essential step in the maturation of proteins in eubacteria, by removing the formyl group from the N-terminal methionine residue of ribosome-synthesized polypeptides. In spite of this important role in translation, the enzyme had so far eluded characterization because of its instability. We report the isolation of the deformylase gene of Escherichia coli, def, by overexpression of a genomic library from a high-copy-number plasmid and selection for utilization of the substrate analogue formyl-leucyl-methionine as a source of methionine. The def gene encodes a 169 amino acid polypeptide that bears no obvious resemblance to other known proteins. It forms an operon with the fmt gene, that encodes the initiator methionyl-tRNA(i) transformylase, which was recently characterized (Guillon et al., J. Bacteriol., 174, 4294-4301, 1992). This operon was mapped at min 72 of the E. coli chromosome. The def gene could be inactivated if the fmt gene was also inactivated, or if biosynthesis of N10-formyl-tetrahydrofolate, the formyl donor in methionyl-tRNA(i) transformylation, was blocked by trimethoprim. These findings designate deformylase as a target for antibacterial chemotherapy. Images PMID:8112305

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

    PubMed Central

    Pike, Schuyler T.; Rajendra, Rashmi; Artzt, Karen; Appling, Dean R.

    2010-01-01

    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 C1-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. PMID:19948730

  4. [Peptide-deformylase inhibitors, a new class of antibiotics].

    PubMed

    Dubreuil, Luc

    2002-11-30

    PEPTIDE-DEFORMYLASE: During protein synthesis in bacteria, a transformylase coding the fmt gene provides a formyl group on methionine before binding to the ARNm-ARNt complex. This tormylated methionine initiates the protein synthesis. The adjunction of an amino acid to the peptide chain leads to a peptide associated with a formylated methionine. The final stage requires a metallo-enzyme, peptide deformylase, which releases the peptide and regenerates the methionin. PEPTIDE-DEFORMYLASE INHIBITORS (PDF): Often rejected by the efflux pumps of Gram negative bacteria, PDF inhibitors are administered in the form of pro-drugs, capable of acting even in the bacteria that have lost their transformylase gene. TWO PRODUCTS: These are VCR 4887 developed by Versicor and Novartis and BB 83698 developed by British Biotechnology Genesoft. They are presently in the process of clinical predevelopment. They represent an important innovation and widen the range of new antibiotic classes. PMID:12497724

  5. Peptide deformylase inhibitors as potent antimycobacterial agents.

    PubMed

    Teo, Jeanette W P; Thayalan, Pamela; Beer, David; Yap, Amelia S L; Nanjundappa, Mahesh; Ngew, Xinyi; Duraiswamy, Jeyaraj; Liung, Sarah; Dartois, Veronique; Schreiber, Mark; Hasan, Samiul; Cynamon, Michael; Ryder, Neil S; Yang, Xia; Weidmann, Beat; Bracken, Kathryn; Dick, Thomas; Mukherjee, Kakoli

    2006-11-01

    Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC50) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC90 values of <1 microM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of < or =5 x 10(-7) in M. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents. PMID:16966397

  6. A survey of polypeptide deformylase function throughout the eubacterial lineage.

    PubMed

    Mazel, D; Coïc, E; Blanchard, S; Saurin, W; Marlière, P

    1997-03-14

    N-terminal formylation of ribosome-synthesized polypeptides is assumed to be among the most conserved features that distinguish the eubacterial line of descent from other living phyla. In order to assess the ancientness of this trait, def genes encoding polypeptide deformylase were characterized from four eubacterial species, Lactococcus lactis, Bacillus subtilis, Calothrix PCC7601 and Thermotoga maritima, taking advantage of the conditional viability of the def mutants of Escherichia coli. Altogether, eight sequences of polypeptide deformylase have been obtained from all the eubacterial sources which were investigated, either through systematic genome sequence analysis or through genetic screening, yielding a highly homologous family. A gene putatively encoding Met-tRNAi formyltransferase, fmt, was found downstream of the deformylase gene except in L. lactis, Mycoplasma genitalium, Calothrix PCC7601 and T. maritima. These results argue strongly for the ancestral character of N-terminal formylation in eubacteria. Most of the wide deviations of amino acid usage observed in def- and fmt-encoded proteins among species is best accounted for by the nucleotide composition of genomes. Furthermore, the species of origin of each protein appears to be more recognizable than its function, considering only its amino acid composition. PMID:9086272

  7. Drug forecast – the peptide deformylase inhibitors as antibacterial agents

    PubMed Central

    Guay, David R P

    2007-01-01

    The relatively rapid development of microbial resistance after the entry of every new antimicrobial into the marketplace necessitates a constant supply of new agents to maintain effective pharmacotherapy. Despite extensive efforts to identify novel lead compounds from molecular targets, only the peptide deformylase inhibitors (PDIs) have shown any real promise, with some advancing to phase I human trials. Bacterial peptide deformylase, which catalyzes the removal of the N-formyl group from N-terminal methionine following translation, is essential for bacterial protein synthesis, growth, and survival. The majority of PDIs are pseudopeptide hydroxamic acids and two of these (IV BB-83698 and oral NVP LBM-415) entered phase I human trials. However, agents to the present have suffered from major potential liabilities. Their in vitro activity has been limited to gram-positive aerobes and some anaerobes and has been quite modest against the majority of such species (MIC90 values ranging from 1–8 mg/L). They have exerted bacteriostatic, not bacteriocidal, activity, thus reducing their potential usefulness in the management of serious infections in the immunocompromised. The relative ease with which microorganisms have been able to develop resistance and the multiple available mechanisms of resistance (mutations in fmt, defB, folD genes; AcrAB/TolC efflux pump; overexpression of peptide deformylase) are worrisome. These could portend a short timespan of efficacy after marketing. Despite these current liabilities, further pursuit of more potent and broader spectrum PDIs which are less susceptible to bacterial mechanisms of resistance is still warranted. PMID:18472972

  8. New peptide deformylase inhibitors design, synthesis and pharmacokinetic assessment.

    PubMed

    Lv, Fengping; Chen, Chen; Tang, Yang; Wei, Jianhai; Zhu, Tong; Hu, Wenhao

    2016-08-01

    The docking approach for the screening of designed small molecule ligands, led to the identification of a critical arginine residue in peptide deformylase for spiro cyclopropyl PDF inhibitor's extra hydrophobic binding, providing us a useful tool for searching more efficient PDF inhibitors to fight for horrifying antibiotics resistance. Further synthetic modification was undertaken to optimize the potency of amide compounds. To lower metabolic susceptibility and in turn reduce unwanted metabolic toxicity that was observed clinically, while retaining desired antibacterial activity, the use of azoles as amide bioisosteres had also been investigated. After the completion of chemical synthesis, all the compounds were evaluated through in vitro antibacterial activity assay, some of which were further subject to in vivo rat pharmacokinetic assessment. Those findings in this letter showed that spiro cyclopropyl proline N-formyl hydroxylamines, and especially the bioisosteric azoles, can represent a promising class of PDF inhibitors. PMID:27293070

  9. Catalysis of Methyl Group Transfers Involving Tetrahydrofolate and B12

    PubMed Central

    Ragsdale, Stephen W.

    2011-01-01

    This review focuses on the reaction mechanism of enzymes that use B12 and tetrahydrofolate (THF) to catalyze methyl group transfers. It also covers the related reactions that use B12 and tetrahydromethanopterin (THMPT), which is a THF analog used by archaea. In the past decade, our understanding of the mechanisms of these enzymes has increased greatly because the crystal structures for three classes of B12-dependent methyltransferases have become available and because biophysical and kinetic studies have elucidated the intermediates involved in catalysis. These steps include binding of the cofactors and substrates, activation of the methyl donors and acceptors, the methyl transfer reaction itself, and product dissociation. Activation of the methyl donor in one class of methyltransferases is achieved by an unexpected proton transfer mechanism. The cobalt (Co) ion within the B12 macrocycle must be in the Co(I) oxidation state to serve as a nucleophile in the methyl transfer reaction. Recent studies have uncovered important principles that control how this highly reducing active state of B12 is generated and maintained. PMID:18804699

  10. Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms

    PubMed Central

    Giglione, Carmela; Serero, Alexandre; Pierre, Michèle; Boisson, Bertrand; Meinnel, Thierry

    2000-01-01

    The N-terminal protein processing pathway is an essential mechanism found in all organisms. However, it is widely believed that deformylase, a key enzyme involved in this process in bacteria, does not exist in eukaryotes, thus making it a target for antibacterial agents such as actinonin. In an attempt to define this process in higher eukaryotes we have used Arabidopsis thaliana as a model organism. Two deformylase cDNAs, the first identified in any eukaryotic system, and six distinct methionine aminopeptidase cDNAs were cloned. The corresponding proteins were characterized in vivo and in vitro. Methionine aminopeptidases were found in the cytoplasm and in the organelles, while deformylases were localized in the organelles only. Our work shows that higher plants have a much more complex machinery for methionine removal than previously suspected. We were also able to identify deformylase homologues from several animals and clone the corresponding cDNA from human cells. Our data provide the first evidence that lower and higher eukaryotes, as well as bacteria, share a similar N-terminal protein processing machinery, indicating universality of this system. PMID:11060042

  11. Structure analysis of peptide deformylases from Streptococcus pneumoniae, Staphylococcus aureus, Thermotoga maritima and Pseudomonas aeruginosa: snapshots of the oxygen sensitivity of peptide deformylase.

    PubMed

    Kreusch, Andreas; Spraggon, Glen; Lee, Chris C; Klock, Heath; McMullan, Daniel; Ng, Ken; Shin, Tanya; Vincent, Juli; Warner, Ian; Ericson, Christer; Lesley, Scott A

    2003-07-01

    Peptide deformylase (PDF) has received considerable attention during the last few years as a potential target for a new type of antibiotics. It is an essential enzyme in eubacteria for the removal of the formyl group from the N terminus of the nascent polypeptide chain. We have solved the X-ray structures of four members of this enzyme family, two from the Gram-positive pathogens Streptococcus pneumoniae and Staphylococcus aureus, and two from the Gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa. Combined with the known structures from the Escherichia coli enzyme and the recently solved structure of the eukaryotic deformylase from Plasmodium falciparum, a complete picture of the peptide deformylase structure and function relationship is emerging. This understanding could help guide a more rational design of inhibitors. A structure-based comparison between PDFs reveals some conserved differences between type I and type II enzymes. Moreover, our structures provide insights into the known instability of PDF caused by oxidation of the metal-ligating cysteine residue. PMID:12823970

  12. Flavimycins A and B, dimeric 1,3-dihydroisobenzofurans with peptide deformylase inhibitory activity from Aspergillus flavipes.

    PubMed

    Kwon, Yun-Ju; Sohn, Mi-Jin; Kim, Chang-Jin; Koshino, Hiroyuki; Kim, Won-Gon

    2012-02-24

    Flavimycins A (1) and B (2), novel dimeric 1,3-dihydroisobenzofurans, were isolated as inhibitors of peptide deformylase from cultures of Aspergillus flavipes. Their chemical structures were established by NMR and MS data analysis. Compounds 1 and 2 exist as epimeric mixtures at C-1 through fast hemiacetal-aldehyde tautomerism. Compounds 1 and 2 inhibited Staphylococcus aureus peptide deformylase with IC₅₀ values of 35.8 and 100.1 μM, respectively. Consistent with their PDF inhibition, 1 showed two times stronger antibacterial activity than 2 on S. aureus including MRSA, with MIC values of 32-64 μg/mL. PMID:22329646

  13. In Vitro and Intracellular Activities of Peptide Deformylase Inhibitor GSK1322322 against Legionella pneumophila Isolates

    PubMed Central

    Dubois, Jacques; Dubois, Maïtée; Martel, Jean-François; Aubart, Kelly

    2014-01-01

    GSK1322322, a novel peptide deformylase inhibitor currently in development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia, showed poor in vitro activity against a panel of 50 Legionella pneumophila strains, with MICs ranging from 1 to 16 μg/ml and an MIC90 of 16 μg/ml, but very potent intracellular activity, with the minimum extracellular concentrations capable of inhibiting intracellular proliferation (MIECs) ranging from 0.12 to 2 μg/ml and 98% of the strains being inhibited by concentrations of ≤1 μg/ml. PMID:25348534

  14. Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics.

    PubMed

    Lee, Mona D; She, Yuhong; Soskis, Michael J; Borella, Christopher P; Gardner, Jeffrey R; Hayes, Paula A; Dy, Benzon M; Heaney, Mark L; Philips, Mark R; Bornmann, William G; Sirotnak, Francis M; Scheinberg, David A

    2004-10-01

    Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics. PMID:15489958

  15. Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics

    PubMed Central

    Lee, Mona D.; She, Yuhong; Soskis, Michael J.; Borella, Christopher P.; Gardner, Jeffrey R.; Hayes, Paula A.; Dy, Benzon M.; Heaney, Mark L.; Philips, Mark R.; Bornmann, William G.; Sirotnak, Francis M.; Scheinberg, David A.

    2004-01-01

    Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics. PMID:15489958

  16. Tetrahydrofolate enzyme levels in Acetobacterium woodii and their implication in the synthesis of acetate from CO2.

    PubMed Central

    Tanner, R S; Wolfe, R S; Ljungdahl, L G

    1978-01-01

    Acetate synthesis from CO2 by Acetobacterium woodii may occur as in homoacetate-fermenting clostridia, as indicated by high levels of enzymes of the tetrahydrofolate pathway and by pyruvate-dependent formation of acetate from methyl-B12 and methyltetrahydrofolate. PMID:659361

  17. Tetrahydrofolate enzyme levels in Acetobacterium woodii and their implication in the synthesis of acetate from CO2.

    PubMed

    Tanner, R S; Wolfe, R S; Ljungdahl, L G

    1978-05-01

    Acetate synthesis from CO2 by Acetobacterium woodii may occur as in homoacetate-fermenting clostridia, as indicated by high levels of enzymes of the tetrahydrofolate pathway and by pyruvate-dependent formation of acetate from methyl-B12 and methyltetrahydrofolate. PMID:659361

  18. mTORC1 Induces Purine Synthesis Through Control of the Mitochondrial Tetrahydrofolate Cycle

    PubMed Central

    Ricoult, Stéphane J.H.; Asara, John M.; Manning, Brendan D.

    2016-01-01

    In response to growth signals, mTOR complex 1 (mTORC1) stimulates anabolic processes underlying cell growth. We found that mTORC1 increases metabolic flux through the de novo purine synthesis pathway in various mouse and human cells, thereby influencing the nucleotide pool available for nucleic acid synthesis. mTORC1 had transcriptional effects on multiple enzymes contributing to purine synthesis, with expression of the mitochondrial tetrahydrofolate (mTHF) cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) being closely associated with mTORC1 signaling in both normal and cancer cells. MTHFD2 expression and purine synthesis were stimulated by ATF4, which was activated by mTORC1 independent of its canonical induction downstream of eIF2α phosphorylation. Thus, mTORC1 stimulates the mTHF cycle, which contributes one-carbon units to enhance production of purine nucleotides in response to growth signals. PMID:26912861

  19. Discovery and refinement of a new structural class of potent peptide deformylase inhibitors.

    PubMed

    Boularot, Adrien; Giglione, Carmela; Petit, Sylvain; Duroc, Yann; Alves de Sousa, Rodolphe; Larue, Valéry; Cresteil, Thierry; Dardel, Frédéric; Artaud, Isabelle; Meinnel, Thierry

    2007-01-11

    New classes of antibiotics are urgently needed to counter increasing levels of pathogen resistance. Peptide deformylase (PDF) was originally selected as a specific bacterial target, but a human homologue, the inhibition of which causes cell death, was recently discovered. We developed a dual-screening strategy for selecting highly effective compounds with low inhibition effect against human PDF. We selected a new scaffold in vitro that discriminated between human and bacterial PDFs. Analyses of structure-activity relationships identified potent antibiotics such as 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (6b) with the same mode of action in vivo as previously identified PDF inhibitors but without the apoptotic effects of these inhibitors in human cells. PMID:17201406

  20. In Vivo Characterization of the Peptide Deformylase Inhibitor LBM415 in Murine Infection Models ▿

    PubMed Central

    Osborne, Colin S.; Neckermann, Georg; Fischer, Evelin; Pecanka, Robert; Yu, Donghui; Manni, Kari; Goldovitz, Julie; Amaral, Kerri; Dzink-Fox, JoAnn; Ryder, Neil S.

    2009-01-01

    LBM415 is an antibacterial agent belonging to the peptide deformylase inhibitor class of compounds. It has previously been shown to demonstrate good activity in vitro against a range of pathogens. In this study, the in vivo efficacy of LBM415 was evaluated in various mouse infection models. We investigated activity against a systemic infection model caused by intraperitoneal inoculation of Staphylococcus aureus (methicillin [meticillin] susceptible [MSSA] and methicillin resistant [MRSA]) and Streptococcus pneumoniae (penicillin susceptible [PSSP] and multidrug resistant [MDRSP]), a thigh infection model caused by intramuscular injection of MRSA, and a lung infection produced by intranasal inoculation of PSSP. In the systemic MSSA and MRSA infections, LBM415 was equivalent to linezolid and vancomycin. In the systemic PSSP infection, LBM415 was equivalent to linezolid, whereas against systemic MDRSP infection, the LBM415 50% effective dose (ED50) was 4.8 mg/kg (dosed subcutaneously) and 36.6 mg/kg (dosed orally), compared to 13.2 mg/kg for telithromycin and >60 mg/kg for penicillin V and clarithromycin. In the MRSA thigh infection, LBM415 significantly reduced thigh bacterial levels compared to those of untreated mice, with levels similar to those after treatment with linezolid at the same dose levels. In the pneumonia model, the ED50 to reduce the bacterial lung burden by >4 log10 in 50% of treated animals was 23.3 mg/kg for LBM415, whereas moxifloxacin showed an ED50 of 14.3 mg/kg. In summary, LBM415 showed in vivo efficacy in sepsis and specific organ infection models irrespective of resistance to other antibiotics. Results suggest the potential of peptide deformylase inhibitors as a novel class of therapeutic agents against antibiotic-resistant pathogens. PMID:19596876

  1. Microbiome changes in healthy volunteers treated with GSK1322322, a novel antibiotic targeting bacterial peptide deformylase.

    PubMed

    Arat, Seda; Spivak, Aaron; Van Horn, Stephanie; Thomas, Elizabeth; Traini, Christopher; Sathe, Ganesh; Livi, George P; Ingraham, Karen; Jones, Lori; Aubart, Kelly; Holmes, David J; Naderer, Odin; Brown, James R

    2015-02-01

    GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study

  2. Microbiome Changes in Healthy Volunteers Treated with GSK1322322, a Novel Antibiotic Targeting Bacterial Peptide Deformylase

    PubMed Central

    Arat, Seda; Spivak, Aaron; Van Horn, Stephanie; Thomas, Elizabeth; Traini, Christopher; Sathe, Ganesh; Livi, George P.; Ingraham, Karen; Jones, Lori; Aubart, Kelly; Holmes, David J.; Naderer, Odin

    2014-01-01

    GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study

  3. Inhibition of chlamydial infection in the genital tract of female mice by topical application of a peptide deformylase inhibitor.

    PubMed

    Balakrishnan, Amit; Wang, Lingling; Li, Xiaojin; Ohman-Strickland, Pamela; Malatesta, Paul; Fan, Huizhou

    2009-01-01

    Chlamydia trachomatis is an obligate intracellular bacterium responsible for a number of health problems, including sexually transmitted infection in humans. We recently discovered that C. trachomatis infection in cell culture is highly susceptible to inhibitors of peptide deformylase, an enzyme that removes the N-formyl group from newly synthesized polypeptides. In this study, one of the deformylase inhibitors, GM6001, was tested for potential antichlamydial activity using a murine genital C. muridarum infection model. Topical application of GM6001 significantly reduced C. muridarum loading in BALB/c mice that were vaginally infected with the pathogen. In striking contrast, growth of the probiotic Lactobacillus plantarum is strongly resistant to the PDF inhibitor. GM6001 demonstrated no detectable toxicity against host cells. On the basis of these data and our previous observations, we conclude that further evaluation of PDF inhibitors for prevention and treatment of sexually transmitted chlamydial infection is warranted. PMID:17936604

  4. mTORC1 induces purine synthesis through control of the mitochondrial tetrahydrofolate cycle.

    PubMed

    Ben-Sahra, Issam; Hoxhaj, Gerta; Ricoult, Stéphane J H; Asara, John M; Manning, Brendan D

    2016-02-12

    In response to growth signals, mechanistic target of rapamycin complex 1 (mTORC1) stimulates anabolic processes underlying cell growth. We found that mTORC1 increases metabolic flux through the de novo purine synthesis pathway in various mouse and human cells, thereby influencing the nucleotide pool available for nucleic acid synthesis. mTORC1 had transcriptional effects on multiple enzymes contributing to purine synthesis, with expression of the mitochondrial tetrahydrofolate (mTHF) cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) being closely associated with mTORC1 signaling in both normal and cancer cells. MTHFD2 expression and purine synthesis were stimulated by activating transcription factor 4 (ATF4), which was activated by mTORC1 independent of its canonical induction downstream of eukaryotic initiation factor 2α eIF2α phosphorylation. Thus, mTORC1 stimulates the mTHF cycle, which contributes one-carbon units to enhance production of purine nucleotides in response to growth signals. PMID:26912861

  5. Variations in metabolic pathways create challenges for automated metabolic reconstructions: Examples from the tetrahydrofolate synthesis pathway

    PubMed Central

    de Crécy-Lagard, Valérie

    2014-01-01

    The availability of thousands of sequenced genomes has revealed the diversity of biochemical solutions to similar chemical problems. Even for molecules at the heart of metabolism, such as cofactors, the pathway enzymes first discovered in model organisms like Escherichia coli or Saccharomyces cerevisiae are often not universally conserved. Tetrahydrofolate (THF) (or its close relative tetrahydromethanopterin) is a universal and essential C1-carrier that most microbes and plants synthesize de novo. The THF biosynthesis pathway and enzymes are, however, not universal and alternate solutions are found for most steps, making this pathway a challenge to annotate automatically in many genomes. Comparing THF pathway reconstructions and functional annotations of a chosen set of folate synthesis genes in specific prokaryotes revealed the strengths and weaknesses of different microbial annotation platforms. This analysis revealed that most current platforms fail in metabolic reconstruction of variant pathways. However, all the pieces are in place to quickly correct these deficiencies if the different databases were built on each other's strengths. PMID:25210598

  6. Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers.

    PubMed

    Berman, M H; Frazer, A C

    1992-03-01

    DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells. PMID:1575495

  7. Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers

    SciTech Connect

    Berman, M.H.; Frazer, A.C. )

    1992-03-01

    DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 {plus minus} 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent K{sub m} values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.

  8. Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch

    SciTech Connect

    Huang, Lili; Ishibe-Murakami, Satoko; Patel, Dinshaw J.; Serganov, Alexander

    2011-09-15

    Tetrahydrofolate (THF), a biologically active form of the vitamin folate (B{sub 9}), is an essential cofactor in one-carbon transfer reactions. In bacteria, expression of folate-related genes is controlled by feedback modulation in response to specific binding of THF and related compounds to a riboswitch. Here, we present the X-ray structures of the THF-sensing domain from the Eubacterium siraeum riboswitch in the ligand-bound and unbound states. The structure reveals an 'inverted' three-way junctional architecture, most unusual for riboswitches, with the junction located far from the regulatory helix P1 and not directly participating in helix P1 formation. Instead, the three-way junction, stabilized by binding to the ligand, aligns the riboswitch stems for long-range tertiary pseudoknot interactions that contribute to the organization of helix P1 and therefore stipulate the regulatory response of the riboswitch. The pterin moiety of the ligand docks in a semiopen pocket adjacent to the junction, where it forms specific hydrogen bonds with two moderately conserved pyrimidines. The aminobenzoate moiety stacks on a guanine base, whereas the glutamate moiety does not appear to make strong interactions with the RNA. In contrast to other riboswitches, these findings demonstrate that the THF riboswitch uses a limited number of available determinants for ligand recognition. Given that modern antibiotics target folate metabolism, the THF riboswitch structure provides insights on mechanistic aspects of riboswitch function and may help in manipulating THF levels in pathogenic bacteria

  9. Structure-Based Drug Design of Small Molecule Peptide Deformylase Inhibitors to Treat Cancer.

    PubMed

    Gao, Jian; Wang, Tao; Qiu, Shengzhi; Zhu, Yasheng; Liang, Li; Zheng, Youguang

    2016-01-01

    Human peptide deformylase (HsPDF) is an important target for anticancer drug discovery. In view of the limited HsPDF, inhibitors were reported, and high-throughput virtual screening (HTVS) studies based on HsPDF for developing new PDF inhibitors remain to be reported. We reported here on diverse small molecule inhibitors with excellent anticancer activities designed based on HTVS and molecular docking studies using the crystal structure of HsPDF. The compound M7594_0037 exhibited potent anticancer activities against HeLa, A549 and MCF-7 cell lines with IC50s of 35.26, 29.63 and 24.63 μM, respectively. Molecular docking studies suggested that M7594_0037 and its three derivatives could interact with HsPDF by several conserved hydrogen bonds. Moreover, the pharmacokinetic and toxicity properties of M7594_0037 and its derivatives were predicted using the OSIRIS property explorer. Thus, M7594_0037 and its derivatives might represent a promising scaffold for the further development of novel anticancer drugs. PMID:27023495

  10. Pharmacokinetics in Animals and Humans of a First-in-Class Peptide Deformylase Inhibitor

    PubMed Central

    Ramanathan-Girish, Sandhya; McColm, Juliet; Clements, John M.; Taupin, Phil; Barrowcliffe, Sue; Hevizi, John; Safrin, Sharon; Moore, Clive; Patou, Gary; Moser, Heinz; Gadd, Alison; Hoch, Ute; Jiang, Vernon; Lofland, Denene; Johnson, Kirk W.

    2004-01-01

    BB-83698, a potent and selective inhibitor of peptide deformylase, was the first compound of this novel antibacterial class to progress to clinical trials. Single- and/or multiple-dose studies with doses ranging from 10 to 50 mg of BB-83698/kg of body weight were done with mice, rats, and dogs. Intravenous pharmacokinetics were characterized by low to moderate clearances and moderate volumes of distribution for all species. In dogs, but not in rodents, central nervous system (CNS) effects were dose limiting for intravenously administered BB-83698 and were suspected to be related to a high maximum concentration of the agent in plasma (Cmax) rather than to total systemic exposure. Controlled infusion studies with dogs demonstrated that CNS effects could be avoided without compromising systemic exposure by reducing the Cmax. A randomized, double-blind, placebo-controlled, five-way-crossover, single-dose-escalation, phase I study to explore the safety, tolerability, and pharmacokinetics of intravenous BB-83698 at doses ranging from 10 to 475 mg was performed with healthy male volunteers. Systemic exposures were generally in linear relationships with administered doses in animals and humans. Pharmacokinetics were consistent, predictable, and exhibited good allometric scaling among all species (r2 >0.98). Moreover, BB-83698 dosing in humans proceeded to a predicted efficacious exposure (the area under the concentration-time curve/MIC ratio, up to 184) without any clinically significant adverse effects. PMID:15561864

  11. Structure and Activity of Human Mitochondrial Peptide Deformylase, a Novel Cancer Target

    SciTech Connect

    Escobar-Alvarez, Sindy; Goldgur, Yehuda; Yang, Guangli; Ouerfelli, Ouathek; Li, Yueming; Scheinberg, David A.

    2009-07-21

    Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 A), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 A) identified the substrate-binding site. A defined S1' pocket, but no S2' or S3' substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2' and S3' binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2'and P3' positions of a formylated peptide substrate to turnover.

  12. Structure and activity of human mitochondrial peptide deformylase, a novel cancer target.

    PubMed

    Escobar-Alvarez, Sindy; Goldgur, Yehuda; Yang, Guangli; Ouerfelli, Ouathek; Li, Yueming; Scheinberg, David A

    2009-04-17

    Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 A), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 A) identified the substrate-binding site. A defined S1' pocket, but no S2' or S3' substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2' and S3' binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2'and P3' positions of a formylated peptide substrate to turnover. PMID:19236878

  13. Pharmacokinetics in animals and humans of a first-in-class peptide deformylase inhibitor.

    PubMed

    Ramanathan-Girish, Sandhya; McColm, Juliet; Clements, John M; Taupin, Phil; Barrowcliffe, Sue; Hevizi, John; Safrin, Sharon; Moore, Clive; Patou, Gary; Moser, Heinz; Gadd, Alison; Hoch, Ute; Jiang, Vernon; Lofland, Denene; Johnson, Kirk W

    2004-12-01

    BB-83698, a potent and selective inhibitor of peptide deformylase, was the first compound of this novel antibacterial class to progress to clinical trials. Single- and/or multiple-dose studies with doses ranging from 10 to 50 mg of BB-83698/kg of body weight were done with mice, rats, and dogs. Intravenous pharmacokinetics were characterized by low to moderate clearances and moderate volumes of distribution for all species. In dogs, but not in rodents, central nervous system (CNS) effects were dose limiting for intravenously administered BB-83698 and were suspected to be related to a high maximum concentration of the agent in plasma (Cmax) rather than to total systemic exposure. Controlled infusion studies with dogs demonstrated that CNS effects could be avoided without compromising systemic exposure by reducing the Cmax. A randomized, double-blind, placebo-controlled, five-way-crossover, single-dose-escalation, phase I study to explore the safety, tolerability, and pharmacokinetics of intravenous BB-83698 at doses ranging from 10 to 475 mg was performed with healthy male volunteers. Systemic exposures were generally in linear relationships with administered doses in animals and humans. Pharmacokinetics were consistent, predictable, and exhibited good allometric scaling among all species (r2 >0.98). Moreover, BB-83698 dosing in humans proceeded to a predicted efficacious exposure (the area under the concentration-time curve/MIC ratio, up to 184) without any clinically significant adverse effects. PMID:15561864

  14. Genome-wide identification and in silico analysis of poplar peptide deformylases.

    PubMed

    Liu, Chang-Cai; Liu, Bao-Guang; Yang, Zhi-Wei; Li, Chun-Ming; Wang, Bai-Chen; Yang, Chuan-Ping

    2012-01-01

    Peptide deformylases (PDF) behave as monomeric metal cation hydrolases for the removal of the N-formyl group (Fo). This is an essential step in the N-terminal Met excision (NME) that occurs in these proteins from eukaryotic mitochondria or chloroplasts. Although PDFs have been identified and their structure and function have been characterized in several herbaceous species, it remains as yet unexplored in poplar. Here, we report on the first identification of two genes (PtrPDF1A and PtrPDF1B) respectively encoding two putative PDF polypeptides in Populus trichocarpa by genome-wide investigation. One of them (XP_002300047.1) encoded by PtrPDF1B (XM_002300011.1) was truncated, and then revised into a complete sequence based on its ESTs support with high confidence. We document that the two PDF1s of Populus are evolutionarily divergent, likely as a result of independent duplicated events. Furthermore, in silico simulations demonstrated that PtrPDF1A and PtrPDF1B should act as similar PDF catalytic activities to their corresponding PDF orthologs in Arabidopsis. This result would be value of for further assessment of their biological activities in poplar, and further experiments are now required to confirm them. PMID:22606033

  15. Understanding the Origins of Time-Dependent Inhibition by Polypeptide Deformylase Inhibitors

    SciTech Connect

    Totoritis, Rachel; Duraiswami, Chaya; Taylor, Amy N.; Kerrigan, John J.; Campobasso, Nino; Smith, Katherine J.; Ward, Paris; King, Bryan W.; Murrayz-Thompson, Monique; Jones, Amber D.; Van Aller, Glenn S.; Aubart, Kelly M.; Zalacain, Magdalena; Thrall, Sara H.; Meek, Thomas D.; Schwartz, Benjamin

    2012-03-15

    The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.

  16. Ligand and Structure-Based Approaches for the Identification of Peptide Deformylase Inhibitors as Antibacterial Drugs.

    PubMed

    Gao, Jian; Liang, Li; Zhu, Yasheng; Qiu, Shengzhi; Wang, Tao; Zhang, Ling

    2016-01-01

    Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins, which makes it an important antibacterial drug target. Given the importance of PDF inhibitors like actinonin in antibacterial drug discovery, several reported potent PDF inhibitors were used to develop pharmacophore models using the Galahad module of Sybyl 7.1 software. Generated pharmacophore models were composed of two donor atom centers, four acceptor atom centers and two hydrophobic groups. Model-1 was screened against the Zinc database and several compounds were retrieved as hits. Compounds with Qfit values of more than 60 were employed to perform a molecular docking study with the receptor Escherichia coli PDF, then compounds with docking score values of more than 6 were used to predict the in silico pharmacokinetic and toxicity risk via OSIRIS property explorer. Two known PDF inhibitors were also used to perform a molecular docking study with E. coli PDF as reference molecules. The results of the molecular docking study were validated by reproducing the crystal structure of actinonin. Molecular docking and in silico pharmacokinetic and toxicity prediction studies suggested that ZINC08740166 has a relatively high docking score of 7.44 and a drug score of 0.78. PMID:27428963

  17. Caffeic acid phenethyl ester (CAPE), an active component of propolis, inhibits Helicobacter pylori peptide deformylase activity.

    PubMed

    Cui, Kunqiang; Lu, Weiqiang; Zhu, Lili; Shen, Xu; Huang, Jin

    2013-05-31

    Helicobacter pylori (H. pylori) is a major causative factor for gastrointestinal illnesses, H. pylori peptide deformylase (HpPDF) catalyzes the removal of formyl group from the N-terminus of nascent polypeptide chains, which is essential for H. pylori survival and is considered as a promising drug target for anti-H. pylori therapy. Propolis, a natural antibiotic from honeybees, is reported to have an inhibitory effect on the growth of H. pylori in vitro. In addition, previous studies suggest that the main active constituents in the propolis are phenolic compounds. Therefore, we evaluated a collection of phenolic compounds derived from propolis for enzyme inhibition against HpPDF. Our study results show that Caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, is a competitive inhibitor against HpPDF, with an IC50 value of 4.02 μM. Furthermore, absorption spectra and crystal structural characterization revealed that different from most well known PDF inhibitors, CAPE block the substrate entrance, preventing substrate from approaching the active site, but CAPE does not have chelate interaction with HpPDF and does not disrupt the metal-dependent catalysis. Our study provides valuable information for understanding the potential anti-H. pylori mechanism of propolis, and CAPE could be served as a lead compound for further anti-H. pylori drug discovery. PMID:23611786

  18. Peptide deformylase: a new target in antibacterial, antimalarial and anticancer drug discovery.

    PubMed

    Sangshetti, Jaiprakash N; Khan, Firoz A Kalam; Shinde, Devanand B

    2015-01-01

    Peptide deformylase (PDF) is a class of metalloenzyme responsible for catalyzing the removal of the N-formyl group from N-terminal methionine following translation. PDF inhibitors are moving into new phase of drug development. Initially, PDF was considered as an important target in antibacterial drug discovery; however genome database searches have revealed PDF-like sequences in parasites (P. falciparum) and human, widening the utility of this target in antimalarial and anticancer drug discovery along with antibacterial. Using structural and mechanistic information together with high throughput screening, several types of chemical classes of PDF inhibitors with improved efficacy and specificity have been identified. Various drugs like, GSK-1322322 (Phase II), BB-83698 (Phase I), and LBM-415 (Phase I) have entered into clinical developments. Developments in the field have prompted us to review the current aspects of PDFs, especially their structures, different classes of PDF inhibitors, and molecular modeling studies. In nut shell, this review enlightens PDF as a versatile target along with its inhibitors and future perspectives of different PDF inhibitors. PMID:25174923

  19. Safety, tolerability, and pharmacokinetics of oral and intravenous administration of GSK1322322, a peptide deformylase inhibitor.

    PubMed

    Naderer, Odin J; Jones, Lori S; Zhu, John; Kurtinecz, Milena; Dumont, Etienne

    2013-11-01

    GSK1322322 is the first in a new class of antibiotics that targets peptide deformylase (PDF), an essential bacterial enzyme required for protein maturation. This randomized, double-blind, placebo-controlled, eight-cohort phase I trial enrolled 62 healthy volunteers to assess safety, tolerability, and pharmacokinetic profiles of GSK1322322. GSK1322322 was administered as a single oral or intravenous (IV) dose, escalating from 500 to 3,000 mg or repeat IV doses escalating from 500 to 1,500 mg twice daily. Upon repeat IV administration, GSK1322322 exhibits linear pharmacokinetics over time upon repeat doses as shown by time-invariant pharmacokinetics. A dose-proportional increase in area under concentration-time curve was observed after single or repeat IV dosing, whereas clearance at steady state remained generally unchanged across doses. There was minimal accumulation of GSK1322322 after repeat IV twice-daily administration. After oral tablet doses of GSK1322322 1,000 and 1,500 mg, absolute bioavailability was 69% and 56%, respectively. GSK1322322 administration at single and repeat IV doses and at supratherapeutic single IV doses of 2,000 and 3,000 mg was associated with mild-to-moderate drug-related adverse events. On the basis of the pharmacokinetics and tolerability demonstrated in this study, GSK1322322 has the potential to become the first-in-class PDF inhibitor for clinical use. PMID:23907665

  20. Ligand and Structure-Based Approaches for the Identification of Peptide Deformylase Inhibitors as Antibacterial Drugs

    PubMed Central

    Gao, Jian; Liang, Li; Zhu, Yasheng; Qiu, Shengzhi; Wang, Tao; Zhang, Ling

    2016-01-01

    Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins, which makes it an important antibacterial drug target. Given the importance of PDF inhibitors like actinonin in antibacterial drug discovery, several reported potent PDF inhibitors were used to develop pharmacophore models using the Galahad module of Sybyl 7.1 software. Generated pharmacophore models were composed of two donor atom centers, four acceptor atom centers and two hydrophobic groups. Model-1 was screened against the Zinc database and several compounds were retrieved as hits. Compounds with Qfit values of more than 60 were employed to perform a molecular docking study with the receptor Escherichia coli PDF, then compounds with docking score values of more than 6 were used to predict the in silico pharmacokinetic and toxicity risk via OSIRIS property explorer. Two known PDF inhibitors were also used to perform a molecular docking study with E. coli PDF as reference molecules. The results of the molecular docking study were validated by reproducing the crystal structure of actinonin. Molecular docking and in silico pharmacokinetic and toxicity prediction studies suggested that ZINC08740166 has a relatively high docking score of 7.44 and a drug score of 0.78. PMID:27428963

  1. DMSP: tetrahydrofolate methyltransferase from the marine sulfate-reducing bacterium strain WN

    NASA Astrophysics Data System (ADS)

    Jansen, M.; Hansen, T. A.

    2000-08-01

    Dimethylsulfoniopropionate (DMSP), an important compatible solute of many marine algae, can be metabolised by bacteria via cleavage to dimethylsulfide and acrylate or via an initial demethylation. This is the first report on the purification of an enzyme that specifically catalyses the demethylation of DMSP. The enzyme was isolated from the sulfate-reducing bacterium strain WN, which grows on DMSP and demethylates it to methylthiopropionate. DMSP:tetrahydrofolate (THF) methyltransferase from strain WN was purified 76-fold [to a specific activity of 40.5 μmol min -1 (mg protein) -1]. SDS polyacrylamide gel electrophoresis showed two bands of approximately 10 and 35 kDa; in particular the 35 kDa polypeptide became significantly enriched during the purification. Storage of the purified fraction at -20°C under nitrogen resulted in a 99% loss of activity in two days. The activity could be partially restored by addition of 200 μM cyanocobalamin, hydroxocobalamin or coenzyme B 12. ATP did not have any positive effect on activity. Reduction of the assay mixture by titanium(III)nitrilotriacetic acid slightly stimulated the activity. Gel filtration chromatography revealed a native molecular mass between 45 and 60 kDa for the DMSP:THF methyltransferase. The enzyme was most active at 35°C and pH 7.8. Glycine betaine, which can be considered an N-containing structural analogue of DMSP, did not serve as a methyl donor for DMSP:THF methyltransferase. Various sulfur-containing DMSP-analogues were tested but only methylethylsulfoniopropionate served as methyl donor. None of these compounds inhibited methyl transfer from DMSP to THF. Strain WN did not grow on any of the sulfur-containing DMSP-analogues.

  2. New Antibiotic Molecules: Bypassing the Membrane Barrier of Gram Negative Bacteria Increases the Activity of Peptide Deformylase Inhibitors

    PubMed Central

    Mamelli, Laurent; Petit, Sylvain; Chevalier, Jacqueline; Giglione, Carmela; Lieutaud, Aurélie; Meinnel, Thierry; Artaud, Isabelle; Pagès, Jean-Marie

    2009-01-01

    Background Multi-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol. Methodology/Principal Findings To determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the lastest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step. Conclusions/Significance Our results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound. PMID:19649280

  3. An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway.

    PubMed

    Serero, Alexandre; Giglione, Carmela; Sardini, Alessandro; Martinez-Sanz, Juan; Meinnel, Thierry

    2003-12-26

    Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated. PMID:14532271

  4. Comparative analysis of the antibacterial activity of a novel peptide deformylase inhibitor, GSK1322322.

    PubMed

    O'Dwyer, Karen; Hackel, Meredith; Hightower, Sarah; Hoban, Daryl; Bouchillon, Samuel; Qin, Donghui; Aubart, Kelly; Zalacain, Magdalena; Butler, Deborah

    2013-05-01

    GSK1322322 is a novel peptide deformylase (PDF) inhibitor being developed for the intravenous and oral treatment of acute bacterial skin and skin structure infections and hospitalized patients with community-acquired pneumonia. The activity of GSK1322322 was tested against a global collection of clinical isolates of Haemophilus influenzae (n = 2,370), Moraxella catarrhalis (n = 115), Streptococcus pneumoniae (n = 947), Streptococcus pyogenes (n = 617), and Staphylococcus aureus (n = 940), including strains resistant to one or more marketed antibiotics. GSK1322322 had an MIC(90) of 1 μg/ml against M. catarrhalis and 4 μg/ml against H. influenzae, with 88.8% of β-lactamase-positive strains showing growth inhibition at that concentration. All S. pneumoniae strains were inhibited by ≤ 4 μg/ml of GSK1322322, with an MIC(90) of 2 μg/ml. Pre-existing resistance mechanisms did not affect its potency, as evidenced by the MIC(90) of 1 μg/ml for penicillin, levofloxacin, and macrolide-resistant S. pneumoniae. GSK1322322 was very potent against S. pyogenes strains, with an MIC(90) of 0.5 μg/ml, irrespective of their macrolide resistance phenotype. This PDF inhibitor was also active against S. aureus strains regardless of their susceptibility to methicillin, macrolides, or levofloxacin, with an MIC(90) of 4 μg/ml in all cases. Time-kill studies showed that GSK1322322 had bactericidal activity against S. pneumoniae, H. influenzae, S. pyogenes, and S. aureus, demonstrating a ≥ 3-log(10) decrease in the number of CFU/ml at 4× MIC within 24 h in 29 of the 33 strains tested. Given the antibacterial potency demonstrated against this panel of organisms, GSK1322322 represents a valuable alternative therapy for the treatment of infectious diseases caused by drug-resistant pathogens. PMID:23478958

  5. Structures of Staphylococcus aureus peptide deformylase in complex with two classes of new inhibitors.

    PubMed

    Lee, Sang Jae; Lee, Seung-Jae; Lee, Seung Kyu; Yoon, Hye-Jin; Lee, Hyung Ho; Kim, Kyeong Kyu; Lee, Bong Jin; Lee, Byung Il; Suh, Se Won

    2012-07-01

    Peptide deformylase (PDF) catalyzes the removal of the formyl group from the N-terminal methionine residue in newly synthesized polypeptides, which is an essential process in bacteria. Four new inhibitors of PDF that belong to two different classes, hydroxamate/pseudopeptide compounds [PMT387 (7a) and PMT497] and reverse-hydroxamate/nonpeptide compounds [PMT1039 (15e) and PMT1067], have been developed. These compounds inhibited the growth of several pathogens involved in respiratory-tract infections, such as Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae, and leading nosocomial pathogens such as Staphylococcus aureus and Klebsiella pneumoniae with a minimum inhibitory concentration (MIC) in the range 0.1-0.8 mg ml(-1). Interestingly, the reverse-hydroxamate/nonpeptide compounds showed a 250-fold higher antimicrobial activity towards S. aureus, although the four compounds showed similar K(i) values against S. aureus PDF enzymes, with K(i) values in the 11-85 nM range. To provide a structural basis for the discovery of additional PDF inhibitors, the crystal structures of S. aureus PDF in complex with the four inhibitors were determined at resolutions of 1.90-2.30 Å. The inhibitor-bound structures displayed distinct deviations depending on the inhibitor class. The distance between the Zn(2+) ion and the carbonyl O atom of the hydroxamate inhibitors (or the hydroxyl O atom of the reverse-hydroxamate inhibitors) appears to be correlated to S. aureus inhibition activity. The structural information reported in this study should aid in the discovery of new PDF inhibitors that can be used as novel antibacterial drugs. PMID:22751663

  6. Tetrahydrofolate and tetrahydromethanopterin compared: functionally distinct carriers in C1 metabolism.

    PubMed Central

    Maden, B E

    2000-01-01

    In most organisms, tetrahydrofolate (H(4)folate) is the carrier of C(1) fragments between formyl and methyl oxidation levels. The C(1) fragments are utilized in several essential biosynthetic processes. In addition, C(1) flux through H(4)folate is utilized for energy metabolism in some groups of anaerobic bacteria. In methanogens and several other Archaea, tetrahydromethanopterin (H(4)MPT) carries C(1) fragments between formyl and methyl oxidation levels. At first sight H(4)MPT appears to resemble H(4)folate at the sites where C(1) fragments are carried. However, the two carriers are functionally distinct, as discussed in the present review. In energy metabolism, H(4)MPT permits redox-flux features that are distinct from the pathway on H(4)folate. In the reductive direction, ATP is consumed in the entry of carbon from CO(2) into the H(4)folate pathway, but not in entry into the H(4)MPT pathway. In the oxidative direction, methyl groups are much more readily oxidized on H(4)MPT than on H(4)folate. Moreover, the redox reactions on H(4)MPT are coupled to more negative reductants than the pyridine nucleotides which are generally used in the H(4)folate pathway. Thermodynamics of the reactions of C(1) reduction via the two carriers differ accordingly. A major underlying cause of the thermodynamic differences is in the chemical properties of the arylamine nitrogen N(10) on the two carriers. In H(4)folate, N(10) is subject to electron withdrawal by the carbonyl group of p-aminobenzoate, but in H(4)MPT an electron-donating methylene group occurs in the corresponding position. It is also proposed that the two structural methyl groups of H(4)MPT tune the carrier's thermodynamic properties through an entropic contribution. H(4)MPT appears to be unsuited to some of the biosynthetic functions of H(4)folate, in particular the transfer of activated formyl groups, as in purine biosynthesis. Evidence bearing upon whether H(4)MPT participates in thymidylate synthesis is discussed

  7. How water molecules affect the catalytic activity of hydrolases - A XANES study of the local structures of peptide deformylase

    NASA Astrophysics Data System (ADS)

    Cui, Peixin; Wang, Yu; Chu, Wangsheng; Guo, Xiaoyun; Yang, Feifei; Yu, Meijuan; Zhao, Haifeng; Dong, Yuhui; Xie, Yaning; Gong, Weimin; Wu, Ziyu

    2014-12-01

    Peptide deformylase (PDF) is a prokaryotic enzyme that catalyzes the deformylation of nascent peptides generated during protein synthesis and water molecules play a key role in these hydrolases. Using X-ray absorption near edge spectroscopy (XANES) and ab initio calculations we accurately probe the local atomic environment of the metal ion binding in the active site of PDF at different pH values and with different metal ions. This new approach is an effective way to monitor existing correlations among functions and structural changes. We show for the first time that the enzymatic activity depends on pH values and metal ions via the bond length of the nearest coordinating water (Wat1) to the metal ion. Combining experimental and theoretical data we may claim that PDF exhibits an enhanced enzymatic activity only when the distance of the Wat1 molecule with the metal ion falls in the limited range from 2.15 to 2.55 Å.

  8. The structure of a tetrahydrofolate-sensing riboswitch reveals two ligand binding sites in a single aptamer.

    PubMed

    Trausch, Jeremiah J; Ceres, Pablo; Reyes, Francis E; Batey, Robert T

    2011-10-12

    Transport and biosynthesis of folate and its derivatives are frequently controlled by the tetrahydrofolate (THF) riboswitch in Firmicutes. We have solved the crystal structure of the THF riboswitch aptamer in complex with folinic acid, a THF analog. Uniquely, this structure reveals two molecules of folinic acid binding to a single structured domain. These two sites interact with ligand in a similar fashion, primarily through recognition of the reduced pterin moiety. 7-deazaguanine, a soluble analog of guanine, binds the riboswitch with nearly the same affinity as its natural effector. However, 7-deazaguanine effects transcriptional termination to a substantially lesser degree than folinic acid, suggesting that the cellular guanine pool does not act upon the THF riboswitch. Under physiological conditions the ligands display strong cooperative binding, with one of the two sites playing a greater role in eliciting the regulatory response, which suggests that the second site may play another functional role. PMID:21906956

  9. A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.

    PubMed

    Waller, Jeffrey C; Alvarez, Sophie; Naponelli, Valeria; Lara-Nuñez, Aurora; Blaby, Ian K; Da Silva, Vanessa; Ziemak, Michael J; Vickers, Tim J; Beverley, Stephen M; Edison, Arthur S; Rocca, James R; Gregory, Jesse F; de Crécy-Lagard, Valérie; Hanson, Andrew D

    2010-06-01

    Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes. PMID:20489182

  10. Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applicatons in agriculture

    SciTech Connect

    Dirk, Lynnette M.A.; Schmidt, Jack J.; Cai, Yiying; Barnes, Jonathan C.; Hanger, Katherine M.; Nayak, Nihar R.; Williams, Mark A.; Grossman, Robert B.; Houtz, Robert L.; Rodgers, David W.

    2008-07-28

    The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 {angstrom} (1 {angstrom}=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr{sup 178} as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr{sup 2} in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr{sup 178} to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr{sup 178} can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

  11. 1H, 13C and 15N NMR assignments of the E. coli peptide deformylase in complex with a natural inhibitor called actinonin.

    PubMed

    Larue, Valéry; Seijo, Bili; Tisne, Carine; Dardel, Frédéric

    2009-06-01

    In eubacteria, the formyl group of nascent polypeptides is removed by peptide deformylase protein (PDF). This is the reason why PDF has received special attention in the course of the search for new antibacterial agents. We observed by NMR that actinonin, a natural inhibitor, induced drastic changes in the HSQC spectrum of E. coli PDF. We report here the complete NMR chemical shift assignments of PDF resonances bound to actinonin. PMID:19636969

  12. Assessment of Folic Acid Supplementation in Pregnant Women by Estimation of Serum Levels of Tetrahydrofolic Acid, Dihydrofolate Reductase, and Homocysteine

    PubMed Central

    Saxena, Vartika; Mirza, Anissa Atif; Kumari, Ranjeeta; Sharma, Kapil; Bharadwaj, Jyoti

    2016-01-01

    Background. Status of folic acid use in pregnant women of the hilly regions in North India was little known. This study was carried out to assess the folic acid use and estimate folate metabolites in pregnant women of this region. Materials and Methods. This cross-sectional study is comprised of 76 pregnant women, whose folic acid supplementation was assessed by a questionnaire and serum levels of homocysteine, tetrahydrofolic acid (THFA), and dihydrofolate reductase (DHFR) were estimated using Enzyme Linked Immunoassays. Results. The study data revealed awareness of folic acid use during pregnancy was present in 46.1% and 23.7% were taking folic acid supplements. The study depicted that there was no statistically significant difference between serum levels of THFA and DHFR in pregnant women with and without folic acid supplements (p = 0.790). Hyperhomocysteinemia was present in 15.78% of the participants. Conclusion. Less awareness about folic acid supplementation and low use of folic acid by pregnant women were observed in this region. Sufficient dietary ingestion may suffice for the escalated requirements in pregnancy, but since this cannot be ensured, hence folic acid supplementation should be made as an integral part of education and reproductive health programs for its better metabolic use, growth, and development of fetus. PMID:27064332

  13. Polymorphisms in the methylene tetrahydrofolate reductase and methionine synthase reductase genes and their correlation with unexplained recurrent spontaneous abortion susceptibility.

    PubMed

    Zhu, L

    2015-01-01

    We aimed to explore the correlation between unexplained recurrent spontaneous abortion and polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes. A case control study was conducted in 118 patients with unexplained recurrent spontaneous abortion (abortion group) and 174 healthy women (control group). The genetic material was extracted from the oral mucosal epithelial cells obtained from all subjects. The samples were subjected to fluorescence quantitative PCR to detect the single nucleotide polymorphisms (SNPs) in the MTHFR (C677T and A1298C) and MTRR (A66G) gene loci. The distribution frequency (18/118, 15.3%) of the MTHFR 677TT genotype was significantly higher in the abortion group (χ2 = 11.006, P = 0.004) than in the control group (2/174, 1.1%); on the other hand, the distribution frequency of the MTHFR A1298C genotype did not significantly differ between the abortion and control groups (χ(2) = 0.441, P = 0.507). The distribution frequency of the MTRR A66G genotype was also significantly higher in the abortion group (14/118, 11.9%; χ(2) = 10.503, P = 0.005) than in the control group (8/174, 4.6%). The MTHFR C677T and MTRR A66G polymorphisms are significantly correlated with the occurrence of spontaneous abortion. PMID:26345779

  14. How water molecules affect the catalytic activity of hydrolases--a XANES study of the local structures of peptide deformylase.

    PubMed

    Cui, Peixin; Wang, Yu; Chu, Wangsheng; Guo, Xiaoyun; Yang, Feifei; Yu, Meijuan; Zhao, Haifeng; Dong, Yuhui; Xie, Yaning; Gong, Weimin; Wu, Ziyu

    2014-01-01

    Peptide deformylase (PDF) is a prokaryotic enzyme that catalyzes the deformylation of nascent peptides generated during protein synthesis and water molecules play a key role in these hydrolases. Using X-ray absorption near edge spectroscopy (XANES) and ab initio calculations we accurately probe the local atomic environment of the metal ion binding in the active site of PDF at different pH values and with different metal ions. This new approach is an effective way to monitor existing correlations among functions and structural changes. We show for the first time that the enzymatic activity depends on pH values and metal ions via the bond length of the nearest coordinating water (Wat1) to the metal ion. Combining experimental and theoretical data we may claim that PDF exhibits an enhanced enzymatic activity only when the distance of the Wat1 molecule with the metal ion falls in the limited range from 2.15 to 2.55 Å. PMID:25503313

  15. Synthesis, antibacterial activity, and biological evaluation of formyl hydroxyamino derivatives as novel potent peptide deformylase inhibitors against drug-resistant bacteria.

    PubMed

    Yang, Shouning; Shi, Wei; Xing, Dong; Zhao, Zheng; Lv, Fengping; Yang, Liping; Yang, Yushe; Hu, Wenhao

    2014-10-30

    Peptide deformylase (PDF) has been identified as a promising target for novel antibacterial agents. In this study, a series of novel formyl hydroxyamino derivatives were designed and synthesized as PDF inhibitors and their antibacterial activities were evaluated. Among the potent PDF inhibitors (1o, 1q, 1o', 1q', and 1x), in vivo studies showed that compound 1q possesses mild toxicity, a good pharmacokinetic profile and protective effects. The good in vivo efficacy and low toxicity suggest that this class of compounds has potential for development and use in future antibacterial drugs. PMID:25151577

  16. [Relationship between hyperhomocysteinemia and C677T polymorphism of methylene tetrahydrofolate reductase gene in a healthy Algerian population].

    PubMed

    Hambaba, L; Abdessemed, S; Yahia, M; Laroui, S; Rouabah, F

    2008-01-01

    Plasmatic homocysteine concentration depends mostly on 5,10 methylene tetrahydrofolate reductase (MTHFR) polymorphisms, a key enzyme in folate metabolism. The most common point mutation C677T is associated to cardiovascular and neurological pathologies; its ethnic repartition is quite heterogenic. In the present study, we proposed to describe the genotypic and allelic frequencies of C677T polymorphism and its influence on plasmatic homocysteine level in a healthy Algerian population. The investigation was turned on 100 apparently healthy voluntary subjects. Homocysteine concentration was determined using an immunoassay by fluorescence polarisation on IMx. Genotypes were determined by RT-PCR (Light cycle 480). Mean homocysteine concentration value was 14,69 +/- 7,30 micromol/L. 41% of people sample show a moderate hyperhomocysteinemia (>15 micromol/L). For the MTHFR C677T, estimated frequency of the allele T in the 100 people sample was about 35,5% with genotypic frequency of 6%. Plasmatic homocysteine is significantly higher in people carrying allele T: (CC vs CT: 11,8 +/- 2,97 micromol/L vs 15,47 +/- 6,74 micromol/L, p = 0,0004); (CC vs TT: 11,8 +/- 2,97 micromol/L vs 30,05 +/- 13,35 micromol/L, p = 0,01) and (CT vs TT: 15,47 +/- 6,74 micromol/L vs 30,05 +/- 13,35 micromol/L, p = 0,021). Our study shows an intermediate allelic frequency that joins the North-South world gradient and a high hyperhomocysteinemia prevalence. C677T polymorphism of MTHFR seems playing a predominant role in the moderate hyperhomocyteinemia. These two observations should be taken into consideration in the evaluation of morbid and/or lethal pathologies predisposition in the Algerian population. PMID:19091662

  17. Crystallization and preliminary X-ray crystallographic analysis of peptide deformylase (PDF) from Bacillus cereus in ligand-free and actinonin-bound forms

    SciTech Connect

    Park, Joon Kyu; Moon, Jin Ho; Kim, Jae-Hong; Kim, Eunice EunKyeong

    2005-01-01

    Peptide deformylase (PDF) from B. cereus has been overexpressed, purified and crystallized in ligand-free and actinonin-bound forms. Diffraction data have been collected from these crystals to 1.7 and 2.0 Å resolution, respectively. In bacteria, protein expression initiates with an N-formyl group and this needs to be removed in order to ensure proper bacterial growth. These formylation and deformylation processes are unique to eubacteria; therefore, inhibition of these would provide a novel antibacterial therapy. Deformylation is carried out by peptide deformylase (PDF). PDF from Bacillus cereus, one of the major pathogenic bacteria, was cloned into expression plasmid pET-28a (Novagen), overexpressed in Escherichia coli BL21 (DE3) and purified to high quality. Crystals have been obtained of both ligand-free PDF and PDF to which actinonin, a highly potent naturally occurring inhibitor, is bound. Both crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.72, b = 44.04, c = 85.19 Å and a = 41.31, b = 44.56, c = 84.47 Å, respectively. Diffraction data were collected to 1.7 Å resolution for the inhibitor-free crystals and to 2.0 Å resolution for the actinonin-bound crystals.

  18. Single-dose safety, tolerability, and pharmacokinetics of the antibiotic GSK1322322, a novel peptide deformylase inhibitor.

    PubMed

    Naderer, Odin J; Dumont, Etienne; Zhu, John; Kurtinecz, Milena; Jones, Lori S

    2013-05-01

    GSK1322322 is a potent inhibitor of peptide deformylase, an essential bacterial enzyme required for protein maturation. GSK1322322 is active against community-acquired skin and respiratory tract pathogens, including methicillin-resistant Staphylococcus aureus, multidrug-resistant Streptococcus pneumoniae, and atypical pathogens. This phase I, randomized, double-blind, placebo-controlled, 2-part, single-dose, dose escalation study (first time in humans) evaluated the safety, tolerability, and pharmacokinetics of GSK1322322 (powder-in-bottle formulation) in healthy volunteers. In part A, dose escalation included GSK1322322 doses of 100, 200, 400, 800, and 1,500 mg under fasting conditions and 800 mg administered with a high-fat meal. In part B, higher doses of GSK1322322 (2,000, 3,000, and 4,000 mg) were evaluated under fasting conditions. Of the 39 volunteers enrolled in the study, 29 and 10 volunteers were treated with GSK1322322 and placebo, respectively. Upon single-dose administration, GSK1322322 was absorbed rapidly, with median times to maximum plasma concentration (T(max)) ranging from 0.5 to 1.0 h. The maximum observed plasma concentration (C(max)) and exposure (area under the concentration-time curve [AUC]) of GSK1322322 were greater than dose proportional between 100 and 1,500 mg and less than dose proportional between 1,500 and 4,000 mg. Administration of the drug with a high-fat meal reduced the rate of absorption (reduced C(max) and delayed T(max)) without affecting the extent of absorption (no effect on AUC). GSK1322322 was generally well tolerated, with all adverse events being mild to moderate in intensity during both parts of the study. The most frequently reported adverse event was headache. Data from this study support further evaluation of GSK1322322. PMID:23403431

  19. Structure-activity relationship analysis of the peptide deformylase inhibitor 5-bromo-1H-indole-3-acetohydroxamic acid.

    PubMed

    Petit, Sylvain; Duroc, Yann; Larue, Valéry; Giglione, Carmela; Léon, Carole; Soulama, Coralie; Denis, Alexis; Dardel, Frédéric; Meinnel, Thierry; Artaud, Isabelle

    2009-02-01

    The lead compound 5-bromoindolyl-3-acetohydroxamic acid (10) was recently identified as a potent inhibitor of bacterial peptide deformylases (PDFs). The synthesis and associated activities of new variants were investigated at position 5 to optimize the fit at the S1' subsite and at position 1 to improve both potency and antibacterial activity. A morphomimetic series, termed "reverse-indole" was synthesized. The indole derivatives remain selective in vitro inhibitors of PDF2 over PDF1. Bromide is the best group at position 5 and cannot be replaced by bulkier substituents. In this series, an N-benzyl group at position 1 in 19 e improves the potency relative to 10. In the case of PDF1, and unlike PDF2, potency is increased as the alkyl chain becomes longer and more ramified. These data support the results of NMR footprinting experiments that were performed with (15)N-labeled Ni-PDF and the corresponding 3-acetic acid derivatives. Most of the compounds have antibacterial activities toward B. subtilis, but are inefficient toward E. coli owing to active removal by the major efflux pumps. Among the reverse-indole derivatives, 23 c, which is the exact mirror image of 19 e, shows strong potency in vitro against PDF2, but little against PDF1, although this compound displays significant antibacterial activity toward an efflux-minus mutant of E. coli. All the compounds were assessed with major pathogenic bacteria, but most of them are inefficient antibacterial agents. The reverse-indole compounds 23 a and 23 c have potency against S. pneumoniae that is similar to that of actinonin. PMID:19053131

  20. Potent sub-MIC effect of GSK1322322 and other peptide deformylase inhibitors on in vitro growth of Staphylococcus aureus.

    PubMed

    Butler, Deborah; Chen, Dongzhao; O'Dwyer, Karen; Lewandowski, Thomas; Aubart, Kelly; Zalacain, Magdalena

    2014-01-01

    Peptide deformylase (PDF), a clinically unexploited antibacterial target, plays an essential role in protein maturation. PDF inhibitors, therefore, represent a new antibiotic class with a unique mode of action that provides an alternative therapy for the treatment of infections caused by drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). GSK1322322 is a novel PDF inhibitor that is in phase II clinical development for the treatment of lower respiratory tract and skin infections. We have discovered that PDF inhibitors can prevent S. aureus in vitro growth for up to 6 h at concentrations 8- to 32-fold below their MICs. This phenomenon seems specific to PDF inhibitors, as none of the antimicrobial agents with alternative mechanisms of action tested show such a potent and widespread effect. It also appears limited to S. aureus, as PDF inhibitors do not show such an inhibition of growth at sub-MIC levels in Streptococcus pneumoniae or Haemophilus influenzae. Analysis of the effect of GSK1322322 on the early growth of 100 randomly selected S. aureus strains showed that concentrations equal to or below 1/8× MIC inhibited growth of 91% of the strains tested for 6 h, while the corresponding amount of moxifloxacin or linezolid only affected the growth of 1% and 6% of strains, respectively. Furthermore, the sub-MIC effect demonstrated by GSK1322322 appears more substantial on those strains at the higher end of the MIC spectrum. These effects may impact the clinical efficacy of GSK1322322 in serious infections caused by multidrug-resistant S. aureus. PMID:24165188

  1. Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor GSK1322322 in Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae.

    PubMed

    Min, Sharon; Ingraham, Karen; Huang, Jianzhong; McCloskey, Lynn; Rilling, Sarah; Windau, Anne; Pizzollo, Jason; Butler, Deborah; Aubart, Kelly; Miller, Linda A; Zalacain, Magdalena; Holmes, David J; O'Dwyer, Karen

    2015-08-01

    The continuous emergence of multidrug-resistant pathogenic bacteria is compromising the successful treatment of serious microbial infections. GSK1322322, a novel peptide deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated safety and efficacy in human proof-of-concept clinical studies. In vitro studies were performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major pathogens that cause respiratory tract and skin infections. Resistance to GSK1322322 occurred at high frequency through loss-of-function mutations in the formyl-methionyl transferase (FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4 strains) and via missense mutations in Streptococcus pneumoniae (6/21 strains), but the mutations were associated with severe in vitro and/or in vivo fitness costs. The overall FoR to GSK1322322 was very low in Haemophilus influenzae, with only one PDF mutant being identified in one of four strains. No target-based mutants were identified from S. pyogenes, and only one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S. pneumoniae, PDF mutants were isolated from only six of 21 strains tested; an additional 10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants characterized from those three organisms (35/37 mutants) carried mutations in residues at or in close proximity to one of three highly conserved motifs that are part of the active site of the PDF protein, with 30 of the 35 mutations occurring at position V71 (using the S. pneumoniae numbering system). PMID:26014938

  2. Plasma homocysteine levels, methylene tetrahydrofolate reductase A1298C gene polymorphism and risk of retinal vein thrombosis.

    PubMed

    Ghaznavi, Habib; Soheili, Zahra; Samiei, Shahram; Soltanpour, Mohammad Soleiman

    2016-09-01

    There are limited data regarding the role of methylene tetrahydrofolate reductase (MTHFR) A1298C polymorphism and hyperhomocysteinemia as risk factors for retinal vein thrombosis (RVT) in Iranians. This study aimed to examine a possible association between fasting plasma total homocysteine (tHcy) levels, MTHFR A1298C polymorphism and RVT development in Iranian patients. Our study population consisted of 73 patients with a diagnosis of RVT (52.7 ± 16.2 years) and 73 age and sex-matched healthy controls (49.1 ± 14.6 years). Genotyping for the MTHFR A1298Cpolymorphism was conducted by PCR-RFLP technique and plasma tHcy levels were measured by an enzyme immunoassay method. Fasting plasma tHcy levels were 20.29 ± 8.5 μmol/l in RVT patients and 10.9 ± 3.1 μmol/l in control subjects. The number of cases with abnormal tHcy values (hyperhomocysteinemia) was significantly higher in the RVT patients than control subjects (P = 0.0001). The prevalence of MTHFR 1298CC homozygote genotype was similar in RVT patients and controls (17.8 vs.15.1%, P = 0.45). There were no significant differences in genotype distribution of MTHFR A1298C polymorphism between males and females in both RVT patients and controls (P > 0.05). The frequency of the 1298C allele was 39.1 and 35.6% in patients and controls, respectively, and did not differ significantly between them (P = 0.23). Moreover, heterozygote and homozygote genotypes in the RVT patients had significantly higher abnormal tHcy values than corresponding genotypes in control subjects (P < 0.001). Our study demonstrated that hyperhomocysteinemia but not homozygosity for MTHFR A1298C polymorphism is a significant risk factor for RVT in the Iranian population. PMID:26650461

  3. Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis.

    PubMed

    Bao, Xiaofeng; Pachikara, Niseema D; Oey, Christopher B; Balakrishnan, Amit; Westblade, Lars F; Tan, Ming; Chase, Theodore; Nickels, Bryce E; Fan, Huizhou

    2011-09-01

    Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ⁶⁶-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability. PMID:21719536

  4. Internal jugular vein thrombosis due to heterozygote methylene tetrahydrofolate reductase (MTHFR) 1298C and Factor V G1691A mutations after a minor trauma

    PubMed Central

    Gumussoy, Murat; Arslan, Ilker B.; Cukurova, Ibrahim; Uluyol, Sinan

    2014-01-01

    Internal jugular vein thrombosis usually appears in central venous catheterization, distant malignancies, hypercoagulation, infections, or secondary to ovarian hyperstimulation syndrome. A 44-year-old female patient presented to us with sore throat, and pain and swelling on the right side of her neck. She had a history of simple neck trauma 10 days ago. Ultrasonography and computed tomography showed bilateral multiple lymphadenopathies and right internal jugular vein thrombosis. Patient was put on parenteral antibiotherapy and oral anticoagulant treatment. Genomic DNA tests for hypercoagulation revealed methylene tetrahydrofolate reductase 1298C heterozygote mutation and Factor V G1691A (Leiden) mutation. Patient has been under clinical control for 1 year and does not have any complaints. In this article, diagnosis, treatment, and the etiology of internal jugular vein thrombosis, which is a rare and potentially fatal condition, have been discussed through this case. PMID:25937730

  5. Comparative Genomics Guided Discovery of Two Missing Archaeal Enzyme Families Involved in the Biosynthesis of the Pterin Moiety of Tetrahydromethanopterin and Tetrahydrofolate

    PubMed Central

    2012-01-01

    C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H4-MPT) or tetrahydrofolate (H4-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2′,3′-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586–37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658–6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function. PMID:22931285

  6. Comparative genomics guided discovery of two missing archaeal enzyme families involved in the biosynthesis of the pterin moiety of tetrahydromethanopterin and tetrahydrofolate.

    PubMed

    de Crécy-Lagard, Valérie; Phillips, Gabriela; Grochowski, Laura L; El Yacoubi, Basma; Jenney, Francis; Adams, Michael W W; Murzin, Alexey G; White, Robert H

    2012-11-16

    C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H(4)-MPT) or tetrahydrofolate (H(4)-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2',3'-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586-37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658-6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function. PMID:22931285

  7. Expression of the tetrahydrofolate-dependent nitric oxide synthase from the green alga Ostreococcus tauri increases tolerance to abiotic stresses and influences stomatal development in Arabidopsis.

    PubMed

    Foresi, Noelia; Mayta, Martín L; Lodeyro, Anabella F; Scuffi, Denise; Correa-Aragunde, Natalia; García-Mata, Carlos; Casalongué, Claudia; Carrillo, Néstor; Lamattina, Lorenzo

    2015-06-01

    Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants. NO plays a crucial role in growth and development, from germination to senescence, and is also involved in plant responses to biotic and abiotic stresses. In animals, NO is synthesized by well-described nitric oxide synthase (NOS) enzymes. NOS activity has also been detected in higher plants, but no gene encoding an NOS protein, or the enzymes required for synthesis of tetrahydrobiopterin, an essential cofactor of mammalian NOS activity, have been identified so far. Recently, an NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) has been discovered and characterized. Arabidopsis thaliana plants were transformed with OtNOS under the control of the inducible short promoter fragment (SPF) of the sunflower (Helianthus annuus) Hahb-4 gene, which responds to abiotic stresses and abscisic acid. Transgenic plants expressing OtNOS accumulated higher NO concentrations compared with siblings transformed with the empty vector, and displayed enhanced salt, drought and oxidative stress tolerance. Moreover, transgenic OtNOS lines exhibited increased stomatal development compared with plants transformed with the empty vector. Both in vitro and in vivo experiments indicate that OtNOS, unlike mammalian NOS, efficiently uses tetrahydrofolate as a cofactor in Arabidopsis plants. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool to improve plant fitness under adverse growth conditions. PMID:25880454

  8. Correlation between the 677C>T polymorphism in the methylene tetrahydrofolate reductase gene and serum homocysteine levels in coronary heart disease.

    PubMed

    Chen, Y Y; Wang, B N; Yu, X P

    2016-01-01

    The aim of the current study was to explore the correlation between serum homocysteine (HCY) levels and the methylene tetrahydrofolate reductase (MTHFR) gene 677C/T polymorphism and coronary heart disease (CHD). We consecutively enrolled 208 patients with CHD confirmed by CTA or coronary angiography from our hospital. An additional 200 healthy volunteers were enrolled as the control group. Serum HCY levels, MTHFR C677T genotype, and other related indicators were evaluated for the two groups. Compared to those in the control group, the serum HCY levels in the CHD patients were significantly higher (P < 0.05). The proportion of individuals with the heterozygous MTHFR CT genotype and homozygous mutant TT genotype among CHD patients was significantly higher than that in the control group (P < 0.05). In the acute coronary syndrome (ACS) subgroup, the proportion of those with the CT and TT genotypes was significantly higher than that of the stable CHD subgroup (P < 0.05). In summary, serum HCY levels were elevated in CHD patients, and the frequency of the CT and TT genotypes were also significantly increased, especially among the ACS subgroup. Taken together, this suggests that serum HCY levels and MTHFR C677T genotypes are correlated with CHD. PMID:27051002

  9. Bacterial Peptide deformylase inhibition of cyano substituted biaryl analogs: Synthesis, in vitro biological evaluation, molecular docking study and in silico ADME prediction.

    PubMed

    Khan, Firoz A Kalam; Patil, Rajendra H; Shinde, Devanand B; Sangshetti, Jaiprakash N

    2016-08-15

    Herein, we report the synthesis and screening of cyano substituted biaryl analogs 5(a-m) as Peptide deformylase (PDF) enzyme inhibitors. The compounds 5a (IC50 value=13.16μM), 5d (IC50 value=15.66μM) and 5j (IC50 value=19.16μM) had shown good PDF inhibition activity. The compounds 5a (MIC range=11.00-15.83μg/mL), 5b (MIC range=23.75-28.50μg/mL) and 5j (MIC range=7.66-16.91μg/mL) had also shown potent antibacterial activity when compared with ciprofloxacin (MIC range=25-50μg/mL). Thus, the active derivatives were not only potent PDF inhibitors but also efficient antibacterial agents. In order to gain more insight on the binding mode of the compounds with PDF, the synthesized compounds 5(a-m) were docked against PDF enzyme of Escherichia coli and compounds exhibited good binding properties. In silico ADME properties of synthesized compounds were also analyzed and showed potential to develop as good oral drug candidates. PMID:27269198

  10. Metalloprotease inhibitors GM6001 and TAPI-0 inhibit the obligate intracellular human pathogen Chlamydia trachomatis by targeting peptide deformylase of the bacterium.

    PubMed

    Balakrishnan, Amit; Patel, Bhairavi; Sieber, Stephan A; Chen, Ding; Pachikara, Niseema; Zhong, Guangming; Cravatt, Benjamin F; Fan, Huizhou

    2006-06-16

    Chlamydia trachomatis is an obligate intracellular bacterium responsible for a number of human diseases. The mechanism underlying the intracellular parasitology of Chlamydiae remains poorly understood. In searching for host factors required for chlamydial infection, we discovered that C. trachomatis growth was effectively inhibited with GM6001 and TAPI-0, two compounds known as specific inhibitors of matrix metalloproteases. The inhibition was independent of chlamydial entry of the cell, suggesting that the loss of extracellular metalloprotease activities of the host cell is unlikely to be the mechanism for the growth suppression. Nucleotide sequences of candidate metalloprotease genes remained unchanged in a chlamydial variant designated GR10, which had been selected for resistance to the inhibitors. Nevertheless, GR10 displayed a single base mutation in the presumable promoter region of the gene for peptide deformylase (PDF), a metal-dependent enzyme that removes the N-formyl group from newly synthesized bacterial proteins. The mutation correlated with an increased PDF expression level and resistance to actinonin, a known PDF inhibitor with antibacterial activity, as compared with the parental strain. Recombinant chlamydial PDF was covalently labeled with a hydroxamate-based molecular probe designated AspR1, which was developed for the detection of metalloproteases. The AspR1 labeling of the chlamydial PDF became significantly less efficient in the presence of excessive amounts of GM6001 and TAPI-0. Finally, the PDF enzyme activity was efficiently inhibited with GM6001 and TAPI-0. Taken together, our results suggest that the metalloprotease inhibitors suppress chlamydial growth by targeting the bacterial PDF. These findings have important biochemical and medical implications. PMID:16565079

  11. The crystal structure of mitochondrial (Type 1A) peptide deformylase provides clear guidelines for the design of inhibitors specific for the bacterial forms.

    PubMed

    Fieulaine, Sonia; Juillan-Binard, Céline; Serero, Alexandre; Dardel, Frédéric; Giglione, Carmela; Meinnel, Thierry; Ferrer, Jean-Luc

    2005-12-23

    Peptide deformylase (PDF) inhibitors have a strong potential to be used as a new class of antibiotics. However, recent studies have shown that the mitochondria of most eukaryotes, including humans, contain an essential PDF, PDF1A. The crystal structure of the Arabidopsis thaliana PDF1A (AtPDF1A), considered representative of PDF1As in general, has been determined. This structure displays several similarities to that of known bacterial PDFs. AtPDF1A behaves as a dimer, with the C-terminal residues responsible for linking the two subunits. This arrangement is similar to that of Leptospira interrogans PDF, the only other dimeric PDF identified to date. AtPDF1A is the first PDF for which zinc has been identified as the catalytic ion. However, the zinc binding pocket does not differ from the binding pockets of PDFs with iron rather than zinc. The crystal structure of AtPDF1A in complex with a substrate analog revealed that the substrate binding pocket of PDF1A displays strong modifications. The S1' binding pocket is significantly narrower, due to the creation of a floor from residues present in all PDF1As but not in bacterial PDFs. A true S3' pocket is created by the residues of a helical CD-loop, which is very long in PDF1As. Finally, these modified substrate binding pockets modify the position of the substrate in the active site. These differences provide guidelines for the design of bacterial PDF inhibitors that will not target mitochondrial PDFs. PMID:16192279

  12. Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart.

    PubMed

    Fieulaine, Sonia; Desmadril, Michel; Meinnel, Thierry; Giglione, Carmela

    2014-02-01

    Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of the N-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeable in vivo and display similar properties in vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate. PMID:24531459

  13. Pharmacokinetics/Pharmacodynamics of Peptide Deformylase Inhibitor GSK1322322 against Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in Rodent Models of Infection.

    PubMed

    Hoover, Jennifer; Lewandowski, Thomas; Straub, Robert J; Novick, Steven J; DeMarsh, Peter; Aubart, Kelly; Rittenhouse, Stephen; Zalacain, Magdalena

    2016-01-01

    GSK1322322 is a novel inhibitor of peptide deformylase (PDF) with good in vitro activity against bacteria associated with community-acquired pneumonia and skin infections. We have characterized the in vivo pharmacodynamics (PD) of GSK1322322 in immunocompetent animal models of infection with Streptococcus pneumoniae and Haemophilus influenzae (mouse lung model) and with Staphylococcus aureus (rat abscess model) and determined the pharmacokinetic (PK)/PD index that best correlates with efficacy and its magnitude. Oral PK studies with both models showed slightly higher-than-dose-proportional exposure, with 3-fold increases in area under the concentration-time curve (AUC) with doubling doses. GSK1322322 exhibited dose-dependent in vivo efficacy against multiple isolates of S. pneumoniae, H. influenzae, and S. aureus. Dose fractionation studies with two S. pneumoniae and S. aureus isolates showed that therapeutic outcome correlated best with the free AUC/MIC (fAUC/MIC) index in S. pneumoniae (R(2), 0.83), whereas fAUC/MIC and free maximum drug concentration (fCmax)/MIC were the best efficacy predictors for S. aureus (R(2), 0.9 and 0.91, respectively). Median daily fAUC/MIC values required for stasis and for a 1-log10 reduction in bacterial burden were 8.1 and 14.4 for 11 S. pneumoniae isolates (R(2), 0.62) and 7.2 and 13.0 for five H. influenzae isolates (R(2), 0.93). The data showed that for eight S. aureus isolates, fAUC correlated better with efficacy than fAUC/MIC (R(2), 0.91 and 0.76, respectively), as efficacious AUCs were similar for all isolates, independent of their GSK1322322 MIC (range, 0.5 to 4 μg/ml). Median fAUCs of 2.1 and 6.3 μg · h/ml were associated with stasis and 1-log10 reductions, respectively, for S. aureus. PMID:26482300

  14. Pharmacokinetics/Pharmacodynamics of Peptide Deformylase Inhibitor GSK1322322 against Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in Rodent Models of Infection

    PubMed Central

    Lewandowski, Thomas; Straub, Robert J.; Novick, Steven J.; DeMarsh, Peter; Aubart, Kelly; Rittenhouse, Stephen; Zalacain, Magdalena

    2015-01-01

    GSK1322322 is a novel inhibitor of peptide deformylase (PDF) with good in vitro activity against bacteria associated with community-acquired pneumonia and skin infections. We have characterized the in vivo pharmacodynamics (PD) of GSK1322322 in immunocompetent animal models of infection with Streptococcus pneumoniae and Haemophilus influenzae (mouse lung model) and with Staphylococcus aureus (rat abscess model) and determined the pharmacokinetic (PK)/PD index that best correlates with efficacy and its magnitude. Oral PK studies with both models showed slightly higher-than-dose-proportional exposure, with 3-fold increases in area under the concentration-time curve (AUC) with doubling doses. GSK1322322 exhibited dose-dependent in vivo efficacy against multiple isolates of S. pneumoniae, H. influenzae, and S. aureus. Dose fractionation studies with two S. pneumoniae and S. aureus isolates showed that therapeutic outcome correlated best with the free AUC/MIC (fAUC/MIC) index in S. pneumoniae (R2, 0.83), whereas fAUC/MIC and free maximum drug concentration (fCmax)/MIC were the best efficacy predictors for S. aureus (R2, 0.9 and 0.91, respectively). Median daily fAUC/MIC values required for stasis and for a 1-log10 reduction in bacterial burden were 8.1 and 14.4 for 11 S. pneumoniae isolates (R2, 0.62) and 7.2 and 13.0 for five H. influenzae isolates (R2, 0.93). The data showed that for eight S. aureus isolates, fAUC correlated better with efficacy than fAUC/MIC (R2, 0.91 and 0.76, respectively), as efficacious AUCs were similar for all isolates, independent of their GSK1322322 MIC (range, 0.5 to 4 μg/ml). Median fAUCs of 2.1 and 6.3 μg · h/ml were associated with stasis and 1-log10 reductions, respectively, for S. aureus. PMID:26482300

  15. Cellular pharmacokinetics and intracellular activity of the novel peptide deformylase inhibitor GSK1322322 against Staphylococcus aureus laboratory and clinical strains with various resistance phenotypes: studies with human THP-1 monocytes and J774 murine macrophages.

    PubMed

    Peyrusson, Frédéric; Butler, Deborah; Tulkens, Paul M; Van Bambeke, Françoise

    2015-09-01

    GSK1322322 is a peptide deformylase inhibitor active against Staphylococcus aureus strains resistant to currently marketed antibiotics. Our aim was to assess the activity of GSK1322322 against intracellular S. aureus using an in vitro pharmacodynamic model and, in parallel, to examine its cellular pharmacokinetics and intracellular disposition. For intracellular activity analysis, we used an established model of human THP-1 monocytes and tested one fully susceptible S. aureus strain (ATCC 25923) and 8 clinical strains with resistance to oxacillin, vancomycin, daptomycin, macrolides, clindamycin, linezolid, or moxifloxacin. Uptake, accumulation, release, and subcellular distribution (cell fractionation) of [(14)C]GSK1322322 were examined in uninfected murine J774 macrophages and uninfected and infected THP-1 monocytes. GSK1322322 demonstrated a uniform activity against the intracellular forms of all S. aureus strains tested, disregarding their resistance phenotypes, with a maximal relative efficacy (E max) of a 0.5 to 1 log10 CFU decrease compared to the original inoculum within 24 h and a static concentration (C s) close to its MIC in broth. Influx and efflux were very fast (<5 min to equilibrium), and accumulation was about 4-fold, with no or a minimal effect of the broad-spectrum eukaryotic efflux transporter inhibitors gemfibrozil and verapamil. GSK1322322 was recovered in the cell-soluble fraction and was dissociated from the main subcellular organelles and from bacteria (in infected cells). The results of this study show that GSK1322322, as a typical novel deformylase inhibitor, may act against intracellular forms of S. aureus. They also suggest that GSK1322322 has the ability to freely diffuse into and out of eukaryotic cells as well as within subcellular compartments. PMID:26169402

  16. Cellular Pharmacokinetics and Intracellular Activity of the Novel Peptide Deformylase Inhibitor GSK1322322 against Staphylococcus aureus Laboratory and Clinical Strains with Various Resistance Phenotypes: Studies with Human THP-1 Monocytes and J774 Murine Macrophages

    PubMed Central

    Peyrusson, Frédéric; Butler, Deborah; Tulkens, Paul M.

    2015-01-01

    GSK1322322 is a peptide deformylase inhibitor active against Staphylococcus aureus strains resistant to currently marketed antibiotics. Our aim was to assess the activity of GSK1322322 against intracellular S. aureus using an in vitro pharmacodynamic model and, in parallel, to examine its cellular pharmacokinetics and intracellular disposition. For intracellular activity analysis, we used an established model of human THP-1 monocytes and tested one fully susceptible S. aureus strain (ATCC 25923) and 8 clinical strains with resistance to oxacillin, vancomycin, daptomycin, macrolides, clindamycin, linezolid, or moxifloxacin. Uptake, accumulation, release, and subcellular distribution (cell fractionation) of [14C]GSK1322322 were examined in uninfected murine J774 macrophages and uninfected and infected THP-1 monocytes. GSK1322322 demonstrated a uniform activity against the intracellular forms of all S. aureus strains tested, disregarding their resistance phenotypes, with a maximal relative efficacy (Emax) of a 0.5 to 1 log10 CFU decrease compared to the original inoculum within 24 h and a static concentration (Cs) close to its MIC in broth. Influx and efflux were very fast (<5 min to equilibrium), and accumulation was about 4-fold, with no or a minimal effect of the broad-spectrum eukaryotic efflux transporter inhibitors gemfibrozil and verapamil. GSK1322322 was recovered in the cell-soluble fraction and was dissociated from the main subcellular organelles and from bacteria (in infected cells). The results of this study show that GSK1322322, as a typical novel deformylase inhibitor, may act against intracellular forms of S. aureus. They also suggest that GSK1322322 has the ability to freely diffuse into and out of eukaryotic cells as well as within subcellular compartments. PMID:26169402

  17. 5-methyl-tetrahydrofolate and the S-adenosylmethionine cycle in C57BL/6J mouse tissues: gender differences and effects of arylamine N-acetyltransferase-1 deletion.

    PubMed

    Witham, Katey L; Butcher, Neville J; Sugamori, Kim S; Brenneman, Debbie; Grant, Denis M; Minchin, Rodney F

    2013-01-01

    Folate catabolism involves cleavage of the C(9)-N(10) bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions. PMID:24205029

  18. 5-Methyl-Tetrahydrofolate and the S-Adenosylmethionine Cycle in C57BL/6J Mouse Tissues: Gender Differences and Effects of Arylamine N-Acetyltransferase-1 Deletion

    PubMed Central

    Witham, Katey L.; Butcher, Neville J.; Sugamori, Kim S.; Brenneman, Debbie; Grant, Denis M.; Minchin, Rodney F.

    2013-01-01

    Folate catabolism involves cleavage of the C9-N10 bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions. PMID:24205029

  19. Pharmacogenetic evaluation of ABCB1, Cyp2C9, Cyp2C19 and methylene tetrahydrofolate reductase polymorphisms in teratogenicity of anti-epileptic drugs in women with epilepsy

    PubMed Central

    Jose, Manna; Banerjee, Moinak; Mathew, Anila; Bharadwaj, Tashi; Vijayan, Neetha; Thomas, Sanjeev V.

    2014-01-01

    Aim: Pregnancy in women with epilepsy (WWE) who are on anti-epileptic drugs (AEDs) has two- to three-fold increased risk of fetal malformations. AEDs are mostly metabolized by Cyp2C9, Cyp2C19 and Cyp3A4 and transported by ABCB1. Patients on AED therapy can have folate deficiency. We hypothesize that the polymorphisms in ABCB1, Cyp2C9, Cyp2C19 and methylene tetrahydrofolate reductase (MTHFR) might result in differential expression resulting in differential drug transport, drug metabolism and folate metabolism, which in turn may contribute to the teratogenic impact of AEDs. Materials and Methods: The ABCB1, Cyp2C9, Cyp2C19 and MTHFR polymorphisms were genotyped for their role in teratogenic potential and the nature of teratogenecity in response to AED treatment in WWE. The allelic, genotypic associations were tested in 266 WWE comprising of 143 WWE who had given birth to babies with WWE-malformation (WWE-M) and 123 WWE who had normal offsprings (WWE-N). Results: In WWE-M, CC genotype of Ex07 + 139C/T was overrepresented (P = 0.0032) whereas the poor metabolizer allele *2 and *2 *2 genotype of CYP2C219 was significantly higher in comparison to WWE-N group (P = 0.007 and P = 0.005, respectively). All these observations were independent of the nature of malformation (cardiac vs. non cardiac malformations). Conclusion: Our study indicates the possibility that ABCB1 and Cyp2C19 may play a pivotal role in the AED induced teratogenesis, which is independent of nature of malformation. This is one of the first reports indicating the pharmacogenetic role of Cyp2C19 and ABCB1 in teratogenesis of AED in pregnant WWE. PMID:25221392

  20. Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation.

    PubMed

    Chaudhari, Snehal N; Mukherjee, Madhumati; Vagasi, Alexandra S; Bi, Gaofeng; Rahman, Mohammad M; Nguyen, Christine Q; Paul, Ligi; Selhub, Jacob; Kipreos, Edward T

    2016-07-11

    Here we describe an in vitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation. PMID:27404357

  1. Quantitative flux analysis reveals folate-dependent NADPH production

    NASA Astrophysics Data System (ADS)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

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

  2. 10-Formyl-2,4,6,8,12-penta­nitro-2,4,6,8,10,12-hexa­azatetra­cyclo­[5.5.0.03,11.05,9]dodeca­ne

    PubMed Central

    Jin, Shaohua; Chen, Shusen; Chen, Huaxiong; Li, Lijie; Shi, Yanshan

    2009-01-01

    The title compound, C7H7N11O11 (PNMFIW), is a caged heterocycle substituted with five nitro and one formyl groups. It is related to the hexa­azaisowurtzitane family of high-density high-energy polycyclic cage compounds. Four nitro groups are appended to the four N atoms of the two five-membered rings, while a nitro group and a formyl are attached to the two N atoms of the six-membered ring. PMID:21578838

  3. Division of labour: how does folate metabolism partition between one-carbon metabolism and amino acid oxidation?

    PubMed

    Brosnan, Margaret E; MacMillan, Luke; Stevens, Jennifer R; Brosnan, John T

    2015-12-01

    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. PMID:26567272

  4. Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli.

    PubMed

    Nagy, P L; Marolewski, A; Benkovic, S J; Zalkin, H

    1995-03-01

    The enzyme encoded by Escherichia coli purU has been overproduced, purified, and characterized. The enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate (formyl-FH4) to FH4 and formate. Formyl-FH4 hydrolase thus generates the formate that is used by purT-encoded 5'-phosphoribosylglycinamide transformylase for step three of de novo purine nucleotide synthesis. Formyl-FH4 hydrolase, a hexamer with 32-kDa subunits, is activated by methionine and inhibited by glycine. Heterotropic cooperativity is observed for activation by methionine in the presence of glycine and for inhibition by glycine in the presence of methionine. These results, along with previous mutant analyses, lead to the conclusion formyl-FH4 hydrolase is a regulatory enzyme whose main function is to balance the pools of FH4 and C1-FH4 in response to changing growth conditions. The enzyme uses methionine and glycine to sense the pools of C1-FH4 and FH4, respectively. PMID:7868604

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

    PubMed

    Ducker, Gregory S; Chen, Li; Morscher, Raphael J; Ghergurovich, Jonathan M; Esposito, Mark; Teng, Xin; Kang, Yibin; Rabinowitz, Joshua D

    2016-06-14

    One-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here, we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-tetrahydrofolate (10-formyl-THF), and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis, with the mitochondrial pathway required in nutrient-poor conditions. PMID:27211901

  6. Role of Hyperhomocysteinemia and Methylene Tetrahydrofolate Reductase C677T Polymorphism in Idiopathic Portal Vein Thrombosis

    PubMed Central

    Ghaznavi, Habib; Soheili, Zahra; Samiei, Shahram; Soltanpour, Mohammad Soleiman

    2016-01-01

    Purpose: Portal vein thrombosis (PVT) is a rare and life-threatening vascular disorder characterized by obstruction or narrowing of the portal vein. Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has been studied in PVT patients with conflicting results. In the present study the association of hyperhomocysteinemia and MTHFR C677T polymorphism with PVT risk was investigated in Iranians. Materials and Methods: Our study population consisted of 10 idiopathic PVT patients and 80 healthy control subjects matched for age and sex. MTHFR C677T polymorphism was genotyped by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) technique and plasma total homocysteine (tHcy) levels were determined by enzyme immunoassay method. Results: Mean plasma tHcy levels were significantly higher in PVT patients (20.2±6.8) than control subjects (10.9±4.7) (P=0.001). Moreover, plasma tHcy levels were significantly higher in 677T allele carriers relative to 677C allele carriers in both PVT patients (P=0.01) and control subjects (P=0.03). Neither homozygote nor heterozygote genotypes of MTHFR C677T polymorphism correlated significantly with PVT risk (P>0.05). Moreover, MTHFR C677T polymorphism didn’t increase the risk of PVT under dominant (CT+TT vs. CC) or recessive (TT vs. CC+CT) genetic models analyzed (P>0.05). The difference in frequency of minor 677T allele between PVT patients and control subjects was not statistically significant (P>0.05). Conclusion: Based on the current study, we suggest that hyperhomocysteinemia constitutes a significant and common risk factor for PVT. Also, MTHFR C677T polymorphism is not a risk factor for PVT but is a contributing factor for elevated plasma tHcy levels. PMID:27051654

  7. Association of Methylene Tetrahydrofolate Reductase Polymorphism with BMD and Homocysteine in Premenopausal North Indian Women

    PubMed Central

    Pandey, Sanjeev Kumar; Singh, Ankur; Polipalli, Sunil Kumar; Gupta, Sangeeta; Kapoor, Seema

    2013-01-01

    Background and Aim: Osteoporosis (OP) is a common nutrigenomic disease associated with various genetic components. Observational studies have indicated that mildly elevated homocysteine was a strong risk factor for osteoporotic fractures. Yet there is no clear biologic mechanism for an effect of homocysteine on bone.The aim of this study was to investigate the association of MTHFR C677T and A1298C polymorphisms, and to verify the association of these polymorphisms with bone mineral density and homocysteine in premenopausal women of northern India. Material and Methods: We included 402 north Indian patients with altered BMD, both Osteopenic (OPN) and Osteoporosis, and normal controls. Genotype identification for MTHFR C677T and A1298C polymorphisms were analyzed by PCR-RFLP method, correlated with Bone Mineral Density (BMD), Homocysteine (Hcy), Folate and Vitamin B12. Results: The study groups did not differ in terms of age, weight and body mass indices. Prevalence of Genotype frequencies (GFs) for MTHFRC677T OP were (n: 402): CC 361 (89.8%), CT 25 (6.22%), TT 16 (3.98%) and that for MTHFR A1298C were (n: 402) AA 353(87.81%), AC 29(7.21%), CC 20(4.98%). Folate was significantly lower in the OP group than those in both the other groups, while there was no significant difference in Hcy in the OP group relative to OPN, as compared to controls. Conclusion: The GFs for MTHFR C677T and A1298C polymorphisms were not different between both groups. In conclusion, polymorphism of the MTHFR 677T is associated with small differences in BMD with folate levels. Further, more investigations should be done in larger studies for other epigenetic pathways, that may increase the risk of Osteoporosis. PMID:24551672

  8. Synthesis and in vitro antibacterial activity of oxazolidine LBM-415 analogs as peptide deformylase inhibitors.

    PubMed

    Yu, Linliang; Zhou, Weicheng; Wang, Zhenyu

    2011-03-01

    The drug resistant bacteria pose a severe threat to human health. The increasing resistance of those pathogens to traditional antibacterial therapy renders the identification of new antibacterial agents with novel antibacterial mechanisms an urgent need. In this study, a series of (2S)-N-substituted-1-[(formyhydroxyamino)methyl]-1-oxohexyl]-2-oxazolidinecarboxamides were designed, synthesized and evaluated for in vitro antibacterial activity. Most of these compounds displayed good activities against Gram-positive organisms comparable to reference agent LBM-415. PMID:21288715

  9. Acylprolinamides: a new class of peptide deformylase inhibitors with in vivo antibacterial activity.

    PubMed

    Axten, Jeffrey M; Medina, Jesús R; Blackledge, Charles W; Duquenne, Céline; Grant, Seth W; Bobko, Mark A; Peng, Tony; Miller, William H; Pinckney, Theresa; Gallagher, Timothy F; Kulkarni, Swarupa; Lewandowski, Thomas; Van Aller, Glenn S; Zonis, Rimma; Ward, Paris; Campobasso, Nino

    2012-06-15

    A new class of PDF inhibitor with potent, broad spectrum antibacterial activity is described. Optimization of blood stability and potency provided compounds with improved pharmacokinetics that were suitable for in vivo experiments. Compound 5c, which has robust antibacterial activity, demonstrated efficacy in two respiratory tract infection models. PMID:22579486

  10. Cyanobacterial aldehyde deformylase oxygenation of aldehydes yields n-1 aldehydes and alcohols in addition to alkanes

    PubMed Central

    Aukema, Kelly G.; Makris, Thomas M.; Stoian, Sebastian A.; Richman, Jack E.; Münck, Eckard; Lipscomb, John D.; Wackett, Lawrence P.

    2013-01-01

    Aldehyde-deformylating oxygenase (ADO) catalyzes O2-dependent release of the terminal carbon of a biological substrate, octadecanal, to yield formate and heptadecane in a reaction that requires external reducing equivalents. We show here that ADO also catalyzes incorporation of an oxygen atom from O2 into the alkane product to yield alcohol and aldehyde products. Oxygenation of the alkane product is much more pronounced with C9-10 aldehyde substrates, so that use of nonanal as the substrate yields similar amounts of octane, octanal, and octanol products. When using doubly-labeled [1,2-13C]-octanal as the substrate, the heptane, heptanal and heptanol products each contained a single 13C-label in the C-1 carbons atoms. The only one-carbon product identified was formate. [18O]-O2 incorporation studies demonstrated formation of [18O]-alcohol product, but rapid solvent exchange prevented similar determination for the aldehyde product. Addition of [1-13C]-nonanol with decanal as the substrate at the outset of the reaction resulted in formation of [1-13C]-nonanal. No 13C-product was formed in the absence of decanal. ADO contains an oxygen-bridged dinuclear iron cluster. The observation of alcohol and aldehyde products derived from the initially formed alkane product suggests a reactive species similar to that formed by methane monooxygenase (MMO) and other members of the bacterial multicomponent monooxygenase family. Accordingly, characterization by EPR and Mössbauer spectroscopies shows that the electronic structure of the ADO cluster is similar, but not identical, to that of MMO hydroxylase component. In particular, the two irons of ADO reside in nearly identical environments in both the oxidized and fully reduced states, whereas those of MMOH show distinct differences. These favorable characteristics of the iron sites allow a comprehensive determination of the spin Hamiltonian parameters describing the electronic state of the diferrous cluster for the first time for any biological system. The nature of the diiron cluster and the newly recognized products from ADO catalysis hold implications for the mechanism of C-C bond cleavage. PMID:24490119

  11. Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice.

    PubMed

    Momb, Jessica; Lewandowski, Jordan P; Bryant, Joshua D; Fitch, Rebecca; Surman, Deborah R; Vokes, Steven A; Appling, Dean R

    2013-01-01

    Maternal supplementation with folic acid is known to reduce the incidence of neural tube defects (NTDs) by as much as 70%. Despite the strong clinical link between folate and NTDs, the biochemical mechanisms through which folic acid acts during neural tube development remain undefined. The Mthfd1l gene encodes a mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase, termed MTHFD1L. This gene is expressed in adults and at all stages of mammalian embryogenesis with localized regions of higher expression along the neural tube, developing brain, craniofacial structures, limb buds, and tail bud. In both embryos and adults, MTHFD1L catalyzes the last step in the flow of one-carbon units from mitochondria to cytoplasm, producing formate from 10-formyl-THF. To investigate the role of mitochondrial formate production during embryonic development, we have analyzed Mthfd1l knockout mice. All embryos lacking Mthfd1l exhibit aberrant neural tube closure including craniorachischisis and exencephaly and/or a wavy neural tube. This fully penetrant folate-pathway mouse model does not require feeding a folate-deficient diet to cause this phenotype. Maternal supplementation with sodium formate decreases the incidence of NTDs and partially rescues the growth defect in embryos lacking Mthfd1l. These results reveal the critical role of mitochondrially derived formate in mammalian development, providing a mechanistic link between folic acid and NTDs. In light of previous studies linking a common splice variant in the human MTHFD1L gene with increased risk for NTDs, this mouse model provides a powerful system to help elucidate the specific metabolic mechanisms that underlie folate-associated birth defects, including NTDs. PMID:23267094

  12. Natural folates from biofortified tomato and synthetic 5-methyl-tetrahydrofolate display equivalent bioavailability in a murine model.

    PubMed

    Castorena-Torres, Fabiola; Ramos-Parra, Perla A; Hernández-Méndez, Rogelio V; Vargas-García, Andrés; García-Rivas, Gerardo; de la Garza, Rocío I Díaz

    2014-03-01

    Folate deficiency is a global health problem related to neural tube defects, cardiovascular disease, dementia, and cancer. Considering that folic acid (FA) supply through industrialized foods is the most successful intervention, limitations exist for its complete implementation worldwide. Biofortification of plant foods, on the other hand, could be implemented in poor areas as a complementary alternative. A biofortified tomato fruit that accumulates high levels of folates was previously developed. In this study, we evaluated short-term folate bioavailability in rats infused with this folate-biofortified fruit. Fruit from tomato segregants hyperaccumulated folates during an extended ripening period, ultimately containing 3.7-fold the recommended dietary allowance in a 100-g portion. Folate-depleted Wistar rats separated in three groups received a single dose of 1 nmol of folate/g body weight in the form of lyophilized biofortified tomato fruit, FA, or synthetic 5-CH3-THF. Folate bioavailability from the biofortified tomato was comparable to that of synthetic 5-CH3-THF, with areas under the curve (AUC(0-∞)) of 2,080 ± 420 and 2,700 ± 220 pmol · h/mL, respectively (P = 0.12). Whereas, FA was less bioavailable with an AUC(0-∞) of 750 ± 10 pmol · h/mL. Fruit-supplemented animals reached maximum levels of circulating folate in plasma at 2 h after administration with a subsequent steady decline, while animals treated with FA and synthetic 5-CH3-THF reached maximum levels at 1 h. Pharmacokinetic parameters revealed that biofortified tomato had slower intestinal absorption than synthetic folate forms. This is the first study that demonstrates the bioavailability of folates from a biofortified plant food, showing its potential to improve folate deficiency. PMID:24445671

  13. An Ancient Riboswitch Class in Bacteria Regulates Purine Biosynthesis and One-carbon Metabolism

    PubMed Central

    Kim, Peter B.; Nelson, James W.; Breaker, Ronald R.

    2015-01-01

    SUMMARY Over thirty years ago, ZTP (5-amino-4-imidazole carboxamide riboside 5'-triphosphate), a modified purine biosynthetic intermediate, was proposed to signal 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria. However, the mechanisms by which this putative alarmone or its precursor ZMP (5-aminoimidazole-4-carboxamide ribonucleotide, also known as AICAR) brings about any metabolic changes remain unexplained. Herein we report the existence of a widespread riboswitch class that is most commonly associated with genes related to de novo purine biosynthesis and one carbon metabolism. Biochemical data confirms that members of this riboswitch class selectively bind ZMP and ZTP with nanomolar affinity, while strongly rejecting numerous natural analogs. Indeed, increases in the ZMP/ZTP pool, caused by folate stress in bacterial cells, trigger changes in the expression of a reporter gene fused to representative ZTP riboswitches in vivo. The wide distribution of this riboswitch class suggests that ZMP/ZTP signaling is important for species in numerous bacterial lineages. PMID:25616067

  14. The toxicity of methanol

    SciTech Connect

    Tephly, T.R. )

    1991-01-01

    Methanol toxicity in humans and monkeys is characterized by a latent period of many hours followed by a metabolic acidosis and ocular toxicity. This is not observed in most lower animals. The metabolic acidosis and blindness is apparently due to formic acid accumulation in humans and monkeys, a feature not seen in lower animals. The accumulation of formate is due to a deficiency in formate metabolism which is, in turn, related, in part, to low hepatic tetrahydrofolate (H{sub 4}folate). An excellent correlation between hepatic H{sub 4} folate and formate oxidation rates has been shown within and across species. Thus, humans and monkeys possess low hepatic H{sub 4}folate levels, low rates of formate oxidation and accumulation of formate after methanol. Formate, itself, produces blindness in monkeys in the absence of metabolic acidosis. In addition to low hepatic H{sub 4}folate concentrations, monkeys and humans also have low hepatic 10-formyl H{sub 4}folate dehydrogenase levels, the enzyme which is the ultimate catalyst for conversion of formate to carbon dioxide. This review presents the basis for the role of folic acid-dependent reactions in the regulation of methanol toxicity.

  15. Metal Ion-Mediated Nucleobase Recognition by the ZTP Riboswitch.

    PubMed

    Trausch, Jeremiah J; Marcano-Velázquez, Joan G; Matyjasik, Michal M; Batey, Robert T

    2015-07-23

    The ZTP riboswitch is a widespread family of regulatory RNAs that upregulate de novo purine synthesis in response to increased intracellular levels of ZTP or ZMP. As an important intermediate in purine biosynthesis, ZMP also serves as a proxy for the concentration of N10-formyl-tetrahydrofolate, a key component of one-carbon metabolism. Here, we report the structure of the ZTP riboswitch bound to ZMP at a resolution of 1.80 Å. The RNA contains two subdomains brought together through a long-range pseudoknot further stabilized through helix-helix packing. ZMP is bound at the subdomain interface of the RNA through a set of interactions with the base, ribose sugar, and phosphate moieties of the ligand. Unique to nucleobase recognition by RNAs, the Z base is inner-sphere coordinated to a magnesium cation bound by two backbone phosphates. This interaction, along with steric hindrance by the backbone, imparts specificity over chemically similar compounds such as ATP/AMP. PMID:26144884

  16. 5,10-methylene tetrahydrofolate reductase C677T gene polymorphism, homocysteine concentration and the extent of premature coronary artery disease in southern Iran.

    PubMed

    Senemar, Sara; Saffari, Babak; Sharifkazemi, Mohammad Bagher; Bahari, Marzieh; Jooyan, Najmeh; Dehaghani, Elham Davoudi; Yavarian, Majid

    2013-01-01

    Elevated level of plasma homocysteine (Hcy) has been identified as an independent risk factor for coronary artery disease (CAD). Furthermore, numerous studies have documented the influences of a common polymorphism (C677T) of methylenetetrahydrofolate reductase (MTHFR) on homocysteine levels. However the relationship between this mutation and cardiovascular diseases (CVD) has remained as a controversial issue. The present study was undertaken to investigate the relationship between C677T polymorphism of MTHFR gene, plasma total Hcy levels and the number of affected vessels as a criterion for the extent of CAD. MTHFR genotypes and plasma homocysteine (HCY) concentrations were examined in 231 patients and 300 healthy subjects who underwent diagnostic coronary angiography. A multiple linear regression analysis was performed to identify the predictors of Hcy levels whereas logistic regression model was built to determine the association of Hcy quartiles with the risk of CAD adjusted for risk factors. The prevalence of MTHFR genotypes was similar between CAD patients and non-CAD individuals while the geometric mean of Hcy values was significantly higher in patient group (14.13 ± 4.11 μmol/l) than in control group (10.19 ± 3.52 μmol/l) (P < 0.001). Moreover, unlike the MTHFR polymorphism, Hcy concentration increased with increasing number of stenosed vessels and the CAD risk increased about 2 folds in the top two Hcy quartiles (≥ 17.03 and 13.20-17.02 μmol/l) compared with the lowest quartile (≤ 9.92 μmol/l) after controlling for conventional risk factors (P<0.001 for both). Our data suggest that hyperhomocysteinaemia (HHcy) is significantly associated to CAD risk increase as well as to the extent of coronary atherosclerosis. PMID:26417236

  17. Genetic variation in Glutathione S-Transferase Omega-1, Arsenic Methyltransferase and Methylene-tetrahydrofolate Reductase, arsenic exposure and bladder cancer: a case–control study

    PubMed Central

    2012-01-01

    Background Ingestion of groundwater with high concentrations of inorganic arsenic has been linked to adverse health outcomes, including bladder cancer, however studies have not consistently observed any elevation in risk at lower concentrations. Genetic variability in the metabolism and clearance of arsenic is an important consideration in any investigation of its potential health risks. Therefore, we examined the association between genes thought to play a role in the metabolism of arsenic and bladder cancer. Methods Single nucleotide polymorphisms (SNPs) in GSTO-1, As3MT and MTHFR were genotyped using DNA from 219 bladder cancer cases and 273 controls participating in a case–control study in Southeastern Michigan and exposed to low to moderate (<50 μg/L) levels of arsenic in their drinking water. A time-weighted measure of arsenic exposure was constructed using measures from household water samples combined with past residential history, geocoded and merged with archived arsenic data predicted from multiple resources. Results While no single SNP in As3MT was significantly associated with bladder cancer overall, several SNPs were associated with bladder cancer among those exposed to higher arsenic levels. Individuals with one or more copies of the C allele in rs11191439 (the Met287Thr polymorphism) had an elevated risk of bladder cancer (OR = 1.17; 95% CI = 1.04-1.32 per 1 μg/L increase in average exposure). However, no association was observed between average arsenic exposure and bladder cancer among TT homozygotes in the same SNP. Bladder cancer cases were also 60% less likely to be homozygotes for the A allele in rs1476413 in MTHFR compared to controls (OR = 0.40; 95% CI = 0.18-0.88). Conclusions Variation in As3MT and MTHFR is associated with bladder cancer among those exposed to relatively low concentrations of inorganic arsenic. Further investigation is warranted to confirm these findings. PMID:22747749

  18. The urinary excretion of orally administered pteroyl-l-glutamic acid by the rat

    PubMed Central

    Blair, J. A.; Dransfield, E.

    1971-01-01

    1. The urinary excretion of folates after oral administration of [2-14C]pteroyl-l-glutamic acid was studied by assaying the radioactivity in the urine and in materials purified and characterized by t.l.c. 2. Radioactivity excreted was 6.8, 5.9 and 30.7% of the oral dose in the first 24h after doses of 3.1, 32 and 320μg/kg respectively. 3. Extensive decomposition of urinary folates to pteroyl-l-glutamic acid was prevented by antioxidants or collection of urine frozen. 4. At the three dosages, two major and one minor radioactive compounds were isolated. One of the major metabolites was 5-methyltetrahydropteroylglutamic acid. The others were unidentified but were not pteroylglutamic acid, 7,8-dihydro-, 5,6,7,8-tetrahydro-, 5- or 10-formyl-tetrahydro-, 5,10-methylidyne-tetrahydro-, 5-formimidoyl-tetrahydro-, 5,10-methylene-tetrahydro-, 5-methyltetrahydro-pteroylglutamic acid, nor any decomposition products of these compounds formed during isolation. Labelled unconjugated pteridines were absent. 5. Labelled pteroyl-l-glutamic acid was displaced by oral administration of unlabelled pteroyl-l-glutamic acid (1.6mg/kg) when given 3.5h after, but not when given 24h after the labelled dose. 6. The results show that orally administered [2-14C]pteroyl-l-glutamic acid is absorbed without metabolism and is then metabolized into naturally occurring tetrahydro-folates. 7. These findings are discussed with reference to previous work. PMID:5124394

  19. Strand-specific RNA-seq analysis of the Lactobacillus delbrueckii subsp. bulgaricus transcriptome.

    PubMed

    Zheng, Huajun; Liu, Enuo; Shi, Tao; Ye, Luyi; Konno, Tomonobu; Oda, Munehiro; Ji, Zai-Si

    2016-02-01

    Lactobacillus delbrueckii subsp. bulgaricus 2038 (Lb. bulgaricus 2038) is an industrial bacterium that is used as a starter for dairy products. We proposed several hypotheses concerning its industrial features previously. Here, we utilized RNA-seq to explore the transcriptome of Lb. bulgaricus 2038 from four different growth phases under whey conditions. The most abundantly expressed genes in the four stages were mainly involved in translation (for the logarithmic stage), glycolysis (for control/lag stages), lactic acid production (all the four stages), and 10-formyl tetrahydrofolate production (for the stationary stage). The high expression of genes like d-lactate dehydrogenase was thought as a result of energy production, and consistent expression of EPS synthesis genes, the restriction-modification (RM) system and the CRISPR/Cas system were validated for explaining the advantage of this strain in yoghurt production. Several postulations, like NADPH production through GapN bypass, converting aspartate into carbon-skeleton intermediates, and formate production through degrading GTP, were proved not working under these culture conditions. The high expression of helicase genes and co-expressed amino acids/oligopeptides transporting proteins indicated that the helicase might mediate the strain obtaining nitrogen source from the environment. The transport system of Lb. bulgaricus 2038 was found to be regulated by antisense RNA, hinting the potential application of non-coding RNA in regulating lactic acid bacteria (LAB) gene expression. Our study has primarily uncovered Lb. bulgaricus 2038 transcriptome, which could gain a better understanding of the regulation system in Lb. bulgaricus and promote its industrial application. PMID:26675359

  20. Investigation of metabolism and disposition of GSK1322322, a peptidase deformylase inhibitor, in healthy humans using the entero-test for biliary sampling.

    PubMed

    Mamaril-Fishman, Donna; Zhu, John; Lin, Min; Felgate, Clive; Jones, Lori; Stump, Patrick; Pierre, Esaie; Bowen, Chester; Naderer, Odin; Dumont, Etienne; Patel, Parul; Gorycki, Peter D; Wen, Bo; Chen, Liangfu; Deng, Yanli

    2014-08-01

    GSK1322322 (N-((R)-2-(cyclopentylmethyl)-3-(2-(5-fluoro-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-methylpyrimidin-4-yl)hydrazinyl)-3-oxopropyl)-N-hydroxy-formamide) is an antibiotic in development by GlaxoSmithKline. In this study, we investigated the metabolism and disposition of [(14)C]GSK1322322 in healthy humans and demonstrated the utility of the Entero-Test in a human radiolabel study. We successfully collected bile from five men using this easy-to-use device after single i.v. (1000 mg) or oral administration (1200 mg in a solution) of [(14)C]GSK1322322. GSK1322322 had low plasma clearance (23.6 liters/hour) with a terminal elimination half-life of ∼4 hours after i.v. administration. After oral administration, GSK1322322 was readily and almost completely absorbed (time of maximal concentration of 0.5 hour; bioavailability 97%). GSK1322322 predominated in the systemic circulation (>64% of total plasma radioactivity). An O-glucuronide of GSK1322322 (M9) circulated at levels between 10% and 15% of plasma radioactivity and was pharmacologically inactive. Humans eliminated the radioactive dose in urine and feces at equal proportions after both i.v. and oral doses (∼45%-48% each). Urine contained mostly unchanged GSK1322322, accounting for 30% of the dose. Bile contained mostly M9, indicating that glucuronidation was likely a major pathway in humans (up to 30% of total dose). In contrast, M9 was found in low amounts in feces, indicating its instability in the gastrointestinal tract. Therefore, without the Entero-Test bile data, the contribution of glucuronidation would have been notably underestimated. An unusual N-dehydroxylated metabolite (a secondary amide) of GSK1322322 was observed primarily in the feces and was most likely formed by gut microbes. PMID:24872378

  1. Phosphate Triester Hydrolysis Promoted by an N2S (thiolate) Zinc Complex: Mechanistic Implications for the Metal-Dependent Reactivity of Peptide Deformylase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The zinc(II) complex (PATH)ZnOH, where PATH is an N2S(thiolate) ligand, has been investigated for its ability to promote the hydrolysis of the phosphate triester tris(4-nitrophenyl) phosphate (TNP). The hydrolysis of TNP was examined as a function of PATH-zinc(II) complex concentration, substrate co...

  2. Initiation of Protein Synthesis by Folate-Sufficient and Folate-Deficient Streptococcus faecalis R: Partial Purification and Properties of Methionyl-Transfer Ribonucleic Acid Synthetase and Methionyl-Transfer Ribonucleic Acid Formyltransferase

    PubMed Central

    Samuel, Charles E.; Rabinowitz, Jesse C.

    1974-01-01

    RNAfMet are competitive inhibitors of both plus- and minus-folate S. faecalis formyltransferase; folic acid, pteroic acid, aminopterin, and Met-tRNAmMet are not inhibitory. These results indicate that the presence or absence of folic acid in the culture medium of S. faecalis has no apparent effect on either methionyl-tRNA synthetase or methionyl-tRNA formyltransferase, the two enzymes directly involved in the formation of formylmethionyl-tRNAfMet. Therefóre, the lack of N-formylation of Met-tRNAfMet in minus-folate S. faecalis is due to the absence of the formyl donor, a 10-formyl-tetrahydropteroyl derivative. Although the general properties of S. faecalis methionyl-tRNA synthetase are similar to those of other aminoacyl-tRNA synthetases, S. faecalis methionyl-tRNA formyltransferase differs from other previously described transformylases in certain kinetic parameters. PMID:4206871

  3. Folate composition of ten types of mushrooms determined by liquid chromatography-mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    White button, crimini, shiitake, maitake, enoki, oyster, chanterelle, morel, portabella, and uv-treated portabella mushrooms were sampled from U.S. retail outlets and major producers. Folate (5-methyltetrahydrofolate [5MTHF], 10-formyl folate [10FF], 5-formyltetrahydrofolate [5FTHF]) was analyzed u...

  4. 1,3,4-Oxadiazole Derivatives: Synthesis, Characterization, Antimicrobial Potential, and Computational Studies

    PubMed Central

    Kajal, Anu; Saini, Vipin; Prasad, Deo Nanadan

    2014-01-01

    We report the synthesis and biological assessment of 1,3,4-oxadiazole substituted 24 derivatives as novel, potential antibacterial agents. The structures of the newly synthesized derivatives were established by the combined practice of UV, IR, 1H NMR, 13C NMR, and mass spectrometry. Further these synthesized derivatives were subjected to antibacterial activity against all the selected microbial strains in comparison with amoxicillin and cefixime. The antibacterial activity of synthesized derivatives was correlated with their physicochemical and structural properties by QSAR analysis using computer assisted multiple regression analysis and four sound predictive models were generated with good R2, R adj 2, and Fischer statistic. The derivatives with potent antibacterial activity were subjected to molecular docking studies to investigate the interactions between the active derivatives and amino acid residues existing in the active site of peptide deformylase to assess their antibacterial potential as peptide deformylase inhibitor. PMID:25147788

  5. Identification of novel aminopiperidine derivatives for antibacterial activity against Gram-positive bacteria.

    PubMed

    Lee, Hee-Yeol; An, Kyung-Mi; Jung, Juyoung; Koo, Je-Min; Kim, Jeong-Geun; Yoon, Jong-Min; Lee, Myong-Jae; Jang, HyeonSoo; Lee, Hong-Sub; Park, Soobong; Kang, Jae-Hoon

    2016-07-01

    We have previously reported amidopiperidine derivatives as a novel peptide deformylase (PDF) inhibitor and evaluated its antibacterial activity against Gram-positive bacteria, but poor pharmacokinetic profiles have resulted in low efficacy in in vivo mouse models. In order to overcome these weaknesses, we newly synthesized aminopiperidine derivatives with remarkable antimicrobial properties and oral bioavailability, and also identified their in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP). PMID:27173797

  6. Evaluation of ascorbic acid in protecting labile folic acid derivatives.

    PubMed Central

    Wilson, S D; Horne, D W

    1983-01-01

    The use of ascorbic acid as a reducing agent to protect labile, reduced derivatives of folic acid has been evaluated by high-performance liquid chromatographic separations and Lactobacillus casei microbiological assay of eluate fractions. Upon heating for 10 min at 100 degrees C, solutions of tetrahydropteroylglutamic acid (H4PteGlu) in 2% sodium ascorbate gave rise to 5,10-methylene-H4PteGlu and 5-methyl-H4PteGlu. H2PteGlu acid gave rise to 5-methyl-H4PteGlu and PteGlu. 10-Formyl-H4PteGlu gave rise to 5-formyl-H4PteGlu and 10-formyl-PteGlu. 5-Formyl-H4-PteGlu gave rise to a small amount of 10-formyl-PteGlu. 5-Methyl-H4PteGlu and PteGlu appeared stable to these conditions. These interconversions were not seen when solutions of these folate derivatives were kept at 0 degrees C in 1% ascorbate. These observations indicate that elevated temperatures are necessary for the interconversions of folates in ascorbate solutions. Assays of ascorbic acid solutions indicated the presence of formaldehyde (approximately equal to 6 mM). This was confirmed by the identification of 3,5-diacetyl-1,4-dihydrolutidine by UV, visible, and fluorescence spectroscopy and by thin-layer chromatography of chloroform extracts of the reaction mixture of ascorbic acid solutions, acetylacetone, and ammonium acetate. These results indicate that solutions of sodium ascorbate used at elevated temperatures are not suitable for extracting tissue for the subsequent assay of the individual folic acid derivatives. PMID:6415653

  7. Formation of 10-Formylfolic Acid, a Potent Inhibitor of Dihydrofolate Reductase, in Rat Liver Slices Incubated with Folic Acid

    PubMed Central

    d'Urso-Scott, M.; Uhoch, J.; Bertino, J. R.

    1974-01-01

    During investigation of folate polyglutamate biosynthesis in rat liver slices utilizing [2-14C]folic acid, a folate compound that behaved like a polyglutamate form in the Sephadex G-15 gel filtration system was found to accumulate. Subsequent chromatographic, spectral, chemical, and enzymic studies have indicated that the compound formed in liver slices incubated with [14C]folic acid with and without methotrexate was 10-formyl folate. This folate is of interest in that it is the most potent natural inhibitor of dihydrofolate reductase known and may be capable of serving a regulatory function within the cell. PMID:4527808

  8. Relationship between the 19 base pair deletion polymorphism in DHFR and unmetabolized folic and in plasma and RBC folate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: A 19 base pair (bp) deletion allele of dihydrofolate reductase (DHFR), an enzyme that makes folic acid metabolically active and reduces dihydrofolate to tetrahydrofolate to stimulate folate turnover, has been implicated in folate related health outcomes. Objective: Examine the effect ...

  9. Lentils (Lens culinaris L.), a rich source of folates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pulses contain folates in the form of reduced tetrahydrofolate which is the biologically active form absorbed in the jejunum. Genetic biofortification potential of US-grown lentils (Lens culinaris L.) with the bioavailable form of folate has not been widely studied. The objectives of this study wer...

  10. Liquid chromatographic determination of folate monoglutamates in fish, meat, egg, and dairy products consumed in Finland.

    PubMed

    Vahteristo, L T; Ollilainen, V; Varo, P

    1997-01-01

    A liquid chromatographic (LC) method with fluorescence and UV detection was used to determine the folate contents of fish, meat, fish and meat products, chicken, eggs, and milk consumed in Finland. 5-Methyltetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid from 24 commodities obtained from supermarkets, retail stores, and different outlets in the Helsinki area were analyzed. Pooled samples were extracted at pH 6.0 in the presence of antioxidants and deconjugated with hog kidney deconjugase. Very low levels of folates were detected in meat and meat products. Fresh fish, fish sticks, and chicken meat contained reasonable amounts (3-13 micrograms/100 g) of tetrahydrofolate and 5-methyltetrahydrofolate. Egg yolk contained high concentrations of 5-methyltetrahydrofolate (140-150 micrograms/100 g); 10-formylfolic acid was also detected (14-17 micrograms/100 g). Between-species differences in folate monoglutamate distributions were observed. The highest levels of tetrahydrofolate, > 5 micrograms/100 g, were found in chicken meat and fillets of rainbow trout, whitefish, and baltic herring. Tetrahydrofolate was most abundant in fresh fish. LC was well suited for analyzing folate compositions of meat, fish, and other foods of animal origin. Recovery of added folates ranged from 49 to 96%. PMID:9086593

  11. A 19-base pair deletion polymorphism in dihydrofolate reductase is associated with increased unmetabolized folic acid in plasma and decreased red blood cell folate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dihydrofolate reductase (DHFR) catalyzes the reduction of folic acid to tetrahydrofolate (THF). A 19-bp noncoding deletion allele maps to intron 1, beginning 60 bases from the splice donor site, and has been implicated in neural tube defects and cancer, presumably by influencing folate metabolism. T...

  12. Status of vitamin B-12 and B-6 but not of folate, homocysteine and the methylenetetrahydrofolate reductase C677T polymorphism are associated with impaired cognition and depression in adults

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The C677T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene differs in frequency in different ethnic groups which have differing prevalence of age-related cognitive impairments. We used a battery of neuropsychological tests to examine association of the MTHFR C677T polymorphism w...

  13. Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine forming Met, which is then used for the syn...

  14. Prevalence of MTHFR C677T Polymorphism in North Indian Mothers Having Babies with Trisomy 21 Down Syndrome

    ERIC Educational Resources Information Center

    Kohli, Utkarsh; Arora, Sadhna; Kabra, Madhulika; Ramakrishnan, Lakshmy; Gulati, Sheffali; Pandey, Ravindra

    2008-01-01

    Recent studies have evaluated possible links between polymorphisms in maternal folate metabolism genes and Down syndrome. Some of these studies show a significantly increased prevalence of the C677T polymorphism of the 5,10-methylene tetrahydrofolate reductase (NADPH) gene (MTHFR) among mothers who have had babies with Down syndrome. This study…

  15. Prevalence of MTHFR C677T and MS A2756G polymorphisms in major depressive disorder, and their impact on response to fluoxetine treatment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To examine the prevalence of the C677T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene and the A2756G polymorphism of methionine synthase (MS), and their impact on antidepressant response. We screened 224 subjects (52% female, mean age 39 +/- 11 years) with SCID-diagnosed major...

  16. A case of recurrent pancytopenia in a patient with acute promyelocytic leukemia on maintenance chemotherapy and concomitant methyltetrahydrofolate reductase and thiopurine S-methyltransferase mutation - review of literature.

    PubMed

    Keung, Yi-Kong; Keung, Lap-Woon; Hong-Lung Hu, Eddie

    2016-06-01

    Pharmacogenetics is a study of how genetic variation of an individual affects the drug response. We report a case of recurrent pancytopenia resulting from maintenance chemotherapy in a patient with acute promyelocytic leukemia and two pharmacogenetic mutations, namely, methylene tetrahydrofolate reductase C677T homozygous mutation and thiopurine methyltransferase mutation. PMID:25791511

  17. Corrinoid-Dependent Methyl Transfer Reactions Are Involved in Methanol and 3,4-Dimethoxybenzoate Metabolism by Sporomusa ovata

    PubMed Central

    Stupperich, Erhard; Konle, Ralph

    1993-01-01

    Washed and air-oxidized proteins from Sporomusa ovata cleaved the C-O bond of methanol or methoxyaromatics and transferred the methyl to dl-tetrahydrofolate. The reactions strictly required a reductive activation by titanium citrate, catalytic amounts of ATP, and the addition of dl-tetrahydrofolate. Methylcorrinoid-containing proteins carried the methanol methyl, which was transferred to dl-tetrahydrofolate at a specific rate of 120 nmol h-1 mg of protein-1. Tetrahydrofolate methylation diminished after the addition of 1-iodopropane or when the methyl donor methanol was replaced by 3,4-dimethoxybenzoate. However, whole Sporomusa cells utilize the methoxyl groups of 3,4-dimethoxybenzoate as a carbon source by a sequential O demethylation to 4-hydroxy-3-methoxybenzoate and 3,4-dihydroxybenzoate. The in vitro O demethylation of 3,4-[4-methoxyl-14C]dimethoxybenzoate proceeded via two distinct corrinoid-containing proteins to form 5-[14C]methyltetrahydrofolate at a specific rate of 200 nmol h-1 mg of protein-1. Proteins from 3,4-dimethoxybenzoate-grown cells efficiently used methoxybenzoates with vicinal substituents only, but they were unable to activate methanol. These results emphasized that specific enzymes are involved in methanol activation as well as in the activation of various methoxybenzoates and that similar corrinoid-dependent methyl transfer pathways are employed in 5-methyl-tetrahydrofolate formation from these substrates. Methyl-tetrahydrofolate could be demethylated by a distinct methyl transferase. That enzyme activity was present in washed and air-oxidized cell extracts from methanol-grown cells and from 3,4-dimethoxybenzoate-grown cells. It used cob(I)alamin as the methyl acceptor in vitro, which was methylated at a rate of 48 nmol min-1 mg of protein-1 even when ATP was omitted from the assay mixture. This methyl-cob(III)alamin formation made possible a spectrophotometric quantification of the preceding methyl transfers from methanol or

  18. Side chain modified 5-deazafolate and 5-deazatetrahydrofolate analogues as mammalian folylpolyglutamate synthetase and glycinamide ribonucleotide formyltransferase inhibitors: synthesis and in vitro biological evaluation.

    PubMed

    Rosowsky, A; Forsch, R A; Reich, V E; Freisheim, J H; Moran, R G

    1992-05-01

    5-Deazafolate and 5-deazatetrahydrofolate (DATHF) analogues with the glutamic acid side chain replaced by homocysteic acid (HCysA), 2-amino-4-phosphonobutanoic acid (APBA), and ornithine (Orn) were synthesized as part of a larger program directed toward inhibitors of folylpolyglutamate synthetase (FPGS) as probes of the FPGS active site and as potential therapeutic agents. The tetrahydro compounds were also of interest as non-polyglutamatable inhibitors of the purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFT). Reductive coupling of N2-acetamido-6-formylpyrido[2,3-d]pyrimidin-4(3H)-one with 4-aminobenzoic acid, followed by N10-formylation, mixed anhydride condensation of the resultant N2-acetyl-N10-formyl-5- deazapteroic acid with L-homocysteic acid, and removal of the N2-acetyl and N10-formyl groups with NaOH, afforded N-(5-deazapteroyl)-L-homocysteic acid (5-dPteHCysA). Mixed anhydride condensation of N2-acetyl-N10-formyl- 5-deazapteroic acid with methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid, followed by consecutive treatment with Me3SiBr and NaOH, yielded D,L-2-[(5-deazapteroyl)amino]-4-phosphonobutanoic acid (5-dPteAPBA). Treatment with NaOH alone led to retention of one ethyl ester group on the phosphonate moiety. Catalytic hydrogenation of N2-acetyl-N10-formyl-5-deazapteroic acid followed by mixed anhydride condensation with methyl L-homocysteate and deprotection with NaOH afforded N-(5,6,7,8-tetrahydro-5-deazapteroyl)-L-homocysteic acid (5-dH4PteHCysA). Similar chemistry starting from methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid and methyl N delta-(benzyloxycarbonyl)-L-ornithinate yielded D,L-2-[(5-deaza-5,6,7,8-tetrahydropteroyl)amino]-4-phosphonobut ano ic acid (5-dH4Pte-APBA) and N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine (5-dH4PteOrn), respectively. The 5-deazafolate analogues were inhibitors of mouse liver FPGS, and the DATHF analogues inhibited both mouse FPGS and mouse leukemic cell GARFT

  19. Neither Folic Acid Supplementation nor Pregnancy Affects the Distribution of Folate Forms in the Red Blood Cells of Women1–3

    PubMed Central

    Hartman, Brenda A.; Fazili, Zia; Pfeiffer, Christine M.; O’Connor, Deborah L.

    2016-01-01

    It is not known whether folate metabolism is altered during pregnancy to support increased DNA and RNA biosynthesis. By using a state-of-the-art LC tandem mass spectrometry technique, the aim of this study was to investigate differences in RBC folate forms between pregnant and nonpregnant women and between nonpregnant women consuming different concentrations of supplemental folic acid. Forms of folate in RBCs were used to explore potential shifts in folate metabolism during early erythropoiesis. Total RBC folate and folate forms [tetrahydrofolate; 5-methyltetrahydrofolate (5-methyl-THF); 4α-hydroxy-5-methyl-tetrahydrofolate (an oxidation product of 5-methyl-THF); 5-formyl-tetrahydrofolate; and 5,10-methenyl-tetrahydrofolate] were measured in 4 groups of women (n = 26): pregnant women (PW) (30–36 wk of gestation) consuming 1 mg/d of folic acid, and nonpregnant women consuming 0 mg/d (NPW-0), 1 mg/d (NPW-1), and 5 mg/d (NPW-5) folic acid. The mean ± SD RBC folate concentration of the NPW-0 group (890 ± 530 nmol/L) was lower than the NPW-1 (1660 ± 350 nmol/L) and NPW-5 (1980 ± 570 nmol/L) groups as assessed by microbiologic assay (n = 26, P < 0.0022). No difference was found between the NPW-1 and NPW-5 groups. We detected 5-methyl-THF [limit of detection (LOD) = 0.06 nmol/L] in all groups and tetrahydrofolate (LOD = 0.2 nmol/L) in most women regardless of methylenetetrahydrofolate reductase genotype. Most women consuming folic acid supplements had detectable concentrations of 5,10-methenyl-tetrahydrofolate (LOD = 0.31 nmol/L). However, there was no difference in the relative distribution of 5-methyl-THF (83–84%), sum of non-methyl folates (0.6–3%), or individual non-methyl folate forms in RBCs across groups. We conclude that although folic acid supplementation in nonpregnant women increases RBC total folate and the concentration of individual folate forms, it does not alter the relative distribution of folate forms. Similarly, distribution of RBC folate

  20. In vivo oxidation of the methyl group of hepatic 5-methyltetrahydrofolate.

    PubMed

    Lumb, M; Chanarin, I; Deacon, R; Perry, J

    1988-11-01

    Methionine given parenterally to rats caused rapid disappearance of methyltetrahydrofolate from the liver and a corresponding rise in tetrahydrofolate and formyl-tetrahydrofolate concentrations. When [14C]H3--H4folate was given, methionine caused an increased [14C]0(2) excretion, indicating that oxidation of the methyl group had occurred. Methionine was more effective than S-adenosylmethionine at causing oxidation, but serine was ineffective. The lowest dose of methionine to produce an effect was 0.5 mumol, which is less than the daily dietary intake in a rat. The data suggest that the concentration of methylfolate in rat livers is controlled by the concentrations of methionine. PMID:3145288

  1. [Successful direct thrombin inhibitor treatment of a left atrial appendage thrombus developed under rivaroxaban therapy].

    PubMed

    Szegedi, Nándor; Gellér, László; Tahin, Tamás; Merkely, Béla; Széplaki, Gábor

    2016-01-24

    The authors present the history of a 62-year-old man on continuous rivaroxaban therapy who was scheduled for pulmonary vein isolation due to persistent atrial fibrillation. Preoperative transesophageal echocardiography detected the presence of left atrial appendage thrombus. Thrombophilia tests showed that the patient was heterozygous carrier of the methylene-tetrahydrofolate reductase gene mutation. The authors hypothesized that a direct thrombin inhibitor might exert a more appropriate effect against thrombosis in this case and, therefore, a switch to dabigatran was performed. After two months of anticoagulation with the direct thrombin inhibitor and folic acid supplementation the thrombus resolved. The authors underline that thrombus formation may develop in atrial fibrillation even if the patient is adequately treated with rivaroxaban. This case suggests, that methylene-tetrahydrofolate reductase gene mutation may modulate the efficacy of direct Xa factor inhibitors. According to this case history, dabigatran may be an effective therapeutic option in resolving established thrombus. PMID:26772828

  2. Whipple's disease, genomics, and drug therapy

    SciTech Connect

    Cannon, William R.

    2003-05-31

    The recent articles concerning the release of the genome for Tropheryma whipplei [1, 2], the causative agent of Whipple's disease, anticipate new medical discoveries and conclusions that will be drawn from the decoding of the genome. Although the reports mention that genes for key metabolic processes were missing, we were nevertheless surprised to find that the genome does not contain the coding sequence for dihydrofolate reductase (DHFR). This is significant because competitive inhibition of DHFR by trimethoprim is the mode of action of this antibacterial agent. Lacking an adequate population for clinical studies, retrospective analyses and patient series ([3, 4] references therein) have concluded that the drug combination of trimethoprim and sulfamethoxazole are the preferred treatment regimes. The treatment goal is to disrupt purine and pyrimidine synthesis, and hence replication, by shutting down tetrahydrofolate biosynthesis. However, while the use of trimethoprim will affect the host, thereby indirectly affect the bacterium through a reduced tetrahydorfolate pool, it is unlikely that trimethoprim has any effect on tetrahydrofolate production in T. whipplei. To be sure that there weren't any weak homologues to DHFR or that the DHFR gene was somehow missed due to being part of a multi-functional enzyme, we performed a sequence search (TBLASTN) of the T. whipplei genome using the DHFR protein sequence from the fellow actinomycete, Mycobaterium tuberculosis, as the target sequence. No close or distant homologues were found. While some bacterial plasmids code for a type II DHFR that has no homology to the more common type I DHFR found in most species, type II DHFR does not bind trimethoprim and tetrahydrofolate production is unaffected by the presence of trimethoprim. Furthermore, the genome additionally lacks a gene for thymidylate synthetase, another key enzyme in the folate-one carbon pathway that utilizes the DHFR product tetrahydrofolate. Lacking randomized

  3. Hyperhomocysteinaemia, Helicobacter pylori, and coronary heart disease.

    PubMed

    Sung, J J; Sanderson, J E

    1996-10-01

    Hyperhomocysteinaemia and Helicobacter pylori infection have recently been implicated in the pathogenesis of coronary artery disease. These two risk factors, though they seem unrelated, could be linked by a deficiency of vitamins and folate caused by chronic gastritis in H pylori infection. This nutritional defect could lead to failure of methylation by 5-methyl-tetrahydrofolic acid and thus exacerbate the accumulation of homocysteine in susceptible patients. Homocysteine is toxic to endothelial cells and results in coronary artery disease. PMID:8983673

  4. Chloromethane-Induced Genes Define a Third C1 Utilization Pathway in Methylobacterium chloromethanicum CM4

    PubMed Central

    Studer, Alex; McAnulla, Craig; Büchele, Rainer; Leisinger, Thomas; Vuilleumier, Stéphane

    2002-01-01

    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. PMID:12057941

  5. Simple procedure for the synthesis of high specific activity tritiated (6S)-5-formyltetrahydrofolate

    SciTech Connect

    Moran, R.G.; Colman, P.D.

    1982-05-01

    The 5-position of tetrahydrofolate was found to be unusually reactive with low concentrations of formic acid in the presence of a water-soluble carbodiimide. The product of this reaction has neutral and acid ultraviolet spectra and chromatographic behavior consistent with its identity as 5-formyltetrahydrofolate (leucovoriun). When enzymatically synthesized (6S)-tetrahydrofolate was used as starting material, the product supported the growth of folate-depleted L1210 cells at one-half the concentration required for authentic (6R,S)-leucovorin. This reaction has been used to produce high specific activity (44 Ci/mmol) (/sup 3/H)(6S)-5-formyltetrahydrofolate in high yield. Experiments with (/sup 14/C)formic acid indicate that 1 mol of formate reacted per mol of tetrahydrofolate but that no reaction occurred with a variety of other folate compounds. (6S)-5-Formyltetrahydrofolate, labeled in the formyl group with /sup 14/C, has also been synthesized using this reaction. These easily produced, labeled folates should allow close examination of the transport and utilization of leucovorin and of the mechanism of reversal of methotrexate toxicity by reduced folate cofactors.

  6. The Search for Herbal Antibiotics: An In-Silico Investigation of Antibacterial Phytochemicals.

    PubMed

    Snow Setzer, Mary; Sharifi-Rad, Javad; Setzer, William N

    2016-01-01

    Recently, the emergence and spread of pathogenic bacterial resistance to many antibiotics (multidrug-resistant strains) have been increasing throughout the world. This phenomenon is of great concern and there is a need to find alternative chemotherapeutic agents to combat these antibiotic-resistant microorganisms. Higher plants may serve as a resource for new antimicrobials to replace or augment current therapeutic options. In this work, we have carried out a molecular docking study of a total of 561 antibacterial phytochemicals listed in the Dictionary of Natural Products, including 77 alkaloids (17 indole alkaloids, 27 isoquinoline alkaloids, 4 steroidal alkaloids, and 28 miscellaneous alkaloids), 99 terpenoids (5 monoterpenoids, 31 sesquiterpenoids, 52 diterpenoids, and 11 triterpenoids), 309 polyphenolics (87 flavonoids, 25 chalcones, 41 isoflavonoids, 5 neoflavonoids, 12 pterocarpans, 10 chromones, 7 condensed tannins, 11 coumarins, 30 stilbenoids, 2 lignans, 5 phenylpropanoids, 13 xanthones, 5 hydrolyzable tannins, and 56 miscellaneous phenolics), 30 quinones, and 46 miscellaneous phytochemicals, with six bacterial protein targets (peptide deformylase, DNA gyrase/topoisomerase IV, UDP-galactose mutase, protein tyrosine phosphatase, cytochrome P450 CYP121, and NAD⁺-dependent DNA ligase). In addition, 35 known inhibitors were docked with their respective targets for comparison purposes. Prenylated polyphenolics showed the best docking profiles, while terpenoids had the poorest. The most susceptible protein targets were peptide deformylases and NAD⁺-dependent DNA ligases. PMID:27626453

  7. Interplay between trigger factor and other protein biogenesis factors on the ribosome

    NASA Astrophysics Data System (ADS)

    Bornemann, Thomas; Holtkamp, Wolf; Wintermeyer, Wolfgang

    2014-06-01

    Nascent proteins emerging from translating ribosomes in bacteria are screened by a number of ribosome-associated protein biogenesis factors, among them the chaperone trigger factor (TF), the signal recognition particle (SRP) that targets ribosomes synthesizing membrane proteins to the membrane and the modifying enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). Here, we examine the interplay between these factors both kinetically and at equilibrium. TF rapidly scans the ribosomes until it is stabilized on ribosomes presenting TF-specific nascent chains. SRP binding to those complexes is strongly impaired. Thus, TF in effect prevents SRP binding to the majority of ribosomes, except those presenting SRP-specific signal sequences, explaining how the small amount of SRP in the cell can be effective in membrane targeting. PDF and MAP do not interfere with TF or SRP binding to translating ribosomes, indicating that nascent-chain processing can take place before or in parallel with TF or SRP binding.

  8. Structural reconstruction of the catalytic center of LiPDF through multiple scattering calculation with MXAN

    NASA Astrophysics Data System (ADS)

    Guo, Xiaoyun; Chu, Wangsheng; Ma, Sixuan; Gong, Weimin; Benfatto, Maurizio; Hu, Tiandou; Xie, Yaning; Wu, ZiYu

    2006-11-01

    Peptide deformylase (PDF, EC 3.5.1.27) is essential for the normal growth of eubacterium but not for mammalians. Recently, PDF has been studied as a target for new antibiotics. In this paper, X-ray absorption spectroscopy was employed to determine the local structure around the zinc ion of PDF from Leptospira Interrogans in dry powder, because it is very difficult to obtain the crystallized sample of LiPDF. We performed X-ray absorption near edge structure (XANES) calculation and reconstructed successfully the local geometry of the active center, and the results from calculations show that a water molecule (Wat1) has moved towards the zinc ion and lies in the distance range to coordinate with the zinc ion weakly. In addition, the sensitivity of theoretical spectra to the different ligand bodies was evaluated in terms of goodness-of-fit.

  9. Effect of metal ion on the structure and function of LiPDF: The study of the fine structure around the metal site using XANES

    NASA Astrophysics Data System (ADS)

    Wang, Yu; Chu, Wangsheng; Yang, Feifei; Yu, Meijuan; Zhao, Haifeng; Gong, Weimin; Dong, Yuhui; Xie, Yaning; Wu, Ziyu

    2010-07-01

    We used X-ray absorption near edge structure (XANES) spectroscopy to investigate the metal-dependent enzymatic activity of the peptide deformylase from Leptospira interrogans ( LiPDF). Ab initio full multiple scattering calculations performed by MXAN are applied to obtain the local structure of the cobalt-containing LiPDF (Co- LiPDF) and zinc-containing LiPDF (Zn- LiPDF) around the metal sites in pH9.0 buffer solution. The result shows the cobalt-wat1 (the bond water molecule) distance of Co- LiPDF is 1.89 Å, much shorter than that of Zn- LiPDF, 2.50 Å. That is an essential factor for its low catalytic activity.

  10. New pathway for the biodegradation of indole in Aspergillus niger

    SciTech Connect

    Kamath, A.; Vaidyanathan, C.S. )

    1990-01-01

    Indole and its derivatives form a class of toxic recalcitrant environmental pollutants. The growth of Aspergillus niger was inhibited by very low concentrations (0.005 to 0.02%) of indole, even when 125- to 500-fold excess glucose was present in the medium. When 0.02% indole was added, the fungus showed a lag phase for about 30 h and the uptake of glucose was inhibited. Indole was metabolized by a new pathway via indoxyl (3-hydroxyindole), N-formylanthranilic acid, anthranilic acid, 2,3-dihydroxybenzoic acid, and catechol, which was further degraded by an ortho cleavage. The enzymes N-formylanthranilate deformylase, anthranilate hydroxylase, 2,3-dihydroxybenzoate decarboxylase, and catechol dioxygenase were induced by indole as early as after 5 h of growth, and their activities were demonstrated in a cell-free system.

  11. Quantification of key red blood cell folates from subjects with defined MTHFR 677C>T genotypes using stable isotope dilution liquid chromatography/mass spectrometry

    PubMed Central

    Huang, Yuehua; Khartulyari, Stefanie; Morales, Megan E.; Stanislawska-Sachadyn, Anna; Von Feldt, Joan M.; Whitehead, Alexander S.; Blair, Ian A.

    2014-01-01

    Red blood cell (RBC) folate levels are established at the time of erythropoiesis and therefore provide a surrogate biomarker for the average folate status of an individual over the preceding four months. Folates are present as folylpolyglutamates, highly polar molecules that cannot be secreted from the RBCs, and must be converted into their monoglutamate forms prior to analysis. This was accomplished using an individual’s plasma pteroylpolyglutamate hydrolase by lysing the RBCs in whole blood at pH 5 in the presence of ascorbic acid. Quantitative conversion of formylated tetrahydrofolate derivatives into the stable 5,10-methenyltetrahydrofolate (5,10-MTHF) form was conducted at pH 1.5 in the presence of [13C5]-5-formyltetrahydrofolate. The resulting [13C5]-5,10-MTHF was then used as an internal standard for the formylated forms of tetrahydrofolate that had been converted into 5,10-MTHF as well any 5,10-MTHF that had been present in the original sample. A stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry method was validated and then used for the accurate and precise quantification of RBC folic acid, 5-methyltetrahydrofolate (5-MTHF), tetrahydrofolate (THF), and 5,10-MTHF. The method was sensitive and robust and was used to assess the relationship between different methylenetetrahydrofolate reductase (MTHFR) 677C>T genotypes and RBC folate phenotypes. Four distinct RBC folate phenotypes could be identified. These were classified according to the relative amounts of individual RBC folates as type I (5-MTHF >95%; THF <5%; 5,10-MTHF <5%), type II (5-MTHF <95%; THF 5% to 20%; 5,10-MTHF <5%), type III (5-MTHF >55%; THF >20%; 5,10-MTHF >5%), and type IV (5-MTHF <55%; THF >20%; 5,10-MTHF >5%). PMID:18634122

  12. Mthfd1 is a modifier of chemically induced intestinal carcinogenesis

    PubMed Central

    MacFarlane, Amanda J.; Perry, Cheryll A.; McEntee, Michael F.; Lin, David M.; Stover, Patrick J.

    2011-01-01

    The causal metabolic pathways underlying associations between folate and risk for colorectal cancer (CRC) have yet to be established. Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate and methionine. Methionine is converted to S-adenosylmethionine (AdoMet), the major one-carbon donor for cellular methylation reactions. Impairments in folate metabolism can modify DNA synthesis, genomic stability and gene expression, characteristics associated with tumorigenesis. The Mthfd1 gene product, C1-tetrahydrofolate synthase, is a trifunctional enzyme that generates one-carbon substituted tetrahydrofolate cofactors for one-carbon metabolism. In this study, we use Mthfd1gt/+ mice, which demonstrate a 50% reduction in C1-tetrahydrofolate synthase, to determine its influence on tumor development in two mouse models of intestinal cancer, crosses between Mthfd1gt/+ and Apcmin/+ mice and azoxymethane (AOM)-induced colon cancer in Mthfd1gt/+ mice. Mthfd1 hemizygosity did not affect colon tumor incidence, number or load in Apcmin/+ mice. However, Mthfd1 deficiency increased tumor incidence 2.5-fold, tumor number 3.5-fold and tumor load 2-fold in AOM-treated mice. DNA uracil content in the colon was lower in Mthfd1gt/+ mice, indicating that thymidylate biosynthesis capacity does not play a significant role in AOM-induced colon tumorigenesis. Mthfd1 deficiency-modified cellular methylation potential, as indicated by the AdoMet: S-adenosylhomocysteine ratio and gene expression profiles, suggesting that changes in the transcriptome and/or decreased de novo purine biosynthesis and associated mutability cause cellular transformation in the AOM CRC model. This study emphasizes the impact and complexity of gene–nutrient interactions with respect to the relationships among folate metabolism and colon cancer initiation and progression. PMID:21156972

  13. Bipartite Structure of the ade3 Locus of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Jones, Elizabeth W.

    1977-01-01

    Forty ade3 mutants were examined with respect to their growth requirements, levels of the tetrahydrofolate interconversion enzymes, and/or map positions. Four deletions were detected. Mutations that result in a requirement for adenine and histidine map in one region of the locus; those which result in a requirement for adenine only map in a quite separate region of the locus, a region not disclosed in previous studies. No correlation was observed between growth properties of the strains and enzyme levels. PMID:324867

  14. Mutants of the Formyltetrahydrofolate Interconversion Pathway of SACCHAROMYCES CEREVISIAE

    PubMed Central

    McKenzie, K. Q.; Jones, Elizabeth W.

    1977-01-01

    Thirteen mutants of Saccharomyces cerevisiae that lack one or more of the three enzyme activities of the pathway for interconversion of tetrahydrofolate coenzymes at the formate level of oxidation have been isolated. They do not require adenine. All fail to complement mutations in the ade3 locus. Mutations that greatly reduce activity for one enzyme also reduce activity for the other two interconversion enzymes. The three enzyme activities cochromatograph on TEAE-cellulose columns. A mutation that eliminates synthetase activity also alters the chromatographic behavior of the remaining cyclohydrolase and dehydrogenase activities. It is suggested that the three activities reside in an enzyme complex encoded by the ade3 locus. PMID:328341

  15. Catheter-Directed Thrombolysis of Inferior Vena Cava Thrombosis in a 13-Day-Old Neonate and Review of Literature

    SciTech Connect

    Khan, Jawad U.; Takemoto, Clifford M.; Casella, James F.; Streiff, Michael B.; Nwankwo, Ikechi J.; Kim, Hyun S.

    2008-07-15

    Complete inferior vena cava thrombosis (IVC) in neonates is uncommon, but may cause significant morbidity. A 13-day-old neonate suffered IVC thrombosis secondary to antithrombin III deficiency, possibly contributed to by a mutation in the methyl tetrahydrofolate reductase gene. Catheter-directed thrombolysis (CDT) with recombinant tissue plasminogen activator (rt-PA, Alteplase) was used successfully to treat extensive venous thrombosis in this neonate without complications. We also review the literature on CDT for treatment of IVC thrombosis in critically ill neonates and infants.

  16. The role of purine degradation in methane biosynthesis and energy production in Methanococcus vannielii. Progress report

    SciTech Connect

    DeMoll, E.

    1998-11-01

    Firstly, characterization of a purine degrading pathway in Methanococcus vannielii was determined. The pathway is similar to that described for Clostridia. The M. vannielli pathway differs in a few respects from the Clostridial pathway. The pathway of Clostridia uses tetrahydrofolic acid (THF), whereas the pathway of M. vannielii uses tetrahydromethanopterin (H{sub 4}MPt) as a cofactor in the transfer of both the formimino moiety of formiminoglycine and apparently in the cleavage of glycine by a glycin decarboxylase type mechanism that is dependent upon at least H{sub 4}MPt and either NAD{sup +} or NADP{sup +}. Secondly, the relationship of purine degradation to methanogenesis was investigated.

  17. Interaction of Human Hemoglobin with Methotrexate

    NASA Astrophysics Data System (ADS)

    Zaharia, M.; Gradinaru, R.

    2015-05-01

    This study focuses on the interaction between methotrexate and human hemoglobin using steady-state ultraviolet-visible and fluorescence quenching methods. Fluorescence quenching was found to be valuable in assessing drug binding to hemoglobin. The quenching of methotrexate is slightly smaller than the quenching observed with related analogs (dihydrofolate and tetrahydrofolate). The quenching studies were performed at four different temperatures and various pH values. The number of binding sites for tryptophan is ~1. Parameter-dependent assays revealed that electrostatic forces play an essential role in the methotrexate-hemoglobin interaction. Furthermore, the complex was easily eluted using gel filtration chromatography.

  18. The reductive acetyl coenzyme A pathway: sequence and heterologous expression of active methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum.

    PubMed Central

    Roberts, D L; Zhao, S; Doukov, T; Ragsdale, S W

    1994-01-01

    The methyltransferase (MeTr) from Clostridium thermoaceticum transfers the N5-methyl group of (6S)-methyltetrahydrofolate to the cobalt center of a corrinoid/iron-sulfur protein in the acetyl coenzyme A pathway. MeTr was purified to homogeneity and shown to lack metals. The acsE gene encoding MeTr was sequenced and actively expressed in Escherichia coli at a level of 9% of cell protein. Regions in the sequence of MeTr and the E. coli cobalamin-dependent methionine synthase were found to share significant homology, suggesting that they may represent tetrahydrofolate-binding domains. PMID:7928975

  19. Two parallel pathways in the kinetic sequence of the Dihydrofolate Reductase from Mycobacterium tuberculosis

    PubMed Central

    Czekster, Clarissa M.; Vandemeulebroucke, An; Blanchard, John S.

    2011-01-01

    Dihydrofolate reductase from Mycobacterium tuberculosis catalyzes the NAD(P)H dependent reduction of dihydrofolate, yielding NAD(P)+ and tetrahydrofolate, the primary one carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. Previously, steady-state studies revealed that the chemical step significantly contributes to the steady state turnover number, but that a step after the chemical step was likely limiting the reaction rate. Here, we report the first pre-steady state investigation of the kinetic sequence of the MtDHFR aiming to identify kinetic intermediates, and the identity of the rate limiting steps. This kinetic analysis suggests a kinetic sequence comprising two parallel pathways with a rate determining product release. Although product release is likely occurring in a random fashion, there is a slight preference for the release of THF first, a kinetic sequence never observed for a wild type dihydrofolate reductase of any organism studied to date. Temperature studies were conducted to determine the magnitude of the energetic barrier posed by the chemical step, and the pH dependence of the chemical step was studied, demonstrating an acidic shift from the pKa observed under steady-state. The rate constants obtained here were combined with the activation energy for the chemical step to compare energy profiles for each kinetic sequence. The two parallel pathways are discussed, as well as their implications on the catalytic cycle of this enzyme. PMID:21744813

  20. Mammalian folylpoly-. gamma. -glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

    SciTech Connect

    Cichowicz, D.J.; Shane, B.

    1987-01-27

    Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of M/sub r/ 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K/sup +/ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the K/sub m/ value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.

  1. The Molecular Basis of Folate Salvage in Plasmodium falciparum

    PubMed Central

    Salcedo-Sora, J. Enrique; Ochong, Edwin; Beveridge, Susan; Johnson, David; Nzila, Alexis; Biagini, Giancarlo A.; Stocks, Paul A.; O'Neill, Paul M.; Krishna, Sanjeev; Bray, Patrick G.; Ward, Stephen A.

    2011-01-01

    Tetrahydrofolates are essential cofactors for DNA synthesis and methionine metabolism. Malaria parasites are capable both of synthesizing tetrahydrofolates and precursors de novo and of salvaging them from the environment. The biosynthetic route has been studied in some detail over decades, whereas the molecular mechanisms that underpin the salvage pathway lag behind. Here we identify two functional folate transporters (named PfFT1 and PfFT2) and delineate unexpected substrate preferences of the folate salvage pathway in Plasmodium falciparum. Both proteins are localized in the plasma membrane and internal membranes of the parasite intra-erythrocytic stages. Transport substrates include folic acid, folinic acid, the folate precursor p-amino benzoic acid (pABA), and the human folate catabolite pABAGn. Intriguingly, the major circulating plasma folate, 5-methyltetrahydrofolate, was a poor substrate for transport via PfFT2 and was not transported by PfFT1. Transport of all folates studied was inhibited by probenecid and methotrexate. Growth rescue in Escherichia coli and antifolate antagonism experiments in P. falciparum indicate that functional salvage of 5-methyltetrahydrofolate is detectable but trivial. In fact pABA was the only effective salvage substrate at normal physiological levels. Because pABA is neither synthesized nor required by the human host, pABA metabolism may offer opportunities for chemotherapeutic intervention. PMID:21998306

  2. Cyanocobalamin [c-lactam] inhibits vitamin B12 and causes cytotoxicity in HL60 cells: methionine protects cells completely.

    PubMed

    Matthews, J H

    1997-06-15

    The [c-lactam] derivative of cobalamin antagonizes vitamin B12 in vivo. Therefore, we investigated its effects in tissue culture to develop a model in which to study vitamin B12-deficient hemopoiesis. HL60 cells were cultured in medium containing either methionine or L-homocysteine thiolactone, and various concentrations of 5-methyltetrahydrofolate or pteroylglutamic acid. In medium with L-homocysteine thiolactone, 5-methyltetrahydrofolate, and dialyzed serum, cyanocobalamin [c-lactam] caused cell death, reversible by additional vitamin B12. Pteroylglutamic acid did not prevent this cytotoxic effect. Methionine completely protected cells against cyanocobalamin [c-lactam] for periods of up to 4 months of culture, irrespective of the folate source. Cyanocobalamin [c-lactam] reversibly impaired the incorporation of 5-[14CH3]-tetrahydrofolate and [1-(14)C] propionic acid by intact cells, consistent with inhibition of methionine synthase and methylmalonyl-CoA mutase. A substantial proportion of 5-[14CH3]-tetrahydrofolate uptake could not be suppressed by methionine and may, therefore, have occurred outside of the methionine synthase pathway. These findings are the first indication that cyanocobalamin [c-lactam] antagonizes vitamin B12 in vitro and causes cell death from methionine deficiency. The model should be valuable for investigating the biochemical pathology of vitamin B12-deficient hemopoiesis. The results suggest that methylfolate is not trapped when methionine synthase is inhibited in HL60 cells, but they do not disprove the methylfolate trap hypothesis as applied to normal blood cells. PMID:9192785

  3. Relative bioavailability of folate from the traditional food plant Moringa oleifera L. as evaluated in a rat model.

    PubMed

    Saini, R K; Manoj, P; Shetty, N P; Srinivasan, K; Giridhar, P

    2016-01-01

    Moringa oleifera is an affordable and rich source of dietary folate. Quantification of folate by HPLC showed that 5-formyl-5,6,7,8-tetrahydrofolic acid (502.1 μg/100 g DW) and 5,6,7,8-tetrahydrofolic acid (223.9 μg/100 g DW) as the most dominant forms of folate in M. oleifera leaves. The bioavailability of folate and the effects of folate depletion and repletion on biochemical and molecular markers of folate status were investigated in Wistar rats. Folate deficiency was induced by keeping the animals on a folate deficient diet with 1 % succinyl sulfathiazole (w/w). After the depletion period, animals were repleted with different levels of folic acid and M. oleifera leaves as a source of folate. Feeding the animals on a folate deficient diet for 7 weeks caused a significant (3.4-fold) decrease in serum folate content, compared to non-depleted control animals. Relative bioavailability of folate from dehydrated leaves of M. oleifera was 81.9 %. During folate depletion and repletion, no significant changes in liver glycine N-methyl transferase and 5-methyltetrahydrofolate-homocysteine methyltransferase expression were recorded. In RDA calculations, only 50 % of natural folate is assumed to be bioavailable. Therefore, the bioavailability of folate from Moringa is much higher, suggesting that M. oleifera based food can be used as a significant source of folate. PMID:26787970

  4. Orphan Toxin OrtT (YdcX) of Escherichia coli Reduces Growth during the Stringent Response

    PubMed Central

    Islam, Sabina; Benedik, Michael J.; Wood, Thomas K.

    2015-01-01

    Toxin/antitoxin (TA) systems are nearly universal in prokaryotes; toxins are paired with antitoxins which inactivate them until the toxins are utilized. Here we explore whether toxins may function alone; i.e., whether a toxin which lacks a corresponding antitoxin (orphan toxin) is physiologically relevant. By focusing on a homologous protein of the membrane-damaging toxin GhoT of the Escherichia coli GhoT/GhoS type V TA system, we found that YdcX (renamed OrtT for orphan toxin related to tetrahydrofolate) is toxic but is not part of TA pair. OrtT is not inactivated by neighboring YdcY (which is demonstrated to be a protein), nor is it inactivated by antitoxin GhoS. Also, OrtT is not inactivated by small RNA upstream or downstream of ortT. Moreover, screening a genomic library did not identify an antitoxin partner for OrtT. OrtT is a protein and its toxicity stems from membrane damage as evidenced by transmission electron microscopy and cell lysis. Furthermore, OrtT reduces cell growth and metabolism in the presence of both antimicrobials trimethoprim and sulfamethoxazole; these antimicrobials induce the stringent response by inhibiting tetrahydrofolate synthesis. Therefore, we demonstrate that OrtT acts as an independent toxin to reduce growth during stress related to amino acid and DNA synthesis. PMID:25643179

  5. Hyperhomocysteinemia-induced upper extremity deep vein thrombosis and pulmonary embolism in a patient with methyltetrahydrofolate reductase mutation: a case report and literature review.

    PubMed

    Gao, Lin; Kolanuvada, Bangaruraju; Naik, Geetha; Zhang, Yingzhong; Zhao, Min; Sun, Lili; Alaie, Dariush; Petrillo, Richard L

    2016-09-01

    The study highlights pulmonary embolism and deep vein thrombosis by methylene tetrahydrofolate reductase (MTHFR) deficiency-related hyperhomocysteinemia occurring in rare locations of left veins superior to the heart extensively. A 59-year-old white man with history of leg pain, smoking, weight loss, benign prostatic hyperplasia, lipoma and panic attack presented with shortness of breath and chest pain for 2 days precipitated by not feeling well for months. The diagnostic workup revealed pulmonary embolism and deep vein thrombosis in the left subclavian vein which extended throughout the left brachiocephalic vein to the superior vena cava and left jugular vein. Further workup showed moderate hyperhomocysteinemia with normal levels of vitamin B6, B12 and folic acid. Methylene tetrahydrofolate reductase genetic study found the patient to be homozygous for G677T variant. He was started on low-molecular-weight heparin and was discharged on oral anticoagulant. No recurrent thrombotic episodes were witnessed after 4 months of follow-up after discharge. PMID:26650456

  6. Folate metabolism in malaria

    PubMed Central

    Ferone, Robert

    1977-01-01

    It is known that malaria parasites are inhibited by sulfonamides and antifolate compounds, require 4-aminobenzoic acid for growth, and respond only partly to intact folic and folinic acids. Biochemical data obtained during the last decade on the synthesis of nucleic acid precursors and on folate enzymes in malaria support the hypothesis that malaria parasites are similar to microorganisms that synthesize folate cofactors de novo. Sulfa drugs inhibit plasmodial dihydropteroate synthase (EC 2.5.1.15). Pyrimethamine and many other antifolate compounds bind to tetrahydrofolate dehydrogenase (EC 1.5.1.3) of the parasite more tightly than to the host enzyme. However, the metabolic consequences of the depletion of folate cofactors as a result of drug inhibition are not yet known. Other areas to be studied are the origin of the pteridine moiety of folates, the addition of glutamate(s) in folate cofactor biosynthesis, the means by which intact, exogenous folates affect malarial growth, and demonstration of the enzymes and reactions involving N5-methyl tetrahydrofolate. PMID:338184

  7. Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase.

    PubMed Central

    Rothenberg, S P; Iqbal, M P

    1982-01-01

    A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of [3H]methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme. Images PMID:6952216

  8. Stage specific gene expression and cellular localization of two isoforms of the serine hydroxymethyltransferase in the protozoan parasite Leishmania.

    PubMed

    Gagnon, Dominic; Foucher, Aude; Girard, Isabelle; Ouellette, Marc

    2006-11-01

    Serine hydroxymethyltransferase (SHMT) catalyses the reversible conversion of serine and tetrahydrofolate to glycine and methylene-tetrahydrofolate. The recent completion of the genome sequence of Leishmania major revealed the presence of two genes coding for two isoforms of this protein. In silico analysis showed that one isoform had an extension at its N-terminus and was predicted to localize to the mitochondrion. The situation is different in other kinetoplastid parasites with only one SHMT encoding gene in Trypanosoma cruzi and no SHMT encoding gene in Trypanosoma brucei. The two L. major SHMT genes were cloned in frame with the green fluorescent protein and the resulting fusion proteins showed differential localization: the short form (SHMT-S) was found in the cytosol while the long one (SHMT-L) was found in an organelle that has hallmarks of the parasite mitochondrion. Indeed, SHMT-L had a similar cellular fractionation pattern as the mitochondrial HSP60 as determined by digitonin fractionation. Both SHMT-S and SHMT-L genes were expressed preferentially in the amastigote stage of the parasite and the RNA levels of SHMT-L could be modulated by glycine, serine, and folate. Overexpression of SHMT-S increased resistance to the antifolate methotrexate and to a lower level to the inhibitor thiosemicarbazide in a rich folate containing medium. These findings suggest that folate metabolism is compartmentalised in Leishmania and that SHMT RNA levels are responsive to environmental conditions. PMID:16876889

  9. Metabolomic fingerprint reveals that metformin impairs one-carbon metabolism in a manner similar to the antifolate class of chemotherapy drugs.

    PubMed

    Corominas-Faja, Bruna; Quirantes-Piné, Rosa; Oliveras-Ferraros, Cristina; Vazquez-Martin, Alejandro; Cufí, Sílvia; Martin-Castillo, Begoña; Micol, Vicente; Joven, Jorge; Segura-Carretero, Antonio; Menendez, Javier A

    2012-07-01

    Metabolomic fingerprint of breast cancer cells treated with the antidiabetic drug metformin revealed a significant accumulation of 5-formimino-tetrahydrofolate, one of the tetrahydrofolate forms carrying activated one-carbon units that are essential for the de novo synthesis of purines and pyrimidines. De novo synthesis of glutathione, a folate-dependent pathway interconnected with one-carbon metabolism was concomitantly depleted in response to metformin. End-product reversal studies demonstrated that thymidine alone leads to a significant but incomplete protection from metformin's cytostatic effects. The addition of the substrate hypoxanthine for the purine salvage pathway produces major rightward shifts in metformin's growth inhibition curves. Metformin treatment failed to activate the DNA repair protein ATM kinase and the metabolic tumor suppressor AMPK when thymidine and hypoxanthine were present in the extracellular milieu. Our current findings suggest for the first time that metformin can function as an antifolate chemotherapeutic agent that induces the ATM/AMPK tumor suppressor axis secondarily following the alteration of the carbon flow through the folate-related one-carbon metabolic pathways. PMID:22837425

  10. B vitamin polymorphisms and behavior: evidence of associations with neurodevelopment, depression, schizophrenia, bipolar disorder and cognitive decline.

    PubMed

    Mitchell, E Siobhan; Conus, Nelly; Kaput, Jim

    2014-11-01

    The B vitamins folic acid, vitamin B12 and B6 are essential for neuronal function, and severe deficiencies have been linked to increased risk of neurodevelopmental disorders, psychiatric disease and dementia. Polymorphisms of genes involved in B vitamin absorption, metabolism and function, such as methylene tetrahydrofolate reductase (MTHFR), cystathionine β synthase (CβS), transcobalamin 2 receptor (TCN2) and methionine synthase reductase (MTRR), have also been linked to increased incidence of psychiatric and cognitive disorders. However, the effects of these polymorphisms are often quite small and many studies failed to show any meaningful or consistent associations. This review discusses previous findings from clinical studies and highlights gaps in knowledge. Future studies assessing B vitamin-associated polymorphisms must take into account not just traditional demographics, but subjects' overall diet, relevant biomarkers of nutritional status and also analyze related genetic factors that may exacerbate behavioral effects or nutritional status. PMID:25173634

  11. [Stroke and iridodonesis revealing a homocystinuria caused by a compound heterozygous mutation of cystathionine beta-synthase].

    PubMed

    Lefaucheur, R; Triquenot-Bagan, A; Quillard, M; Genevois, O; Hannequin, D

    2008-01-01

    Iridodonesis or tremulous iris is a clinical sign of ectopia lentis which is frequently associated with homocystinuria. We present a forty-two-year-old woman victim of a left middle cerebral artery ischemic stroke. The clinical examination found bilateral iridodonesis and laboratory tests showed an increased level of serum homocysteine and homocystinuria. Homocystinuria was caused by a compound heterozygous I278T and D444N mutation of cystathionine beta-synthase (CBS) gene and also a C667T heterozygous polymorphism of methylene-tetrahydrofolate-reductase gene. This case was atypical because of the incomplete phenotype, development of complications in adulthood and the association of a rare compound heterozygous mutation of the CBS gene. PMID:18805305

  12. Isolation and characterization of a human intestinal bacterium, Eubacterium sp. ARC-2, capable of demethylating arctigenin, in the essential metabolic process to enterolactone.

    PubMed

    Jin, Jong-Sik; Zhao, Yu-Feng; Nakamura, Norio; Akao, Teruaki; Kakiuchi, Nobuko; Hattori, Masao

    2007-05-01

    Plant lignans, such as pinoresinol diglucoside, secoisolariciresinol diglucoside and arctiin, are metabolized to mammalian lignans, enterolactone or enterodiol, by human intestinal bacteria. Their metabolic processes include deglucosylation, ring cleavage, demethylation, dehydroxylation and oxidation. Here we isolated an intestinal bacterium capable of demethylating arctigenin, an aglycone of arctiin, to 2,3-bis(3,4-dihydroxybenzyl)butyrolactone (1) from human feces, and identified as an Eubacterium species (E. sp. ARC-2), which is similar to Eubacterium limosum on the basis of morphological and biochemical properties and 16S rRNA gene sequencing. By incubating with E. sp. ARC-2, arctigenin was converted to 1 through stepwise demethylation. Demethylation of arctigenin by E. sp. ARC-2 was tetrahydrofolate- and ATP-dependent, indicating that the reaction was catalyzed by methyltransferase. Moreover, E. sp. ARC-2 transformed secoisolariciresinol to 2,3-bis(3,4-dihydroxybenzyl)-1,4-butanediol by demethylation. PMID:17473433

  13. Distribution of pteroylglutamates in rat liver during regeneration after partial hepatectomy.

    PubMed Central

    Marchetti, M; Tolomelli, B; Formiggini, G; Bovina, C; Barbiroli, B

    1980-01-01

    1. Liver pteroylpolyglutamate distribution was studied during regeneration after partial hepatectomy in rats maintained under controlled feeding conditions. 2. Pteroylhexaglutamate, pteroylpentaglutamate and pteroyltetraglutamate concentrations decrease from 12 to 72 h after operation, then increase and reach normal values at 180 h. Pteroyltriglutamate concentration, already high at 12 h, remains so in the subsequent periods. Pteroyldiglutamate concentration was unchanged. Monoglutamate concentrations at first decrease, and at 180 h exceed normal values. 3. The decrease in polyglutamate derivatives with a high number of glutamate residues, at present considered to be the coenzyme forms of folate, could be related not to a decreased synthesis, but to a greater requirement for these compounds during the early periods of regeneration, when biosynthetic processes are markedly increased. It is indeed probable that the increased availability of the preferred substrate of pteroylpolyglutamate synthetase, i.e. tetrahydrofolate, enhances conversion of folate into coenzyme forms. PMID:7396880

  14. Bilateral Jugular Vein and Sigmoid Sinus Thrombosis Related to an Inherited Coagulopathy: An Unusual Presentation

    PubMed Central

    Altıntaş, Özge; Baran, Gözde; Mehdi, Elnur; Asil, Talip

    2014-01-01

    Internal jugular vein thrombosis (IJVT) is a rare condition associated with malignancy, coagulopathy, and trauma. The optimal management of any IJVT must be individualized and depends on the condition of the patient. Case Presentation. We report the case of a 42-year-old woman with a history of a first trimester spontaneous abortion. Apart from a tension-type headache, she had no neurological symptoms. She reported an incidental diagnosis of right-sided IJVT when she was evaluated for hyperthyroidism ultrasonographically. On ultrasonography, we observed bilateral jugular vein thrombosis. The patient was started on oral warfarin. Seven months later, when she was adequately anticoagulated, she developed a second thrombosis. According to the etiological workup, she had a mutation in the homozygous methylene tetrahydrofolate reductase (MTHFR) gene and reduced protein C levels and activity. Conclusion. This report illustrates an unusual presentation of a rare condition. In this case, the etiology was associated with the coagulopathy, which occurred despite adequate anticoagulation. PMID:25221687

  15. Subchorionic hematoma associated with thrombophilia: report of three cases.

    PubMed

    Heller, Debra S; Rush, Demaretta; Baergen, Rebecca N

    2003-01-01

    Subchorionic hematomas (SCHs) are associated with poor reproductive outcome including spontaneous abortions and stillbirth. Although many associations with maternal and prenatal factors have been reported, an underlying etiology has not been elucidated. We report three cases of SCHs associated with thrombophilias in the mother. One patient suffered a fetal demise at 30 wk gestational age, and two patients had second trimester losses. The mother of the 30-wk fetus was homozygous for mutations on the methylene-tetrahydrofolate reductase gene C677T. The other two patients had Protein S deficiency. SCHs may be associated with abnormal coagulative states suggesting that the underlying etiology of SCH may be related to hypercoagulability in the maternal circulation. The presence of a SCH may be the first indicator of an underlying thrombophilia and, thus, it is suggested that women who have placentas showing SCH should undergo a thrombophilia workup. PMID:12658542

  16. Incomplete Wood–Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi

    PubMed Central

    Zhuang, Wei-Qin; Yi, Shan; Bill, Markus; Brisson, Vanessa L.; Feng, Xueyang; Men, Yujie; Conrad, Mark E.; Tang, Yinjie J.; Alvarez-Cohen, Lisa

    2014-01-01

    The acetyl-CoA “Wood–Ljungdahl” pathway couples the folate-mediated one-carbon (C1) metabolism to either CO2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood–Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH3-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH3-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood–Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities. PMID:24733917

  17. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    PubMed Central

    Giancaspero, Teresa A.; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M.; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-01-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD “chaperone.” The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells. PMID:25954742

  18. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis.

    PubMed

    Giancaspero, Teresa A; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-01-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells. PMID:25954742

  19. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

    PubMed Central

    Ticak, Tomislav; Kountz, Duncan J.; Girosky, Kimberly E.; Krzycki, Joseph A.; Ferguson, Donald J.

    2014-01-01

    COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptors such as nitrate or fumarate, producing dimethylglycine and CO2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. In addition, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. They are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health. PMID:25313086

  20. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    NASA Astrophysics Data System (ADS)

    Giancaspero, Teresa Anna; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina Maria; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-04-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC 2.7.7.2) in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD "chaperone". The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.-) and dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4), respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

  1. Oxalyl-Coenzyme A Reduction to Glyoxylate Is the Preferred Route of Oxalate Assimilation in Methylobacterium extorquens AM1

    PubMed Central

    Schneider, Kathrin; Skovran, Elizabeth

    2012-01-01

    Oxalate catabolism is conducted by phylogenetically diverse organisms, including Methylobacterium extorquens AM1. Here, we investigate the central metabolism of this alphaproteobacterium during growth on oxalate by using proteomics, mutant characterization, and 13C-labeling experiments. Our results confirm that energy conservation proceeds as previously described for M. extorquens AM1 and other characterized oxalotrophic bacteria via oxalyl-coenzyme A (oxalyl-CoA) decarboxylase and formyl-CoA transferase and subsequent oxidation to carbon dioxide via formate dehydrogenase. However, in contrast to other oxalate-degrading organisms, the assimilation of this carbon compound in M. extorquens AM1 occurs via the operation of a variant of the serine cycle as follows: oxalyl-CoA reduction to glyoxylate and conversion to glycine and its condensation with methylene-tetrahydrofolate derived from formate, resulting in the formation of C3 units. The recently discovered ethylmalonyl-CoA pathway operates during growth on oxalate but is nevertheless dispensable, indicating that oxalyl-CoA reductase is sufficient to provide the glyoxylate required for biosynthesis. Analysis of an oxalyl-CoA synthetase- and oxalyl-CoA-reductase-deficient double mutant revealed an alternative, although less efficient, strategy for oxalate assimilation via one-carbon intermediates. The alternative process consists of formate assimilation via the tetrahydrofolate pathway to fuel the serine cycle, and the ethylmalonyl-CoA pathway is used for glyoxylate regeneration. Our results support the notion that M. extorquens AM1 has a plastic central metabolism featuring multiple assimilation routes for C1 and C2 substrates, which may contribute to the rapid adaptation of this organism to new substrates and the eventual coconsumption of substrates under environmental conditions. PMID:22493020

  2. Development of nitrilase promoter-derived inducible vectors for Streptomyces.

    PubMed

    Matsumoto, Masako; Hashimoto, Yoshiteru; Saitoh, Yuki; Kumano, Takuto; Kobayashi, Michihiko

    2016-06-01

    An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes. PMID:26923287

  3. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    DOE PAGESBeta

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products inmore » BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.« less

  4. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    SciTech Connect

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

  5. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxygenase with a bacterial type-I fatty acid synthase in E. coli.

    PubMed

    Coursolle, Dan; Lian, Jiazhang; Shanklin, John; Zhao, Huimin

    2015-09-01

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg L(-1) long chain alcohol/alkane products including a 57 mg L(-1) titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system. PMID:26135500

  6. Quantitative investigation of two metallohydrolases by X-ray absorption spectroscopy near-edge spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhao, W.; Chu, W. S.; Yang, F. F.; Yu, M. J.; Chen, D. L.; Guo, X. Y.; Zhou, D. W.; Shi, N.; Marcelli, A.; Niu, L. W.; Teng, M. K.; Gong, W. M.; Benfatto, M.; Wu, Z. Y.

    2007-09-01

    The last several years have witnessed a tremendous increase in biological applications using X-ray absorption spectroscopy (BioXAS), thanks to continuous advancements in synchrotron radiation (SR) sources and detector technology. However, XAS applications in many biological systems have been limited by the intrinsic limitations of the Extended X-ray Absorption Fine Structure (EXAFS) technique e.g., the lack of sensitivity to bond angles. As a consequence, the application of the X-ray absorption near-edge structure (XANES) spectroscopy changed this scenario that is now continuously changing with the introduction of the first quantitative XANES packages such as Minut XANES (MXAN). Here we present and discuss the XANES code MXAN, a novel XANES-fitting package that allows a quantitative analysis of experimental data applied to Zn K-edge spectra of two metalloproteins: Leptospira interrogans Peptide deformylase ( LiPDF) and acutolysin-C, a representative of snake venom metalloproteinases (SVMPs) from Agkistrodon acutus venom. The analysis on these two metallohydrolases reveals that proteolytic activities are correlated to subtle conformation changes around the zinc ion. In particular, this quantitative study clarifies the occurrence of the LiPDF catalytic mechanism via a two-water-molecules model, whereas in the acutolysin-C we have observed a different proteolytic activity correlated to structural changes around the zinc ion induced by pH variations.

  7. Docking modes of BB-3497 into the PDF active site--a comparison of the pure MM and QM/MM based docking strategies.

    PubMed

    Kumari, Tripti; Issar, Upasana; Kakkar, Rita

    2014-01-01

    Peptide deformylase (PDF) has emerged as an important antibacterial drug target. Considerable effort is being directed toward developing peptidic and non-peptidic inhibitors for this metalloprotein. In this work, the known peptidic inhibitor BB-3497 and its various ionization and tautomeric states are evaluated for their inhibition efficiency against PDF using a molecular mechanics (MM) approach as well as a mixed quantum mechanics/molecular mechanics (QM/MM) approach, with an aim to understand the interactions in the binding site. The evaluated Gibbs energies of binding with the mixed QM/MM approach are shown to have the best predictive power. The experimental pose is found to have the most negative Gibbs energy of binding, and also the smallest strain energy. A quantum mechanical evaluation of the active site reveals the requirement of strong chelation by the ligand with the metal ion. The investigated ligand chelates the metal ion through the two oxygens of its reverse hydroxamate moiety, particularly the N-O(-) oxygen, forming strong covalent bonds with the metal ion, which is penta-coordinated. In the uninhibited state, the metal ion is tetrahedrally coordinated, and hence chelation with the inhibitor is associated with an increase of the metal ion coordination. Thus, the strong binding of the ligand at the binding site is accounted for. PMID:25994638

  8. Proteome-wide analysis of the amino terminal status of Escherichia coli proteins at the steady-state and upon deformylation inhibition.

    PubMed

    Bienvenut, Willy V; Giglione, Carmela; Meinnel, Thierry

    2015-07-01

    A proteome wide analysis was performed in Escherichia coli to identify the impact on protein N-termini of actinonin, an antibiotic specifically inhibiting peptide deformylase (PDF). A strategy and tool suite (SILProNaQ) was employed to provide large-scale quantitation of N-terminal modifications. In control conditions, more than 1000 unique N-termini were identified with 56% showing initiator methionine removal. Additional modifications corresponded to partial or complete Nα-acetylation (10%) and N-formyl retention (5%). Among the proteins undergoing these N-terminal modifications, 140 unique N-termini from translocated membrane proteins were highlighted. The very early time-course impact of actinonin was followed after addition of bacteriostatic concentrations of the drug. Under these conditions, 26% of all proteins did not undergo deformylation any longer after 10 min, a value reaching more than 60% of all characterized proteins after 40 min of treatment. The N-formylation ratio measured on individual proteins increased with the same trend. Upon early PDF inhibition, two major categories of proteins retained their N-formyl group: a large number of inner membrane proteins and many proteins involved in protein synthesis including factors assisting the nascent chains in early cotranslational events. All MS data have been deposited in the ProteomeXchange with identifiers PXD001979, PXD002012 and PXD001983 (http://proteomecentral.proteomexchange.org/dataset/PXD001979, http://proteomecentral.proteomexchange.org/dataset/PXD002012 and http://proteomecentral.proteomexchange.org/dataset/PXD001983). PMID:26017780

  9. Staphylococcus aureus formyl-methionyl transferase mutants demonstrate reduced virulence factor production and pathogenicity.

    PubMed

    Lewandowski, Thomas; Huang, Jianzhong; Fan, Frank; Rogers, Shannon; Gentry, Daniel; Holland, Reannon; Demarsh, Peter; Aubart, Kelly; Zalacain, Magdalena

    2013-07-01

    Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. PMID:23571548

  10. Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

    PubMed Central

    Lewandowski, Thomas; Huang, Jianzhong; Fan, Frank; Rogers, Shannon; Gentry, Daniel; Holland, Reannon; DeMarsh, Peter; Zalacain, Magdalena

    2013-01-01

    Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. PMID:23571548

  11. Mononuclear Iron Enzymes Are Primary Targets of Hydrogen Peroxide Stress*

    PubMed Central

    Anjem, Adil; Imlay, James A.

    2012-01-01

    This study tested whether nonredox metalloenzymes are commonly charged with iron in vivo and are primary targets of oxidative stress because of it. Indeed, three sample mononuclear enzymes, peptide deformylase, threonine dehydrogenase, and cytosine deaminase, were rapidly damaged by micromolar hydrogen peroxide in vitro and in live Escherichia coli. The first two enzymes use a cysteine residue to coordinate the catalytic metal atom; it was quantitatively oxidized by the radical generated by the Fenton reaction. Because oxidized cysteine can be repaired by cellular reductants, the effect was to avoid irreversible damage to other active-site residues. Nevertheless, protracted H2O2 exposure gradually inactivated these enzymes, consistent with the overoxidation of the cysteine residue to sulfinic or sulfonic forms. During H2O2 stress, E. coli defended all three proteins by inducing MntH, a manganese importer, and Dps, an iron-sequestration protein. These proteins appeared to collaborate in replacing the iron atom with nonoxidizable manganese. The implication is that mononuclear metalloproteins are common targets of H2O2 and that both structural and metabolic arrangements exist to protect them. PMID:22411989

  12. Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein.

    PubMed

    Sachleben, Joseph R; McElroy, Craig A; Gollnick, Paul; Foster, Mark P

    2010-08-31

    Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT(12), composed of a tetramer of trimers, AT(3). Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT(3)), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT(3) to form inactive AT(12) is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK(a) for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors "active" trimeric AT and protonation favors "inactive" dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH. PMID:20713740

  13. Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

    PubMed

    Besold, Angelique N; Widger, Leland R; Namuswe, Frances; Michalek, Jamie L; Michel, Sarah L J; Goldberg, David P

    2016-04-22

    Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(ii). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(ii)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(ii) and Co(ii) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(ii) and Zn(ii) and a pentacoordinate geometry for Co(ii)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(ii)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(ii)-CP-1(CAHH) and Co(ii)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(ii) complexes. PMID:26936488

  14. Acyl-Carbon Bond Cleaving Cytochrome P450 Enzymes: CYP17A1, CYP19A1 and CYP51A1.

    PubMed

    Akhtar, Muhammad; Wright, J Neville

    2015-01-01

    Cytochrome P450 (P450 or CYP) enzymes in their resting state contain the heme-iron in a high-spin FeIII state. Binding of a substrate to a P450 enzyme allows transfer of the first electron, producing a Fe(II) species that reacts with oxygen to generate a low-spin iron superoxide intermediate (FeIII-O-O•) ready to accept the second electron to produce an iron peroxy anion intermediate (a, FeIII-O-O-). In classical monooxygenation reactions, the peroxy anion upon protonation fragments to form the reactive Compound I intermediate (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). However, when the substrate projects a carbonyl functionality, of the type b, at the active site as is the case for reactions catalyzed by CYP17A1, CYP19A1 and CYP51A1, the peroxy anion (FeIII-O-O-) is trapped, yielding a tetrahedral intermediate (c) that fragments to an acyl-carbon cleavage product (d plus an acid). Analogous acyl-carbon cleavage reactions are also catalyzed by certain hepatic P450s and CYP125A1 from Mycobacterium tuberculosis. A further improvisation on the theme is provided by aldehyde deformylases that convert long-chain aliphatic aldehydes to hydrocarbons. CYP17A1 is involved in the biosynthesis of corticoids as well as androgens. The flux toward these two classes of hormones seems to be regulated by cytochrome b 5, at the level of the acyl-carbon cleavage reaction. It is this regulation of CYP17A1 that provides a safety mechanism, ensuring that during corticoid biosynthesis, which requires 17α-hydroxylation by CYP17A1, androgen formation is avoided (Fig. 4.1). PMID:26002733

  15. Genome sequences of two closely related Vibrio parahaemolyticus phages, VP16T and VP16C.

    PubMed

    Seguritan, Victor; Feng, I-Wei; Rohwer, Forest; Swift, Mark; Segall, Anca M

    2003-11-01

    Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced. The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes. Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database. Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit). Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon. In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins. Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus. Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other. We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V. parahaemolyticus strain 16 host. Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V. parahaemolyticus. The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages. PMID:14563879

  16. Safety, Tolerability, and Efficacy of GSK1322322 in the Treatment of Acute Bacterial Skin and Skin Structure Infections

    PubMed Central

    Corey, Ralph; O'Riordan, William D.; Dumont, Etienne; Jones, Lori S.; Kurtinecz, Milena; Zhu, John Z.

    2014-01-01

    GSK1322322 represents a new class of antibiotics that targets an essential bacterial enzyme required for protein maturation, peptide deformylase. This multicenter, randomized, phase IIa study compared the safety, tolerability, and efficacy of GSK1322322 at 1,500 mg twice daily (b.i.d.) with that of linezolid at 600 mg b.i.d. in patients suspected of having Gram-positive acute bacterial skin and skin structure infections (ABSSSIs). The primary endpoint was assessment of the safety of GSK1322322, and a key secondary endpoint was the number of subjects with a ≥20% decrease in lesion area from the baseline at 48 and 72 h after treatment initiation. GSK1322322 administration was associated with mild-to-moderate drug-related adverse events, most commonly, nausea, vomiting, diarrhea, and headache. Adverse events (86% versus 74%) and withdrawals (28% versus 11%) were more frequent in the GSK1322322-treated group. Treatment with GSK1322322 and linezolid was associated with ≥20% decreases from the baseline in the lesion area in 73% (36/49) and 92% (24/26) of the patients, respectively, at the 48-h assessment and in 96% (44/46) and 100% (25/25) of the patients, respectively, at the 72-h assessment. Reductions in exudate/pus, pain, and skin infection scores were comparable between the GSK1322322 and linezolid treatments. The clinical success rates within the intent-to-treat population and the per-protocol population that completed this study were 67 and 91%, respectively, in the GSK1322322-treated group and 89 and 100%, respectively, in the linezolid-treated group. These results will be used to guide dose selection in future studies with GSK1322322 to optimize its tolerability and efficacy in patients with ABSSSIs. (This study has been registered at ClinicalTrials.gov under registration no. NCT01209078 and at http://www.gsk-clinicalstudyregister.com [PDF113414].) PMID:25136015

  17. Utility of the clinical practice of administering thrombophilic screening and antithrombotic prophylaxis with low-molecular-weight heparin to healthy donors treated with G-CSF for mobilization of peripheral blood stem cells.

    PubMed

    Martino, Massimo; Luise, Francesca; Oriana, Vincenzo; Console, Giuseppe; Moscato, Tiziana; Mammì, Corrado; Messina, Giuseppe; Massara, Elisabetta; Irrera, Giuseppe; Piromalli, Angela; Lombardo, Vincenzo Trapani; Laganà, Carmelo; Iacopino, Pasquale

    2007-01-01

    The aim of the study was to verify the utility of the clinical practice of administering thrombophilic screening and antithrombotic prophylaxis with low-molecular-weight heparin to healthy donors receiving granulocyte colony-stimulating factor to mobilize peripheral blood stem cells. Thrombophilia screening comprised of testing for factor V Leiden G1691A, prothrombin G20210A, the thermolabile variant (C677T) of the methylene tetrahydrofolate reductase gene, protein C, protein S, factor VIII and homocysteine plasmatic levels, antithrombin III activity, and acquired activated protein C resistance. We investigated prospectively 72 white Italian healthy donors, 39 men and 33 women, with a median age of 42 years (range, 18-65). Five donors (6.9%) were heterozygous carriers of Factor V Leiden G1691A; two healthy donors had the heterozygous prothrombin G20210A gene mutation; C677T mutation in the methylene tetrahydrofolate reductase gene was present in 34 (47.2%) donors in heterozygous and in 7 donors (9.7%) in homozygous. Acquired activated protein C resistance was revealed in 8 donors of the study (11.1%). The protein C plasmatic level was decreased in 3 donors (4.2%); the protein S level was decreased in 7 donors (9.7%). An elevated factor VIII dosage was shown in 10 donors (13.9%) and hyperhomocysteinemia in 9 donors (12.5%). Concentration of antithrombin III was in the normal range for all study group donors. The factor V Leiden mutation was combined with the heterozygous prothrombin G20210A in 2 cases and with protein S deficiency in one case; 2 healthy donors presented an associated deficiency of protein C and protein S. Although none of these healthy subjects had a previous history of thrombosis, low-molecular-weight heparin was administered to all donors during granulocyte colony-stimulating factor administration to prevent thrombotic events. No donor experienced short or long-term thrombotic diseases after a median follow-up of 29.2 months. Our data do not

  18. Significant Impact of the MTHFR Polymorphisms and Haplotypes on Male Infertility Risk

    PubMed Central

    Gupta, Nishi; Sarkar, Saumya; David, Archana; Gangwar, Pravin Kumar; Gupta, Richa; Khanna, Gita; Sankhwar, Satya Narayan; Khanna, Anil; Rajender, Singh

    2013-01-01

    Background Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate and affects the activity of cellular cycles participating in nucleotide synthesis, DNA repair, genome stability, maintenance of methyl pool, and gene regulation. Genetically compromised MTHFR activity has been suggested to affect male fertility. The objective of the present study was to find the impact on infertility risk of c.203G>A, c.1298A>C, and c.1793G>A polymorphisms in the MTHFR gene. Methods PCR-RFLP and DNA sequencing were used to genotype the common SNPs in the MTHFR gene in 630 infertile and 250 fertile males. Chi-square test was applied for statistical comparison of genotype data. Linkage disequilibrium between the SNPs and the frequency of common haplotypes were assessed using Haploview software. Biochemical levels of total homocysteine (tHcy) and folic acid were measured. Meta-analysis on c.1298A>C polymorphism was performed using data from ten studies, comprising 2734 cases and 2737 controls. Results c.203G>A and c.1298A>C were found to be unrelated to infertility risk. c.1793G>A was protective against infertility (P = 0.0008). c.677C>T and c.1793G>A were in significant LD (D’ = 0.9). Folic acid and tHcy level did not correlate with male infertility. Pooled estimate on c.1298A>C data from all published studies including our data showed no association of this polymorphism with male infertility (Odds ratio = 1.035, P = 0.56), azoospermia (Odds ratio = 0.97, P = 0.74), or oligoasthenoteratozoospermia (Odds ratio = 0.92, p = 0.29). Eight haplotypes with more than 1% frequency were detected, of which CCGA was protective against infertility (p = 0.02), but the significance of the latter was not seen after applying Bonferroni correction. Conclusion Among MTHFR polymorphisms, c.203G>A and c.1298A>C do not affect infertility risk and c.1793G>A is protective against infertility. Haplotype analysis

  19. [Cerebral venous sinus thrombosis associated with hyperhomocysteinemia due to combined deficiencies of folate and vitamin B12].

    PubMed

    Kanaya, Yuhei; Neshige, Shuichiro; Takemaru, Makoto; Shiga, Yuji; Takeshima, Shinichi; Kuriyama, Masaru

    2016-01-01

    A 63-year-old man was admitted to our hospital because of convulsive seizures. Radiological examinations revealed cerebral venous sinus thrombosis in the anterior part of the superior sagittal sinus. He had marked hyperhomocysteinemia (93.5 nmol/ml) due to combined deficiencies of folate and vitamin B12. He was T/T homozygous for methylene tetrahydrofolate reductase C677T polymorphism. He received a supplement therapy of vitamins. First, he was administered folate orally. After 3 months, the serum level of homocysteine decreased to 22.6 nmol/ml (an 86% reduction), but was still above the normal level. Next, an additional supplement therapy of vitamin B12 lowered the homocysteine level to normal (12.3 nmol/ml) after 4 months. These results showed that the increase of homocysteine levels in this patient was mainly caused by the deficiency of folate. Additionally, acquired risk factors like vitamin deficiencies increased the level of serum homocysteine to almost 100 nmol/ml. PMID:26797484

  20. Hemolysis and hyperhomocysteinemia caused by cobalamin deficiency: three case reports and review of the literature.

    PubMed

    Acharya, Utkarsh; Gau, Jen-Tzer; Horvath, William; Ventura, Paolo; Hsueh, Chung-Tsen; Carlsen, Wayne

    2008-01-01

    Concurrent hemolysis in patients with vitamin B12 deficiency is a well-recognized phenomenon and has been attributed to intramedullary destruction of erythrocytes (ineffective erythropoiesis). Recent studies revealed that homocysteine increased the risk of hemolysis in vitamin B12 deficiency in vitro and there is a high frequency (30%) of vitamin B12 deficiency in asymptomatic patients with homozygous methylene tetrahydrofolate reductase (MTHFR) C677T mutation, a known cause of hyperhomocysteinemia. Here we report three patients with MTHFR mutations and vitamin B12 deficiency presenting with hemolytic anemia and severely elevated homocysteine levels. Patients demonstrated complete resolution of hemolysis with simultaneous normalization of serum homocysteine levels after vitamin B12 treatments. We reviewed pertinent literature, and hypothesized that hemolytic anemia may be more prevalent in patients who have a coexisting MTHFR gene mutation and vitamin B12 deficiency possibly related to severely elevated homocysteine levels. The hemolysis in these cases occurred predominantly in peripheral blood likely due to the combined effects of structurally defective erythrocytes and homocysteine-induced endothelial damage with microangiopathy. PMID:19094231

  1. Pathophysiology, prevention, and potential treatment of neural tube defects.

    PubMed

    Manning, S M; Jennings, R; Madsen, J R

    2000-01-01

    Neural tube defects (NTD) remain a major cause of morbidity in spite of the reduction in liveborn incidence with periconceptional folic acid. However, the etiology remains unknown. This article reviews studies that address causation and potential treatment of NTD in humans and in animal models that resemble aspects of the common human NTD. Studies of nutritional markers of vitamin B12 and folic acid support a defect in homocysteine metabolism; a thermolabile variant of methylene tetrahydrofolate reductase, an enzyme that remethylates homocysteine to methionine, correlates with a risk of NTD in some human populations. Numerous mouse mutant models of NTD exist, attesting to the ease of disruption of neurulation, and a genetic basis for this malformation. Of these models, the curly tail mouse mutant most closely resembles the common human NTD. Folic acid does not prevent NTD in this model; however inositol supplementation does result in a significant reduction in incidence. Recent advances in fetal surgery, and evidence from mechanically created myelomeningocele in large animals amenable to surgical intervention suggest that the handicaps associated with myelomeningocele and associated Chiari Type II malformation may be prevented by in utero NTD closure. Success will depend on preservation of neurological tissue until such intervention is possible. Further research in animal models at the genetic and cellular levels, together with technological surgical advances, provide hope that prevention of more NTD and the associated handicaps may be possible. MRDD Research Reviews 6:6-14, 2000. PMID:10899792

  2. Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation1[C][W][OA

    PubMed Central

    Hung, Chiu-Yueh; Fan, Longjiang; Kittur, Farooqahmed S.; Sun, Kehan; Qiu, Jie; Tang, She; Holliday, Bronwyn M.; Xiao, Bingguang; Burkey, Kent O.; Bush, Lowell P.; Conkling, Mark A.; Roje, Sanja; Xie, Jiahua

    2013-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-to-nornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and S-adenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine N-demethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions. PMID:23221678

  3. The human flavoproteome

    PubMed Central

    Lienhart, Wolf-Dieter; Gudipati, Venugopal; Macheroux, Peter

    2013-01-01

    Vitamin B2 (riboflavin) is an essential dietary compound used for the enzymatic biosynthesis of FMN and FAD. The human genome contains 90 genes encoding for flavin-dependent proteins, six for riboflavin uptake and transformation into the active coenzymes FMN and FAD as well as two for the reduction to the dihydroflavin form. Flavoproteins utilize either FMN (16%) or FAD (84%) while five human flavoenzymes have a requirement for both FMN and FAD. The majority of flavin-dependent enzymes catalyze oxidation–reduction processes in primary metabolic pathways such as the citric acid cycle, β-oxidation and degradation of amino acids. Ten flavoproteins occur as isozymes and assume special functions in the human organism. Two thirds of flavin-dependent proteins are associated with disorders caused by allelic variants affecting protein function. Flavin-dependent proteins also play an important role in the biosynthesis of other essential cofactors and hormones such as coenzyme A, coenzyme Q, heme, pyridoxal 5′-phosphate, steroids and thyroxine. Moreover, they are important for the regulation of folate metabolites by using tetrahydrofolate as cosubstrate in choline degradation, reduction of N-5.10-methylenetetrahydrofolate to N-5-methyltetrahydrofolate and maintenance of the catalytically competent form of methionine synthase. These flavoenzymes are discussed in detail to highlight their role in health and disease. PMID:23500531

  4. An omics approach to rational feed: Enhancing growth in CHO cultures with NMR metabolomics and 2D-DIGE proteomics.

    PubMed

    Blondeel, Eric J M; Ho, Raymond; Schulze, Steffen; Sokolenko, Stanislav; Guillemette, Simon R; Slivac, Igor; Durocher, Yves; Guillemette, J Guy; McConkey, Brendan J; Chang, David; Aucoin, Marc G

    2016-09-20

    Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities. PMID:27496566

  5. Formyltetrahydrofolate Synthetase Gene Diversity in the Guts of Higher Termites with Different Diets and Lifestyles ▿ †

    PubMed Central

    Ottesen, Elizabeth A.; Leadbetter, Jared R.

    2011-01-01

    In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The “higher” termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the “lower” termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches. PMID:21441328

  6. Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay.

    PubMed Central

    Ha, T; Morgan, S L; Vaughn, W H; Eto, I; Baggott, J E

    1990-01-01

    The colorimetric assay for 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (phosphoribosylamino-imidazolecarboxamide formyltransferase; EC 2.1.2.3) has been extensively modified. The modified assay is based upon the short-term permanganate oxidation of the folate product, tetrahydrofolate (H4folate) to p-aminobenzoyl glutamate (pABG). The modified assay was used to detect the transformylase activity in crude extracts of peripheral-blood mononuclear cells (PBMCs). Azathioprine and its metabolite, thioinosinic acid (tIMP), are competitive inhibitors (with respect to AICAR) of the chicken liver transformylase and the transformylase from PBMCs of the MRL/lpr mouse, an animal model of systemic autoimmune disease. The Ki values of tIMP and azathioprine for the chicken liver enzyme are 39 +/- 4 microM and 120 +/- 10 microM, whereas the Ki values for the enzyme from PBMCs of the MRL/lpr mouse are 110 +/- 20 microM and 90 +/- 14 microM respectively. The anti-inflammatory drugs ibuprofen and naproxen are also inhibitors of the transformylase. PMID:2268263

  7. Folic acid mitigated cardiac dysfunction by normalizing the levels of tissue inhibitor of metalloproteinase and homocysteine-metabolizing enzymes postmyocardial infarction in mice

    PubMed Central

    Qipshidze, Natia; Tyagi, Neetu; Sen, Utpal; Givvimani, Srikanth; Metreveli, Naira; Lominadze, David

    2010-01-01

    Myocardial infarction (MI) results in significant metabolic derangement, causing accumulation of metabolic by product, such as homocysteine (Hcy). Hcy is a nonprotein amino acid generated during nucleic acid methylation and demethylation of methionine. Folic acid (FA) decreases Hcy levels by remethylating the Hcy to methionine, by 5-methylene tetrahydrofolate reductase (5-MTHFR). Although clinical trials were inconclusive regarding the role of Hcy in MI, in animal models, the levels of 5-MTHFR were decreased, and FA mitigated the MI injury. We hypothesized that FA mitigated MI-induced injury, in part, by mitigating cardiac remodeling during chronic heart failure. Thus, MI was induced in 12-wk-old male C57BL/J mice by ligating the left anterior descending artery, and FA (0.03 g/l in drinking water) was administered for 4 wk after the surgery. Cardiac function was assessed by echocardiography and by a Millar pressure-volume catheter. The levels of Hcy-metabolizing enzymes, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 5-MTHFR, were estimated by Western blot analyses. The results suggest that FA administered post-MI significantly improved cardiac ejection fraction and induced tissue inhibitor of metalloproteinase, CBS, CSE, and 5-MTHFR. We showed that FA supplementation resulted in significant improvement of myocardial function after MI. The study eluted the importance of homocysteine (Hcy) metabolism and FA supplementation in cardiovascular disease. PMID:20802128

  8. Optical observation of correlated motions in dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2015-03-01

    Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

  9. Role of Long-Range Protein Dynamics in Different Thymidylate Synthase Catalyzed Reactions

    PubMed Central

    Abeysinghe, Thelma; Kohen, Amnon

    2015-01-01

    Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein’s dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-mono­phosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function. PMID:25837629

  10. Correlated Protein Motion Measurements of Dihydrofolate Reductase Crystals

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2014-03-01

    We report the first direct measurements of the long range structural vibrational modes in dihydrofolate reductase (DHFR). DHFR is a universal housekeeping enzyme that catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetra-hydrofolate, with the aid of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). This crucial enzymatic role as the target for anti-cancer [methotrexate (MTX)], and other clinically useful drugs, has made DHFR a long-standing target of enzymological studies. The terahertz (THz) frequency range (5-100 cm-1), corresponds to global correlated protein motions. In our lab we have developed Crystal Anisotropy Terahertz Microscopy (CATM), which directly measures these large scale intra-molecular protein vibrations, by removing the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for mouse DHFR with the ligand binding of NADPH and MTX single crystals as well as Escherichia coli DHFR with the ligand binding of NADPH and MTX single crystals. This work is supported by NSF grant MRI2 grant DBI2959989.

  11. In situ enrichment of folate by microorganisms in beta-glucan rich oat and barley matrices.

    PubMed

    Kariluoto, Susanna; Edelmann, Minnamari; Nyström, Laura; Sontag-Strohm, Tuula; Salovaara, Hannu; Kivelä, Reetta; Herranen, Mirkka; Korhola, Matti; Piironen, Vieno

    2014-04-17

    The objective was to study folate production of yeast strains, bacteria isolated from oat bran, and selected lactic acid bacteria as well as one propionibacterium in oat and barley based models. Simultaneously, we aimed at sustaining the stability of viscosity, representing the physicochemical state of beta-glucan. Total folate contents were determined microbiologically and vitamers for selected samples by UHPLC. Folate in yeast cells comprised mainly 5-methyltetrahydrofolate and tetrahydrofolate. Folate production by microbes in YPD medium was different to that in cereal fermentations where vitamers included 5-methyltetrahydrofolate, 5,10-methenyltetrahydrofolate and formylated derivatives. Microbes producing significant amounts of folate without affecting viscosity were Saccharomyces cerevisiae ALKO743 and Candida milleri ABM4949 among yeasts and Pseudomonas sp. ON8 and Janthinobacterium sp. RB4 among bacteria. Net folate production was up to 120 ng/g after 24 h fermentation and could increase during 2-week storage. Glucose addition increased the proportion of 5-methyltetrahydrofolate. Streptococcus thermophilus ABM5097, Lactobacillus reuteri, and Propionibacterium sp. ABM5378 produced folate but in lower concentrations. Both endogenous and added microbes contribute to folate enhancement. Selection of microbes with folate producing capability and limited hydrolytic activity will enable the development of products rich in folate and beta-glucan. PMID:24561828

  12. Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase

    SciTech Connect

    Rothenbery, S.P.; Iqbal, M.P.

    1982-01-01

    A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahdrofolate:NADP/sup +/ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of (/sup 3/H)methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme.

  13. Formate metabolism in the cobalamin-inactivated rat.

    PubMed

    Deacon, R; Perry, J; Lumb, M; Chanarin, I

    1990-03-01

    Endogenous formate levels in blood and liver were assayed in rats both after inactivation of cobalamin (Cbl) by exposure to N2O as well as in air-breathing controls. The uptake of [14C]formate by tetrahydrofolate (H4folate) in bone marrow cells and liver homogenate and the incorporation of [14C]formate into purine, pyrimidine, methionine, serine and choline, was measured. There was a significant accumulation of endogenous formate following Cbl inactivation. There was impaired utilization of [14C]formate for single unit carbon (C1 unit) transfers mediated by folate in Cbl-inactivated tissues, other than for synthesis of adenine. The impairment was not accompanied by any accumulation of labelled methylH4folate indicating that methylfolate trapping played no part in impaired single carbon unit transfer. The effect of Cbl lack was a failure to form formylH4folate so that formate accumulated. The reason for this is not known. PMID:2334642

  14. Gene-Nutrient Interaction between Folate and Dihydrofolate Reductase in Risk for Adenomatous Polyp Occurrence: A Preliminary Report.

    PubMed

    Choi, Jeong-hwa; Yates, Zoe; Martin, Charlotte; Boyd, Lyndell; Ng, Xiaowei; Skinner, Virginia; Wai, Ron; Veysey, Martin; Lucock, Mark

    2015-01-01

    Folate and related gene variants are significant risk factors in the aetiology of colorectal cancer. Dihydrofolate reductase (DHFR) is critical in the metabolism of synthetic folic acid (pteroylmonoglutamatamic, PteGlu) to tetrahydrofolate following absorption. Therefore, the 19bp deletion variant of DHFR may lead to the alteration of folate-related colorectal disease susceptibility. This study examined the association between PteGlu and 19bp del-DHFR, and adenomatous polyp (AP) occurrence, an antecedent of colorectal cancer. A total of 199 subjects (162 controls and 37 AP cases) were analysed to determine dietary intake of total folate, natural methylfolate and synthetic PteGlu, level of erythrocyte folate and plasma homocysteine (tHcy), and genotype of 19bp del-DHFR. Dietary folate intake, erythrocyte folate, tHcy and 19bp del-DHFR variants did not independently predict the occurrence of AP. However, a gene-nutrient interaction was observed when subjects were stratified according to dietary folate intake. In subjects with a folate intake above the median value due to significant dietary PteGlu content, the presence of the 19bp-deletion allele decreased the risk for AP (OR=0.35, 95% CI: 0.13-0.97). However, such association was not evident in individuals with a folate intake below the median value. In conclusion, the finding suggests that folate nutrition and 19bp del-DHFR variation may interact to modify AP risk. PMID:26875486

  15. Turnover of the methyl moiety of 5-methyltetrahydropteroylglutamic acid in the cobalamin-inactivated rat.

    PubMed

    Lumb, M; Chanarin, I; Perry, J; Deacon, R

    1985-11-01

    The metabolism of the methyl group of 5-methyltetrahydrofolate was studied in rats in which cobalamin had been inactivated by exposure to nitrous oxide and in air-breathing control animals. Methylfolate labeled with [14C] in the methyl group and with [3H] in the pteridine-PABA portion was injected and the disappearance of [14C]H3- relative to [3H]folate was measured in liver. The half-time of the methyl group in the livers of control rats was two hours. There was no turnover of the methyl group for the first 72 hours after cobalamin inactivation. After 72 hours, there was a slow turnover of the methyl group, with a half-time of 43 hours. In control rats, it is assumed that the methyl group was metabolized by transfer to homocysteine to form methionine. In cobalamin-inactivated rats, it was shown that methylfolate was used as the substrate for forming folate polyglutamate, and analogues with 3, 4, and 5 glutamic acid residues were present. It is likely that oxidation of the methyl group by methylene tetrahydrofolate reductase occurs from folate polyglutamate containing six and seven glutamic acid residues, (Brody et al, Biochemistry 21: 276, 1982), since we were unable to demonstrate labeled methyl in longer chain analogues. PMID:4052631

  16. Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis

    SciTech Connect

    Doan,L.; Martucci, W.; Vargo, M.; Atreya, C.; Anderson, K.

    2007-01-01

    Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency {approx}200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is {approx}80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.

  17. [The influence of hereditary thrombophilic mechanisms on the degree of permanent intravascular coagulation in patients with artificial heart valves].

    PubMed

    Vavilova, T V; Sirotkina, O V; Razorenov, G I; Razorenova, T S; Emanuél', V L; Gritsenko, V V; Orlovskiĭ, P I; Doĭnikov, D N; Sharafutdinov, V E; Karpov, S A; Kuznetsov, A A; Kadinskaia, M I

    2004-01-01

    The genotyping of 40 patients with artificial heart valves (AHV) was performed after prosthesis of the mitral and aotic valves with bicuspid AHV (Medinzh-2 and CarboMedics). The patients took phenylin and varfarin. The patients' genotype was estimated by the thrombophylic genes: factor V Leiden (FVL), prothrombin G20210A, methylene tetrahydrofolate reductase C677T, G/A--455FGB, 4G/5G PAI-1, PI A1/A2 GPIIIa. The genes determining the thrombocytic activity or the vascular wall state substantially influence the third degree of the intensity of the permanent intravascular coagulation (PIC-3) independent of the degree of correction of hemostasis of oral anticoagulants. The addition of anti-aggregants to therapy is the only that can normalize functional activity of thrombocytes in patients with AHV having such defects. The laboratory detection of the genetic defects is of great practical importance for the determination of risk groups of formation of PIC-3 and the strategy of antithrombotic protection of patients with AHV. PMID:15651704

  18. Toward cell circuitry: Topological analysis of enzyme reaction networks via reaction route graphs

    NASA Astrophysics Data System (ADS)

    Datta, Ravindra; Vilekar, Saurabh A.; Fishtik, Ilie; Dittami, James P.

    2008-05-01

    The first step toward developing complete cell circuitry is to build quantitative networks for enzyme reactions. The conventional King-Altman-Hill (KAH) algorithm for topological analysis of enzyme networks, adapted from electrical networks, is based on “Reaction Graphs” that, unlike electrical circuits, are not quantitative, being straightforward renderings of conventional schematics of reaction mechanisms. Therefore, we propose the use of “Reaction Route (RR) Graphs” instead, as a more suitable graph-theoretical representation for topological analysis of enzyme reaction networks. The RR Graphs are drawn such that they are not only useful for visualizing the various reaction routes or pathways, but unlike Reaction Graphs possess network properties consistent with requisite kinetic, mass balance, and thermodynamic constraints. Therefore, they are better than the conventional Reaction Graphs for topological representation and analysis of enzyme reactions, both via the KAH methodology as well as via numerical matrix inversion. The difference between the two is highlighted based on the example of a single enzyme reaction network for the conversion of 7,8-dihydrofolate and NADPH into 5,6,7,8-tetrahydrofolate and NADP +, catalyzed by the enzyme dihydrofolate reductase.

  19. Design and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbes.

    PubMed

    Bar-Even, Arren; Noor, Elad; Flamholz, Avi; Milo, Ron

    2013-01-01

    Electrosynthesis is a promising approach that enables the biological production of commodities, like fuels and fine chemicals, using renewably produced electricity. Several techniques have been proposed to mediate the transfer of electrons from the cathode to living cells. Of these, the electroproduction of formate as a mediator seems especially promising: formate is readily soluble, of low toxicity and can be produced at relatively high efficiency and at reasonable current density. While organisms that are capable of formatotrophic growth, i.e. growth on formate, exist naturally, they are generally less suitable for bulk cultivation and industrial needs. Hence, it may be helpful to engineer a model organism of industrial relevance, such as E. coli, for growth on formate. There are numerous metabolic pathways that can potentially support formatotrophic growth. Here we analyze these diverse pathways according to various criteria including biomass yield, thermodynamic favorability, chemical motive force, kinetics and the practical challenges posed by their expression. We find that the reductive glycine pathway, composed of the tetrahydrofolate system, the glycine cleavage system, serine hydroxymethyltransferase and serine deaminase, is a promising candidate to support electrosynthesis in E. coli. The approach presented here exemplifies how combining different computational approaches into a systematic analysis methodology provides assistance in redesigning metabolism. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. PMID:23123556

  20. The Mitochondrial Folylpolyglutamate Synthetase Gene Is Required for Nitrogen Utilization during Early Seedling Development in Arabidopsis1[C][W][OA

    PubMed Central

    Jiang, Ling; Liu, Yanyan; Sun, Hong; Han, Yueting; Li, Jinglai; Li, Changkun; Guo, Wenzhu; Meng, Hongyan; Li, Sha; Fan, Yunliu; Zhang, Chunyi

    2013-01-01

    Investigations into the biochemical processes and regulatory mechanisms of nitrogen (N) utilization can aid in understanding how N is used efficiently in plants. This report describes a deficiency in N utilization in an Arabidopsis (Arabidopsis thaliana) transfer DNA insertion mutant of the mitochondrial folylpolyglutamate synthetase gene DFC, which catalyzes the conjugation of glutamate residues to the tetrahydrofolate during folate synthesis. The mutant seedlings displayed several metabolic changes that are typical of plant responses to low-N stress, including increased levels of starch and anthocyanin synthesis as well as decreased levels of soluble protein and free amino acid, as compared with those in wild-type seedlings when external N was sufficient. More striking changes were observed when dfc seedlings were grown under N-limited conditions, including shorter primary roots, fewer lateral roots, higher levels of glycine and carbon-N ratios, and lower N content than those in wild-type seedlings. Gene expression studies in mutant seedlings revealed altered transcript levels of several genes involved in folate biosynthesis and N metabolism. The biochemical and metabolic changes also suggested that N assimilation is drastically perturbed due to a loss of DFC function. The observation that elevated CO2 partly rescued the dfc phenotypes suggests that the alterations in N metabolism in dfc may be mainly due to a defect in photorespiration. These results indicate that DFC is required for N utilization in Arabidopsis and provide new insight into a potential interaction between folate and N metabolism. PMID:23129207

  1. Systematic integration of molecular profiles identifies miR-22 as a regulator of lipid and folate metabolism in breast cancer cells.

    PubMed

    Koufaris, C; Valbuena, G N; Pomyen, Y; Tredwell, G D; Nevedomskaya, E; Lau, C-He; Yang, T; Benito, A; Ellis, J K; Keun, H C

    2016-05-01

    Dysregulated microRNA (miRNA) mediate malignant phenotypes, including metabolic reprogramming. By performing an integrative analysis of miRNA and metabolome data for the NCI-60 cell line panel, we identified an miRNA cluster strongly associated with both c-Myc expression and global metabolic variation. Within this cluster the cancer-associated and cardioprotective miR-22 was shown to repress fatty acid synthesis and elongation in tumour cells by targeting ATP citrate lyase and fatty acid elongase 6, as well as impairing mitochondrial one-carbon metabolism by suppression of methylene tetrahydrofolate dehydrogenase/cyclohydrolase. Across several data sets, expression of these target genes were associated with poorer outcomes in breast cancer patients. Importantly, a beneficial effect of miR-22 on clinical outcomes in breast cancer was shown to depend on the expression levels of the identified target genes, demonstrating the relevance of miRNA/mRNA interactions to disease progression in vivo. Our systematic analysis establishes miR-22 as a novel regulator of tumour cell metabolism, a function that could contribute to the role of this miRNA in cellular differentiation and cancer development. Moreover, we provide a paradigmatic example of effect modification in outcome analysis as a consequence of miRNA-directed gene targeting, a phenomenon that could be exploited to improve patient prognosis and treatment. PMID:26477310

  2. Serine Hydroxymethyltransferase 1 and 2: Gene Sequence Variation and Functional Genomic Characterization

    PubMed Central

    Hebbring, Scott J.; Chai, Yubo; Ji, Yuan; Abo, Ryan P.; Jenkins, Gregory D.; Fridley, Brooke; Zhang, Jianping; Eckloff, Bruce W.; Wieben, Eric D.; Weinshilboum, Richard M.

    2012-01-01

    Serine hydroxymethyltransferase (SHMT) catalyzes the transfer of a beta carbon from serine to tetrahydrofolate (THF) to form glycine and 5,10-methylene-THF. This reaction plays an important role in neurotransmitter synthesis and metabolism. We set out to resequence SHMT1 and SHMT2, followed by functional genomic studies. We identified 87 and 60 polymorphisms in SHMT1 and SHMT2, respectively. We observed no significant functional effect of the 13 nonsynonymous SNPs in these genes, either on catalytic activity or protein quantity. We imputed additional variants across the two genes using “1000 Genomes” data, and identified 14 variants that were significantly associated (p-value < 1.0E-10) with SHMT1 mRNA expression in lymphoblastoid cell lines. Many of these SNPs were also significantly correlated with basal SHMT1 protein expression in 268 human liver biopsy samples. Reporter gene assays suggested that the SHMT1 promoter SNP, rs669340, contributed to this variation. Finally, SHMT1 and SHMT2 expression were significantly correlated with those of other Folate and Methionine Cycle genes at both the mRNA and protein levels. These experiments represent a comprehensive study of SHMT1 and SHMT2 gene sequence variation and its functional implications. In addition, we obtained preliminary indications that these genes may be co-regulated with other Folate and Methionine Cycle genes. PMID:22220685

  3. Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

    SciTech Connect

    Prasannan, Priya; Suliman, Huda S.; Robertus, Jon D.

    2009-05-15

    Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.

  4. The association between circulating total folate and folate vitamers with overall survival after postmenopausal breast cancer diagnosis.

    PubMed

    McEligot, Archana Jaiswal; Ziogas, Argyrios; Pfeiffer, Christine M; Fazili, Zia; Anton-Culver, Hoda

    2015-01-01

    We studied the relationship between plasma total folate and folate vitamer concentrations [5-methyltetrahydrofolic acid, pteroylglutamic acid (folic acid) and tetrahydrofolic acid] with overall survival after breast cancer diagnosis. A secondary aim was to assess the relationship between folic acid supplement use with circulating total folate and folate vitamer concentrations. Participants were postmenopausal women diagnosed with breast cancer (n = 498) with an average follow-up of 6.7 yr. Plasma total folate and folate vitamers were measured by isotope-dilution LC-MS/MS in samples collected at or postdiagnosis. Cox proportional multivariate hazards models (controlled for stage, age at diagnosis, body mass index, parity, hormone replacement therapy use, treatment, alcohol use, folic acid use, and energy intake), were used to assess overall survival after breast cancer diagnosis. We found that the relative risk of dying for women with plasma total folate concentrations in the highest quartile was 59% lower (hazard ratio: 0.41, 95% confidence interval: 0.19-0.90) compared with the lowest quartile. Data on supplement use showed that women taking folic acid supplements had significantly higher circulating total folate and folate vitamer concentrations (P < 0.0001), suggesting that increased folate consumption through diet and/or supplementation may improve prognosis after breast cancer diagnosis. PMID:25647689

  5. Comprehensive metabolic profiling of mono- and polyglutamated folates and their precursors in plant and animal tissue using liquid chromatography/negative ion electrospray ionisation tandem mass spectrometry.

    PubMed

    Garratt, Lee C; Ortori, Catharine A; Tucker, Gregory A; Sablitzky, Fred; Bennett, Malcolm J; Barrett, David A

    2005-01-01

    This work reports the use of reversed-phase ion-pair chromatography coupled to electrospray ionisation mass spectrometry for the simultaneous profiling of folate-based metabolites including natural folates, their polyglutamatyl derivatives and their biosynthetic precursors in plant and animal tissue. A simple sample preparation method, using 0.1% citric acid and ascorbic acid in ice-cold methanol, was used to extract and stabilise the folates, and three internal standards were used. Chromatography was on a C18 column using slow gradient elution with a mobile phase consisting of methanol/water with 5 mM dimethylhexylamine. Mass spectrometric detection was performed by multiple reaction monitoring in seven separate time windows in negative ion mode over the 25 min run time. Full, quantitative analysis was obtained for 16 folates and a 'semi-quantitative' analysis was possible for all other folates with up to eight conjugated glutamate residues by reference to structurally related calibration standards. The precision, accuracy and recovery of the method were generally within the accepted guidelines for a quantitative bioanalytical method and the method was linear over the range 0.2 to 10 ng of individual folate per sample. The method was applied to profile mono- and polyglutamated tetrahydrofolates (including subcellular analysis) in a range of plant species, including Arabidopsis, spinach, Brassica and wheat; the technique was also successfully applied to the profiling of folates in mouse tissue. PMID:16047318

  6. Computational identification of obligatorily autocatalytic replicators embedded in metabolic networks

    PubMed Central

    Kun, Ádám; Papp, Balázs; Szathmáry, Eörs

    2008-01-01

    Background If chemical A is necessary for the synthesis of more chemical A, then A has the power of replication (such systems are known as autocatalytic systems). We provide the first systems-level analysis searching for small-molecular autocatalytic components in the metabolisms of diverse organisms, including an inferred minimal metabolism. Results We find that intermediary metabolism is invariably autocatalytic for ATP. Furthermore, we provide evidence for the existence of additional, organism-specific autocatalytic metabolites in the forms of coenzymes (NAD+, coenzyme A, tetrahydrofolate, quinones) and sugars. Although the enzymatic reactions of a number of autocatalytic cycles are present in most of the studied organisms, they display obligatorily autocatalytic behavior in a few networks only, hence demonstrating the need for a systems-level approach to identify metabolic replicators embedded in large networks. Conclusion Metabolic replicators are apparently common and potentially both universal and ancestral: without their presence, kick-starting metabolic networks is impossible, even if all enzymes and genes are present in the same cell. Identification of metabolic replicators is also important for attempts to create synthetic cells, as some of these autocatalytic molecules will presumably be needed to be added to the system as, by definition, the system cannot synthesize them without their initial presence. PMID:18331628

  7. Experimental and Metabolic Modeling Evidence for a Folate-Cleaving Side-Activity of Ketopantoate Hydroxymethyltransferase (PanB)

    PubMed Central

    Thiaville, Jennifer J.; Frelin, Océane; García-Salinas, Carolina; Harrison, Katherine; Hasnain, Ghulam; Horenstein, Nicole A.; Díaz de la Garza, Rocio I.; Henry, Christopher S.; Hanson, Andrew D.; de Crécy-Lagard, Valérie

    2016-01-01

    Tetrahydrofolate (THF) and its one-carbon derivatives, collectively termed folates, are essential cofactors, but are inherently unstable. While it is clear that chemical oxidation can cleave folates or damage their pterin precursors, very little is known about enzymatic damage to these molecules or about whether the folate biosynthesis pathway responds adaptively to damage to its end-products. The presence of a duplication of the gene encoding the folate biosynthesis enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (FolK) in many sequenced bacterial genomes combined with a strong chromosomal clustering of the folK gene with panB, encoding the 5,10-methylene-THF-dependent enzyme ketopantoate hydroxymethyltransferase, led us to infer that PanB has a side activity that cleaves 5,10-methylene-THF, yielding a pterin product that is recycled by FolK. Genetic and metabolic analyses of Escherichia coli strains showed that overexpression of PanB leads to accumulation of the likely folate cleavage product 6-hydroxymethylpterin and other pterins in cells and medium, and—unexpectedly—to a 46% increase in total folate content. In silico modeling of the folate biosynthesis pathway showed that these observations are consistent with the in vivo cleavage of 5,10-methylene-THF by a side-activity of PanB, with FolK-mediated recycling of the pterin cleavage product, and with regulation of folate biosynthesis by folates or their damage products. PMID:27065985

  8. Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS †

    PubMed Central

    Choi, Sung-Chan; Chase, Theodore; Bartha, Richard

    1994-01-01

    The synthesis of methylmercury by Desulfovibrio desulfuricans LS was investigated on the basis of 14C incorporation from precursors and the measurement of relevant enzyme activities in cell extracts. The previously observed incorporation of C-3 from serine into methylmercury was confirmed by measurement of relatively high activities of serine hydroxymethyltransferase and other enzymes of this pathway. High rates of label incorporation into methylmercury from H14COO- and H14CO3- prompted the assay of enzymes of the acetyl coenzyme A (CoA) synthase pathway. These enzymes were found to be present but at activity levels much lower than those reported for acetogens. Propyl iodide inhibited methylmercury and acetyl-CoA syntheses to similar extents, and methylmercury synthesis was found to compete with acetyl-CoA synthesis for methyl groups. On the basis of these findings, we propose that in methylmercury synthesis by D. desulfuricans LS the methyl group is transferred from CH3-tetrahydrofolate via methylcobalamin. The methyl group may originate from C-3 of serine or from formate via the acetyl-CoA synthase pathway. These pathways are not unique to D. desulfuricans LS, and thus the ability of this bacterium to methylate mercury is most likely associated with the substrate specificity of its enzymes. PMID:16349435

  9. Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

    PubMed Central

    Doré, J.; Bryant, M. P.

    1990-01-01

    Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-14C] pyruvate, and chemically synthesized O-[methyl-14C]vanillate were used to further investigate the catabolism of one-carbon (C1) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO2 was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO2 readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO2 and pyruvate predominantly labeled the carboxyl group of acetate. No CO2 was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate. PMID:16348178

  10. Characterization of a folate-induced hypermotility response after bilateral injection into the rat nucleus accumbens

    SciTech Connect

    Stephens, R.L. Jr.

    1986-01-01

    The objective of these studies was to pharmacologically characterize the mechanism responsible for a folate-induced stimulation of locomotor activity in rats after bilateral injection into the nucleus accumbens region of the brain. Folic acid (FA) and 5-formyltetrahydrofolic acid (FTHF) produced this hypermotility response after intra-accumbens injection, while other reduced folic acid derivatives dihydrofolic acid, tetrahydrofolic acid, and 5-methyltetrahydrofolic acid were ineffective. Studies were designed to determine the role of catecholamines in the nucleus accumbens in the folate-induced hypermotility response. The findings suggest that the folate-induced response is dependent on intact neuronal dopamine stores, and is mediated by stimulation of dopamine receptors of the nucleus accumbens. However the folates do not appear to enhance dopaminergic neutransmission. Thus, FA and FTHF were inefficient at 1 mM concentrations in stimulating /sup 3/H-dopamine release from /sup 3/H-dopamine preloaded nucleus accumbens slices or dopamine from endogenous stores. Pteroic acid, the chemical precursor of folic acid which lacks the glutamate moiety, was ineffective in producing a stimulation of locomotor activity after intra-accumbens injection. Since glutamate is an excitatory amino acid (EAA), compounds characterized as EAA receptor antagonists were utilized to determine if the folate-induced hypermotility response is mediated by activation of EAA receptors in the nucleus accumbens. These results suggest that activation of quisqualate receptors of the nucleus accumbens may mediate the folate-induced hypermotility response.

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

    PubMed

    Väremo, Leif; Scheele, Camilla; Broholm, Christa; Mardinoglu, Adil; Kampf, Caroline; Asplund, Anna; Nookaew, Intawat; Uhlén, Mathias; Pedersen, Bente Klarlund; Nielsen, Jens

    2015-05-12

    Skeletal myocytes are metabolically active and susceptible to insulin resistance and are thus implicated in type 2 diabetes (T2D). This complex disease involves systemic metabolic changes, and their elucidation at the systems level requires genome-wide data and biological networks. Genome-scale metabolic models (GEMs) provide a network context for the integration of high-throughput data. We generated myocyte-specific RNA-sequencing data and investigated their correlation with proteome data. These data were then used to reconstruct a comprehensive myocyte GEM. Next, we performed a meta-analysis of six studies comparing muscle transcription in T2D versus healthy subjects. Transcriptional changes were mapped on the myocyte GEM, revealing extensive transcriptional regulation in T2D, particularly around pyruvate oxidation, branched-chain amino acid catabolism, and tetrahydrofolate metabolism, connected through the downregulated dihydrolipoamide dehydrogenase. Strikingly, the gene signature underlying this metabolic regulation successfully classifies the disease state of individual samples, suggesting that regulation of these pathways is a ubiquitous feature of myocytes in response to T2D. PMID:25937284

  12. Computer simulation of protein systems

    NASA Technical Reports Server (NTRS)

    Osguthorpe, D. J.; Dauber-Osguthorpe, P.; Wolff, J.; Kitson, D. H.; Hagler, A. T.

    1984-01-01

    Ligand binding to dihydrofolate reductase (DHFR) is discussed. This is an extremely important enzyme, as it is the target of several drugs (inhibitors) which are used clinically as antibacterials, antiprotozoals and in cancer chemotherapy. DHFR catalyzes the NADPH (reduced nicotinamide adenine dinucleotide phosphate) dependent reduction of dihydrofolate to tetrahydrofolate, which is used in several pathways of purine and pyrimidine iosynthesis, including that of thymidylate. Since DNA synthesis is dependent on a continuing supply of thymidylate, a blockade of DHFR resulting in a depletion of thymidylate can lead to the cessation of growth of a rapidly proliferating cell line. DHFR exhibits a significant species to species variability in its sensitivity to various inhibitors. For example, trimethoprim, an inhibitor of DHFR, binds to bacterial DHFR's 5 orders of magnitude greater than to vertebrate DHFR's. The structural mechanics, dynamics and energetics of a family of dihydrofolate reductases are studied to rationalize the basis for the inhibitor of these enyzmes and to understand the molecular basis of the difference in the binding constants between the species. This involves investigating the conformational changes induced in the protein on binding the ligand, the internal strain imposed by the enzyme on the ligand, the restriction of fluctuations in atom positions due to binding and the consequent change in entropy.

  13. Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

    PubMed Central

    Howell, E E; Foster, P G; Foster, L M

    1988-01-01

    The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium. Images PMID:2838456

  14. Folate content in tomato ( Lycopersicon esculentum ). influence of cultivar, ripeness, year of harvest, and pasteurization and storage temperatures.

    PubMed

    Iniesta, M Dolores; Pérez-Conesa, Darío; García-Alonso, Javier; Ros, Gaspar; Periago, M Jesús

    2009-06-10

    The effects of cultivar, on-vine ripening, and year of harvest on the folate content of raw tomatoes were studied. Folate content in hot-break tomato puree (HTP) subjected to pasteurization at different temperatures and its evolution during the shelf life of tomato juice were also investigated. 5-Methyltetrahydrofolate (5-CH(3)-H(4)-folate) was the only folate compound identified in raw tomatoes and HTP, but tetrahydrofolate (H(4)-folate) was 10% of the folate detected in tomato juice. The content of folates in raw tomatoes ranged from 4.1 to 35.3 microg/100 g of fresh weight and was highly influenced by all of the factors studied. No clear trend of folate content with ripening stage was observed. The extractability of 5-CH(3)-H(4)-folate from HTP increased significantly after pasteurization at 98 degrees C for 40 s, but higher temperatures decreased its content. Tomato juice showed folate losses during storage independent of the storage temperature. Folate losses were higher when tomato juice was packed in glass bottles than in Tetra Pak. PMID:19449809

  15. MTHFD1 controls DNA methylation in Arabidopsis

    PubMed Central

    Groth, Martin; Moissiard, Guillaume; Wirtz, Markus; Wang, Haifeng; Garcia-Salinas, Carolina; Ramos-Parra, Perla A.; Bischof, Sylvain; Feng, Suhua; Cokus, Shawn J.; John, Amala; Smith, Danielle C.; Zhai, Jixian; Hale, Christopher J.; Long, Jeff A.; Hell, Ruediger; Díaz de la Garza, Rocío I.; Jacobsen, Steven E.

    2016-01-01

    DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases. PMID:27291711

  16. Sulfur deficiency changes mycosporine-like amino acid (MAA) composition of Anabaena variabilis PCC 7937: a possible role of sulfur in MAA bioconversion.

    PubMed

    Singh, Shailendra P; Klisch, Manfred; Sinha, Rajeshwar P; Häder, Donat-Peter

    2010-01-01

    In the present investigation we show for the first time that bioconversion of a primary mycosporine-like amino acid (MAA) into a secondary MAA is regulated by sulfur deficiency in the cyanobacterium Anabaena variabilis PCC 7937. This cyanobacterium synthesizes the primary MAA shinorine (RT = 2.2 min, lambda(max) = 334 nm) under normal conditions (PAR + UV-A + UV-B); however, under sulfur deficiency, a secondary MAA palythine-serine (RT = 3.9 min, lambda(max) = 320 nm) appears. Addition of methionine to sulfur-deficient cultures resulted in the disappearance of palythine-serine, suggesting the role of primary MAAs under sulfur deficiency in recycling of methionine by donating the methyl group from the glycine subunit of shinorine to tetrahydrofolate to regenerate the methionine from homocysteine. This is also the first report for the synthesis of palythine-serine by cyanobacteria which has so far been reported only from corals. Addition of methionine also affected the conversion of mycosporine-glycine into shinorine, consequently, resulted in the appearance of mycosporine-glycine (RT = 3.6 min, lambda(max) = 310 nm). Our results also suggest that palythine-serine is synthesized from shinorine. Based on these results we propose that glycine decarboxylase is the potential enzyme that catalyzes the bioconversion of shinorine to palythine-serine by decarboxylation and demethylation of the glycine unit of shinorine. PMID:20456655

  17. Role of long-range protein dynamics in different thymidylate synthase catalyzed reactions.

    PubMed

    Abeysinghe, Thelma; Kohen, Amnon

    2015-01-01

    Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein's dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-mono-phosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function. PMID:25837629

  18. Modulation of the cytotoxicity of 3'-azido-3'-deoxythymidine and methotrexate after transduction of folate receptor cDNA into human cervical carcinoma: identification of a correlation between folate receptor expression and thymidine kinase activity.

    PubMed

    Sun, X L; Jayaram, H N; Gharehbaghi, K; Li, Q J; Xiao, X; Antony, A C

    1999-02-15

    Cervical carcinoma is an AIDS-defining illness. The expression of folate receptors (FRs) in cervical carcinoma (HeLa-IU1) cells was modulated by stable transduction of FR cDNA encapsidated in recombinant adeno-associated virus-2 in the sense and antisense orientation (sense and antisense cells, respectively). Although sense cells proliferated slower than antisense or untransduced cells in vivo and in vitro in 2% (but not 10%) FCS, [methyl-3H]thymidine incorporation into DNA was significantly increased in sense cells in 10% serum; therefore, the basis for this discrepancy was investigated. The activity of thymidine kinase (TK) was subsequently directly correlated with the extent of FR expression in single cell-derived clones of transduced cells. This elevated TK activity was not a result of recruitment of the salvage pathway based on the presence of adequate dTTP pools, normal thymidylate synthase (TS) activity, persistence of increased thymidine incorporation despite the exogenous provision of excess 5,10-methylene-tetrahydrofolate, and documentation of adequate folates in sense cells. The increase in TK activity conferred significant biological properties to sense cells (but not antisense or untransduced cells) as demonstrated by augmented phosphorylation of 3'-azido-3'-deoxythymidine (AZT) and concomitantly greater sensitivity to the cytotoxic effects of AZT. Conversely, sense cells were highly resistant to methotrexate, but this was reversed by the addition of AZT. The direct correlation of FR expression and TK activity indicates a previously unrecognized consequence of FR overexpression. PMID:10029088

  19. Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors.

    PubMed

    Capasso, Clemente; Supuran, Claudiu T

    2014-06-01

    Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families. PMID:23627736

  20. In Vivo Analysis of Folate Coenzymes and Their Compartmentation in Saccharomyces Cerevisiae

    PubMed Central

    McNeil, J. B.; Bognar, A. L.; Pearlman, R. E.

    1996-01-01

    In eukaryotes, enzymes responsible for the interconversion of one-carbon units exist in parallel in both mitochondria and the cytoplasm. Strains of Saccharomyces cerevisiae were constructed that possess combinations of gene disruptions at the SHM1 [mitochondrial serine hydroxymethyltransferase (SHMTm)], SHM2 [cytoplasmic SHMT (SHMTc)], MIS1 [mitochondrial C(1)-tetrahydrofolate synthase (C(1)-THFSm)], ADE3 [cytoplasmic C(1)-THF synthase (C(1)-THFSc)], GCV1 [glycine cleavage system (GCV) protein T], and the GLY1 (involved in glycine synthesis) loci. Analysis of the in vivo growth characteristics and phenotypes was used to determine the contribution to cytoplasmic nucleic acid and amino acid anabolism by the mitochondrial enzymes involved in the interconversion of folate coenzymes. The data indicate that mitochondria transport formate to the cytoplasmic compartment and mitochondrial synthesis of formate appears to rely primarily on SHMTm rather than the glycine cleavage system. The glycine cleavage system and SHMTm cooperate to specifically synthesize serine. With the inactivation of SHM1, however, the glycine cleavage system can make an observable contribution to the level of mitochondrial formate. Inactivation of SHM1, SHM2 and ADE3 is required to render yeast auxotrophic for TMP and methionine, suggesting that TMP synthesized in mitochondria may be available to the cytoplasmic compartment. PMID:8852837

  1. MTHFD1 controls DNA methylation in Arabidopsis.

    PubMed

    Groth, Martin; Moissiard, Guillaume; Wirtz, Markus; Wang, Haifeng; Garcia-Salinas, Carolina; Ramos-Parra, Perla A; Bischof, Sylvain; Feng, Suhua; Cokus, Shawn J; John, Amala; Smith, Danielle C; Zhai, Jixian; Hale, Christopher J; Long, Jeff A; Hell, Ruediger; Díaz de la Garza, Rocío I; Jacobsen, Steven E

    2016-01-01

    DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases. PMID:27291711

  2. The metabolism of 5-methyltetrahydropteroyl-L-glutamic acid and its oxidation products in the rat.

    PubMed Central

    Kennelly, J C; Blair, J A; Pheasant, A E

    1982-01-01

    Folate metabolism in the rat was investigated using radiolabelled 5-methyltetrahydropteroylglutamate (5-CH3-H4PteGlu) and its oxidation products. 5-CH3-H4PteGlu is absorbed completely from the intestine, although in some preparations it is an equimolecular mixture of C-6 epimers, only one of which is naturally present in biological systems. The methyl group is incorporated into non-folate compounds, including methionine and creatine. No evidence was observed for the oxidation of the methyl group of 5-CH3-H4PteGlu to form other folate types. The tetrahydrofolate moiety of 5-CH3-H4PteGlu is metabolized in a similar manner to folic acid, forming formyl folates and tissue polyglutamates, and is catabolized by scission. The triazine oxidation product of 5-CH3-H4PteGlu is not metabolized by the rat or its gut microflora. 5-Methyl-5,6-dihydropteroylglutamate, however, is assimilated into the folate pool, but is substantially broken down by passage through the gut. The possible implication of this in scorbutic diets is discussed. PMID:7150248

  3. Identification and characterization of trimethylamine N-oxide (TMAO) demethylase and TMAO permease in Methylocella silvestris BL2.

    PubMed

    Zhu, Yijun; Jameson, Eleanor; Parslow, Rosemary A; Lidbury, Ian; Fu, Tiantian; Dafforn, Timothy R; Schäfer, Hendrik; Chen, Yin

    2014-10-01

    Methylocella silvestris, an alphaproteobacterium isolated from a forest soil, can grow on trimethylamine N-oxide (TMAO) as a sole nitrogen source; however, the molecular and biochemical mechanisms underpinning its growth remain unknown. Marker-exchange mutagenesis enabled the identification of several genes involved in TMAO metabolism, including Msil_3606, a permease of the amino acids-polyamine (APC) superfamily, and Msil_3603, consisting of an N-terminal domain of unknown function (DUF1989) and a C-terminal tetrahydrofolate-binding domain. Null mutants of Msil_3603 and Msil_3606 can no longer grow on TMAO. Purified Msil_3603 from recombinant Escherichia coli can convert TMAO to dimethylamine and formaldehyde (1 TMAO → 1 dimethylamine + 1 formaldehyde), confirming that it encodes a bona fide TMAO demethylase (Tdm). Tdm of M. silvestris and eukaryotic Tdms have no sequence homology and contrasting characteristics. Recombinant Tdm of M. silvestris appears to be hexameric, has a high affinity for TMAO (Km = 3.3 mM; Vmax = 21.7 nmol min(-1)  mg(-1) ) and only catalyses demethylation of TMAO and a structural homologue, dimethyldodecylamine N-oxide. Our study has contributed to the understanding of the genetic and biochemical mechanisms for TMAO degradation in M. silvestris. PMID:25088783

  4. Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii.

    PubMed

    Shanmugasundaram, T; Ragsdale, S W; Wood, H G

    1988-07-01

    Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum. PMID:2855585

  5. Methylenetetrahydrofolate dehydrogenase from Clostridium formicoaceticum and methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase (combined) from Clostridium thermoaceticum

    SciTech Connect

    Ljungdahl, L.G.; O'Brien, W.E.; Moore, M.R.; Liu, M.T.

    1980-01-01

    Methylenetetrahydrofolate dehydrogenase is widely distributed and has been found in every cell type investigated. The NAD-specific enzyme has been purified to homogeneity from Clostridium formicoaceticum and the NADP-specific enzyme has been obtained from Clostridium thermoaceticum. Other sources of the NADP-specific enzyme are Streptococcus species, Escherichia coli, Clostridium cylindrosporum, Salmonella typhimurium, yeast, liver from various animals, calf thymus, and plants. The NAD-specific enzyme has been demonstrated in Acetobacterium woodii, some methane bacteria, and in Ehrlich ascites tumor cells. Of considerable interest are the observations that in porcine and ovine livers, as well as in yeast, methylenetetrahydrofolate dehydrogenase purified to homogeneity also contains methylenetetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities. Now it appears that the purified methylenetetrahydrofolate dehydrogenase from C. thermoaceticum also has cyclohydrolase but not synthetase activity. Methylenetetrahydrofolate dehydrogenase has been discussed previously in this series, as has methenyltetrahydrofolate cyclohydrolase. In C. formicoaceticum and C. thermoaceticum these tetrahydrofolate-dependent enzymes participate in a sequence of metabolic reactions by which carbon dioxide is reduced to the methyl group of 5-methyltetrahydrofolate which in turn is utilized for the synthesis of acetate. This pathway provides the mechanism for disposing of reducing equivalents generated in glycolysis.

  6. Effects of methotrexate on the developments of heart and vessel in zebrafish.

    PubMed

    Sun, Shuna; Gui, Yonghao; Wang, Yuexiang; Qian, Linxi; Liu, Xuefei; Jiang, Qiu; Song, Houyan

    2009-01-01

    Methotrexate (MTX), an antagonist of folic acid, can inhibit dihydrofolate reductase (DHFR) which is of great importance in the synthesis of tetrahydrofolic acid and embryonic development. In this study, we found that after being exposed to 1.5 mM MTX at 6-10 hours post-fertilization, zebrafish embryos fail to form normal cardiovascular system. In MTX-treated embryos, the morphological development of ventricle and atrium was disrupted, the cardiac twist was abnormal, the heart rate and ventricular shortening fraction were reduced, and the vascular development was disrupted. We also found that either microinjection with dhfr-gfp mRNA or treatment with folinic acid calcium salt pentahydrate (CF) could cause improved development in the heart and vessels in MTX-treated embryos, which proved that MTX induced the malformations by inhibiting DHFR. The transcript levels of genes such as hand2, mef2a, mef2c, and flk-1 were reduced in MTXtreated embryos. Compared with the MTX-treated group, the transcript levels of hand2, mef2a, mef2c, and flk-1 were increased in the MTX 1 dhfr-gfp mRNA injected group and in the MTX 1 CF group. Our results indicated that the disrupted development of the heart and vessels in MTX-treated embryos is related to the reduced transcript levels of hand2, mef2a, mef2c, and flk-1. PMID:19129954

  7. Structure of the cobalamin-binding protein of a putative O-demethylase from Desulfitobacterium hafniense DCB-2

    SciTech Connect

    Sjuts, Hanno; Dunstan, Mark S.; Fisher, Karl; Leys, David

    2013-08-01

    The first crystal structure of the vitamin B12-binding protein from a three-component O-demethylase enzyme system is reported. During O-demethylation methyl groups are transferred from phenyl methyl ethers to tetrahydrofolate via methyl-B12 intermediates. This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5 Å resolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on conformation similar to other cobalamin-binding domains that catalyse methyl-transfer reactions. EPR spectroscopy of CobDH confirms cobalamin binding and reveals the presence of a cob(III)alamin superoxide, indicating binding of oxygen to the fully oxidized cofactor. These data provide the first structural insights into the methyltransferase reactions that occur during O-demethylation by D. hafniense.

  8. Biochemical and histochemical features of human cultured cells (EUE) adapted to hypertonic medium.

    PubMed

    Bolognani, L; Fantin, A M; Conti, A M; Gervaso, M V; Salè, M F

    1978-01-01

    EUE cells from a human heteroploid line cultured in hypertonic medium (0.274 M NaCl) modify their lipid pattern: sulfolipid concentration reaches 86 to 90 microgram/mg protein whilst it ranges between 19 to 32 microgram/mg in cells cultured in isotonic medium. Ganglioside concentration reaches 2.6 nmoles of sialic acid/mg protein (after 75 days) and 13 (after 85 days) in hypertonic saline medium. Whilst it is 0.5 in isotonic medium. Phospholipid concentration does not show any similar change. Cytoenzymatic analysis reveals that dehydrogenases (lactate, G-6-P dehydrogenases, tetrahydrofolate reductase and NADH diaphorase) appear strongly enhanced in cells grown on hypertonic medium. On the contrary higher acid phosphatase and ATPase activity was demonstrable in cells grown on isotonic medium. These results are similar (except for ATPase activity) to those observed in salt secreting glands involved in strong osmotic work. The results are discussed in relation to the problem of energy supply in cells performing osmotic work. PMID:151474

  9. Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

    PubMed

    Strickland, Kyle C; Hoeferlin, L Alexis; Oleinik, Natalia V; Krupenko, Natalia I; Krupenko, Sergey A

    2010-01-15

    4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions. PMID:19933275

  10. Inherited prothrombotic risk factors and cerebral venous thrombosis.

    PubMed

    Hillier, C E; Collins, P W; Bowen, D J; Bowley, S; Wiles, C M

    1998-10-01

    Fifteen patients with cerebral venous thrombosis were ascertained retrospectively. Their case notes were reviewed, and stored or new blood was assayed for factor V Leiden (FVL) mutation, prothrombin gene mutation 20201A, and 5,10 methylene tetrahydrofolate reductase (MTHFR) C677T mutation. A clinical risk factor was identified in 13 patients--the oral contraceptive pill (5), puerperium (1), HRT (1), mastoiditis (1), dehydration (1), lumbar puncture and myelography (1), carcinoma (1), lupus anticoagulant (2). In addition, two patients had the FVL mutation and five (one of whom also had the FVL mutation) were homozygous for the MTHFR mutation. The latter showed a higher than expected frequency compared to 300 healthy controls from South Wales (OR 3.15.95% Cl 1.01-9.83). No patient had the prothrombin 20201A mutation. Two patients died and three had a monocular visual deficit following anticoagulation (13) or thrombolytic (2) treatment, but there was no association between the presence of a primary prothrombotic risk factor and outcome. These results confirm the importance of investigating patients for both clinical predisposing factors and primary prothrombotic states. PMID:10024925

  11. MTHFR C677T and A1298C polymorphisms as predictors of radiotherapy response in head and neck squamous cell carcinoma.

    PubMed

    Anders, Q S; Stur, E; Agostini, L P; Garcia, F M; Reis, R S; Santos, J A; Mendes, S O; Maia, L L; Peterle, G T; Stange, V; Carvalho, M B; Tajara, E H; Santos, M; Silva-Conforti, A M A; Louro, I D

    2015-01-01

    The C677T and A1298C polymorphisms in methylene-tetrahydrofolate reductase (MTHFR), which regulates the release of active folate in the body, may have reduced activity. Given that folate participates in important intracellular pathways, such as nucleotide synthesis and biomolecule methylation, it seems plausible that patients with head and neck squamous cell carcinoma (HNSCC) may respond differently to radiotherapy treatments, based on genetic polymor-phisms. Therefore, this study sought to understand the role of these polymorphisms in HNSCC patient radiotherapy response. Genotypes were detected by PCR-RFLP after extraction of DNA from peripheral blood lymphocytes. Survival curves were analyzed by the Kaplan- Meier model, and significant differences were analyzed by the Wil-coxon test. Response to radiotherapy in patients with laryngeal SCC was significantly associated with the MTHFR C677T polymorphism (P = 0.030). Indeed, the presence of at least one T allele decreases the mortality rate up to 3-fold. Therefore, we propose that MTHFR C677T may represent a putative biomarker for radiotherapy prognosis in la-ryngeal SCC patients. PMID:26535623

  12. Evolution in an oncogenic bacterial species with extreme genome plasticity: Helicobacter pylori East Asian genomes

    PubMed Central

    2011-01-01

    Background The genome of Helicobacter pylori, an oncogenic bacterium in the human stomach, rapidly evolves and shows wide geographical divergence. The high incidence of stomach cancer in East Asia might be related to bacterial genotype. We used newly developed comparative methods to follow the evolution of East Asian H. pylori genomes using 20 complete genome sequences from Japanese, Korean, Amerind, European, and West African strains. Results A phylogenetic tree of concatenated well-defined core genes supported divergence of the East Asian lineage (hspEAsia; Japanese and Korean) from the European lineage ancestor, and then from the Amerind lineage ancestor. Phylogenetic profiling revealed a large difference in the repertoire of outer membrane proteins (including oipA, hopMN, babABC, sabAB and vacA-2) through gene loss, gain, and mutation. All known functions associated with molybdenum, a rare element essential to nearly all organisms that catalyzes two-electron-transfer oxidation-reduction reactions, appeared to be inactivated. Two pathways linking acetyl~CoA and acetate appeared intact in some Japanese strains. Phylogenetic analysis revealed greater divergence between the East Asian (hspEAsia) and the European (hpEurope) genomes in proteins in host interaction, specifically virulence factors (tipα), outer membrane proteins, and lipopolysaccharide synthesis (human Lewis antigen mimicry) enzymes. Divergence was also seen in proteins in electron transfer and translation fidelity (miaA, tilS), a DNA recombinase/exonuclease that recognizes genome identity (addA), and DNA/RNA hybrid nucleases (rnhAB). Positively selected amino acid changes between hspEAsia and hpEurope were mapped to products of cagA, vacA, homC (outer membrane protein), sotB (sugar transport), and a translation fidelity factor (miaA). Large divergence was seen in genes related to antibiotics: frxA (metronidazole resistance), def (peptide deformylase, drug target), and ftsA (actin-like, drug target

  13. Impact of thrombophilic genes mutations on thrombosis risk in Egyptian nonmetastatic cancer patients.

    PubMed

    Wahba, Mona Ahmed; Ismail, Mona Ahmed; Saad, Abeer Attia; Habashy, Deena Mohamed; Hafeez, Zeinab Mohamed Abdel; Boshnak, Noha Hussein

    2015-04-01

    Venous thromboembolism (VTE) is a common complication in cancer patients. Several genetic risk factors related to thrombophilia are known; however, their contributions to thrombotic tendency in cancer patients have conflicting results. We aimed to determine the prevalence of factor V Leiden (FVL), prothrombin (PTH) G20210A and methylene tetrahydrofolate reductase (MTHFR) C677T gene polymorphisms in Egyptian nonmetastatic cancer patients and their influence on thrombosis risk in those patients. Factor V Leiden, PTH G20210A and MTHFR C677T polymorphisms were detected in 40 cancer patients with VTE (group 1) and 40 cancer patients with no evidence of VTE (group 2) by PCR-based DNA analysis. Factor V and MTHFR mutations were higher in group 1 than in group 2 (factor V heterozygous mutation: 20 vs. 7.5%, homozygous mutation: 10 vs. 2.5%; MTHFR heterozygous mutation: 40 vs. 25%, homozygous mutation 5 vs. 0%, respectively) (P = 0.03). Mortality rate was higher in group 1 (75%) than in group 2 (25%; P < 0.001). No difference was found between those groups regarding PTH mutation (P = 1). Mortality rate was higher in the presence of homozygous and heterozygous factor V mutation (100 and 82%, respectively) compared to the wild type (41%) (P = 0.0006). Having any of the three studied gene mutations worsened the overall survival (P = 0.0003). Cox regression proved that both thrombosis and presence of factor V mutation are independent factors affecting survival in cancer patients (P < 0.001 and P = 0.01, respectively). In conclusion, there is an association between factor V and MTHFR mutations and risk of VTE in Egyptian cancer patients. Thrombosis and presence of factor V mutation are independent factors that influence survival in those patients. PMID:25565385

  14. The C677T variant in MTHFR modulates associations between blood-based and cerebrospinal fluid biomarkers of neurodegeneration

    PubMed Central

    Roussotte, Florence F.; Narr, Katherine L.; Small, Gary W.

    2016-01-01

    The C677T functional variant in the methylene-tetrahydrofolate reductase (MTHFR) gene results in reduced enzymatic activity and elevated blood levels of homocysteine. Plasma levels of apolipoprotein E (ApoE) are negatively correlated with cerebral amyloid burden, but plasma homocysteine concentrations are associated with increased amyloid-β (Aβ) deposition in the brain. Here, we sought to determine whether associations between low plasma ApoE levels and elevated in-vivo amyloid burden were modulated by carrying the C677T variant. We tested this hypothesis in a large sample of elderly participants from the Alzheimer’s Disease Neuroimaging Initiative. We used general linear models to examine associations between plasma homocysteine concentrations, circulating ApoE levels, cerebrospinal fluid concentrations of Aβ, and their modulation by MTHFR and ApoE genotype. Age, sex, and dementia status were included as covariates in all analyses. Higher circulating levels of ApoE predicted increased cerebrospinal fluid concentrations of Aβ, indicating lower in-vivo burden, in C-allele carriers, but not in homozygotes at the C677T variant, who showed significant elevations in plasma homocysteine levels. This modulation by the MTHFR genotype did not remain significant after controlling for ApoE genotype. In T-homozygotes who do not carry the ApoE-ε4 allele, the relationship between low plasma ApoE levels and an increased risk of dementia is likely obscured by the presence of elevated plasma homocysteine. This report suggests the value of genotyping patients at the C677T functional variant when using plasma ApoE levels as a preclinical biomarker for Alzheimer’s disease. PMID:27380243

  15. Genetic and Biochemical Determinants of Serum Concentrations of Monocyte Chemoattractant Protein-1, a Potential Neural Tube Defect Risk Factor

    PubMed Central

    Lu, Zhi-Yong; Morales, Megan; Khartulyari, Stephanie; Mei, Minghua; Murphy, Kristen M.; Stanislawska-Sachadyn, Anna; Summers, Carolyn M.; Huang, Yuehua; Von Feldt, Joan M; Blair, Ian A.; Mitchell, Laura E.; Whitehead, Alexander S.

    2010-01-01

    Background Women with the AA genotype at the (−2518) A>G promoter polymorphism of CCL-2, which encodes the potent pro-inflammatory chemokine monocyte chemoattractant protein 1 (MCP-1), may be at increased risk for having offspring affected by spina bifida. As the A allele at this locus has been associated with decreased transcription of MCP-1 mRNA relative to the G allele, the observed genetic association suggests that the risk of spina bifida may be increased in the offspring of women with low MCP-1 levels. The present study was undertaken to identify potential determinants of MCP-1 levels in women of reproductive age. Methods A small cohort of Caucasian and African-American women of reproductive age was recruited to participate in an exploratory investigation of the determinants of several disease-related, biochemical phenotypes, including MCP-1. Subjects completed a brief questionnaire and provided a fasting blood sample for biochemical and genetic studies. Potential biochemical, genetic and lifestyle factors were assessed for their association with MCP-1 levels using linear regression analyses. Results In this cohort, MCP-1 levels were significantly higher in Caucasians as compared to African-Americans. Further, among women of both races, there was evidence that MCP-1 levels were associated with smoking status, MTHFR 677C>T genotype and red blood cell tetrahydrofolate levels. Conclusions The results of these analyses indicate that, if maternal CCL-2 genotype is related to the risk of spina bifida, this relationship is likely to be more complex than initially hypothesized, perhaps depending upon folate intake, MTHFR 677C>T genotype, the distribution of folate derivatives, and immune/inflammatory activity. PMID:18937353

  16. Exploration and comparison of inborn capacity of aerobic and anaerobic metabolisms of Saccharomyces cerevisiae for microbial electrical current production

    PubMed Central

    Mao, Longfei; Verwoerd, Wynand S

    2013-01-01

    Saccharomyces cerevisiae possesses numerous advantageous biological features, such as being robust, easily handled, mostly non-pathogenic and having high catabolic rates, etc., which can be considered as merits for being used as a promising biocatalyst in microbial fuel cells (MFCs) for electricity generation. Previous studies have developed efficient MFC configurations to convert metabolic electron shuttles, such as cytoplasmic NADH, into usable electric current. However, no studies have elucidated the maximum potential of S. cerevisiae for current output and the underlying metabolic pathways, resulting from the interaction of thousands of reactions inside the cell during MFC operation. To address these two key issues, this study used in silico metabolic engineering techniques, flux balance analysis (FBA), and flux variability analysis with target flux minimization (FATMIN), to model the metabolic perturbation of S. cerevisiae under the MFC-energy extraction. The FBA results showed that, in the cytoplasmic NADH-dependent mediated electron transfer (MET) mode, S. cerevisiae had a potential to produce currents at up to 5.781 A/gDW for the anaerobic and 6.193 A/gDW for the aerobic environments. The FATMIN results showed that the aerobic and anaerobic metabolisms are resilient, relying on six and five contributing reactions respectively for high current production. Two reactions, catalyzed by glutamate dehydrogenase (NAD) (EC 1.4.1.3) and methylene tetrahydrofolate dehydrogenase (NAD) (EC 1.5.1.5), were shared in both current-production modes and contributed to over 80% of the identified maximum current outputs. It is also shown that the NADH regeneration was much less energy costly than biomass production rate. Taken together, our finding suggests that S. cerevisiae should receive more research effort for MFC electricity production. PMID:23969939

  17. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography [Neutron and ultrahigh resolution X-ray crystallography reveals water as the proton donor in the catalytic mechanism of dihydrofolate reductase

    SciTech Connect

    Wan, Qun; Bennett, Brad C.; Wilson, Mark A.; Kovalevsky, Andrey; Langan, Paul; Howell, Elizabeth E.; Dealwis, Chris

    2014-12-01

    Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to the lack of consensus on a catalytic mechanism. To resolve this ambiguity, we conducted neutron and ultrahigh resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of DHFR with folate and NADP+ from E. coli. The neutron data were collected to 2.0 Å resolution using a 3.6 mm3 crystal with the quasi-Laue technique, and the structure reveals that the N3 atom of folate is protonated while Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer due to protonation of the N3 atom, suggesting tautomerization is unnecessary for catalysis. In the 1.05 Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa of the N5 atom of DHF by Asp27, and protonation of N5 by water whose access to the active site is gated by fluctuation of the Met20 side chain even though the Met-20 loop is closed.

  18. Stable Isotope Dilution Assays for Clinical Analyses of Folates and Other One-Carbon Metabolites: Application to Folate-Deficiency Studies

    PubMed Central

    Kopp, Markus; Morisset, Rosalie; Koehler, Peter

    2016-01-01

    Folate deficiency is generally accepted as a potential direct or indirect risk factor for diseases including spina bifida, coronary heart diseases, malfunctions of the central nervous system, and cancer. The direct inclusion of folates in the methylation cycle, including the remethylation of homocysteine and regeneration of S-adenosylmethionine, underlines the importance of these vitamins and other components of one-carbon metabolism. Therefore, the aim of the present study was to develop a multiple stable isotope dilution assay (SIDA) for the respective analytes in plasma and tissue samples to allow for a closer look at the interaction between a severe folate deficiency and local folate status, as well as further interactions with circulating S-adenosylmethionine, S-adenosylhomocysteine, and homocysteine. The analytical methods were based on SIDAs coupled with liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis using the deuterated folates [2H4]-5-methyltetrahydrofolic acid, [2H4]-5-formyltetrahydrofolic acid, [2H4]-tetrahydrofolic acid, [2H4]-10-formylfolic acid, and [2H4]-folic acid and the deuterated one-carbon metabolites [2H4]-homocysteine, [2H4]-S-adenosylhomocysteine, and [2H3]-S-adenosylmethionine as internal standards. Three analytical methods have been developed for the analysis of homocysteine, S-adenosylmethionine, S-adenosylhomocysteine, and six folate vitamers. Validation data for the analysis of C1-metabolites in plasma and tissue samples or folate analysis in tissue samples revealed excellent sensitivity, precision, and recovery for all analytes studied. The miniaturized methods using sample volumes as low as 50 μL and weighed portions of 5–25 mg will allow the assessment of the status of folates and additional biomarkers of impaired one-carbon metabolism during folate deficiency. PMID:27276031

  19. Drugs and vitamin B12 and folate metabolism.

    PubMed

    Lindenbaum, J

    1983-01-01

    Deficiency of either folic acid or vitamin B12 may interfere with DNA synthesis and result in megaloblastic anemia or other conditions. These 2 vitamins have dissimilar molecular structures and are present in different foods; they are also absorbed and metabolized differently. In 201 consecutive cases of megaloblastic anemia, for 90% the cause was alcoholism and poor diet; 0.5% (1 case) was related to oral contraceptives (OCs). Megaloblastic anemia due to folate deficiency has occasionally been reported in patients with inflammatory bowel disease and has been attributed to poor diet, impaired absorption, and increased tissue utilization of folate. Sulfasalazine, a compound containing a sulfa drug and a salicylate that is broken down to its active components by the gut flora, is widely used in the treatment of inflammatory bowel disease and has been shown to impair the absorption of folic acid, polyglutamyl folate, and methyl-tetrahydrofolic acid in patients with these disorders. There is also evidence suggesting an interaction between anticonvulsant drugs and folate balance. A number of cases of megaloblastic anemia due to folate deficiency have been reported in women taking OCs. While in some cases no apparent cause for the megaloblastic anemia other than contraceptive therapy was demonstrated, in many patients other underlying disorders that were likely to disturb folate balance such as celiac disease, decreased dietary vitamin intake, and the administration of other drugs known to affect folate status have also been present. There is no convincing evidence that sex steroids affect folate absorption; about 20% of women taking OCs were found to have mild megaloblastic changes on Papanicolaou smears. These changes disappered after folic acid therapy, suggesting that OCs may cause an increased demand for folate limited to the reproductive system. Another finding is of low serum cobalamin levels in women using OCs; this appears however to be a laboratory abnormality

  20. Structures of dimethylsulfoniopropionate-dependent demethylase from the marine organism Pelagabacter ubique

    SciTech Connect

    Schuller, David J.; Reisch, Chris R.; Moran, Mary Ann; Whitman, William B.; Lanzilotta, William N.

    2012-01-20

    Dimethylsulfoniopropionate (DMSP) is a ubiquitous algal metabolite and common carbon and sulfur source for marine bacteria. DMSP is a precursor for the climatically active gas dimethylsulfide that is readily oxidized to sulfate, sulfur dioxide, methanesulfonic acid, and other products that act as cloud condensation nuclei. Although the environmental importance of DMSP metabolism has been known for some time, the enzyme responsible for DMSP demethylation by marine bacterioplankton, dimethylsufoniopropionate-dependent demethylase A (DmdA, EC 2.1.1.B5), has only recently been identified and biochemically characterized. In this work, we report the structure for the apoenzyme DmdA from Pelagibacter ubique (2.1 {angstrom}), as well as for DmdA co-crystals soaked with substrate DMSP (1.6 {angstrom}) or the cofactor tetrahydrofolate (THF) (1.6 {angstrom}). Surprisingly, the overall fold of the DmdA is not similar to other enzymes that typically utilize the reduced form of THF and in fact is a triple domain structure similar to what has been observed for the glycine cleavage T protein or sarcosine oxidase. Specifically, while the THF binding fold appears conserved, previous biochemical studies have shown that all enzymes with a similar fold produce 5,10-methylene-THF, while DmdA catalyzes a redox-neutral methyl transfer reaction to produce 5-methyl-THF. On the basis of the findings presented herein and the available biochemical data, we outline a mechanism for a redox-neutral methyl transfer reaction that is novel to this conserved THF binding domain.

  1. Mechanism of N[superscript 10]-formyltetrahydrofolate synthetase derived from complexes with intermediates and inhibitors

    SciTech Connect

    Celeste, Lesa R.; Chai, Geqing; Bielak, Magdalena; Minor, Wladek; Lovelace, Leslie L.; Lebioda, Lukasz

    2012-09-05

    N{sup 10}-formyltetrahydrofolate synthetase (FTHFS) is a folate enzyme that catalyzes the formylation of tetrahydrofolate (THF) in an ATP dependent manner. Structures of FTHFS from the thermophilic homoacetogen, Moorella thermoacetica, complexed with (1) a catalytic intermediate-formylphosphate (XPO) and product-ADP; (2) with an inhibitory substrate analog-folate; (3) with XPO and an inhibitory THF analog, ZD9331, were used to analyze the enzyme mechanism. Nucleophilic attack of the formate ion on the gamma phosphate of ATP leads to the formation of XPO and the first product ADP. A channel that leads to the putative formate binding pocket allows for the binding of ATP and formate in random order. Formate binding is due to interactions with the gamma-phosphate moiety of ATP and additionally to two hydrogen bonds from the backbone nitrogen of Ala276 and the side chain of Arg97. Upon ADP dissociation, XPO reorients and moves to the position previously occupied by the beta-phosphate of ATP. Conformational changes that occur due to the XPO presence apparently allow for the recruitment of the third substrate, THF, with its pterin moiety positioned between Phe384 and Trp412. This position overlaps with that of the bound nucleoside, which is consistent with a catalytic mechanism hypothesis that FTHFS works via a sequential ping-pong mechanism. More specifically, a random bi uni uni bi ping-pong ter ter mechanism is proposed. Additionally, the native structure originally reported at a 2.5 {angstrom} resolution was redetermined at a 2.2 {angstrom} resolution.

  2. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography.

    PubMed

    Wan, Qun; Bennett, Brad C; Wilson, Mark A; Kovalevsky, Andrey; Langan, Paul; Howell, Elizabeth E; Dealwis, Chris

    2014-12-23

    Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP(+). The neutron data were collected to 2.0-Å resolution using a 3.6-mm(3) crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed. PMID:25453083

  3. Nutrigenetics and nutraceuticals: the next wave riding on personalized medicine.

    PubMed

    Subbiah, M T Ravi

    2007-02-01

    The Human Genome Project and subsequent identification of single nucleotide polymorphisms (SNPs) within populations has played a major role in predicting individual response to drugs (pharmacogenetics) leading to the concept of "personalized medicine." Nutritional genomics is a recent off-shoot of this genetic revolution that includes (1) nutrigenomics: the study of interaction of dietary components with the genome and the resulting proteonomic and metabolomic changes; and (2) nutrigenetics: understanding the gene-based differences in response to dietary components and developing nutraceuticals that are most compatible with health based on individual genetic makeup. Despite the extensive data on genetic polymorphisms in humans, its translation into medical practice has been slow because of the time required to accumulate population data on SNP incidence, understand the significance of a given SNP in disease, and develop suitable diagnostic tests. Nutrigenomics revitalized the field by showing that nutrients and botanicals can interact with the genome and modify subsequent gene expression, which has provided a great impetus for nutrigenetic research and nutraceutical development based on nutrigenetics. Polymorphisms in methlyene tetrahydrofolate reductase (MTHFR) (involved in folate metabolism), apolipoprotein E (Apo E) and ApoA1 (in cardiovascular disease), and leptin/leptin receptor (obesity) genes are some good examples for understanding basic nutrigenetics. Developing nutraceuticals to prevent and manage thrombosis risk in women with thrombophilic gene mutations are discussed in the context of the opportunities that exist at the nutrigenetic/pharmacogenetic interphase leading to "personalized nutrition." Further research on individual differences in genetic profiles and nutrient requirements will help establish nutrigenetics as an essential discipline for nutrition and dietetics practice. PMID:17240315

  4. Customized nutritional enhancement for pregnant women appears to lower incidence of certain common maternal and neonatal complications: an observational study.

    PubMed

    Stone, Leslie P; Stone, P Michael; Rydbom, Emily A; Stone, Lucas A; Stone, T Elliot; Wilkens, Lindsey E; Reynolds, Kathryn

    2014-11-01

    A retrospective chart review analyzed the effect of customized nutrition on the incidence of pregnancy-induced hypertension (PIH), gestational diabetes (GDM), and small- and large-for-gestational-age (SGA, LGA) neonates, examining consecutive deliveries between January 1, 2011, and Decem ber 31, 2012, at a low-risk community hospital. The population was divided into 3 groups: (1) study group (SG), (2) private practice (PP), and (3) community healthcare clinic (CHCC). All groups received standard perinatal management, but additionally the study group was analyzed for serum zinc, carnitine, total 25-hydroxy cholecalciferol (25 OH-D), methylene tetrahydrofolate reductase, and catechol-O-methyl transferase polymorphisms in the first trimester prior to intervention, with subsequent second trimester and postpartum assessment of zinc, carnitine, and 25 OH-D after intervention. Intervention consisted of trimesterby-trimester nutrition and lifestyle education, supplementation of L-methyl folate, magnesium, essential fatty acids, and probiotics for all SG patients, with targeted supplementation of zinc, carnitine, and 25 OH-D. Because of small case occurrence rates of individual conditions in the study group, unreportable reductions were found, except GDM (SG vs CHCC, P value .046 with 95.38% confidence interval [CI]), and PIH (SG vs PP, P value .0505 with 94.95% CIl). The aggregated occurrence rate of the four conditions, however, was significantly lower in the study population than in either comparison population (PP P value .0154 with 98.46% CI, and CHCC P value .0265 with 97.35% CI). Customized nutritional intervention appears to have significantly reduced adverse perinatal outcomes. Prospective study within larger, at-risk populations is needed to determine whether customized nutrition improves conditions individually. PMID:25568832

  5. Customized Nutritional Enhancement for Pregnant Women Appears to Lower Incidence of Certain Common Maternal and Neonatal Complications: An Observational Study

    PubMed Central

    Stone, P. Michael; Rydbom, Emily A.; Stone, Lucas A.; Stone, T. Elliot; Wilkens, Lindsey E.; Reynolds, Kathryn

    2014-01-01

    A retrospective chart review analyzed the effect of customized nutrition on the incidence of pregnancy-induced hypertension (PIH), gestational diabetes (GDM), and small- and large-for-gestational-age (SGA, LGA) neonates, examining consecutive deliveries between January 1, 2011, and Decem ber 31, 2012, at a low-risk community hospital. The population was divided into 3 groups: (1) study group (SG), (2) private practice (PP), and (3) community healthcare clinic (CHCC). All groups received standard perinatal management, but additionally the study group was analyzed for serum zinc, carnitine, total 25-hydroxy cholecalciferol (25 OH-D), methylene tetrahydrofolate reductase, and catechol-O-methyl transferase polymorphisms in the first trimester prior to intervention, with subsequent second trimester and postpartum assessment of zinc, carnitine, and 25 OH-D after intervention. Intervention consisted of trimesterby-trimester nutrition and lifestyle education, supplementation of L-methyl folate, magnesium, essential fatty acids, and probiotics for all SG patients, with targeted supplementation of zinc, carnitine, and 25 OH-D. Because of small case occurrence rates of individual conditions in the study group, unreportable reductions were found, except GDM (SG vs CHCC, P value .046 with 95.38% confidence interval [CI]), and PIH (SG vs PP, P value .0505 with 94.95% CIl). The aggregated occurrence rate of the four conditions, however, was significantly lower in the study population than in either comparison population (PP P value .0154 with 98.46% CI, and CHCC P value .0265 with 97.35% CI). Customized nutritional intervention appears to have significantly reduced adverse perinatal outcomes. Prospective study within larger, at-risk populations is needed to determine whether customized nutrition improves conditions individually. PMID:25568832

  6. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography

    PubMed Central

    Wan, Qun; Bennett, Brad C.; Wilson, Mark A.; Kovalevsky, Andrey; Langan, Paul; Howell, Elizabeth E.; Dealwis, Chris

    2014-01-01

    Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP+. The neutron data were collected to 2.0-Å resolution using a 3.6-mm3 crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed. PMID:25453083

  7. Candidate-gene analysis of white matter hyperintensities on neuroimaging

    PubMed Central

    Tran, Theresa; Cotlarciuc, Ioana; Yadav, Sunaina; Hasan, Nazeeha; Bentley, Paul; Levi, Christopher; Worrall, Bradford B; Meschia, James F; Rost, Natalia; Sharma, Pankaj

    2016-01-01

    Background White matter hyperintensities (WMH) are a common radiographic finding and may be a useful endophenotype for small vessel diseases. Given high heritability of WMH, we hypothesised that certain genotypes may predispose individuals to these lesions and consequently, to an increased risk of stroke, dementia and death. We performed a meta-analysis of studies investigating candidate genes and WMH to elucidate the genetic susceptibility to WMH and tested associated variants in a new independent WMH cohort. We assessed a causal relationship of WMH to methylene tetrahydrofolate reductase (MTHFR). Methods Database searches through March 2014 were undertaken and studies investigating candidate genes in WMH were assessed. Associated variants were tested in a new independent ischaemic cohort of 1202 WMH patients. Mendelian randomization was undertaken to assess a causal relationship between WMH and MTHFR. Results We identified 43 case-control studies interrogating eight polymorphisms in seven genes covering 6,314 WMH cases and 15,461 controls. Fixed-effects meta-analysis found that the C-allele containing genotypes of the aldosterone synthase CYP11B2 T(−344)C gene polymorphism were associated with a decreased risk of WMH (OR=0.61; 95% CI, 0.44 to 0.84; p=0.003). Using mendelian randomisation the association among MTHFR C677T, homocysteine levels and WMH, approached, but did not reach, significance (expected OR=1.75; 95% CI, 0.90−3.41; observed OR=1.68; 95% CI, 0.97−2.94). Neither CYP11B2 T(−344)C nor MTHFR C677T were significantly associated when tested in a new independent cohort of 1202 patients with WMH. Conclusions There is a genetic basis to WMH but anonymous genome wide and exome studies are more likely to provide novel loci of interest. PMID:25835038

  8. Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA.

    PubMed

    Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P; Shanmugam, Keelnatham T; Ingram, Lonnie O

    2012-06-01

    Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair. PMID:22504824

  9. Insights into the reactivation of cobalamin-dependent methionine synthase

    SciTech Connect

    Koutmos, Markos; Datta, Supratim; Pattridge, Katherine A.; Smith, Janet L.; Matthews, Rowena G.

    2009-12-10

    Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every {approx}2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH{sup CT}) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetH{sup CT} ({sub s-s}MetH{sup CT}) that offer further insight into the reactivation of MetH. The structure of {sub s-s}MetH{sup CT} with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of {sub s-s}MetH{sub CT} with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.

  10. Stable Isotope Dilution Assays for Clinical Analyses of Folates and Other One-Carbon Metabolites: Application to Folate-Deficiency Studies.

    PubMed

    Kopp, Markus; Morisset, Rosalie; Koehler, Peter; Rychlik, Michael

    2016-01-01

    Folate deficiency is generally accepted as a potential direct or indirect risk factor for diseases including spina bifida, coronary heart diseases, malfunctions of the central nervous system, and cancer. The direct inclusion of folates in the methylation cycle, including the remethylation of homocysteine and regeneration of S-adenosylmethionine, underlines the importance of these vitamins and other components of one-carbon metabolism. Therefore, the aim of the present study was to develop a multiple stable isotope dilution assay (SIDA) for the respective analytes in plasma and tissue samples to allow for a closer look at the interaction between a severe folate deficiency and local folate status, as well as further interactions with circulating S-adenosylmethionine, S-adenosylhomocysteine, and homocysteine. The analytical methods were based on SIDAs coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using the deuterated folates [2H4]-5-methyltetrahydrofolic acid, [2H4]-5-formyltetrahydrofolic acid, [2H4]-tetrahydrofolic acid, [2H4]-10-formylfolic acid, and [2H4]-folic acid and the deuterated one-carbon metabolites [2H4]-homocysteine, [2H4]-S-adenosylhomocysteine, and [2H3]-S-adenosylmethionine as internal standards. Three analytical methods have been developed for the analysis of homocysteine, S-adenosylmethionine, S-adenosylhomocysteine, and six folate vitamers. Validation data for the analysis of C1-metabolites in plasma and tissue samples or folate analysis in tissue samples revealed excellent sensitivity, precision, and recovery for all analytes studied. The miniaturized methods using sample volumes as low as 50 μL and weighed portions of 5-25 mg will allow the assessment of the status of folates and additional biomarkers of impaired one-carbon metabolism during folate deficiency. PMID:27276031

  11. One carbon metabolism in SAR11 pelagic marine bacteria.

    PubMed

    Sun, Jing; Steindler, Laura; Thrash, J Cameron; Halsey, Kimberly H; Smith, Daniel P; Carter, Amy E; Landry, Zachary C; Giovannoni, Stephen J

    2011-01-01

    The SAR11 Alphaproteobacteria are the most abundant heterotrophs in the oceans and are believed to play a major role in mineralizing marine dissolved organic carbon. Their genomes are among the smallest known for free-living heterotrophic cells, raising questions about how they successfully utilize complex organic matter with a limited metabolic repertoire. Here we show that conserved genes in SAR11 subgroup Ia (Candidatus Pelagibacter ubique) genomes encode pathways for the oxidation of a variety of one-carbon compounds and methyl functional groups from methylated compounds. These pathways were predicted to produce energy by tetrahydrofolate (THF)-mediated oxidation, but not to support the net assimilation of biomass from C1 compounds. Measurements of cellular ATP content and the oxidation of (14)C-labeled compounds to (14)CO(2) indicated that methanol, formaldehyde, methylamine, and methyl groups from glycine betaine (GBT), trimethylamine (TMA), trimethylamine N-oxide (TMAO), and dimethylsulfoniopropionate (DMSP) were oxidized by axenic cultures of the SAR11 strain Ca. P. ubique HTCC1062. Analyses of metagenomic data showed that genes for C1 metabolism occur at a high frequency in natural SAR11 populations. In short term incubations, natural communities of Sargasso Sea microbial plankton expressed a potential for the oxidation of (14)C-labeled formate, formaldehyde, methanol and TMAO that was similar to cultured SAR11 cells and, like cultured SAR11 cells, incorporated a much larger percentage of pyruvate and glucose (27-35%) than of C1 compounds (2-6%) into biomass. Collectively, these genomic, cellular and environmental data show a surprising capacity for demethylation and C1 oxidation in SAR11 cultures and in natural microbial communities dominated by SAR11, and support the conclusion that C1 oxidation might be a significant conduit by which dissolved organic carbon is recycled to CO(2) in the upper ocean. PMID:21886845

  12. Metabolic Interfaces of Mercury Methylation Proteins in Desulfovibrio sp. ND132

    NASA Astrophysics Data System (ADS)

    Wall, J. D.; Bridou, R.; Smith, S. D.; Mok, K.; Widner, F.; Johs, A.; Parks, J.; Pierce, E. M.; Elias, D. A.; Gilmour, C. C.; Taga, M.

    2015-12-01

    Two genes necessary for microbial production of the neurotoxin methylmercury have been identified; hgcA encoding a corrinoid methyltransferase and hgcB, a ferredoxin-like protein. To date, all microbes possessing orthologs of these genes that have been tested are capable of methylating mercury; whereas, organisms lacking hgcA and hgcB are not. Also of interest is the observation that confirmed mercury-methylating microbes are all considered anaerobes although not members of a specific phylogenetic group. They are found scattered in the genomes of methanogens, Firmicutes, and Deltaproteobacteria. Methylation has not been demonstrated to provide protection of the microbes to mercury exposure. To determine the source of evolutionary pressure for acquisition and maintenance of these genes, we are seeking to understand whether there is a second function of the proteins. We are seeking evidence for the metabolic source(s) of the methyl group and for competing reactions. We have found that deletion of the metH gene encoding a tetrahydrofolate methyltransferase in Desulfovibrio sp. ND132 decreases the mercury methylation capacity by ca. 95%, consistent with an interpretation that this enzyme is involved in the pathway for the methyl group for HgcA. In addition, the corrinoid present in HgcA and the MetH of ND132 is strictly dependent on nicotinate nucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase encoded by the cobT gene, linking methionine biosynthesis with mercury methylation at a second level. Additional methyl transferases have not been found to be necessary for this function. While earlier evidence was provided for an involvement of the CO dehydrogenase/acetylCoA synthase, this enzyme is not universally present in methylating strains unlike the pathway for methionine synthesis.

  13. Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim

    SciTech Connect

    Heaslet, Holly; Harris, Melissa; Fahnoe, Kelly; Sarver, Ronald; Putz, Henry; Chang, Jeanne; Subramanyam, Chakrapani; Barreiro, Gabriela; Miller, J. Richard; Pfizer

    2010-09-02

    Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin-resistant Staphylococcus aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram-positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed 'S1 DHFR.' To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild-type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR.

  14. Toxicity of methotrexate in rats preexposed to nitrous oxide.

    PubMed

    Ermens, A A; Schoester, M; Spijkers, L J; Lindemans, J; Abels, J

    1989-11-15

    Several chemotherapeutic protocols for the treatment of malignancies include administration of methotrexate (MTX) during or shortly after total anesthesia. Clinical observations in patients treated for breast carcinoma or childhood cancer have shown unexpected myelosuppression and mucosal damage. This phenomenon may be attributed to the synergistic effects of nitrous oxide, which inactivates the cobalamin coenzyme of methionine synthase, and MTX, which inhibits dihydrofolate reductase, on folate metabolism. However, no quantitative data on dose-effect relationships are available regarding the combined toxicity of MTX and N2O. We investigated the effect of exposure to N2O on the toxicity of MTX. Groups of male Wistar rats were exposed to either 50% N2O/50% O2 or air for 12-48 h. Subsequently, a single i.p. injection of 10, 20, 40, or 80 mg MTX/kg body weight was given. Gastrointestinal toxicity resulted in diarrhea and weight loss in all groups for 5 days after MTX administration. Concomitantly, bone marrow depression with leukocytopenia and thrombocytopenia occurred. Exposure to N2O did not alter the plasma clearance of MTX. No substantial liver or kidney toxicity could be detected, but the 50% lethal dose for MTX was reduced from 60 mg/kg to 10 mg/kg if rats had been exposed to N2O for 48 h; the main causes of death were dehydration and bleeding. The administration of 5-formyl-tetrahydrofolate (4 x 10 mg i.p.) but not 5-methyltetrahydrofolate protected completely against the lethal effect of the drug combination. Altogether, cytotoxic effects of MTX on proliferating cells are potentiated by N2O. Therefore, the use of this anesthetic shortly before or during MTX administration should be avoided. PMID:2804978

  15. [Metabolic syndrome and prevention of migraine headache].

    PubMed

    Takeshima, Takao

    2009-10-01

    Metabolic syndrome (MetS) are consist of central obesity, diabetes, dyslipidemia and hypertension. Previous studies have reported possible association of migraine and MetS were reviewed. Migraine is a prevalent disabling disorder and have been regarded as an episodic and functional disorder. However, recent evidence suggests that in some cases, the disease may follow a chronic and progressive course. On the basis of available evidence, obesity is considered to be associated with migraine frequency and progression. The association between diabetes and migraine is unclear. Similarly, association of migraine with hypertension is also unclear. Female migraineurs commonly have an unfavorable cholesterol profile, i.e. one with high total cholesterol and low HDL levels. Obesity can be considered as a proinflammatory state in which increased inflammatory mediators, vascular hyperreactivity, plasma calcitonin gene-related peptide (CGRP) concentrations and decreased adiponectin concentrations are observed. These alterations can cause an increase in the frequency of migraine attacks developed of central sensitization, and thereafter, chronic migraine. Migraine and obesity may share some neurobiological abnormalities. Orexins modulate both pain and metabolism. Dysfunction in the orexin pathways seems to be a risk factor for both conditions. The methylene-tetrahydrofolate reductase (MTHFR) gene and the angiotensin converting enzyme (ACE) gene exhibit polymorphism. C677Tmutation in the MTHFR gene and the D-allele of the ACE gene are the shared risk factors for the development of migraine and cardiovascular disease. Certain beta-blockers, Ca blockers, ACE inhibitors, and angionten II receptor blocker (ARB) have excellent efficacy in migraine prophylaxis. The pharmacological mechanism of these agents do not seem to stand on their antihypertensive effect, but the other mechanism of action. Appropriate meal, sleep, and exercise are important for the management of MetS and migraine

  16. Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotoga maritima: Carbon Transfer without Covalent Pyrimidine Activation

    PubMed Central

    2015-01-01

    Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine 5′-monophosphate (dUMP) and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF), synthesizing 2′-deoxythymidine 5′-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH2THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis. PMID:25068636

  17. Production of folate by bacteria isolated from oat bran.

    PubMed

    Kariluoto, Susanna; Edelmann, Minnamari; Herranen, Mirkka; Lampi, Anna-Maija; Shmelev, Anton; Salovaara, Hannu; Korhola, Matti; Piironen, Vieno

    2010-09-30

    Twenty bacteria isolated from three commercial oat bran products were tested for their folate production capability. The bacteria as well as some reference organisms were grown until early stationary phase on a rich medium (YPD), and the amount of total folate in the separated cell mass and the culture medium (supernatant) was determined by microbiological assay. Folate vitamer distribution was determined for eight bacteria including one isolated from rye flakes. For seven bacteria the effect of temperature and pH on folate production was studied in more detail. Relatively large amount of folate was both produced in the cell biomass (up to 20.8microg/g) and released to the culture medium (up to 0.38microg/g) by studied bacteria. The best producers were characterized as Bacillus subtilis ON4, Chryseobacterium sp. NR7, Janthinobacterium sp. RB4, Pantoea agglomerans ON2, and Pseudomonas sp ON8. The level of folate released in culture medium was the highest for B. subtilis ON5, Chryseobacterium sp. NR7, Curtobacterium sp. ON7, Enterococcus durans ON9, Janthinobacterium sp. RB4, Paenibacillus sp. ON10, Propionibacterium sp. RB9, and Staphylococcus kloosii RB7. Marked differences in the distribution of folate vitamers among the bacterial strains were revealed by the HPLC analysis. The main vitamers were tetrahydrofolate, 5,10-methenyltetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate. Increase in the folate content during bacterial growth was accompanied by proportional increase in the 5-methyltetrahydrofolate content and decrease of 5-formyltetrahydrofolate. 10-Formylfolic acid dominated in the culture media of four bacteria, and Janthinobacterium sp. RB4 was also found to excrete 5-methyltetrahydrofolate. Intracellular folate content was higher when the bacteria were grown at 28 degrees C than at 18 degrees C or 37 degrees C and also higher at pH 7 than at pH 5.5. PMID:20708290

  18. [Hyperhomocysteinemia in coronary artery diseases. Apropos of a study on 102 patients].

    PubMed

    Blacher, J; Montalescot, G; Ankri, A; Chadefaux-Vekemans, B; Benzidia, R; Grosgogeat, Y; Kamoun, P; Thomas, D

    1996-10-01

    Homocystein is at the crossroads of the metabolic pathways of sulphuric amino acids. Homocystinuria is a congenital autosomal recessive disease, usually related to cystathionine beta-synthetase deficiency. Children with homozygotic forms of the disease have early vascular complications which represent the main cause of death. Moderately elevated serum homocystein levels are related to two major genetic factors (heterozygotic cystathionine beta-synthetase deficiency and mutation of the 5-10 methylene tetrahydrofolate reductase) and several minor, genetic and non-genetic factors (folic acid, vitamins B6 and B12 and betain deficiencies). Previous studies have suggested that hyperhomocysteinaemia could be a cardiovascular risk factor. This study was based on 222 subjects including 102 consecutive patients with angiographically documented coronary artery disease and 120 control subjects without vascular disease. No relationship was observed between serum homocystein concentrations and the classical cardiovascular risk factors. Coronary patients had higher average homocystein concentrations than control subjects (11.27 +/- 0.52 vs 8.77 +/- 0.31 mumol/l); p < 0.0001): moreover, the prevalence of hyperhomocysteinaemia (> 15.67 mumol/l) was higher in the coronary group (15.7%) than in the controls (2.5%). A significant relationship was also observed between homocystein concentrations and the severity of the coronary disease (defined by a coronary score) and the number of diseased vascular territories. These results underline the relationship between homocystein and vascular risk, especially that of coronary artery disease. The treatment of hyperhomocysteinaemia by folic acid supplements is effective in correcting plasma levels, without side effects and at a relatively low cost. PMID:8952820

  19. Association of PHB 1630 C>T and MTHFR 677 C>T polymorphisms with breast and ovarian cancer risk in BRCA1/2 mutation carriers: results from a multicenter study

    PubMed Central

    Jakubowska, A; Rozkrut, D; Antoniou, A; Hamann, U; Scott, R J; McGuffog, L; Healy, S; Sinilnikova, O M; Rennert, G; Lejbkowicz, F; Flugelman, A; Andrulis, I L; Glendon, G; Ozcelik, H; Thomassen, M; Paligo, M; Aretini, P; Kantala, J; Aroer, B; von Wachenfeldt, A; Liljegren, A; Loman, N; Herbst, K; Kristoffersson, U; Rosenquist, R; Karlsson, P; Stenmark-Askmalm, M; Melin, B; Nathanson, K L; Domchek, S M; Byrski, T; Huzarski, T; Gronwald, J; Menkiszak, J; Cybulski, C; Serrano, P; Osorio, A; Cajal, T R; Tsitlaidou, M; Benítez, J; Gilbert, M; Rookus, M; Aalfs, C M; Kluijt, I; Boessenkool-Pape, J L; Meijers-Heijboer, H E J; Oosterwijk, J C; van Asperen, C J; Blok, M J; Nelen, M R; van den Ouweland, A M W; Seynaeve, C; van der Luijt, R B; Devilee, P; Easton, D F; Peock, S; Frost, D; Platte, R; Ellis, S D; Fineberg, E; Evans, D G; Lalloo, F; Eeles, R; Jacobs, C; Adlard, J; Davidson, R; Eccles, D; Cole, T; Cook, J; Godwin, A; Bove, B; Stoppa-Lyonnet, D; Caux-Moncoutier, V; Belotti, M; Tirapo, C; Mazoyer, S; Barjhoux, L; Boutry-Kryza, N; Pujol, P; Coupier, I; Peyrat, J-P; Vennin, P; Muller, D; Fricker, J-P; Venat-Bouvet, L; Johannsson, O Th; Isaacs, C; Schmutzler, R; Wappenschmidt, B; Meindl, A; Arnold, N; Varon-Mateeva, R; Niederacher, D; Sutter, C; Deissler, H; Preisler-Adams, S; Simard, J; Soucy, P; Durocher, F; Chenevix-Trench, G; Beesley, J; Chen, X; Rebbeck, T; Couch, F; Wang, X; Lindor, N; Fredericksen, Z; Pankratz, V S; Peterlongo, P; Bonanni, B; Fortuzzi, S; Peissel, B; Szabo, C; Mai, P L; Loud, J T; Lubinski, J

    2012-01-01

    Background: The variable penetrance of breast cancer in BRCA1/2 mutation carriers suggests that other genetic or environmental factors modify breast cancer risk. Two genes of special interest are prohibitin (PHB) and methylene-tetrahydrofolate reductase (MTHFR), both of which are important either directly or indirectly in maintaining genomic integrity. Methods: To evaluate the potential role of genetic variants within PHB and MTHFR in breast and ovarian cancer risk, 4102 BRCA1 and 2093 BRCA2 mutation carriers, and 6211 BRCA1 and 2902 BRCA2 carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA) were genotyped for the PHB 1630 C>T (rs6917) polymorphism and the MTHFR 677 C>T (rs1801133) polymorphism, respectively. Results: There was no evidence of association between the PHB 1630 C>T and MTHFR 677 C>T polymorphisms with either disease for BRCA1 or BRCA2 mutation carriers when breast and ovarian cancer associations were evaluated separately. Analysis that evaluated associations for breast and ovarian cancer simultaneously showed some evidence that BRCA1 mutation carriers who had the rare homozygote genotype (TT) of the PHB 1630 C>T polymorphism were at increased risk of both breast and ovarian cancer (HR 1.50, 95%CI 1.10–2.04 and HR 2.16, 95%CI 1.24–3.76, respectively). However, there was no evidence of association under a multiplicative model for the effect of each minor allele. Conclusion: The PHB 1630TT genotype may modify breast and ovarian cancer risks in BRCA1 mutation carriers. This association need to be evaluated in larger series of BRCA1 mutation carriers. PMID:22669161

  20. Effects on growth, antioxidant enzyme activity and levels of extracellular proteins in the green alga Chlorella vulgaris exposed to crude cyanobacterial extracts and pure microcystin and cylindrospermopsin.

    PubMed

    Campos, Alexandre; Araújo, Pedro; Pinheiro, Carlos; Azevedo, Joana; Osório, Hugo; Vasconcelos, Vitor

    2013-08-01

    Toxic cyanobacteria and cyanotoxins have been pointed as important players in the control of phytoplankton diversity and species abundance, causing ecological unbalances and contamination of the environment. In vitro experiments have been undertaken to address the impact of toxic cyanobacteria in green algae. In this regard the aim of this work was to compare the toxicity of two cyanobacteria species, Aphanizomenon ovalisporum and Microcystis aeruginosa, to the green alga Chlorella vulgaris by assessing culture growth when exposed for three and seven days to (I) cyanobacterial cell extracts and (II) pure toxins microcystin-LR (MC-LR) and cylindrospermopsin (CYN). The biochemical response of the green alga to pure toxins was also characterized, through the activity of the antioxidant markers glutathione S-transferase (GST) and glutathione peroxidase (GPx) and the expressed extracellular proteins in seven-day exposed cultures. A. ovalisporum crude extracts were toxic to C. vulgaris. Pure toxins up to 179.0 µg/L, on the other hand, stimulated the green alga growth. Growth results suggest that the toxicity of A. ovalisporum extracts is likely due to a synergistic action of CYN and other metabolites produced by the cyanobacterium. Regarding the green alga antioxidant defense mechanism, CYN at 18.4 and 179.0 µg/L increased the activity of GPx and GST while MC-LR inhibited the enzymes' activity at a concentration of 179.0 µg/L demonstrating a contrasting mode of action. Moreover the identification of F-ATPase subunit, adenylate cyclase, sulfate ABC transporter, putative porin, aspartate aminotransferase, methylene-tetrahydrofolate dehydrogenase and chlorophyll a binding proteins in the culture medium of C. vulgaris indicates that biochemical processes involved in the transport of metabolites, photosynthesis and amino acid metabolism are affected by cyanobacterial toxins and may contribute to the regulation of green alga growth. PMID:23726538

  1. Characterization of an O-Demethylase of Desulfitobacterium hafniense DCB-2

    PubMed Central

    Studenik, Sandra; Vogel, Michaela

    2012-01-01

    Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (∼70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein. PMID:22522902

  2. The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes▿

    PubMed Central

    Schilhabel, Anke; Studenik, Sandra; Vödisch, Martin; Kreher, Sandra; Schlott, Bernhard; Pierik, Antonio Y.; Diekert, Gabriele

    2009-01-01

    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. PMID:19011025

  3. Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death.

    PubMed

    Hemendinger, Richelle A; Armstrong, Edward J; Brooks, Benjamin Rix

    2011-03-15

    Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC₅₀ (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolate (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC₅₀ (concentration at which 50% of maximal cell death is inhibited) of 0.6 μM and 0.4 μM, respectively. In contrast, MTHF (up to 10 μM) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS. PMID:21237187

  4. Comparison of the frequency of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in depressed versus nondepressed patients.

    PubMed

    Lizer, Mitsi H; Bogdan, Renee L; Kidd, Robert S

    2011-11-01

    Numerous studies have found an association between low serum folate levels and incidence of depression. Folic acid supplementation has been successfully used as an adjunct to treat depression in these patients. However, some individuals have a genetic deficiency in the methylene tetrahydrofolate reductase (MTHFR) gene that limits conversion of folic acid to its biologically active form, L-methylfolate. Several studies have identified a higher frequency of genetic variations in the MTHFR gene in depressed patients than in nondepressed controls. This study evaluated the frequency of the most common genetic variation MTHFR C667T in a group of depressed U.S. Caucasians and compared results with those of a control group of nondepressed U.S. Caucasians. Subjects were recruited from a psychiatric practice, an ambulatory care clinic, and the community. Informed consent and a cheek swab sample were obtained from each subject for analysis using real-time polymerase chain reaction (PCR). Allele and genotype frequencies were compared using Pearson X2 analysis. Complete data were obtained for 156 subjects. No significant differences were found in frequency of the MTHFR C667T T allele (0.415 vs 0.365; p=0.408) or the MTHFR C667T TT genotype (20.7% vs 17.6%; p=0.619) between the depressed and non-depressed controls, respectively. Therefore, use of L-methylfolate without an additional indication of need does not appear to be warranted in this group of U.S. Caucasians. Some patients may benefit from L-methylfolate, but an evidence-based approach, such as MTHFR genotyping, should be used to identify these specific patients. Additional research is also needed to confirm the benefit of L-methylfolate in specific patient populations (e.g., MTHFR TT genotype). PMID:22108397

  5. Targeted metabolomics and mathematical modeling demonstrate that vitamin B-6 restriction alters one-carbon metabolism in cultured HepG2 cells

    PubMed Central

    da Silva, Vanessa R.; Ralat, Maria A.; Quinlivan, Eoin P.; DeRatt, Barbara N.; Garrett, Timothy J.; Chi, Yueh-Yun; Frederik Nijhout, H.; Reed, Michael C.

    2014-01-01

    Low vitamin B-6 nutritional status is associated with increased risk for cardiovascular disease and certain cancers. Pyridoxal 5′-phosphate (PLP) serves as a coenzyme in many cellular processes, including several reactions in one-carbon (1C) metabolism and the transsulfuration pathway of homocysteine catabolism. To assess the effect of vitamin B-6 deficiency on these processes and associated pathways, we conducted quantitative analysis of 1C metabolites including tetrahydrofolate species in HepG2 cells cultured in various concentrations of pyridoxal. These results were compared with predictions of a mathematical model of 1C metabolism simulating effects of vitamin B-6 deficiency. In cells cultured in vitamin B-6-deficient medium (25 or 35 nmol/l pyridoxal), we observed >200% higher concentrations of betaine (P < 0.05) and creatinine (P < 0.05) and >60% lower concentrations of creatine (P < 0.05) and 5,10-methenyltetrahydrofolate (P < 0.05) compared with cells cultured in medium containing intermediate (65 nmol/l) or the supraphysiological 2,015 nmol/l pyridoxal. Cystathionine, cysteine, glutathione, and cysteinylglycine, which are components of the transsulfuration pathway and subsequent reactions, exhibited greater concentrations at the two lower vitamin B-6 concentrations. Partial least squares discriminant analysis showed differences in overall profiles between cells cultured in 25 and 35 nmol/l pyridoxal vs. those in 65 and 2,015 nmol/l pyridoxal. Mathematical model predictions aligned with analytically derived results. These data reveal pronounced effects of vitamin B-6 deficiency on 1C-related metabolites, including previously unexpected secondary effects on creatine. These results complement metabolomic studies in humans demonstrating extended metabolic effects of vitamin B-6 insufficiency. PMID:24824655

  6. Is Folate Status a Risk Factor for Asthma or Other Allergic Diseases?

    PubMed Central

    Wang, Ting; Zhang, Hong-Ping; Zhang, Xin; Liang, Zong-An; Ji, Yu-Lin

    2015-01-01

    Purpose It is controversial whether folate status is a risk factor for the development of asthma or other allergic diseases. This study was conducted to investigate whether indirect or direct exposure to folate and impaired folate metabolism, reflected as methylene-tetrahydrofolate reductase (MTHFR) C677T polymorphism, would contribute to the development of asthma and other allergic diseases. Methods Electronic databases were searched to identify all studies assessing the association between folate status and asthma or other allergic diseases. Two reviewers independently assessed the eligibility of studies and extracted data. The relative risk (RR) or odds ratio (OR) with 95% confidence intervals (CI) was calculated and pooled. Results Twenty-six studies (16 cohort, 7 case-control, and 3 cross-sectional studies) were identified. Maternal folic acid supplementation was not associated with the development of asthma, atopic dermatitis (AD), eczema, and sensitization in the offspring, whereas exposure during early pregnancy was related to wheeze occurrence in the offspring (RR=1.06, 95% CI=[1.02-1.09]). The TT genotype of MTHFR C677T polymorphism was at high risk of asthma (OR=1.41, 95% CI=[1.07-1.86]). Conclusions It is indicated that maternal folic acid supplementation during early pregnancy may increase the risk of wheeze in early childhood and that the TT genotype of MTHFR C677T polymorphism impairing folic acid metabolism would be at high risk of asthma development. These results might provide additional information for recommendations regarding forced folate consumption or folic acid supplements during pregnancy based on its well-established benefits for the prevention of congenital malformations. However, currently available evidence is of low quality which is needed to further elucidate. PMID:26333700

  7. Biosynthetic incorporation of telluromethionine into dihydrofolate reductase and crystallographic analysis of the distribution of tellurium atoms in the protein molecule

    SciTech Connect

    Kunkle, M.G.; Lewinski, K.; Boles, J.O.; Dunlap, R.B.; Odom, J.D.; Lebioda, L.

    1994-12-01

    Recent successes in crystallographic studies of proteins with methionine (Met) residues replaced with SeMet, pioneered by Hendrickson and coworkers, inspired us to replace Met with TeMet in Escherichia coli dihydrofolate reductase (DHFR). E. coli DHFR, which catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, consists of 159 residues, 5 of which are Met. TeMet was incorporated into DHFR using the Met auxotroph, E. coli DL41, carrying the expression vector pWT8 with an IPTG inducible promoter and ampicillin resistance gene. The enzyme was purified by successive chromatography on Q-Sepharose and PHenyl Sepharose resins, yielding milligram quantities of homogeneous enzyme with a specific activity of 40 units/mg. TeMet DHFR exhibits kinetic properties similar to those of wt DHFR. Amino acid analysis indicated 3 authentic Met residues in TeMet DHFR, whereas atomic absorption spectroscopy detected 2 Te per protein molecule. Amino acid sequence analysis results suggested that only authentic Met was present in the first three Met positions (1,16,and 20). Crystals of Te-DHFR were grown in the presence of methotrexate from PEG 4000 and were isomorphous with wt-DHFR crystals grown from ethanol. Difference Fourier maps and restrained least-squares refinement show very little, if any, Te in the first three Met positions: Met{sup 1}, Met{sup 16}, and Met{sup 20}, whereas the occupancy of Te in positions 42 and 92 is 0.64. Apparently, the process of folding, subsequent purification, and crystallization select DHFR molecules with Te in Met{sup 42} and Met{sup 92}. Replacing Met with TeMet provides an internal probe that should facilitate structural and mechanistic studies of proteins.

  8. Recurrent IVF failure and hereditary thrombophilia

    PubMed Central

    Safdarian, Leila; Najmi, Zahra; Aleyasin, Ashraf; Aghahosseini, Marzieh; Rashidi, Mandana; Asadollah, Sara

    2014-01-01

    Background: The largest percentage of failed invitro fertilization (IVF (cycles, are due to lack of implantation. As hereditary thrombophilia can cause in placentation failure, it may have a role in recurrent IVF failure. Objective: Aim of this case-control study was to determine whether hereditary thrombophilia is more prevalent in women with recurrent IVF failures. Materials and Methods: Case group comprised 96 infertile women, with a history of recurrent IVF failure. Control group was comprised of 95 healthy women with proven fertility who had conceived spontaneously. All participants were assessed for the presence of inherited thrombophilias including: factor V Leiden, methilen tetrahydrofolate reductase (MTHFR) mutation, prothrombin mutation, homocystein level, protein S and C deficiency, antithrombin III (AT-III) deficiency and plasminogen activator inhibitor-1 (PAI-1) mutation. Presence of thrombophilia was compared between groups. Results: Having at least one thrombophilia known as a risk factor for recurrent IVF failure (95% CI=1.74-5.70, OR=3.15, p=0.00). Mutation of factor V Leiden (95% CI=1.26-10.27, OR=3.06, P=0.01) and homozygote form of MTHFR mutation (95% CI=1.55-97.86, OR=12.33, p=0.05) were also risk factors for recurrent IVF failure. However, we could not find significant difference in other inherited thrombophilia’s. Conclusion: Inherited thrombophilia is more prevalent in women with recurrent IVF failure compared with healthy women. Having at least one thrombophilia, mutation of factor V Leiden and homozygote form of MTHFR mutation were risk factors for recurrent IVF failure. PMID:25114668

  9. MTHFR genetic polymorphism increases the risk of preterm delivery

    PubMed Central

    Nan, Yanrong; Li, Hongmei

    2015-01-01

    Aims: This study aimed to investigate the association between the methylene tetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms and premature delivery susceptibility. Methods: With matched age and gender, 108 premature delivery pregnant women as cases and 108 healthy pregnant women as controls were recruited in this case-control study. The cases and controls had same gestational weeks. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was adopted to analyze C677T and A1298C polymorphisms of the participants. Linkage disequilibrium (LD) and haplotype analysis were conducted by Haploview software. The differences for frequencies of gene type, allele and haplotypes in cases and controls were tested by chi-square test. The relevant risk of premature delivery was represented by odds ratios (ORs) with 95% confidence intervals (95% CIs). Results: TT gene type frequency of C677T polymorphsim was higher in cases than the controls (P=0.004, OR=3.077, 95% CI=1.469-6.447), so was allele T (P=0.002, OR=1.853, 95% CI=1.265-2.716). Whereas, CC gene type of A1298C polymorphism had a lower distribution in cases than the controls (P=0.008, OR=0.095, 95% CI=0.012-0.775), so was allele C (P=0.047, OR=0.610, 95% CI=0.384-0.970). Haplotype analysis and linkage disequilibrium test conducted on the alleles of two polymorphisms in MTHFR gene, we discovered that haplotype T-A had a higher distribution in cases, which indicated that susceptible haplotype T-A was the candidate factor for premature delivery. Conclusions: Gene type TT of MTHFR C677T polymorphism might make premature delivery risk rise while gene type CC of A1298C polymorphism might have protective influence on premature delivery. PMID:26261642

  10. Discovery of a sesamin-metabolizing microorganism and a new enzyme.

    PubMed

    Kumano, Takuto; Fujiki, Etsuko; Hashimoto, Yoshiteru; Kobayashi, Michihiko

    2016-08-01

    Sesamin is one of the major lignans found in sesame oil. Although some microbial metabolites of sesamin have been identified, sesamin-metabolic pathways remain uncharacterized at both the enzyme and gene levels. Here, we isolated microorganisms growing on sesamin as a sole-carbon source. One microorganism showing significant sesamin-degrading activity was identified as Sinomonas sp. no. 22. A sesamin-metabolizing enzyme named SesA was purified from this strain and characterized. SesA catalyzed methylene group transfer from sesamin or sesamin monocatechol to tetrahydrofolate (THF) with ring cleavage, yielding sesamin mono- or di-catechol and 5,10-methylenetetrahydrofolate. The kinetic parameters of SesA were determined to be as follows: Km for sesamin = 0.032 ± 0.005 mM, Vmax = 9.3 ± 0.4 (μmol⋅min(-1)⋅mg(-1)), and kcat = 7.9 ± 0.3 s(-1) Next, we investigated the substrate specificity. SesA also showed enzymatic activity toward (+)-episesamin, (-)-asarinin, sesaminol, (+)-sesamolin, and piperine. Growth studies with strain no. 22, and Western blot analysis revealed that SesA formation is inducible by sesamin. The deduced amino acid sequence of sesA exhibited weak overall sequence similarity to that of the protein family of glycine cleavage T-proteins (GcvTs), which catalyze glycine degradation in most bacteria, archaea, and all eukaryotes. Only SesA catalyzes C1 transfer to THF with ring cleavage reaction among GcvT family proteins. Moreover, SesA homolog genes are found in both Gram-positive and Gram-negative bacteria. Our findings provide new insights into microbial sesamin metabolism and the function of GcvT family proteins. PMID:27444012

  11. Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase*

    PubMed Central

    Maenpuen, Somchart; Amornwatcharapong, Watcharee; Krasatong, Pasupat; Sucharitakul, Jeerus; Palfey, Bruce A.; Yuthavong, Yongyuth; Chitnumsub, Penchit; Leartsakulpanich, Ubolsree; Chaiyen, Pimchai

    2015-01-01

    Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from l-serine to tetrahydrofolate (H4folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH2-H4folate). SHMT is crucial for deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. To define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either l-serine or H4folate. The dissociation constants for the enzyme·l-serine and enzyme·H4folate complexes were determined as 0.18 ± 0.08 and 0.35 ± 0.06 mm, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the kcat value (1.09 ± 0.05 s−1) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development. PMID:25678710

  12. Methylamine Utilization via the N-Methylglutamate Pathway in Methylobacterium extorquens PA1 Involves a Novel Flow of Carbon through C1 Assimilation and Dissimilation Pathways

    PubMed Central

    Nayak, Dipti D.

    2014-01-01

    Methylotrophs grow on reduced single-carbon compounds like methylamine as the sole source of carbon and energy. In Methylobacterium extorquens AM1, the best-studied aerobic methylotroph, a periplasmic methylamine dehydrogenase that catalyzes the primary oxidation of methylamine to formaldehyde has been examined in great detail. However, recent metagenomic data from natural ecosystems are revealing the abundance and importance of lesser-known routes, such as the N-methylglutamate pathway, for methylamine oxidation. In this study, we used M. extorquens PA1, a strain that is closely related to M. extorquens AM1 but is lacking methylamine dehydrogenase, to dissect the genetics and physiology of the ecologically relevant N-methylglutamate pathway for methylamine oxidation. Phenotypic analyses of mutants with null mutations in genes encoding enzymes of the N-methylglutamate pathway suggested that γ-glutamylmethylamide synthetase is essential for growth on methylamine as a carbon source but not as a nitrogen source. Furthermore, analysis of M. extorquens PA1 mutants with defects in methylotrophy-specific dissimilatory and assimilatory modules suggested that methylamine use via the N-methylglutamate pathway requires the tetrahydromethanopterin (H4MPT)-dependent formaldehyde oxidation pathway but not a complete tetrahydrofolate (H4F)-dependent formate assimilation pathway. Additionally, we present genetic evidence that formaldehyde-activating enzyme (FAE) homologs might be involved in methylotrophy. Null mutants of FAE and homologs revealed that FAE and FAE2 influence the growth rate and FAE3 influences the yield during the growth of M. extorquens PA1 on methylamine. PMID:25225269

  13. Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron–sulphur cluster metabolism

    PubMed Central

    Waller, Jeffrey C.; Ellens, Kenneth W.; Alvarez, Sophie; Loizeau, Karen; Ravanel, Stéphane; Hanson, Andrew D.

    2012-01-01

    COG0354 proteins have been implicated in synthesis or repair of iron/sulfur (Fe/S) clusters in all domains of life, and those of bacteria, animals, and protists have been shown to require a tetrahydrofolate to function. Two COG0354 proteins were identified in Arabidopsis and many other plants, one (At4g12130) related to those of α-proteobacteria and predicted to be mitochondrial, the other (At1g60990) related to those of cyanobacteria and predicted to be plastidial. Grasses and poplar appear to lack the latter. The predicted subcellular locations of the Arabidopsis proteins were validated by in vitro import assays with purified pea organelles and by targeting assays in Arabidopsis and tobacco protoplasts using green fluorescent protein fusions. The At4g12130 protein was shown to be expressed mainly in flowers, siliques, and seeds, whereas the At1g60990 protein was expressed mainly in young leaves. The folate dependence of both Arabidopsis proteins was established by functional complementation of an Escherichia coli COG0354 (ygfZ) deletant; both plant genes restored in vivo activity of the Fe/S enzyme MiaB but restoration was abrogated when folates were eliminated by deleting folP. Insertional inactivation of At4g12130 was embryo lethal; this phenotype was reversed by genetic complementation of the mutant. These data establish that COG0354 proteins have a folate-dependent function in mitochondria and plastids, and that the mitochondrial protein is essential. That plants retain mitochondrial and plastidial COG0354 proteins with distinct phylogenetic origins emphasizes how deeply the extant Fe/S cluster assembly machinery still reflects the ancient endosymbioses that gave rise to plants. PMID:21984653

  14. Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization.

    PubMed

    Kneuer, Carsten; Honscha, Kerstin U; Honscha, Walther

    2004-03-01

    Previous Northern blot studies suggested strong expression of a homolog to the sodium-dependent hepatocellular methotrexate transporter in the kidneys. Here, we report on the cloning of the cDNA for the renal methotrexate carrier isoform-1 (RK-MTX-1) and its functional characterization. Sequencing revealed 97% homology to the rat liver methotrexate carrier with an identical open reading frame. Differences were located in the 5'-untranslated region and resulted in the absence of putative regulatory elements (Barbie box, Ah/ARNT receptor) identified in the cDNA for the hepatocellular carrier. For functional characterization, MTX-1 cDNA was stably expressed in Madin-Darby canine kidney (MDCK) cells. A sodium-dependent transport of methotrexate with a K(m) of 41 microM and a V(max) of 337 pmol.mg protein(-1).min(-1) was observed. This uptake was blocked by the reduced folates dihydro- and tetrahydrofolate as well as by methotrexate itself. Folate was inhibiting only weakly, whereas 5-methyltetrahydrofolate was a strong inhibitor. Further inhibitors of the methotrexate transport included the bile acids cholate and taurocholate and xenobiotics like bumetanide and BSP. PAH, ouabain, bumetanide, cholate, taurocholate, and acetyl salicylic acid were tested as potential substrates. However, none of these substances was transported by MTX-1. Furthermore, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells fivefold. Analysis of a fusion protein of RK-MTX-1 and the influenza virus hemagglutinin epitope by immunoblotting revealed a major band at 72 kDa within the cell membrane but not in the soluble fraction of transfected MDCK. Indirect immunofluorescence staining revealed an exclusive localization of the carrier in the plasma membrane, and by confocal laser-scanning microscopy we were able to demonstrate that the protein is expressed in the serosal region of MDCK tubules grown in a morphogenic collagen gel model. PMID:14612385

  15. Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-hydroxyoleic acid

    PubMed Central

    Lladó, Victoria; Terés, Silvia; Higuera, Mónica; Álvarez, Rafael; Noguera-Salva, Maria Antònia; Halver, John E.; Escribá, Pablo V.; Busquets, Xavier

    2009-01-01

    α-Hydroxy-9-cis-octadecenoic acid, a synthetic fatty acid that modifies the composition and structure of lipid membranes. 2-Hydroxyoleic acid (HOA) generated interest due to its potent, yet nontoxic, anticancer activity. It induces cell cycle arrest in human lung cancer (A549) cells and apoptosis in human leukemia (Jurkat) cells. These two pathways may explain how HOA induces regression of a variety of cancers. We showed that HOA repressed the expression of dihydrofolate reductase (DHFR), the enzyme responsible for tetrahydrofolate (THF) synthesis. Folinic acid, which readily produces THF without the participation of DHFR, reverses the antitumor effects of HOA in A549 and Jurkat cells, as well as the inhibitory influence on cyclin D and cdk2 in A549 cells, and on DNA and PARP degradation in Jurkat cells. This effect was very specific, because either elaidic acid (an analog of HOA) or other lipids, failed to alter A549 or Jurkat cell growth. THF is a cofactor necessary for DNA synthesis. Thus, impairment of DNA synthesis appears to be a common mechanism involved in the different responses elicited by cancer cells following treatment with HOA, namely cell cycle arrest or apoptosis. Compared with other antifolates, such as methotrexate, HOA did not directly inhibit DHFR but rather, it repressed its expression, a mode of action that offers certain therapeutic advantages. These results not only demonstrate the effect of a fatty acid on the expression of DHFR, but also emphasize the potential of HOA to be used as a wide-spectrum drug against cancer. PMID:19666584

  16. Reduced folate and serum vitamin metabolites in patients with rectal carcinoma: an open-label feasibility study of pemetrexed with folic acid and vitamin B12 supplementation

    PubMed Central

    Odin, Elisabeth A.; Carlsson, Göran U.; Kurlberg, Göran K.; Björkqvist, Hillevi G.; Tångefjord, Maria T.; Gustavsson, Bengt G.

    2016-01-01

    The objectives of this single-center, open-label, phase II study were to evaluate (a) the feasibility and safety of neoadjuvant administration of pemetrexed with oral folic acid and vitamin B12 (FA/B12) in newly diagnosed patients with resectable rectal cancer and (b) intracellular and systemic vitamin metabolism. Patients were treated with three cycles of pemetrexed (500 mg/m2, every 3 weeks) and FA/B12 before surgery. The reduced folates tetrahydrofolate, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate were evaluated from biopsies in tumor tissue and in adjacent mucosa. Serum levels of homocysteine, cystathionine, and methylmalonic acid were also measured. All 37 patients received three cycles of pemetrexed; 89.2% completed their planned dosage within a 9-week feasibility time frame. Neither dose reductions nor study drug-related serious adverse events were reported. Reduced folate levels were significantly higher in tumor tissue compared with adjacent mucosa at baseline. After FA/B12 administration, tissue levels of reduced folates increased significantly and remained high during treatment in both tumor and mucosa until surgery. Serum levels of cystathionine increased significantly compared with baseline after FA/B12 administration, but then decreased, fluctuating cyclically during pemetrexed therapy. Homocysteine and methylmalonic acid levels decreased significantly after FA/B12 administration, and remained below baseline levels during the study. These results indicate that administration of three neoadjuvant cycles of single-agent pemetrexed, every 3 weeks, with FA/B12 in patients with resectable rectal cancer is feasible and tolerable. Tissue and serum vitamin metabolism results demonstrate the influence of pemetrexed and FA/B12 on vitamin metabolism and warrant further study. PMID:26825869

  17. Functional Promiscuity of the COG0720 Family

    PubMed Central

    Phillips, Gabriela; Grochowski, Laura L.; Bonnett, Shilah; Xu, Huimin; Bailly, Marc; Haas-Blaby, Crysten; El Yacoubi, Basma; Iwata-Reuyl, Dirk; White, Robert H.; de Crécy-Lagard, Valérie

    2011-01-01

    The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G+) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H2NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH4 pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, and phylogenetic co-distribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate, or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis, and resulted in the identification PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families, extensive comparative genomic analysis beyond homology is required to correctly predict function. PMID:21999246

  18. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography [Neutron and ultrahigh resolution X-ray crystallography reveals water as the proton donor in the catalytic mechanism of dihydrofolate reductase

    DOE PAGESBeta

    Wan, Qun; Bennett, Brad C.; Wilson, Mark A.; Kovalevsky, Andrey; Langan, Paul; Howell, Elizabeth E.; Dealwis, Chris

    2014-12-01

    Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to the lack of consensus on a catalytic mechanism. To resolve this ambiguity, we conducted neutron and ultrahigh resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of DHFR with folate and NADP+ from E. coli. The neutron data were collected to 2.0 Å resolution using a 3.6 mm3 crystal with the quasi-Laue technique, and the structuremore » reveals that the N3 atom of folate is protonated while Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer due to protonation of the N3 atom, suggesting tautomerization is unnecessary for catalysis. In the 1.05 Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pKa of the N5 atom of DHF by Asp27, and protonation of N5 by water whose access to the active site is gated by fluctuation of the Met20 side chain even though the Met-20 loop is closed.« less

  19. Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death

    SciTech Connect

    Hemendinger, Richelle A. Armstrong, Edward J.; Brooks, Benjamin Rix

    2011-03-15

    Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC{sub 50} (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolate (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC{sub 50} (concentration at which 50% of maximal cell death is inhibited) of 0.6 {mu}M and 0.4 {mu}M, respectively. In contrast, MTHF (up to 10 {mu}M) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS.

  20. Genetic and Lifestyle Variables Associated with Homocysteine Concentrations and the Distribution of Folate Derivatives in Healthy Premenopausal Women

    PubMed Central

    Summers, Carolyn M.; Mitchell, Laura E.; Stanislawska-Sachadyn, Anna; Baido, Shirley F.; Blair, Ian A.; Von Feldt, Joan M.; Whitehead, Alexander S.

    2014-01-01

    Background Low folate and high homocysteine (Hcy) concentrations are associated with pregnancy-related pathologies such as spina bifida. Polymorphisms in folate/Hcy metabolic enzymes may contribute to this potentially pathogenic biochemical phenotype. Methods The study comprised 26 Caucasian and 23 African-American premenopausal women. Subjects gave fasting blood samples for biochemical phenotyping and genotyping. Total Hcy (tHcy) and both plasma and red blood cell (RBC) folate derivatives [i.e. tetrahydrofolate (THF), 5-methylTHF (5-MTHF), and 5,10-methenylTHF (5,10-MTHF)] were measured using stable isotope dilution liquid chromatography, multiple reaction monitoring, mass spectrometry. Eleven polymorphisms from nine folate/Hcy pathway genes were genotyped. Tests of association between genetic, lifestyle, and biochemical variables were applied. Results In African American women, tHcy concentrations were associated (p<0.05) with total RBC folate, RBC 5-MTHF, B12, and polymorphisms in methionine synthase (MTR) and thymidylate synthase (TYMS). In Caucasian women, tHcy concentrations were not associated with total folate levels, but were associated (p<0.05) with RBC THF, ratios of RBC 5-MTHF: THF, and polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR) and MTR . In African Americans, folate derivative levels were associated with smoking, B12, and polymorphisms in MTR, TYMS, methionine synthase reductase (MTRR), and reduced folate carrier1 (RFC1). In Caucasians, folate derivative levels were associated with vitamin use, B12, and polymorphisms in MTHFR, TYMS, and RFC1. Conclusions Polymorphisms in the folate/Hcy pathway are associated with tHcy and folate derivative levels. In African American and Caucasian women, different factors are associated with folate/Hcy phenotypes and may contribute to race-specific differences in the risks of a range of pregnancy-related pathologies. PMID:20544798

  1. Regulation of C1 metabolism by l-methionine in Saccharomyces cerevisiae

    PubMed Central

    Lor, K. L.; Cossins, E. A.

    1972-01-01

    1. The concentrations of folate derivatives in aerobic cultures of Saccharomyces cerevisiae (A.T.C.C. 9763) were determined by microbiological assay employing Lactobacillus casei (A.T.C.C. 7469) and Pediococcus cerevisiae (A.T.C.C. 8081). Cells cultured in media lacking l-methionine contained higher concentrations of folate derivatives than cells grown in the same media supplemented with 2.5μmol of l-methionine/ml. The concentrations of highly conjugated derivatives were also decreased by supplementing the growth medium with l-methionine. 2. DEAE-cellulose column chromatography of extracts prepared from cells grown under these conditions revealed that the concentrations of methylated tetrahydrofolates were drastically decreased by the methionine supplement. Smaller decreases were also observed in the concentrations of formylated and unsubstituted derivatives. 3. The concentrations of four enzymes of C1 metabolism were compared after 6h of growth in the presence and in the absence of l-methionine (2.5μmol/ml). The specific activities of formyltetrahydrofolate synthetase, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase were not altered by this treatment but that of 5-methyltetrahydrofolate–homocysteine methyltransferase was decreased by approx. 65% when l-methionine was supplied. The activities of 5-methyltetrahydrofolate–homocysteine methyltransferase, serine hydroxymethyltransferase and formyltetrahydrofolate synthetase were not appreciably altered by l-methionine in vitro. In contrast this amino acid was found to inhibit the activity of methylenetetrahydrofolate reductase. 4. Feeding experiments employing sodium [14C]formate indicated that cells grown in the presence of exogenous methionine, although having less ability to convert formate into methionine, readily incorporated 14C into serine and the adenosyl moiety of S-adenosylmethionine. 5. It is suggested that exogenous l-methionine controls C1 metabolism in Saccharomyces

  2. Identification and measurement of the folates in sheep liver

    PubMed Central

    Osborne-White, William S.; Smith, Richard M.

    1973-01-01

    1. Methods are described for the extraction, separation by ion-exchange chromatography and estimation by microbiological assay of the folates in sheep liver. 2. Injection of [2-14C]-pteroylglutamate into a sheep fed on a stock diet led to extensive labelling of chromatographically separable liver folates. About 12% of the label in the liver could not be extracted by the method used. 3. Liver folates were examined in five ewes fed on restricted amounts of a diet of wheaten hay-chaff and gluten and injected weekly with vitamin B12. Chromatographic separation was followed by microbiological assay with Lactobacillus casei, Streptococcus faecalis R. and Pediococcus cerevisiae both before and after treatment of fractions with conjugase (γ-glutamylcarboxypeptidase). Evidence was obtained that the folates present were predominantly polyglutamate forms of tetrahydropteroylglutamate, 5-methyltetrahydropteroylglutamate and 5- (and 10-) formyltetrahydropteroylglutamates. Differences in the responses of the assay organisms permitted quantitative distinction between these three main classes of folates. 4. Methyltetrahydrofolates were eluted in seven successive peaks that were separated by constant increments in the logarithm of eluant [Pi]. A similar relationship existed for seven successive peaks of tetrahydrofolate and may also have existed for each of the two series of formyltetrahydrofolates. 5. Based on these and other observations it is proposed that sheep liver folates consist predominantly of the mono- to hepta-glutamates of each of the reduced pteroates identified. The methods employed allowed quantitative determinations to be made of most of the folates present. The predominant forms were hexaglutamates. 6. Four components active for L. casei were detected that could not be identified. Three of them were polyglutamates. PMID:4204321

  3. Arabidopsis Plastidial Folylpolyglutamate Synthetase Is Required for Seed Reserve Accumulation and Seedling Establishment in Darkness

    PubMed Central

    Meng, Hongyan; Jiang, Ling; Xu, Bosi; Guo, Wenzhu; Li, Jinglai; Zhu, Xiuqing; Qi, Xiaoquan; Duan, Lixin; Meng, Xianbin; Fan, Yunliu; Zhang, Chunyi

    2014-01-01

    Interactions among metabolic pathways are important in plant biology. At present, not much is known about how folate metabolism affects other metabolic pathways in plants. Here we report a T-DNA insertion mutant (atdfb-3) of the plastidial folylpolyglutamate synthetase gene (AtDFB) was defective in seed reserves and skotomorphogenesis. Lower carbon (C) and higher nitrogen (N) content in the mutant seeds than that of the wild type were indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars were detected in the mutant seeds compared with the wild type. Further analysis revealed that atdfb-3 seeds contained less total amino acids and individual Asn and Glu as well as NO3−. These results indicate significant changes in seed storage in the mutant. Defects in hypocotyl elongation were observed in atdfb-3 in darkness under sufficient NO3− conditions, and further enhanced under NO3− limited conditions. The strong expression of AtDFB in cotyledons and hypocotyl during early developmental stage was consistent with the mutant sensitivity to limited NO3− during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restored the hypocotyl length in atdfb-3 seedlings with NO3− as the sole N source. Further study demonstrated that folate profiling and N metabolism were perturbed in atdfb-3 etiolated seedlings. The activity of enzymes involved in N reduction and assimilation was altered in atdfb-3. Taken together, these results indicate that AtDFB is required for seed reserves, hypocotyl elongation and N metabolism in darkness, providing novel insights into potential associations of folate metabolism with seed reserve accumulation, N metabolism and hypocotyl development in Arabidopsis. PMID:25000295

  4. The C677T variant in MTHFR modulates associations between blood-based and cerebrospinal fluid biomarkers of neurodegeneration.

    PubMed

    Roussotte, Florence F; Narr, Katherine L; Small, Gary W; Thompson, Paul M

    2016-08-17

    The C677T functional variant in the methylene-tetrahydrofolate reductase (MTHFR) gene results in reduced enzymatic activity and elevated blood levels of homocysteine. Plasma levels of apolipoprotein E (ApoE) are negatively correlated with cerebral amyloid burden, but plasma homocysteine concentrations are associated with increased amyloid-β (Aβ) deposition in the brain. Here, we sought to determine whether associations between low plasma ApoE levels and elevated in-vivo amyloid burden were modulated by carrying the C677T variant. We tested this hypothesis in a large sample of elderly participants from the Alzheimer's Disease Neuroimaging Initiative. We used general linear models to examine associations between plasma homocysteine concentrations, circulating ApoE levels, cerebrospinal fluid concentrations of Aβ, and their modulation by MTHFR and ApoE genotype. Age, sex, and dementia status were included as covariates in all analyses. Higher circulating levels of ApoE predicted increased cerebrospinal fluid concentrations of Aβ, indicating lower in-vivo burden, in C-allele carriers, but not in homozygotes at the C677T variant, who showed significant elevations in plasma homocysteine levels. This modulation by the MTHFR genotype did not remain significant after controlling for ApoE genotype. In T-homozygotes who do not carry the ApoE-ε4 allele, the relationship between low plasma ApoE levels and an increased risk of dementia is likely obscured by the presence of elevated plasma homocysteine. This report suggests the value of genotyping patients at the C677T functional variant when using plasma ApoE levels as a preclinical biomarker for Alzheimer's disease. PMID:27380243

  5. Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur

    PubMed Central

    Ueki, Iori

    2010-01-01

    Synthesis of cysteine as a product of the transsulfuration pathway can be viewed as part of methionine or homocysteine degradation, with cysteine being the vehicle for sulfur conversion to end products (sulfate, taurine) that can be excreted in the urine. Transsulfuration is regulated by stimulation of cystathionine β-synthase and inhibition of methylene tetrahydrofolate reductase in response to changes in the level of S-adenosylmethionine, and this promotes homocysteine degradation when methionine availability is high. Cysteine is catabolized by several desulfuration reactions that release sulfur in a reduced oxidation state, generating sulfane sulfur or hydrogen sulfide (H2S), which can be further oxidized to sulfate. Cysteine desulfuration is accomplished by alternate reactions catalyzed by cystathionine β-synthase and cystathionine γ-lyase. Cysteine is also catabolized by pathways that require the initial oxidation of the cysteine thiol by cysteine dioxygenase to form cysteinesulfinate. The oxidative pathway leads to production of taurine and sulfate in a ratio of approximately 2:1. Relative metabolism of cysteine by desulfuration versus oxidative pathways is influenced by cysteine dioxygenase activity, which is low in animals fed low-protein diets and high in animals fed excess sulfur amino acids. Thus, desulfuration reactions dominate when cysteine is deficient, whereas oxidative catabolism dominates when cysteine is in excess. In rats consuming a diet with an adequate level of sulfur amino acids, about two thirds of cysteine catabolism occurs by oxidative pathways and one third by desulfuration pathways. Cysteine dioxygenase is robustly regulated in response to cysteine availability and may function to provide a pathway to siphon cysteine to less toxic metabolites than those produced by cysteine desulfuration reactions. PMID:20162368

  6. The basis for folinic acid treatment in neuro-psychiatric disorders.

    PubMed

    Ramaekers, V T; Sequeira, J M; Quadros, E V

    2016-07-01

    Multiple factors such as genetic and extraneous causes (drugs, toxins, adverse psychological events) contribute to neuro-psychiatric conditions. In a subgroup of these disorders, systemic folate deficiency has been associated with macrocytic anemia and neuropsychiatric phenotypes. In some of these, despite normal systemic levels, folate transport to the brain is impaired in the so-called cerebral folate deficiency (CFD) syndromes presenting as developmental and psychiatric disorders. These include infantile-onset CFD syndrome, infantile autism with or without neurologic deficits, a spastic-ataxic syndrome and intractable epilepsy in young children expanding to refractory schizophrenia in adolescents, and finally treatment-resistant major depression in adults. Folate receptor alpha (FRα) autoimmunity with low CSF N(5)-methyl-tetrahydrofolate (MTHF) underlies most CFD syndromes, whereas FRα gene abnormalities and mitochondrial gene defects are rarely found. The age at which FRα antibodies of the blocking type emerge, determines the clinical phenotype. Infantile CFD syndrome and autism with neurological deficits tend to be characterized by elevated FRα antibody titers and low CSF MTHF. In contrast, in infantile autism and intractable schizophrenia, abnormal behavioral signs and symptoms may wax and wane with fluctuating FRα antibody titers over time accompanied by cycling changes in CSF folate, tetrahydrobiopterin (BH4) and neurotransmitter metabolites ranging between low and normal levels. We propose a hypothetical model explaining the pathogenesis of schizophrenia. Based on findings from clinical, genetic, spinal fluid and MRI spectroscopic studies, we discuss the neurochemical changes associated with these disorders, metabolic and regulatory pathways, synthesis and catabolism of neurotransmitters, and the impact of oxidative stress on the pathogenesis of these conditions. A diagnostic algorithm and therapeutic regimens using high dose folinic acid

  7. The pugilistDominant Mutation of Drosophila melanogaster: A Simple-Sequence Repeat Disorder Reveals Localized Transport in the Eye.

    PubMed

    Rong, Yikang S; Golic, Mary M; Golic, Kent G

    2016-01-01

    The pugilist-Dominant mutation results from fusion of a portion of the gene encoding the tri-functional Methylene Tetrahydrofolate Dehydrogenase (E.C.1.5.1.5, E.C.3.5.4.9, E.C.6.3.4.3) to approximately one kb of a heterochromatic satellite repeat. Expression of this fusion gene results in an unusual ring pattern of pigmentation around the eye. We carried out experiments to determine the mechanism for this pattern. By using FLP-mediated DNA mobilization to place different pugD transgenes at pre-selected sites we found that variation in repeat length makes a strong contribution to variability of the pug phenotype. This variation is manifest primarily as differences in the thickness of the pigmented ring. We show that similar phenotypic variation can also be achieved by changing gene copy number. We found that the pugD pattern is not controlled by wingless, which is normally expressed in a similar ring pattern. Finally, we found that physical injury to a pugD eye can lead to pigment deposition in parts of the eye that would not have been pigmented in the absence of injury. Our results are consistent with a model in which a metabolite vital for pigment formation is imported from the periphery of the eye, and pugD limits the extent of its transport towards the center of the eye, thus revealing the existence of a hitherto unknown mechanism of localized transport in the eye. PMID:26999432

  8. Feeding low or pharmacological concentrations of zinc oxide changes the hepatic proteome profiles in weaned piglets.

    PubMed

    Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

    2013-01-01

    Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

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

    PubMed Central

    Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

    2013-01-01

    Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

  10. Antioxidative potential of folate producing probiotic Lactobacillus helveticus CD6.

    PubMed

    Ahire, Jayesh Jagannath; Mokashe, Narendra Uttamrao; Patil, Hemant Jagatrao; Chaudhari, Bhushan Liladhar

    2013-02-01

    Folate producing Lactobacillus sp. CD6 isolated from fermented milk showed 98% similarity with Lactobacillus helveticus based on 16S rRNA gene sequence analysis. It was found to produce a folic acid derivative 5-methyl tetrahydrofolate (5-MeTHF). The intracellular cell-free extract of strain demonstrated antioxidative activity with the inhibition rate of ascorbate autoxidation in the range of 27.5% ± 3.7%. It showed highest metal ion chelation ability for Fe(2+) (0.26 ± 0.06 ppm) as compared to Cu(2+). The DPPH (α,α-Diphenyl-β-Picrylhydrazyl) radical scavenging activity for intact cells were found to be 24.7% ± 10.9% proved its antioxidative potential. Furthermore, it demonstrated 14.89% inhibition of epinephrine autoxidation, 20.9 ± 1.8 μg cysteine equivalent reducing activity and 20.8% ± 0.9% hydroxyl radical scavenging effect. The strain was evaluated for probiotic properties as per WHO and FAO guidelines. It showed 90.61% survival at highly acidic condition (pH 2.0), 90.66% viability in presence of synthetic gastric juice and 68% survivability at 0.5% bile concentration for 24 h. It was susceptible to many antibiotics which reduces the prospect to offer resistance determinants to other organisms if administered in the form of probiotic preparations. It showed in vitro mucus binding and antimicrobial activity against enteric pathogens like Salmonella typhimurium (NCIM 2501), Streptococcus pyogenes (NCIM 2608), and Staphylococcus aureus (NCIM 5021) and moreover it showed non- hemolytic activity on sheep blood agar. PMID:24425884

  11. The In Vitro Redundant Enzymes PurN and PurT Are Both Essential for Systemic Infection of Mice in Salmonella enterica Serovar Typhimurium.

    PubMed

    Jelsbak, Lotte; Mortensen, Mie I B; Kilstrup, Mogens; Olsen, John E

    2016-07-01

    Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundant in vitro in Salmonella enterica serovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that single-gene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ΔpurT mutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ΔpurN mutant was reduced approximately 50% in 20 h. The attenuation of the ΔpurT mutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C1 units in vivo (CI = 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C1 units in vitro The results with the ΔgcvT mutant further revealed that formation of serine by SerA and further conversion of serine into C1 units and glycine by GlyA were not sufficient to ensure C1 formation in S Typhimurium in vivo The results of the present study call for reinvestigations of the concept of metabolic redundancy in S Typhimurium in vivo. PMID:27113361

  12. Folate-dependent enzymes in cultured Chinese hamster ovary cells: impaired mitochondrial serine hydroxymethyltransferase activity in two additional glycine-auxotroph complementation classes

    SciTech Connect

    Taylor, R.T.; Hanna, M.L.

    1982-09-01

    Two glycine-requiring Chinese hamster ovary (CHO) auxotrophs (GLYB and AUXB2) representative of the Gly/sup -/ mutant classes B and C are shown to have defects in folate metabolism. These defects result in 10-fold lower rates of whole cell L-(U-/sup 14/C)serine-to-(/sup 14/C)glycine conversion relative to the parental CHO lines (2 vs 20 nmol/h/10/sup 6/ cells). This restriction in serine hydroxymethyltransferase (SHMT) activity is localized in the mitochondria. Intact mitochondria from GLYB and AUXB2 convert labeled serine to glycine at 1-4% the rate and with only 1-3% of the total capacity of parental CHO mitochondria. Yet, GLYB and AUXB2 contain parental cell amounts of cytosolic and mitochondrial SHMT, the latter displaying normal substrate K/sub m/ values. The whole cell and mitochondrial impairments in glycine formation are corrected in GLYB (but not AUXB2) by a prior growth with 100 ..mu..M dl-folinate. They are also partially restored in spontaneous or chemically induced Gly/sup +/ revertants of GLYB and AUXB2. Subcellular fractionation experiments suggest that a low content (one-fifth parental) of mitochondrial folylpolyglutamates contributes to the auxotrophy of GLYB. These studies demonstrate that mitochondrial SHMT is potentially functional in the Gly/sup -/ mutant classes B (GLYB) and C (AUXB2). The impaired SHMT activity in vivo and in isolated mitochondria may result from a deficiency in mitochondrial recycling of 5,10-methylenetetrahydrofolate back to tetrahydrofolate.

  13. Prebiotic chirality

    NASA Astrophysics Data System (ADS)

    Mekki-Berrada, Ali

    Bringing closer phospholipids each other on a bilayer of liposome, causes their rotation around their fatty acids axis, generating a force which brings closer the two sheets of the bilayer. In this theoretical study I show that for getting the greater cohesion of the liposome, by these forces, the serine in the hydrophilic head must have a L chirality. In the case where the hydrophilic head is absent amino acids with L chirality could contribute to this cohesion by taking the place of L-serine. Some coenzymes having a configuration similar to ethanolamine may also contribute. This is the case of pyridoxamine, thiamine and tetrahydrofolic acid. The grouping of amino acids of L chirality and pyridoxamine on the wall could initialize the prebiotic metabolism of these L amino acids only. This would explain the origin of the homo-chirality of amino acids in living world. Furthermore I show that in the hydrophilic head, the esterification of glycerol-phosphate by two fatty acids go through the positioning of dihydroxyacetone-phosphate and L-glyceraldehyde-3-phosphate, but not of D-glyceraldehyde-3-phosphate, prior their hydrogenation to glycerol-3- phosphate. The accumulation of D-glyceraldehyde-3-phosphate in the cytoplasm displace the thermodynamic equilibria towards the synthesis of D-dATP from D-glyceraldehyde-3-phosphate, acetaldehyde and prebiotic adenine, a reaction which does not require a coenzyme in the biotic metabolism. D-dATP and thiamine, more prebiotic metabolism of L-amino acids on the wall, would initialize D-pentoses phosphate and D-nucleotides pathways from the reaction of D-glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate + prebiotic nucleic bases. The exhaustion of the prebiotic glyceraldehyde (racemic) and the nascent biotic metabolism dominated by D-glyceraldehyde-3-phosphate, would explain the origin of homo-chirality of sugars in living world. References: http://en.wikiversity.org/wiki/Prebiotic_chirality

  14. Detection of intermediates in the oxidative half-reaction of the FAD-dependent thymidylate synthase from Thermotoga maritima: carbon transfer without covalent pyrimidine activation.

    PubMed

    Conrad, John A; Ortiz-Maldonado, Mariliz; Hoppe, Samuel W; Palfey, Bruce A

    2014-08-19

    Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2'-deoxyuridine 5'-monophosphate (dUMP) and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF), synthesizing 2'-deoxythymidine 5'-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH2THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis. PMID:25068636

  15. Association of Aberrations in One Carbon Metabolism with Intimal Medial Thickening in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Dhananjayan, R; Malati, T; Rupasree, Y; Kutala, Vijay Kumar

    2015-07-01

    The present work was aimed to study the association of one carbon genetic variants, hyperhomocysteinemia and oxidative stress markers, i.e., serum nitrite, plasma malondialdehyde (MDA) and glutathione (GSH) on intimal medial thickening (IMT) in patients with type 2 diabetes mellitus (T2D). A total number of 76 subjects from ACS Medical College and Hospital, Chennai, India were included in the study, i.e., Group I (n = 42) of T2D and Group II (n = 34) of age- and sex matched healthy controls. The glycated haemoglobin was measured by ion-exchange resin method; plasma homocysteine by Enzyme Linked Immunosorbant Assay method; serum nitrite (nitric oxide, NO), plasma MDA and GSH by spectrophotometric methods; the IMT by high frequency ultrasound. The polymorphisms of one carbon genetic variants were genotyped using polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism methods. Results indicate that methyltetrahydrofolate homocysteine methyl transferase (MTR) A2756G allele was found to be protective in T2D and the other variants were not significantly associated with T2D. Glutamate carboxypeptidase II (GCP II) C1561T (r = 0.34; p = 0.05) and methylene tetrahydrofolate reductase (MTHFR) C677T (r = 0.35; 0.04) showed positive correlation with plasma homocysteine in T2D cases. In this study, MTR A2756G allele was found to be protective in T2D; GCP II C1561T and MTHFR C677T showed positive association with plasma homocysteine in T2D cases. Among all the genetic variants, MTR A2756G was found influence IMT. RFC 1 G80A and TYMS 5'-UTR 2R3R showed synergistically interact with MTR A2756G in influencing increase in IMT. PMID:26089610

  16. Proteome Analysis of Streptococcus thermophilus Grown in Milk Reveals Pyruvate Formate-Lyase as the Major Upregulated Protein

    PubMed Central

    Derzelle, Sylviane; Bolotin, Alexander; Mistou, Michel-Yves; Rul, Françoise

    2005-01-01

    We investigated the adaptation to milk of Streptococcus thermophilus LMG18311 using a proteomic approach. Two-dimensional electrophoresis of cytosolic proteins were performed after growth in M17 medium or in milk. A major modification of the proteome concerned proteins involved in the supply of amino acids, like the peptidase PepX, and several enzymes involved in amino acid biosynthesis. In parallel, we observed the upregulation of the synthesis of seven enzymes directly involved in the synthesis of purines, as well as formyl-tetrahydrofolate (THF) synthetase and serine hydroxy-methyl transferase, two enzymes responsible for the synthesis of compounds (THF and glycine, respectively) feeding the purine biosynthetic pathway. The analysis also revealed a massive increase in the synthesis of pyruvate formate-lyase (PFL), the enzyme which converts pyruvate into acetyl coenzyme A and formate. PFL has been essentially studied for its role in mixed-acid product formation in lactic acid bacteria during anaerobic fermentation. However, formate is an important methyl group donor for anabolic pathway through the formation of folate derivates. We hypothesized that PFL was involved in purine biosynthesis during growth in milk. We showed that PFL expression was regulated at the transcriptional level and that pfl transcription occurred during the exponential growth phase in milk. The complementation of milk with formate or purine bases was shown to reduce pfl expression, to suppress PFL synthesis, and to stimulate growth of S. thermophilus. These results show a novel regulatory mechanism controlling the synthesis of PFL and suggest an unrecognized physiological role for PFL as a formate supplier for anabolic purposes. PMID:16332852

  17. Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

    PubMed Central

    Arvizu-Flores, Aldo A.; Sugich-Miranda, Rocio; Arreola, Rodrigo; Garcia-Orozco, Karina D.; Velazquez-Contreras, Enrique F.; Montfort, William R.; Maley, Frank; Sotelo-Mundo, Rogerio R.

    2008-01-01

    Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH2THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The kcat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the Km for the (R)-stereoisomer of CH2THF was 300 µM, about 30 fold larger than Km from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH2THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group. PMID:18403248

  18. Folate Intake, MTHFR Polymorphisms, and the Risk of Colorectal Cancer: A Systematic Review and Meta-Analysis

    PubMed Central

    Kennedy, Deborah A.; Stern, Seth J.; Matok, Ilan; Moretti, Myla E.; Sarkar, Moumita; Adams-Webber, Thomasin; Koren, Gideon

    2012-01-01

    Background. The objective was to determine whether relationships exist between the methylene-tetrahydrofolate reductase (MTHFR) polymorphisms and risk of colorectal cancer (CRC) and examine whether the risk is modified by level of folate intake. Methods. MEDLINE, Embase, and SCOPUS were searched to May 2012 using the terms “folic acid,” “folate,” “colorectal cancer,” “methylenetetrahydrofolate reductase,” “MTHFR.” Observational studies were included which (1) assessed the risk of CRC for each polymorphism and/or (2) had defined levels of folate intake for each polymorphism and assessed the risk of CRC. Results. From 910 references, 67 studies met our criteria; hand searching yielded 10 studies. The summary risk estimate comparing the 677CT versus CC genotype was 1.02 (95% CI 0.95–1.10) and for 677TT versus CC was 0.88 (95% CI 0.80–0.96) both with heterogeneity. The summary risk estimates for A1298C polymorphisms suggested no reduced risk. The summary risk estimate for high versus low total folate for the 677CC genotype was 0.70 (95% CI 0.56–0.89) and the 677TT genotype 0.63 (95% CI 0.41–0.97). Conclusion. These results suggest that the 677TT genotype is associated with a reduced risk of developing CRC, under conditions of high total folate intake, and this associated risk remains reduced for both MTHFR 677 CC and TT genotypes. PMID:23125859

  19. Non-growth-associated demethylation of dimethylsulfoniopropionate by (homo)acetogenic bacteria.

    PubMed

    Jansen, M; Hansen, T A

    2001-01-01

    The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662(T), Sporomusa sphaeroides DSM 2875(T), and Acetobacterium woodii DSM 1030(T) were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of E. limosum PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h(-1)) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min(-1) mg of protein(-1), both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min(-1) mg of protein(-1) were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero. PMID:11133459

  20. Genome-guided analysis of physiological and morphological traits of the fermentative acetate oxidizer Thermacetogenium phaeum

    PubMed Central

    2012-01-01

    Background Thermacetogenium phaeum is a thermophilic strictly anaerobic bacterium oxidizing acetate to CO2 in syntrophic association with a methanogenic partner. It can also grow in pure culture, e.g., by fermentation of methanol to acetate. The key enzymes of homoacetate fermentation (Wood-Ljungdahl pathway) are used both in acetate oxidation and acetate formation. The obvious reversibility of this pathway in this organism is of specific interest since syntrophic acetate oxidation operates close to the energetic limitations of microbial life. Results The genome of Th. phaeum is organized on a single circular chromosome and has a total size of 2,939,057 bp. It comprises 3.215 open reading frames of which 75% could be assigned to a gene function. The G+C content is 53.88 mol%. Many CRISPR sequences were found, indicating heavy phage attack in the past. A complete gene set for a phage was found in the genome, and indications of phage action could also be observed in culture. The genome contained all genes required for CO2 reduction through the Wood-Ljungdahl pathway, including two formyl tetrahydrofolate ligases, three carbon monoxide dehydrogenases, one formate hydrogenlyase complex, three further formate dehydrogenases, and three further hydrogenases. The bacterium contains a menaquinone MQ-7. No indications of cytochromes or Rnf complexes could be found in the genome. Conclusions The information obtained from the genome sequence indicates that Th. phaeum differs basically from the three homoacetogenic bacteria sequenced so far, i.e., the sodium ion-dependent Acetobacterium woodii, the ethanol-producing Clostridium ljungdahlii, and the cytochrome-containing Moorella thermoacetica. The specific enzyme outfit of Th. phaeum obviously allows ATP formation both in acetate formation and acetate oxidation. PMID:23259483

  1. Stability of the Heme Environment of the Nitric Oxide Synthase from Staphylococcus aureus in the Absence of Pterin Cofactor

    PubMed Central

    Chartier, François J. M.; Couture, Manon

    2004-01-01

    We have used resonance Raman spectroscopy to probe the heme environment of a recently discovered NOS from the pathogenic bacterium Staphylococcus aureus, named SANOS. We detect two forms of the CO complex in the absence of L-arginine, with νFe-CO at 482 and 497 cm−1 and νC-O at 1949 and 1930 cm−1, respectively. Similarly to mammalian NOS, the binding of L-arginine to SANOS caused the formation of a single CO complex with νFe-CO and νC-O frequencies at 504 and 1917 cm−1, respectively, indicating that L-arginine induced an electrostatic/steric effect on the CO molecule. The addition of pterins to CO-bound SANOS modified the resonance Raman spectra only when they were added in combination with L-arginine. We found that (6R) 5,6,7,8 tetra-hydro-L-biopterin and tetrahydrofolate were not required for the stability of the reduced protein, which is 5-coordinate, and of the CO complex, which does not change with time to a form with a Soret band at 420 nm that is indicative of a change of the heme proximal coordination. Since SANOS is stable in the absence of added pterin, it suggests that the role of the pterin cofactor in the bacterial NOS may be limited to electron/proton transfer required for catalysis and may not involve maintaining the structural integrity of the protein as is the case for mammalian NOS. PMID:15345570

  2. Pyruvate Formate Lyase Acts as a Formate Supplier for Metabolic Processes during Anaerobiosis in Staphylococcus aureus▿

    PubMed Central

    Leibig, Martina; Liebeke, Manuel; Mader, Diana; Lalk, Michael; Peschel, Andreas; Götz, Friedrich

    2011-01-01

    Previous studies demonstrated an upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in Staphylococcus aureus biofilms. To investigate their physiological role, we constructed fdh and pfl deletion mutants (Δfdh and Δpfl). Although formate dehydrogenase activity in the fdh mutant was lost, it showed little phenotypic alterations under oxygen-limited conditions. In contrast, the pfl mutant displayed pleiotropic effects and revealed the importance of formate production for anabolic metabolism. In the pfl mutant, no formate was produced, glucose consumption was delayed, and ethanol production was decreased, whereas acetate and lactate production were unaffected. All metabolic alterations could be restored by addition of formate or complementation of the Δpfl mutant. In compensation reactions, serine and threonine were consumed better by the Δpfl mutant than by the wild type, suggesting that their catabolism contributes to the refilling of formyl-tetrahydrofolate, which acts as a donor of formyl groups in, e.g., purine and protein biosynthesis. This notion was supported by reduced production of formylated peptides by the Δpfl mutant compared to that of the parental strain, as demonstrated by weaker formyl-peptide receptor 1 (FPR1)-mediated activation of leukocytes with the mutant. FPR1 stimulation could also be restored either by addition of formate or by complementation of the mutation. Furthermore, arginine consumption and arc operon transcription were increased in the Δpfl mutant. Unlike what occurred with the investigated anaerobic conditions, a biofilm is distinguished by nutrient, oxygen, and pH gradients, and we thus assume that Pfl plays a significant role in the anaerobic layer of a biofilm. Fdh might be critical in (micro)aerobic layers, as formate oxidation is correlated with the generation of NADH/H+, whose regeneration requires respiration. PMID:21169491

  3. The pugilistDominant Mutation of Drosophila melanogaster: A Simple-Sequence Repeat Disorder Reveals Localized Transport in the Eye

    PubMed Central

    Rong, Yikang S.; Golic, Mary M.; Golic, Kent G.

    2016-01-01

    The pugilist-Dominant mutation results from fusion of a portion of the gene encoding the tri-functional Methylene Tetrahydrofolate Dehydrogenase (E.C.1.5.1.5, E.C.3.5.4.9, E.C.6.3.4.3) to approximately one kb of a heterochromatic satellite repeat. Expression of this fusion gene results in an unusual ring pattern of pigmentation around the eye. We carried out experiments to determine the mechanism for this pattern. By using FLP-mediated DNA mobilization to place different pugD transgenes at pre-selected sites we found that variation in repeat length makes a strong contribution to variability of the pug phenotype. This variation is manifest primarily as differences in the thickness of the pigmented ring. We show that similar phenotypic variation can also be achieved by changing gene copy number. We found that the pugD pattern is not controlled by wingless, which is normally expressed in a similar ring pattern. Finally, we found that physical injury to a pugD eye can lead to pigment deposition in parts of the eye that would not have been pigmented in the absence of injury. Our results are consistent with a model in which a metabolite vital for pigment formation is imported from the periphery of the eye, and pugD limits the extent of its transport towards the center of the eye, thus revealing the existence of a hitherto unknown mechanism of localized transport in the eye. PMID:26999432

  4. Reduced folate and serum vitamin metabolites in patients with rectal carcinoma: an open-label feasibility study of pemetrexed with folic acid and vitamin B12 supplementation.

    PubMed

    Stoffregen, Clemens C; Odin, Elisabeth A; Carlsson, Göran U; Kurlberg, Göran K; Björkqvist, Hillevi G; Tångefjord, Maria T; Gustavsson, Bengt G

    2016-06-01

    The objectives of this single-center, open-label, phase II study were to evaluate (a) the feasibility and safety of neoadjuvant administration of pemetrexed with oral folic acid and vitamin B12 (FA/B12) in newly diagnosed patients with resectable rectal cancer and (b) intracellular and systemic vitamin metabolism. Patients were treated with three cycles of pemetrexed (500 mg/m, every 3 weeks) and FA/B12 before surgery. The reduced folates tetrahydrofolate, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate were evaluated from biopsies in tumor tissue and in adjacent mucosa. Serum levels of homocysteine, cystathionine, and methylmalonic acid were also measured. All 37 patients received three cycles of pemetrexed; 89.2% completed their planned dosage within a 9-week feasibility time frame. Neither dose reductions nor study drug-related serious adverse events were reported. Reduced folate levels were significantly higher in tumor tissue compared with adjacent mucosa at baseline. After FA/B12 administration, tissue levels of reduced folates increased significantly and remained high during treatment in both tumor and mucosa until surgery. Serum levels of cystathionine increased significantly compared with baseline after FA/B12 administration, but then decreased, fluctuating cyclically during pemetrexed therapy. Homocysteine and methylmalonic acid levels decreased significantly after FA/B12 administration, and remained below baseline levels during the study. These results indicate that administration of three neoadjuvant cycles of single-agent pemetrexed, every 3 weeks, with FA/B12 in patients with resectable rectal cancer is feasible and tolerable. Tissue and serum vitamin metabolism results demonstrate the influence of pemetrexed and FA/B12 on vitamin metabolism and warrant further study. PMID:26825869

  5. Physiology and nutrition of Treponema primitia, an H2/CO2-acetogenic spirochete from termite hindguts.

    PubMed

    Graber, Joseph R; Breznak, John A

    2004-03-01

    Treponema primitia strains ZAS-1 and ZAS-2, the first spirochetes to be isolated from termite hindguts (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science 283:686-689, 1999), were examined for nutritional, physiological, and biochemical properties relevant to growth and survival in their natural habitat. In addition to using H(2) plus CO(2) as substrates, these strains were capable of homoacetogenic growth on mono- and disaccharides and (in the case of ZAS-2) methoxylated benzenoids. Cells were also capable of mixotrophic growth (i.e., simultaneous utilization of H(2) and organic substrates). Cell extracts of T. primitia possessed enzyme activities of the Wood/Ljungdahl (acetyl coenzyme A) pathway of acetogenesis, including tetrahydrofolate-dependent enzymes of the methyl group-forming branch. However, a folate compound was required in the medium for growth. ZAS-1 and ZAS-2 growing on H(2) plus CO(2) displayed H(2) thresholds of 650 and 490 ppmv, respectively. Anoxic cultures of ZAS-1 and ZAS-2 maintained growth after the addition of as much as 0.5% (vol/vol) O(2) to the headspace atmosphere. Cell extracts exhibited NADH and NADPH peroxidase and NADH oxidase activities but neither catalase nor superoxide dismutase activity. Results indicate that (i) T. primitia is able to exploit a variety of substrates derived from the food of its termite hosts and in so doing contributes to termite nutrition via acetogenesis, (ii) in situ growth of T. primitia is likely dependent on secretion of a folate compound(s) by other members of the gut microbiota, and (iii) cells possess enzymatic adaptations to oxidative stress, which is likely to be encountered in peripheral regions of the termite hindgut. PMID:15006747

  6. Association between Methylenetetrahydrofolate Reductase (MTHFR) Gene Polymorphisms and Susceptibility to Childhood Acute Lymphoblastic Leukemia in an Iranian Population

    PubMed Central

    Bahari, Gholamreza; Hashemi, Mohammad; Naderi, Majid; Taheri, Mohsen

    2016-01-01

    Background: The present study was aimed to examine the possible association between methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms and childhood acute lymphoblastic leukemia (ALL) in a sample of Iranian population. Subjects and Methods: A total of 220 subjects including 100 children diagnosed with ALL and 120 healthy children participated in the case-control study. The single nucleotide polymorphisms (SNPs) of MTHFR were determined by ARMS-PCR or PCR-RFLP method. Results: Our investigation revealed that rs13306561 both TC and TC + CC genotypes decreased the risk of ALL compared to TT genotype (OR=0.32, 95%CI=0.15-0.68, p=0.002 and OR=0.35, 95%CI=0.17-0.70, p=0.003, respectively). In addition, the rs13306561 C allele decreased the risk of ALL in comparison with T allele (OR=0.42, 95% CI=0.22-0.78, P=0.005). MTHFR rs1801131 (A1298C) polymorphism showed that the AC heterozygous genotype decreased the risk of ALL in comparison with AA homozygous genotype (OR=0.43, 95%CI=0.21-0.90, p=0.037). Neither the overall Chi-square comparison of cases and control subjects (𝜒2=5.54, p=0.063) nor the logistic regression analysis showed significant association between C677T polymorphism and ALL (OR=1.25, 95% CI=0.69-2.23, p=0.552; CT vs. CC). Conclusion: The current investigation findings showed that MTHFR rs1801131 and rs13306561 polymorphisms decreased the risk of ALL in the population which has been studied. Further studies with larger sample sizes and different ethnicities are required to validate our findings. PMID:27489588

  7. The structure of Plasmodium falciparum serine hydroxymethyltransferase reveals a novel redox switch that regulates its activities

    SciTech Connect

    Chitnumsub, Penchit Ittarat, Wanwipa; Jaruwat, Aritsara; Noytanom, Krittikar; Amornwatcharapong, Watcharee; Pornthanakasem, Wichai; Chaiyen, Pimchai; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

    2014-06-01

    The crystal structure of P. falciparum SHMT revealed snapshots of an intriguing disulfide/sulfhydryl switch controlling the functional activity. Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Å resolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similar to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme.

  8. Asparagine 229 in thymidylate synthase contributes to, but is not essential for, catalysis.

    PubMed Central

    Liu, L; Santi, D V

    1993-01-01

    The conserved Asn-229 (N229) of thymidylate synthase (TS, EC 2.1.1.45) provides the only side chain that directly hydrogen bonds with the pyrimidine ring of the substrate dUMP. The carboxamide moiety forms a cyclic hydrogen bond network with the NH-3 and O-4 of the base and is a prime candidate for assisting proton-transfer reactions that occur at O-4 of the pyrimidine ring of dUMP. A complete replacement set of mutants at position 229 of Lactobacillus casei TS (N229 mutants) has been prepared, purified, and characterized. Fifteen of the 19 TS mutants were catalytically active. Steady-state kinetic parameters of N229 mutants varied 17- and 115-fold in the Km values for 5,10-methylene-5,6,7,8-tetrahydrofolate and dUMP, respectively, 1000-fold in kcat values, and 10,000-fold in kcat/Km values. Wild-type TS possesses lower Km and higher kcat and kcat/Km values than any of the TS N229 mutants. We conclude that N229 contributes to, but is not essential for, binding and catalysis. When the wild-type enzyme was not considered, there were excellent correlations between log kcat and the hydrophobicity of the side chains at position 229, in which the more hydrophobic side chains showed higher values. Our results suggest a unique interaction between N229 and the substrates that seems important in appropriately positioning the uracil heterocycle for catalysis. We propose that in the absence of N229, the electrophilic catalyst that transfers protons to the O-4 and stabilizes enol intermediates is a highly conserved molecule of water. PMID:8378336

  9. Stability of the heme environment of the nitric oxide synthase from Staphylococcus aureus in the absence of pterin cofactor.

    PubMed

    Chartier, François J M; Couture, Manon

    2004-09-01

    We have used resonance Raman spectroscopy to probe the heme environment of a recently discovered NOS from the pathogenic bacterium Staphylococcus aureus, named SANOS. We detect two forms of the CO complex in the absence of L-arginine, with nu(Fe-CO) at 482 and 497 cm(-1) and nu(C-O) at 1949 and 1930 cm(-1), respectively. Similarly to mammalian NOS, the binding of L-arginine to SANOS caused the formation of a single CO complex with nu(Fe-CO) and nu(C-O) frequencies at 504 and 1,917 cm(-1), respectively, indicating that L-arginine induced an electrostatic/steric effect on the CO molecule. The addition of pterins to CO-bound SANOS modified the resonance Raman spectra only when they were added in combination with L-arginine. We found that (6R) 5,6,7,8 tetra-hydro-L-biopterin and tetrahydrofolate were not required for the stability of the reduced protein, which is 5-coordinate, and of the CO complex, which does not change with time to a form with a Soret band at 420 nm that is indicative of a change of the heme proximal coordination. Since SANOS is stable in the absence of added pterin, it suggests that the role of the pterin cofactor in the bacterial NOS may be limited to electron/proton transfer required for catalysis and may not involve maintaining the structural integrity of the protein as is the case for mammalian NOS. PMID:15345570

  10. The SU.FOL.OM3 Study: a secondary prevention trial testing the impact of supplementation with folate and B-vitamins and/or Omega-3 PUFA on fatal and non fatal cardiovascular events, design, methods and participants characteristics

    PubMed Central

    Galan, Pilar; Briancon, Serge; Blacher, Jacque; Czernichow, Sébastien; Hercberg, Serge

    2008-01-01

    Background During the last decades, many basic and clinical research have pointed to the role of B vitamins (folate, vitamins B6 and B12) and n-3 fatty acids as nutritional factors that might have a protective effect on the development of cardiovascular diseases (CVD). Methods/design The SU.FOL.OM3 (SUpplementation with FOlate, vitamin B6 and B12 and/or OMega-3 fatty acids) trial is a randomized double-blind, placebo-controlled, secondary-prevention trial designed to test the efficacy of 5-methyl tetra-hydro-folates (5-MTHF) supplementation, in combination with vitamin B6 and B12 and/or n-3 fatty acids, at nutritional doses, on fatal and non fatal ischemic CVD in a 2 × 2 factorial design. A total of 2501 patients aged between 45 and 80 years who had a past history, in the previous year, of myocardial infarction (n = 1151) or instable angina pectoris (n = 711) or an ischemic stroke (n = 639) were included. Subjects have to be supplemented and followed up for five years. Daily supplementation comprised nutritional doses of 5-MTHF (560 μg), vitamin B6 (3 mg) and B12 (20 μg) and/or n-3 fatty acids (600 mg with an EPA:DHA ratio of 2:1). A factorial design 2 × 2 has been applied to investigate the separate effects of the B-vitamins, and the n-3 fatty acids, as well as their interaction as compared to the placebo. The primary endpoint is a combination of myocardial infarction, ischemic stroke and cardiovascular death. Secondary endpoints are events of the composite endpoint taken separately, total mortality, and other cardiovascular events such as acute coronary syndromes, coronary revascularization, cardiac failure, arrhythmia... Conclusion Baseline socio-demographic and medical characteristics of participants are totally comparable in the four randomized groups. Trial registration Current Controlled Trials ISRCTN41926726 PMID:18544171

  11. Synthesis of 5-methyl-5-deaza nonclassical antifolates as inhibitors of dihydrofolate reductases and as potential antipneumocystis, antitoxoplasma, and antitumor agents.

    PubMed

    Gangjee, A; Shi, J; Queener, S F; Barrows, L R; Kisliuk, R L

    1993-10-29

    A series of 2,4-diamino-5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines 4-9 were synthesized as 5-deaza nonclassical antifolates containing trimethoxy, dichloro-, or trichlorophenyl substitutions and a N-H, N-CH3, or N-CHO at the 10-position. The compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (P. carinii), Toxoplasma gondii (T. gondii), rat liver (RL), and Lactobacillus casei (L. casei); as inhibitors of T. gondii and P. carinii cell growth in culture; and as antitumor agents. The compounds were prepared by modifications of procedures for classical 5-deaza folates. 2,4-Diamino-5-methyl-6-[(3',4',5'-trimethoxy-N- methylanilino)methyl]pyrido[2,3-d]pyrimidine (5a) exhibited high potency as well as selectivity (compared to RL DHFR) for P. carinii and T. gondii DHFR. Compound 5a is one of the most potent and selective nonclassical folate inhibitors of T. gondii DHFR known. The N-10 formyl analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]pyrido-[2,3-d]pyrimidine (6a) had decreased potency, but it maintained high selectivity for T. gondii DHFR. The corresponding chloro-substituted analogues maintained potency or had decreased potency; N-10 substitution did not increase potency or selectivity to the extent observed in the 3',4',5'-trimethoxy series. Partial reduction of the B ring to afford the dihydro analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]-5,8-dihydropyrido[2,3-d]pyrimidine (7), its 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine analogue 8, and 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino)methyl]-5,6,7, 8- tetrahydropyrido[2,3-d]pyrimidine (9) resulted in a significant decrease in potency. In T. gondii cell culture inhibitory studies, 2,4-diamino-5-methyl-6-[(3',4',5'- trimethoxyanilino)methyl]pyrido[2,3-d]pyrimidine (4a), 5a, and 6a were less potent compared to their DHFR inhibitory potencies. Against P. carinii cells in culture, 4a and 5a at 10

  12. Effects of folic acid deficiency and MTHFR C677T polymorphism on spontaneous and radiation-induced micronuclei in human lymphocytes.

    PubMed

    Leopardi, Paola; Marcon, Francesca; Caiola, Stefania; Cafolla, Arturo; Siniscalchi, Ester; Zijno, Andrea; Crebelli, Riccardo

    2006-09-01

    Folic acid plays a key role in the maintenance of genomic stability, providing methyl groups for the conversion of uracil to thymine and for DNA methylation. Besides dietary habits, folic acid metabolism is influenced by genetic polymorphism. The C677T polymorphism of the methylene-tetrahydrofolate reductase (MTHFR) gene is associated with a reduction of catalytic activity and is suggested to modify cancer risk differently depending on folate status. In this work the effect of folic acid deficiency on genome stability and radiosensitivity has been investigated in cultured lymphocytes of 12 subjects with different MTHFR genotype (four for each genotype). Cells were grown for 9 days with 12, 24 and 120 nM folic acid and analyzed in a comprehensive micronucleus test coupled with centromere characterization by CREST immunostaining. In other experiments, cells were grown with various folic acid concentrations, irradiated with 0.5 Gy of gamma rays and analyzed in the micronucleus test. The results obtained indicate that folic acid deficiency induces to a comparable extent chromosome loss and breakage, irrespective of the MTHFR genotype. The effect of folic acid was highly significant (P < 0.001) and explained >50% of variance of both types of micronuclei. Also nucleoplasmic bridges and buds were significantly increased under low folate supply; the increase in bridges was mainly observed in TT cells, highlighting a significant effect of the MTHFR genotype (P = 0.006) on this biomarker. Folic acid concentration significantly affected radiation-induced micronuclei (P < 0.001): the increased incidence of radiation-induced micronuclei with low folic acid was mainly accounted for by carriers of the variant MTHFR allele (both homozygotes and heterozygotes), but the overall effect of genotype did not attain statistical significance. Treatment with ionizing radiations also increased the frequency of nucleoplasmic bridges. The effect of folic acid level on this end-point was

  13. Folate status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011-2.

    PubMed

    Pfeiffer, Christine M; Sternberg, Maya R; Fazili, Zia; Lacher, David A; Zhang, Mindy; Johnson, Clifford L; Hamner, Heather C; Bailey, Regan L; Rader, Jeanne I; Yamini, Sedigheh; Berry, R J; Yetley, Elizabeth A

    2015-06-28

    Serum and erythrocyte (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured the serum folate forms (5-methyltetrahydrofolate (5-methylTHF), unmetabolised folic acid (UMFA), non-methyl folate (sum of tetrahydrofolate (THF), 5-formyltetrahydrofolate (5-formylTHF), 5,10-methenyltetrahydrofolate (5,10-methenylTHF)) and MeFox (5-methylTHF oxidation product)) by HPLC-MS/MS and RBC total folate by microbiologic assay in US population ≥ 1 year (n approximately 7500) participating in the National Health and Nutrition Examination Survey 2011-2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37·5 nmol/l; 100 %), UMFA (1·21 nmol/l; 99·9 %), MeFox (1·53 nmol/l; 98·8 %), and THF (1·01 nmol/l; 85·2 %) were mostly detectable. 5-FormylTHF (3·6 %) and 5,10-methenylTHF (4·4 %) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86·7 %); UMFA (4·0 %), non-methyl folate (4·7 %) and MeFox (4·5 %) contributed smaller amounts. Age was positively related to MeFox, but showed a U-shaped pattern for other folates. We generally noted sex and race/ethnic biomarker differences and weak (Spearman's r< 0·4) but significant (P< 0·05) correlations with physiological and lifestyle variables. Fasting, kidney function, smoking and alcohol intake showed negative associations. BMI and body surface area showed positive associations with MeFox but negative associations with other folates. All biomarkers showed significantly higher concentrations with recent folic acid-containing dietary supplement use. These first-time population data for serum folate forms generally show similar associations with demographic, physiological and lifestyle variables as serum total folate. Patterns observed for MeFox may suggest altered folate metabolism dependent on biological

  14. Bridging the gap between gene expression and metabolic phenotype via kinetic models

    PubMed Central

    2013-01-01

    Background Despite the close association between gene expression and metabolism, experimental evidence shows that gene expression levels alone cannot predict metabolic phenotypes, indicating a knowledge gap in our understanding of how these processes are connected. Here, we present a method that integrates transcriptome, fluxome, and metabolome data using kinetic models to create a mechanistic link between gene expression and metabolism. Results We developed a modeling framework to construct kinetic models that connect the transcriptional and metabolic responses of a cell to exogenous perturbations. The framework allowed us to avoid extensive experimental characterization, literature mining, and optimization problems by estimating most model parameters directly from fluxome and transcriptome data. We applied the framework to investigate how gene expression changes led to observed phenotypic alterations of Saccharomyces cerevisiae treated with weak organic acids (i.e., acetate, benzoate, propionate, or sorbate) and the histidine synthesis inhibitor 3-aminotriazole under steady-state conditions. We found that the transcriptional response led to alterations in yeast metabolism that mimicked measured metabolic fluxes and concentration changes. Further analyses generated mechanistic insights of how S. cerevisiae responds to these stresses. In particular, these results suggest that S. cerevisiae uses different regulation strategies for responding to these insults: regulation of two reactions accounted for most of the tolerance to the four weak organic acids, whereas the response to 3-aminotriazole was distributed among multiple reactions. Moreover, we observed that the magnitude of the gene expression changes was not directly correlated with their effect on the ability of S. cerevisiae to grow under these treatments. In addition, we identified another potential mechanism of action of 3-aminotriazole associated with the depletion of tetrahydrofolate. Conclusions Our

  15. Sulfur amino acid metabolism in doxorubicin-resistant breast cancer cells

    SciTech Connect

    Ryu, Chang Seon; Kwak, Hui Chan; Lee, Kye Sook; Kang, Keon Wook; Oh, Soo Jin; Lee, Ki Ho; Kim, Hwan Mook; Ma, Jin Yeul; Kim, Sang Kyum

    2011-08-15

    Although methionine dependency is a phenotypic characteristic of tumor cells, it remains to be determined whether changes in sulfur amino acid metabolism occur in cancer cells resistant to chemotherapeutic medications. We compared expression/activity of sulfur amino acid metabolizing enzymes and cellular levels of sulfur amino acids and their metabolites between normal MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Adr) cells. The S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in MCF-7/Adr cells decreased to {approx} 10% relative to that in MCF-7 cells, which may have resulted from down-regulation of S-adenosylhomocysteine hydrolase. Expression of homocysteine-clearing enzymes, such as cystathionine beta-synthase, methionine synthase/methylene tetrahydrofolate reductase, and betaine homocysteine methyltransferase, was up-regulated in MCF-7/Adr cells, suggesting that acquiring doxorubicin resistance attenuated methionine-dependence and activated transsulfuration from methionine to cysteine. Homocysteine was similar, which is associated with a balance between the increased expressions of homocysteine-clearing enzymes and decreased extracellular homocysteine. Despite an elevation in cysteine, cellular GSH decreased in MCF-7/Adr cells, which was attributed to over-efflux of GSH into the medium and down-regulation of the GSH synthesis enzyme. Consequently, MCF-7/Adr cells were more sensitive to the oxidative stress induced by bleomycin and menadione than MCF-7 cells. In conclusion, our results suggest that regulating sulfur amino acid metabolism may be a possible therapeutic target for chemoresistant cancer cells. These results warrant further investigations to determine the role of sulfur amino acid metabolism in acquiring anticancer drug resistance in cancer cells using chemical and biological regulators involved in sulfur amino acid metabolism. - Research Highlights: > MCF-7/Adr cells showed decreases in cellular GSH

  16. Folate deficiency affects histone methylation.

    PubMed

    Garcia, Benjamin A; Luka, Zigmund; Loukachevitch, Lioudmila V; Bhanu, Natarajan V; Wagner, Conrad

    2016-03-01

    Formaldehyde is extremely toxic reacting with proteins to crosslinks peptide chains. Formaldehyde is a metabolic product in many enzymatic reactions and the question of how these enzymes are protected from the formaldehyde that is generated has largely remained unanswered. Early experiments from our laboratory showed that two liver mitochondrial enzymes, dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SDH) catalyze oxidative demethylation reactions (sarcosine is a common name for monomethylglycine). The enzymatic products of these enzymes were the demethylated substrates and formaldehyde, produced from the removed methyl group. Both DMGDH and SDH contain FAD and both have tightly bound tetrahydrofolate (THF), a folate coenzyme. THF binds reversibly with formaldehyde to form 5,10-methylene-THF. At that time we showed that purified DMGDH, with tightly bound THF, reacted with formaldehyde generated during the reaction to form 5,10-methylene-THF. This effectively scavenged the formaldehyde to protect the enzyme. Recently, post-translational modifications on histone tails have been shown to be responsible for epigenetic regulation of gene expression. One of these modifications is methylation of lysine residues. The first enzyme discovered to accomplish demethylation of these modified histones was histone lysine demethylase (LSD1). LSD1 specifically removes methyl groups from di- and mono-methylated lysines at position 4 of histone 3. This enzyme contained tightly bound FAD and the products of the reaction were the demethylated lysine residue and formaldehyde. The mechanism of LSD1 demethylation is analogous to the mechanism previously postulated for DMGDH, i.e. oxidation of the N-methyl bond to the methylene imine followed by hydrolysis to generate formaldehyde. This suggested that THF might also be involved in the LSD1 reaction to scavenge the formaldehyde produced. Our hypotheses are that THF is bound to native LSD1 by analogy to DMGDH and SDH and

  17. Loss of function of folylpolyglutamate synthetase 1 reduces lignin content and improves cell wall digestibility in Arabidopsis

    SciTech Connect

    Srivastava, Avinash C.; Chen, Fang; Ray, Tui; Pattathil, Sivakumar; Peña, Maria J.; Avci, Utku; Li, Hongjia; Huhman, David V.; Backe, Jason; Urbanowicz, Breeanna; Miller, Jeffrey S.; Bedair, Mohamed; Wyman, Charles E.; Sumner, Lloyd W.; York, William S.; Hahn, Michael G.; Dixon, Richard A.; Blancaflor, Elison B.; Tang, Yuhong

    2015-12-21

    One-carbon (C1) metabolism is important for synthesizing a range of biologically important compounds that are essential for life. In plants, the C1 pathway is crucial for the synthesis of a large number of secondary metabolites, including lignin. Tetrahydrofolate and its derivatives, collectively referred to as folates, are crucial co-factors for C1 metabolic pathway enzymes. Given the link between the C1 and phenylpropanoid pathways, we evaluated whether folylpolyglutamate synthetase (FPGS), an enzyme that catalyzes the addition of a glutamate tail to folates to form folylpolyglutamates, can be a viable target for reducing cell wall recalcitrance in plants. Consistent with its role in lignocellulosic formation, FPGS1 was preferentially expressed in vascular tissues. Total lignin was low in fpgs1 plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 was mainly due to lower guaiacyl (G) lignin levels. Glycome profiling revealed subtle alterations in the cell walls of fpgs1. Further analyses of hemicellulosic polysaccharides by NMR showed that the degree of methylation of 4-O-methyl glucuronoxylan was reduced in the fpgs1 mutant. Microarray analysis and real-time qRT-PCR revealed that transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants. Consistent with the transcript changes of C1-related genes, a significant reduction in S-adenosyl-l-methionine content was detected in the fpgs1 mutant. The modified expression of the various methyltransferases and lignin-related genes indicate possible feedback regulation of C1 pathway-mediated lignin biosynthesis. In conclusion, our observations provide genetic and biochemical support for the importance of folylpolyglutamates in the lignocellulosic pathway and reinforces previous observations that targeting a single FPGS isoform for down-regulation leads to reduced lignin in plants. Because fpgs1 mutants had no dramatic defects in

  18. Loss of function of folylpolyglutamate synthetase 1 reduces lignin content and improves cell wall digestibility in Arabidopsis

    DOE PAGESBeta

    Srivastava, Avinash C.; Chen, Fang; Ray, Tui; Pattathil, Sivakumar; Peña, Maria J.; Avci, Utku; Li, Hongjia; Huhman, David V.; Backe, Jason; Urbanowicz, Breeanna; et al

    2015-12-21

    One-carbon (C1) metabolism is important for synthesizing a range of biologically important compounds that are essential for life. In plants, the C1 pathway is crucial for the synthesis of a large number of secondary metabolites, including lignin. Tetrahydrofolate and its derivatives, collectively referred to as folates, are crucial co-factors for C1 metabolic pathway enzymes. Given the link between the C1 and phenylpropanoid pathways, we evaluated whether folylpolyglutamate synthetase (FPGS), an enzyme that catalyzes the addition of a glutamate tail to folates to form folylpolyglutamates, can be a viable target for reducing cell wall recalcitrance in plants. Consistent with its rolemore » in lignocellulosic formation, FPGS1 was preferentially expressed in vascular tissues. Total lignin was low in fpgs1 plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 was mainly due to lower guaiacyl (G) lignin levels. Glycome profiling revealed subtle alterations in the cell walls of fpgs1. Further analyses of hemicellulosic polysaccharides by NMR showed that the degree of methylation of 4-O-methyl glucuronoxylan was reduced in the fpgs1 mutant. Microarray analysis and real-time qRT-PCR revealed that transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants. Consistent with the transcript changes of C1-related genes, a significant reduction in S-adenosyl-l-methionine content was detected in the fpgs1 mutant. The modified expression of the various methyltransferases and lignin-related genes indicate possible feedback regulation of C1 pathway-mediated lignin biosynthesis. In conclusion, our observations provide genetic and biochemical support for the importance of folylpolyglutamates in the lignocellulosic pathway and reinforces previous observations that targeting a single FPGS isoform for down-regulation leads to reduced lignin in plants. Because fpgs1 mutants had no dramatic

  19. Gene Environment Risk Assessment and Colorectal Cancer Screening in an Average Risk Population: A Randomized, Controlled Trial

    PubMed Central

    Weinberg, David S.; Myers, Ronald E.; Keenan, Eileen; Ruth, Karen; Sifri, Randa; Ziring, Barry; Ross, Eric; Manne, Sharon L.

    2015-01-01

    Background New methods are needed to improve health behaviors such as adherence to colorectal cancer (CRC) screening. There is increasing availability of personalized genetic information to inform medical decisions. It is not known if such information motivates behavioral change. Objective To determine, in average risk persons, if individualized gene-environment risk assessment about CRC susceptibility improves adherence to screening. Design Two-arm, randomized, controlled trial Setting Four medical school affiliated primary care practices Patients 783 patients at average risk for CRC, but not adherent with screening at study entry Intervention Patients were randomized to usual care or to receipt of Gene Environmental Risk Assessment (GERA), which assessed Methylene Tetrahydrofolate Reductase (MTHFR) polymorphisms and serum folate level. Based on pre-specified polymorphism/folate level combinations, GERA participants were told they were at either “elevated” or at “average” risk for CRC. Measurements The primary outcome was receipt of CRC screening within 6 months of study entry. Results CRC screening rates were not statistically significantly different between usual care (35.7%) and GERA (33.1%) arms overall. After adjustment for baseline participant factors, the odds ratio (OR) for screening completion for GERA vs usual care was 0.88 (95% CI 0.64 - 1.22). Within the GERA arm, there was no significant difference in screening rates between GERA average risk (38.1%) and GERA elevated risk (26.9%) groups. Odds ratios for elevated vs. average risk remained non-significant after adjustment for covariates (OR=0.75, 95% CI 0.39 - 1.42). Limitations Only one personalized, gene-environment interaction and only one health behavior, colorectal cancer screening, were assessed. Conclusion In average risk persons, there was no positive association between CRC screening uptake and feedback of a single personalized gene-environment risk assessment (GERA). Additional

  20. Analysis of multiple genetic polymorphisms in aggressive- and slow-growing abdominal aortic aneurysms

    PubMed Central

    Duellman, Tyler; Warren, Christopher L.; Matsumura, Jon; Yang, Jay

    2014-01-01

    Introduction The natural history of abdominal aortic aneurysm (AAA) suggests that some remain slow in growth rate while many develop a more accelerated growth rate reaching a threshold for intervention. We hypothesized that different mechanisms are responsible for AAA that remain slow-growth and never become actionable versus the aggressive-AAA that require intervention may be reflected by distinct associations with genetic polymorphisms. Methods 168 control and 141 AAA subjects all with ultrasound or CT imaging studies covering about 5 years were identified and the AAA growth rate determined from the serial imaging data. Genetic polymorphisms all previously reported as showing significant correlation with AAA: angiotensin 1 receptor (AT1R) (rs5186), interleukin-10 (IL-10) (rs1800896), methyl-tetrahydrofolate reductase (MTHFR) (rs1801133), low density lipoprotein receptor-related protein 1 (LRP1) (rs1466535), angiotensin converting enzyme (ACE) (rs1799752) and several MMP9 SNPs with functional effects on the expression or function were determined by analysis of the genomic DNA. Results AAA subjects were classified as slow-growth rate- (<3.25 mm /yr; n=81) vs. aggressive-AAA (growth rate >3.25 mm /yr, those presenting with a rupture, or those with maximal aortic diameter >5.5 cm (male) or >5.0 cm (female); n=60) and discriminating confounds between the groups identified by logistic regression. Analyses identified MMP9 p-2502 SNP (P=0.029, OR=0.54 (0.31-0.94)) as a significant confound discriminating between control- vs. slow-growth AAA, MMP-9 D165N (P=0.035) and LRP1 (P=0.034) between control vs. aggressive-AAA, and MTHFR (P=0.048, OR=2.99 (1.01-8.86)), MMP9 p-2502 (P=0.037, OR=2.19 (1.05-4.58), and LRP1 (P=0.046, OR= 4.96 (1.03-23.9)) as the statistically significant confounds distinguishing slow- vs. aggressive-AAA. Conclusion Logistic regression identified different genetic confounds for the slow-growth rate-and aggressive-AAA indicating a potential for different

  1. Methanol toxicity and formate oxidation in NEUT2 mice.

    SciTech Connect

    Cook, R. J.; Champion, K. M.; Giometti, C. S.; Biosciences Division; Vanderbilt Univ.

    2001-09-15

    NEUT2 mice are deficient in cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH; EC 1.5.1.6) which catalyzes the oxidation of excess folate-linked one-carbon units in the form of 10-formyltetrahydrofolate to CO{sub 2} and tetrahydrofolate. The absence of FDH should impair the oxidation of formate via the folate-dependent pathway and as a consequence render homozygous NEUT2 mice more susceptible to methanol toxicity. Normal (CB6-F1) and NEUT2 heterozygous and homozygous mice had essentially identical LD50 values for methanol, 6.08, 6.00, and 6.03 g/kg, respectively. Normal mice oxidized low doses of [{sup 14}C]sodium formate (ip 5 mg/kg) to {sup 14}CO{sub 2} at approximately twice the rate of homozygous NEUT2 mice, indicating the presence of another formate-oxidizing system in addition to FDH. Treatment of mice with the catalase inhibitor, 3-aminotriazole (1 g/kg ip) had no effect on the rate of formate oxidation, indicating that at low concentrations formate was not oxidized peroxidatively by catalase. High doses of [{sup 14}C]sodium formate (ip 100 mg/kg) were oxidized to {sup 14}CO{sub 2} at identical rates in normal and NEUT2 homozygous mice. Pretreatment with 3-aminotriazole (1 g/kg ip) in this instance resulted in a 40 and 50% decrease in formate oxidation to CO2 in both normal and homozygous NEUT2 mice, respectively. These results indicate that mice are able to oxidize formate to CO{sub 2} by at least three different routes: (1) folate-dependent via FDH at low levels of formate; (2) peroxidation by catalase at high levels of formate; and (3) by an unknown route(s) which appears to function at both low and high levels of formate. The implications of these observations are discussed in terms of the current hypotheses concerning methanol and formate toxicity in rodents and primates.

  2. Association of Thymidylate Synthase Polymorphisms with Acute Pancreatitis and/or Peripheral Neuropathy in HIV-Infected Patients on Stavudine-Based Therapy

    PubMed Central

    Torres, Ferran; Salazar, Juliana; Gutierrez, Maria del Mar; Mateo, Maria Gracia; Martínez, Esteban; Domingo, Joan Carles; Fernandez, Irene; Villarroya, Francesc; Ribera, Esteban; Vidal, Francesc; Baiget, Montserrat

    2013-01-01

    Background Low expression thymidylate synthase (TS) polymorphism has been associated with increased stavudine triphosphate intracellular (d4T-TP) levels and the lipodystrophy syndrome. The use of d4T has been associated with acute pancreatitis and peripheral neuropathy. However, no relationship has ever been proved between TS polymorphisms and pancreatitis and/or peripheral neuropathy. Methods We performed a case-control study to assess the relationship of TS and methylene-tetrahydrofolate reductase (MTHFR) gene polymorphisms with acute pancreatitis and/or peripheral neuropathy in patients exposed to d4T. Student’s t test, Pearson’s correlations, one-way ANOVA with Bonferroni correction and stepwise logistic regression analyses were done. Results Forty-three cases and 129 controls were studied. Eight patients (18.6%) had acute pancreatitis, and 35 (81.4%) had peripheral neuropathy. Prior AIDS was more frequent in cases than in controls (OR = 2.36; 95%CI 1.10–5.07, P = 0.0247). L7ow expression TS and MTHFR genotype associated with increased activity were more frequent in patients with acute pancreatitis and/or peripheral neuropathy than in controls (72.1% vs. 46.5%, OR = 2.97; 95%CI: 1.33–6.90, P = 0.0062, and 79.1% vs. 56.6%, OR = 2.90, 95%CI: 1.23–7.41, P = 0.0142, respectively). Independent positive or negative predictors for the development of d4T-associated pancreatitis and/or peripheral neuropathy were: combined TS and MTHFR genotypes (reference: A+A; P = 0.002; ORA+B = 0.34 [95%CI: 0.08 to 1.44], ORB+A = 3.38 [95%CI: 1.33 to 8.57], ORB+B = 1.13 [95%CI: 0.34 to 3.71]), nadir CD4 cell count >200 cells/mm3 (OR = 0.38; 95%CI: 0.17–0.86, P = 0.021), and HALS (OR = 0.39 95%CI: 0.18–0.85, P = 0.018). Conclusions Low expression TS plus a MTHFR genotype associated with increased activity is associated with the development of peripheral neuropathy in d4T-exposed patients. PMID:23468971

  3. The association of DNA damage response and nucleotide level modulation with the antibacterial mechanism of the anti-folate drug Trimethoprim

    PubMed Central

    2011-01-01

    Background Trimethoprim is a widely prescribed antibiotic for a variety of bacterial infections. It belongs to a class of anti-metabolites - antifolates - which includes drugs used against malarial parasites and in cancer therapy. However, spread of bacterial resistance to the drug has severely hampered its clinical use and has necessitated further investigations into its mechanism of action and treatment regimen. Trimethoprim selectively starves bacterial cells for tetrahydrofolate, a vital cofactor necessary for the synthesis of several metabolites. The outcome (bacteriostatic or bactericidal) of such starvation, however, depends on the availability of folate-dependent metabolites in the growth medium. To characterize this dependency, we investigated in detail the regulatory and structural components of Escherichia coli cellular response to trimethoprim in controlled growth and supplementation conditions. Results We surveyed transcriptional responses to trimethoprim treatment during bacteriostatic and bactericidal conditions and analyzed associated gene sets/pathways. Concurrent starvation of all folate dependent metabolites caused growth arrest, and this was accompanied by induction of general stress and stringent responses. Three gene sets were significantly associated with the bactericidal effect of TMP in different media including LB: genes of the SOS regulon, genes of the pyrimidine nucleotide biosynthetic pathway and members of the multiple antibiotic resistance (mar) regulon controlled by the MarR repressor. However, the SOS response was identified as the only universal transcriptional signature associated with the loss of viability by direct thymine starvation or by folate stress. We also used genome-wide gene knock-out screen to uncover means of sensitization of bacteria to the drug. We observed that among a number of candidate genes and pathways, the effect of knock-outs in the deoxyribose nucleotide salvage pathway, encoded by the deoCABD operon and

  4. [Pharmacogenetics and pharmacogenomics].

    PubMed

    Bourel, Michel; Ardaillou, Raymond

    2006-01-01

    Genetic polymorphisms can lead to drug adverse effects because certain allelic variants of genes that encode enzymes, targets or carriers involved in drug metabolism, are associated with an increase or a loss of function. Drug metabolism takes place essentially in the liver and is regulated by phase I enzymes (including several cytochrome P450 isoenzymes), the role of which is to make drug metabolites more polar by hydroxylation, and by phase II enzymes that catalyse conjugation reactions. Cytochromes P450 isoenzymes control 80% of oxidative reactions, owing to their low substrate specificity. The most extensively studied polymorphisms are those of CYP2D6 and CYP2C9, which are frequent and affect the metabolism of many drugs. For example, several CYP2C9 gene variants are associated with lower activity of the corresponding enzyme, potentially leading to drug overdose. Thiopurine methyl transferase and UDP-glucuronyl transferase are phase II enzymes that conjugate respectively 6-mercaptopurine metabolites with a methyl radical and metabolites of irinotecan (an anti-tumour drug) with a glucuronyl radical. Mutations in the corresponding genes can, through a loss of function, lead to excessively high levels of active metabolites, with a risk of bone marrow aplasia. The action of vitamin K antagonists is influenced by polymorphisms of vitamine K epoxyde reductase, the target molecule of vitamin K. A mutation in the methylene tetrahydrofolate reductase gene diminishes the folate pool and thereby increases the effects of methotrexate, a folic acid antagonist. Resistance to the anti-platelet effect of aspirin can be due to polymorphisms that affect other platelet aggregation pathways. Genotyping results must be confirmed by phenotyping, which examines the rate of transformation of a drug into its metabolites and shows whether the increase or decrease in this rate is linked to a specific polymorphism. Somatic mutations in malignant tumours can modify the response to

  5. Folic acid metabolism in vitamin B12-deficient sheep. Depletion of liver folates

    PubMed Central

    Smith, Richard M.; Osborne-White, William S.

    1973-01-01

    1. Metabolism of folate was studied in six ewes in an advanced state of vitamin B12 deficiency as judged by voluntary food intake and in their pair-fed controls receiving vitamin B12. A group of four animals that were maintained throughout the experiment at pasture was also studied. 2. After 34–40 weeks on the cobalt-deficient diet urinary excretion of formiminoglutamate by four deficient animals was about 3.2mmol/day and this was not significantly decreased by injection of three of them with about 4.5μg of [2-14C]folate/kg body weight per day for 5 days. Three days after the last injection retention of [2-14C]folate by the livers of the deficient animals (5.5% of the dose) was lower than that of their pair-fed controls (26% of the dose) but there was no evidence of net retention of injected folate in the livers of either group. Urinary excretion of 14C indicated that renal clearance of folate may have been impaired in very severe vitamin B12 deficiency. 3. As estimated by microbiological assays total folates in the livers of animals at pasture (12.9μg/g) included about 24% of 5-methyltetrahydrofolate as compared with about 72% of a total of 12.5μg/g in three further ewes fed on a stock diet of wheaten hay-chaff and lucerne-chaff. Liver folates of vitamin B12-deficient animals (0.5μg/g) included about 88% of 5-methyltetrahydrofolate as compared with about 51% of a total of 5.2μg/g in pair-fed animals treated with vitamin B12. 4. Chromatography of liver folates of the pair-fed animals permitted quantitative estimates of the pteroylglutamates present. The results showed that the vitamin B12-deficient livers were more severely depleted of tetrahydrofolates and formyltetrahydrofolates than of methyltetrahydrofolates and that as the deficiency developed they were more severely depleted of the higher polyglutamates than of the monoglutamate within each of these classes. Results from animals injected with [2-14C]folate indicated an impairment of the exchange

  6. Polymorphisms of Pyrimidine Pathway Enzymes Encoding Genes and HLA-B*40∶01 Carriage in Stavudine-Associated Lipodystrophy in HIV-Infected Patients

    PubMed Central

    Pruvost, Alain; Torres, Ferran; Salazar, Juliana; Gutierrez, Maria del Mar; Domingo, Joan Carles; Fernandez, Irene; Villarroya, Francesc; Vidal, Francesc; Baiget, Montserrat; de la Calle-Martín, Oscar

    2013-01-01

    Purpose To assess in a cohort of Caucasian patients exposed to stavudine (d4T) the association of polymorphisms in pyrimidine pathway enzymes and HLA-B*40∶01 carriage with HIV/Highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome (HALS). Methods Three-hundred and thirty-six patients, 187 with HALS and 149 without HALS, and 72 uninfected subjects were recruited. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Polymorphisms in the thymidylate synthase (TS) and methylene-tetrahydrofolate reductase (MTHFR) genes were determined by direct sequencing, HLA-B genotyping by PCR-SSOr Luminex Technology, and intracellular levels of stavudine triphosphate (d4T-TP) by a LC-MS/MS assay method. Results HALS was associated with the presence of a low expression TS genotype polymorphism (64.7% vs. 42.9%, OR = 2.43; 95%CI: 1.53–3.88, P<0.0001). MTHFR gene polymorphisms and HLA-B*40∶01 carriage were not associated with HALS or d4T-TP intracellular levels. Low and high expression TS polymorphisms had different d4T-TP intracellular levels (25.60 vs. 13.60 fmol/106 cells, P<0.0001). Independent factors associated with HALS were(OR [95%CI]: (a) Combined TS and MTHFR genotypes (p = 0.006, reference category (ref.): ‘A+A’; OR for ‘A+B’ vs. ref.: 1.39 [0.69–2.80]; OR for ‘B+A’ vs. ref.: 2.16 [1.22–3.83]; OR for ‘B+B’ vs. ref.: 3.13, 95%CI: 1.54–6.35), (b) maximum viral load ≥5 log10 (OR: 2.55, 95%CI: 1.56–4.14, P = 0.001), (c) use of EFV (1.10 [1.00–1.21], P = 0.008, per year of use). Conclusion HALS is associated with combined low-expression TS and MTHFR associated with high activity polymorphisms but not with HLA-B*40∶01 carriage in Caucasian patients with long-term exposure to stavudine. PMID:23840581

  7. Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30

    PubMed Central

    Genthe, Nicholas A; Thoden, James B; Benning, Matthew M; Holden, Hazel M

    2015-01-01

    The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N10-formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido-d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed β-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a β-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate. PMID:25752909

  8. Effect of halogen substitutions on dUMP to stability of thymidylate synthase/dUMP/mTHF ternary complex using molecular dynamics simulation.

    PubMed

    Kaiyawet, Nopporn; Rungrotmongkol, Thanyada; Hannongbua, Supot

    2013-06-24

    The stability of the thymidylate synthase (TS)/2-deoxyuridine-5-monophosphate (dUMP)/5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) ternary complex formation and Michael addition are considered as important steps that are involved in the inhibition mechanism of the anticancer prodrug 5-fluorouracil (5-FU). Here, the effect of three different halogen substitutions on the C-5 position of the dUMP (XdUMPs = FdUMP, CldUMP, and BrdUMP), the normal substrate, on the stability of the TS/dUMP and TS/dUMP/mTHF binary and ternary complexes, respectively, was investigated via molecular dynamics simulation. The simulated results revealed that the stability of all the systems was substantially increased by mTHF binding to the catalytic pocket. In the ternary complex, a much greater stabilization of the dUMP and XdUMPs through electrostatic interactions, including charge-charge and hydrogen bond interactions, was found compared to mTHF. An additional unique hydrogen bond between the substituted fluorine of FdUMP and the hydroxyl group of the TS Y94 residue was observed in both the binary and ternary complexes. The distance between the S(-) atom of the TS C146 residue and the C6 atom of dUMP, at <4 Å in all systems, suggested that a Michael addition with the formation of a S-C6 covalent bond potentially occurred, although the hydrogen atom on C6 of dUMP is substituted by a halogen atom. The MM/PBSA binding free energy revealed the significant role of the bridging waters around the ligands in the increased binding affinity (∼10 kcal/mol) of dUMP/XdUMP, either alone or together with mTHF, toward TS. The order of the averaged binding affinity in the ternary systems was found to be CldUMP ≈ FdUMP > dUMP > BrdUMP, suggesting that CldUMP could be a potent candidate TS inhibitor, the same as FdUMP (the metabolite form of 5-FU). PMID:23705822

  9. Polymorphisms of folate metabolic genes and susceptibility to bladder cancer: a case-control study.

    PubMed

    Lin, Jie; Spitz, Margaret R; Wang, Yunfei; Schabath, Matthew B; Gorlov, Ivan P; Hernandez, Ladia M; Pillow, Patricia C; Grossman, H Barton; Wu, Xifeng

    2004-09-01

    Epidemiological studies have shown an association between low folate intake and an increased cancer risk. Major genes involved in folate metabolism include methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS). We investigated joint effects of polymorphisms of the MTHFR (677 C-->T, 1298A-->C) and MS genes (2756 A-->G), dietary folate intake and cigarette smoking on the risk of bladder cancer in a case-control study. The study population consisted of 457 bladder cancer patients and 457 healthy controls, matched to the cases in terms of age, gender and ethnicity. Genotype data were analyzed in a subset of 410 Caucasian cases and 410 controls. Compared with individuals carrying the MTHFR 677 wild-type (CC) and reporting a high folate intake, those carrying the variant genotype (CT or TT) and reporting a low folate intake were at a significantly 3.51-fold increased risk of bladder cancer (95% CI: 1.59-6.52). In contrast, individuals carrying a variant genotype and reporting a high folate intake were at only a 1.39-fold increased risk (95% CI: 0.71-2.70), and those carrying the wild-type and reporting a low folate intake were at only 1.56-fold increased risk (95% CI: 0.82-2.97). The interaction between genetic polymorphisms and folate intake was significant on the multiplicative scale (P = 0.01). When analyzed in the context of smoking status, compared with never smokers with the MTHFR 677 wild-type, the risk increased to 6.56-fold (95% CI: 3.28-13.12) in current smokers carrying the variant genotype. Analyses of the MTHFR 1298, MS 2756 genes revealed similar results. In addition, age at cancer onset in former smokers increased as the proportion of the heteromorphic haplotype in the individual increased (P = 0.005). Our results strongly suggest that polymorphisms of the MTHFR and MS genes act together with low folate intake and smoking to increase bladder cancer risk. These results have important implications for cancer prevention in susceptible

  10. Heterotrimeric NADH-Oxidizing Methylenetetrahydrofolate Reductase from the Acetogenic Bacterium Acetobacterium woodii

    PubMed Central

    Bertsch, Johannes; Öppinger, Christian; Hess, Verena; Langer, Julian D.

    2015-01-01

    ABSTRACT The methylenetetrahydrofolate reductase (MTHFR) of acetogenic bacteria catalyzes the reduction of methylene-THF, which is highly exergonic with NADH as the reductant. Therefore, the enzyme was suggested to be involved in energy conservation by reducing ferredoxin via electron bifurcation, followed by Na+ translocation by the Rnf complex. The enzyme was purified from Acetobacterium woodii and shown to have an unprecedented subunit composition containing the three subunits RnfC2, MetF, and MetV. The stable complex contained 2 flavin mononucleotides (FMN), 23.5 ± 1.2 Fe and 24.5 ± 1.5 S, which fits well to the predicted six [4Fe4S] clusters in MetV and RnfC2. The enzyme catalyzed NADH:methylviologen and NADH:ferricyanide oxidoreductase activity but also methylene-tetrahydrofolate (THF) reduction with NADH as the reductant. The NADH:methylene-THF reductase activity was high (248 U/mg) and not stimulated by ferredoxin. Furthermore, reduction of ferredoxin, alone or in the presence of methylene-THF and NADH, was never observed. MetF or MetVF was not able to catalyze the methylene-THF-dependent oxidation of NADH, but MetVF could reduce methylene-THF using methyl viologen as the electron donor. The purified MTHFR complex did not catalyze the reverse reaction, the endergonic oxidation of methyl-THF with NAD+ as the acceptor, and this reaction could not be driven by reduced ferredoxin. However, addition of protein fractions made the oxidation of methyl-THF to methylene-THF coupled to NAD+ reduction possible. Our data demonstrate that the MTHFR of A. woodii catalyzes methylene-THF reduction according to the following reaction: NADH + methylene-THF → methyl-THF + NAD+. The differences in the subunit compositions of MTHFRs of bacteria are discussed in the light of their different functions. IMPORTANCE Energy conservation in the acetogenic bacterium Acetobacterium woodii involves ferredoxin reduction followed by a chemiosmotic mechanism involving Na

  11. Relationship of the 1793G-A and 677C-T polymorphisms of the 5,10-methylenetetrahydrofolate reductase gene to coronary artery disease.

    PubMed

    Kebert, Cory B; Eichner, June E; Moore, William E; Schechter, Eliot; Yaoi, Takuro; Vogel, Steve; Allen, Richard A; Dunn, S Terence

    2006-01-01

    Numerous studies have investigated the relationship between polymorphisms, in particular 677C-T and 1298A-C, of the methylene-tetrahydrofolate reductase (MTHFR) gene and coronary artery disease (CAD) with conflicting results. This study investigates the potential association of two point mutations in MTHFR, 677C-T and 1793G-A, along with other risk factors, with CAD. This is the first hospital-based study to investigate 1793G-A in this context. Genotype analysis was performed on 729 Caucasians and 66 African Americans undergoing coronary angiography using a novel PCR-based assay involving formation of Holliday junctions. Allelic frequencies for 677C-T were 66.2% C and 33.8% T for Caucasians and 90.9% C and 9.1% T for African Americans. With respect to the 1793G-A polymorphism, allelic frequencies were 94.7% G and 5.3% A for Caucasians and 99.2% G and 0.8% A for African Americans. Disease associations were examined in the Caucasian patients due to their greater genotype variability and larger number in the patient cohort. Results suggest that neither 677CT heterozygotes (OR-1.36; 95% CI 0.95 to 1.96) nor mutant homozygotes (OR-0.73; 95% CI 0.44 to 1.20) have either an increased or decreased risk for CAD compared to the 677CC genotype. Likewise, the 1793GA genotype did not demonstrate a statistically significant association with CAD compared to 1793GG patients (OR-0.79; 95% CI 0.47 to 1.33). Mean homocysteine levels (mumol/L) increased from normal to mutant for 677C-T (677CC: 10.2; 677CT: 11.0; 677TT: 11.6) and normal to heterozygous in 1793G-A (1793GG: 10.7; 1793GA: 11.5). These MTHFR polymorphisms did not contribute to the prediction of clinically defined CAD in Caucasians. PMID:17264399

  12. Relationship of the 1793G-A and 677C-T Polymorphisms of the 5,10-Methylenetetrahydrofolate Reductase Gene to Coronary Artery Disease

    PubMed Central

    Kebert, Cory B.; Eichner, June E.; Moore, William E.; Schechter, Eliot; Yaoi, Takuro; Vogel, Steve; Allen, Richard A.; Dunn, S. Terence

    2006-01-01

    Numerous studies have investigated the relationship between polymorphisms, in particular 677C-T and 1298A-C, of the methylene-tetrahydrofolate reductase (MTHFR) gene and coronary artery disease (CAD) with conflicting results. This study investigates the potential association of two point mutations in MTHFR, 677C-T and 1793G-A, along with other risk factors, with CAD. This is the first hospital-based study to investigate 1793G-A in this context. Genotype analysis was performed on 729 Caucasians and 66 African Americans undergoing coronary angiography using a novel PCR-based assay involving formation of Holliday junctions. Allelic frequencies for 677C-T were 66.2% C and 33.8% T for Caucasians and 90.9% C and 9.1% T for African Americans. With respect to the 1793G-A polymorphism, allelic frequencies were 94.7% G and 5.3% A for Caucasians and 99.2% G and 0.8% A for African Americans. Disease associations were examined in the Caucasian patients due to their greater genotype variability and larger number in the patient cohort. Results suggest that neither 677CT heterozygotes (OR-1.36; 95% CI 0.95 to 1.96) nor mutant homozygotes (OR-0.73; 95% CI 0.44 to 1.20) have either an increased or decreased risk for CAD compared to the 677CC genotype. Likewise, the 1793GA genotype did not demonstrate a statistically significant association with CAD compared to 1793GG patients (OR-0.79; 95% CI 0.47 to 1.33). Mean homocysteine levels (μmol/L) increased from normal to mutant for 677C-T (677CC: 10.2; 677CT: 11.0; 677TT: 11.6) and normal to heterozygous in 1793G-A (1793GG: 10.7; 1793GA: 11.5). These MTHFR polymorphisms did not contribute to the prediction of clinically defined CAD in Caucasians. PMID:17264399

  13. Association of MTHFR genetic polymorphisms with venous thromboembolism in Uyghur population in Xinjiang, China

    PubMed Central

    Li, Zhao; Yadav, Umesh; Mahemuti, Ailiman; Tang, Bao-Peng; Upur, Halmurat

    2015-01-01

    Background: The aim of this study was to reveal the association between Methylene tetrahydrofolate reductase (MTHFR) gene mutations (C677T, A1298C and C1317T) and risk of venous thromboembolism (VTE) in Han and Uyghur population in Xinjiang. Material and method: We conducted a case control study composed of 246 cases, including 86 Uyghur and 160 Han ethnic diagnosed VTE were admitted in the First Affiliated Hospital of Xinjiang Medical University between January 2008 to December 2012, and 292 population including 122 Uyghur ethnic and 170 Han ethnic were studied as controls. To detect the polymorphism of MTHFR gene C677T, A1298T, and C1317T, Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied. Fluorescence polarization immunoassay was adopted to determine the plasma levels Homocysteine (Hcy), folic acid and vitaminB12 (VitB12). The association of the polymorphism of MTHFR and levels Hcy, folic acid and VitB12 with VTE was analyzed. Results: The MTHFR gene C677T genotypes distribution in Uyghur VTE patients and control groups were: TT (27.91% vs. 12.29%), CT (41.86% vs. 52.46%) and CC (30.23% vs. 35.25%), respectively; and in Han VTE patients and control groups were: TT (27.49% vs. 14.71%), CT (44.38% vs. 53.53%) and CC (28.13% vs. 31.76%), respectively, and there were significant differences in TT genotype of MTHFRC677T between VTE patients and controls in both Uyghur and Han ethnic (Uyghur: x2=8.070, P=0.005; Han: x2=8.159, P=0.004). However, there were no significant differences in the MTHFR gene A1298T and C1317T genotyping distribution frequency in Uygur and Han ethnic between VTE patients and controls (P>0.05). Plasma levels of Hcy in MTHFR gene TT genotype were statistically higher than CT and CC genotype (P<0.05). After adjusting for age, gender, smoking, hypertension, hyperlipidemia, diabetes and MTHFR genotype for plasma Hcy levels, multifactor logistic regression analysis showed (OR=1.025, 95% CI 1.003-1.046, P=0

  14. Mthfr as a modifier of the retinal phenotype of Crb1(rd8/rd8) mice.

    PubMed

    Markand, Shanu; Saul, Alan; Tawfik, Amany; Cui, Xuezhi; Rozen, Rima; Smith, Sylvia B

    2016-04-01

    Mutations in crumb homologue 1 (CRB1) in humans are associated with Leber's congenital amaurosis (LCA) and retinitis pigmentosa (RP). There is no clear genotype-phenotype correlation for human CRB1 mutations in RP and LCA. The high variability in clinical features observed in CRB1 mutations suggests that environmental factors or genetic modifiers influence severity of CRB1 related retinopathies. Retinal degeneration 8 (rd8) is a spontaneous mutation in the Crb1 gene (Crb1(rdr/rd8)). Crb1(rdr/rd8) mice present with focal disruption in the outer retina manifesting as white spots on fundus examination. Mild retinal dysfunction with decreased b-wave amplitude has been reported in Crb1(rdr/rd8) mice at 18 months. Methylene tetrahydrofolate reductase (MTHFR) is a crucial enzyme of homocysteine metabolism. MTHFR mutations are prevalent in humans and are linked to a broad spectrum of disorders including cardiovascular and neurodegenerative diseases. We recently reported the retinal phenotype in Mthfr-deficient (Mthfr(+/-)) heterozygous mice. At 24 weeks the mice showed decreased RGC function, thinner nerve fiber layer, focal areas of vascular leakage and 20% fewer cells in the ganglion cell layer (GCL). Considering the variability in CRB1-related retinopathies and the high occurrence of human MTHFR mutations we evaluated whether Mthfr deficiency influences rd8 retinal phenotype. Mthfr heterozygous mice with rd8 mutations (Mthfr(+/-)(rd8/rd8)) and Crb(rd8/rd8) mice (Mthfr(+/+rd8/rd8)) mice were subjected to comprehensive retinal evaluation using ERG, fundoscopy, fluorescein angiography (FA), morphometric and retinal flat mount immunostaining analyses of isolectin-B4 at 8-54 wks. Assessment of retinal function revealed a significant decrease in the a-, b- and c-wave amplitudes in Mthfr(+/-)(rd8/rd8) mice at 52 wks. Fundoscopic evaluation demonstrated the presence of signature rd8 spots in Mthfr(+/+rd8/rd8) mice and an increase in the extent of these rd8 spots in Mthfr

  15. Folate-homocysteine interrelations: potential new markers of folate status.

    PubMed

    Lucock, M D; Daskalakis, I; Schorah, C J; Lumb, C H; Oliver, M; Devitt, H; Wild, J; Dowell, A C; Levene, M I

    1999-05-01

    We report a transient drop in plasma Hcy and Cys following a single oral dose of PteGlu. The thiol change was concomitant with both the peak plasma 5CH3H4PteGlu1 level (by HPLC) and the maximum plasma Lactobacillus casei activity which reflects absorption of unmodified PteGlu. The significant reciprocal association of Hcy with radioassay RBC folate (r = -0.28, 99% CI -0.48, -0.05, P = 0.0016), serum folate (r = -0.37, 99% CI -0.56, -16, P = 0.0001), and vitamin B12 (r = -0.42, 99% CI -0.59, -21, P = 0.0001) is shown and reflects the long-term nutritional effect of B vitamins on this important, potentially atherogenic thiol. These are now well-established associations. We extend the potential for investigation of folate metabolism in health and disease by evaluating a range of new folate indices which are based on erythrocyte coenzymes. These have been looked at independently and in association with established parameters. Erythrocyte methylfolates (mono- to hexaglutamate-5CH3H4PteGlu1-6), formylfolates (tri- to pentaglutamate-5CHOH4PteGlu3-5),formiminotetrahydrofolate (formiminoH4PteGlu1), unsubstituted tetrahydrofolate (H4PteGlu1), andpara-aminobenzoylglutamate (P-ABG) have been measured by HPLC with fluorescence detection. A positive linear association exists between (i) H4PteGlu1 and radioassay RBC folate (r = 0.50, 99% CI 0. 07, 0.77, P = 0.0036), and (ii) H4PteGlu1 and tetraglutamates of both formyl- and methylfolate (r = 0.52, 99% CI 0.10, 0.78, P = 0. 0022, and r = 0.56, 99% CI 0.15, 0.80, P = 0.0009, respectively). Since, in addition, a reciprocal linear association exists between Hcy and tetraglutamyl formylfolate (r = -0.41, 99% CI -0.73, 0.05, P = 0.0206), erythrocyte tetraglutamates may be a good reflection of the bodies' active coenzyme pools. Pentaglutamyl formylfolate, the longest oligo-gamma-glutamyl chain form of this coenzyme may be a good indicator of folate depletion. The abundance of this coenzyme both increases with increasing Hcy (r = 0